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Graduate Theses and Dissertations Graduate School

January 2015

Neuroinflammatory Alterations via CD-36 inTraumatic Brain InjuryDiana G. Hernandez-OntiverosUniversity of South Florida, dagho07@hotmail.com

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Scholar Commons CitationHernandez-Ontiveros, Diana G., "Neuroinflammatory Alterations via CD-36 in Traumatic Brain Injury" (2015). Graduate Theses andDissertations.http://scholarcommons.usf.edu/etd/5699

Neuroinflammatory Alterations via CD-36 in Traumatic Brain Injury

by

Diana G. Hernandez-Ontiveros

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy of Medical Sciences Department of Molecular Pharmacology and Physiology

with a Concentration in Neurosciences Morsani College of Medicine University of South Florida

Major Professor: Paula C. Bickford, Ph.D. Andreas G. Seyfang, Ph.D.

Dominic P. D’Agostino, Ph.D. Daniel Kay-Pong Yip, Ph.D.

Date of Approval: July 8, 2015

Keywords: Fatty acid translocase, inflammation, oxidized low density lipoprotein, macrophages, soluble receptor of advanced end glycation

Copyright © 2015, Diana G. Hernandez-Ontiveros

DEDICATION

I would like to dedicate this work to my parents and family members who have been a

propelling force and anchor throughout my entire professional education in the USA. In addition

to my parents, Francisco and Carmen, I would like to also give recognition to my grandparents,

Paca and Rogelio, for their love, and for playing an active role in my education, and

encouragement to pursue my academic goals. At the same time, to my brother Cesar for his

friendship, constant protection and optimism since we left our hometown. I am deeply thankful

to my husband Anthony, and the “Russo-Scaglione clan” for their love, affection, help, and

motivation to continue moving forward with my life aspirations. I am very grateful and fortunate

for being able to find friends who have helped me along the road: “Muchísimas gracias Coqui

P., el Deland G., el Kentucky G., Amanda G., Drashti, and M. Li.” My profound admiration and

appreciation to all these significant people in my life, who in one way or another have

contributed to my professional education.

ACKNOWLEDGMENTS

I would like to state my sincere gratitude to my mentor Dr. Paula C. Bickford, for allowing

me to learn under her mentorship, for her continuous advice and patience to mentor me

throughout my graduate studies in order to piece together this dissertation and doctorate

degree. This academic endeavor was possible through a common effort from many

collaborators. Thus, I would like to acknowledge every lab member with whom I have teamed up

over the past five years to acquire research experience in the field of neuroscience. Their

contribution has been very valuable in all activities related to this dissertation project. I would

like to thank: faculty, post-docs, graduate and undergraduate students, USF lab technicians and

staff, and all volunteers who have dedicated their time and effort to help complete my

dissertation, in particular Arum Yoo, Lyanne M. Suarez, Jenny Kim, Christian Cerecedo, Daniela

Aguirre Raigoza, Diego Lozano, and Stephanny Reyes. My special thanks to Dr. Mibel Pabon,

Dr. Jared Ehrhart, Dr. Josh Morganti, Dr. Naoki Tajiri, Dr. Byeong Cha, and Amanda Garces for

being there throughout my graduate school, for cheering and encouraging me to improve myself

in my studies and scientific skills. Finally, I particularly would like to thank all faculty members of

my committee: Dr. Dominic P. D’Agostino, Dr. Andreas G. Seyfang, Dr. Daniel K.P. Yip, and Dr.

Stanley M. Stevens Jr., who kindly steered me in the right direction with their useful criticism in

order to improve my knowledge in this field and the quality of this dissertation. My special

thanks to Dr. Eric S. Bennett and Dr. Christopher C. Combie for their support and concern on

the final stages of my dissertation. All of these individuals inspire me to continue expanding my

scientific knowledge and my research abilities in the medical and neurosciences fields.

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TABLE OF CONTENTS

List of Figures ............................................................................................................................ iv Abstract...................................................................................................................................... vi Chapter 1: Introduction ................................................................................................................ 1

1.1 Traumatic Brain Injury (TBI) ....................................................................................... 1 1.1.1 Societal Burden Costs ............................................................................... 10 1.1.2 Gaps in Basic Science Knowledge of TBI ................................................. 10 1.1.3 Gaps in Translational and Clinical Knowledge in TBI ................................ 11 1.2 Cell Death Mechanisms Associated with Secondary Damage in TBI ...................... 13 1.2.1 Neuroinflammation .................................................................................... 17 1.3 CD-36, Fatty Acid Translocase ................................................................................ 21 1.4 Role of CD-36 in Atherosclerosis ............................................................................ 22 1.5 Role of CD-36 in Stroke .......................................................................................... 24 1.6 Role of CD-36 in TBI ............................................................................................... 27 1.7 Role of CD-36 in Other Neurodegenerative Diseases ............................................. 29 1.7.1 Alzheimer’s Disease .................................................................................. 29

1.7.2 Cerebral Amyloid Angiopathy .................................................................... 31 1.7.3 Parkinson’s Disease ............................................................................................ 32

Chapter 2: Materials and Methods ............................................................................................ 34 2.1 Animals ................................................................................................................... 35 2.2 Surgical Procedures ............................................................................................... 35 2.3 Histology.................................................................................................................. 36 2.4 Tissue Collection for Protein Analysis ...................................................................... 37 2.5 Immunohistochemistry ............................................................................................. 38 2.5.1 CD-36/MCP-1 immunohistochemistry in Brain .......................................... 38 2.5.2 CD-36/Iba-1 immunohistochemistry in Brain ............................................. 38 2.5.3 CD-36/NeuN immunohistochemistry in Brain ............................................ 39 2.5.4 CD-36/GFAP immunohistochemistry in Brain ............................................ 39 2.5.5 CD-36 immunohistochemistry in Spleen .................................................... 40 2.6 Laser Scanning Confocal Microscopy ..................................................................... 40 2.7 Fluorescencent Microscopy ..................................................................................... 40 2.8 Statistical Analysis on Indirect Immunofluorescence in Brain .................................. 41 2.9 Statistical Analysis on Fluorescence Intensity in Spleen .......................................... 41 2.10 Tissue Processing ................................................................................................ 41 2.11 Western Blot ......................................................................................................... 42 2.12 Statistical Analysis on Western Blot ...................................................................... 42 2.13 Cell Culture ........................................................................................................... 43 2.13.1 Measurement of Cell Viability: Calcein-AM Fluorescence Dye ................ 43 2.13.2 Measurement of Mitochondrial Activity: MTT Assay ................................ 44

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2.14 Preliminary in vitro Experiments............................................................................. 44 2.14.1 In Vitro study CD-36 and sRAGE expression .......................................... 44 2.14.2 CD-36, Spleen and TBI ........................................................................... 44 2.14.3 TBI and CD-36 Immune Response in Neonatal Rats .............................. 45 2.14.4 sRAGE Modulates CD-36 Expression in Neonatal Spleen and Brain after TBI ........................................................................................ 45

Chapter 3: Inflammatory Role of CD-36 in an Animal Model of TBI .......................................... 46 3.1 Introduction ........................................................................................................... 46

3.1.1 Why CD-36 as a Biomarker of Inflammation? ........................................... 46 3.1.2 Inflammatory Pathways Related to CD-36 ................................................. 47

3.1.3 Neuroinflammation in TBI Accompanied by CD-36 Expression in Brain and Spleen ...................................................................................... 48 3.2 Specific Materials and Methods ............................................................................. 49

3.2.1 Surgical Procedures ................................................................................. 49 3.2.2 Immunohistochemistry of Brain ................................................................ 50 3.2.3 Immunohistochemistry of Spleen ............................................................. 51 3.2.4 Statistical Analysis on Indirect Immunofluorescence in Brain .................... 52 3.2.5 Statistical Analysis of Fluorescence Intensity in Spleen ............................ 53 3.2.6 Western Blot ............................................................................................. 53 3.2.7 Statistical Analysis on Western Blot .......................................................... 54

3.3 Results .................................................................................................................. 55 3.3.1 CD-36 Expression in the TBI Brain ........................................................... 55 3.3.2 CD-36 in the TBI Spleen .......................................................................... 61 3.3.3 Western Blot ............................................................................................ 67 3.3.3.1 Protein Detection in the Brain Cortex ......................................... 67 3.3.3.2 Protein Detection in the Spleen ................................................. 67 3.4 Discussion ............................................................................................................. 72

Chapter 4: Pharmacological Interventions Designed to Abrogate TBI Related Inflammation May Improve Functional Outcome ................................................................................. 78

4.1 Introduction ............................................................................................................ 78 4.2 Specific Materials and Methods .............................................................................. 79 4.2.1 Measurement of Cell Viability: Calcein-AM fluorescence dye .................... 80 4.2.2 Measurement of mitochondrial activity: MTT assay .................................. 80 4.2.3 Data Analysis ........................................................................................... 81 4.3 Results .................................................................................................................. 81 4.4 Discussion ............................................................................................................. 82

Chapter 5: Stem Cell Therapy in Combination with sRAGE May Ameliorate Neuroinflammation in an In-Vitro Cell Model of TBI ............................................................. 88

5.1 Introduction ........................................................................................................... 88 5.2 Specific Materials and Methods .............................................................................. 90 5.2.1 Measurement of Cell Viability: Calcein-AM Fluorescence Dye .................. 91 5.2.2 Measurement of Mitochondrial Activity: MTT Assay ................................. 91 5.2.3 Data Analysis ............................................................................................ 92 5.3 Results .................................................................................................................. 92 5.4 Discussion ............................................................................................................. 93

Chapter 6: Discussion ............................................................................................................... 98

6.1 Relevance of CD-36 ................................................................................................ 98

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6.2 Inflammation and TBI............................................................................................... 98 6.3 Effects of sRAGE in an in Vitro Cell Model of Inflammation .................................. 102 6.4 Therapeutic Effects of Combination Stem Cell Therapy and sRAGE ..................... 103 References ............................................................................................................................ 107 Appendix A: Publications ........................................................................................................ 120 Appendix B: Publisher’s Permission to use Figure 1 ............................................................... 122 About the Author ....................................................................................................... END PAGE

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LIST OF FIGURES

Figure 1: Mechanisms associated with secondary damage in TBI ......................................... 15 Figure 2: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 24 hours post-TBI ................................................................................................ 56 Figure 3: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 48 hours post-TBI ................................................................................................ 57 Figure 4: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 7 days post-TBI.................................................................................................... 58 Figure 5: Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 60 days post-TBI .................................................................................................. 59 Figure 6: A-D Co-localization of CD-36/NeuN at 24 hours after TBI ....................................... 60 Figure 7: A-D Minimal co-localization of CD-36/ GFAP at 24 hours after TBI ....................... 62 Figure 8: Spleen CD-36 immunodetection at 24 hours post TBI ............................................ 63 Figure 9: Spleen CD-36 immunodetection at 48 hours post TBI ............................................ 64 Figure 10: Spleen CD-36 immunodetection at 7 days post TBI. ............................................... 65 Figure 11: Spleen CD-36 immunodetection at 60 days post TBI .............................................. 66 Figure 12: CD-36 brain cortex protein expression at 24 hours post-TBI ................................... 68 Figure 13: CD-36 brain protein expression 48 hours post-TBI .................................................. 69 Figure 14: CD-36 protein expression in spleen 24 hours post-TBI ........................................... 70 Figure 15: CD-36 protein expression in spleen of 48 hours post-TBI ........................................ 71 Figure 16: CD-36 protein expression in spleen of 7 days post-TBI. .......................................... 72 Figure 17: In Vitro hNP1 cells relative cell viability Calcein assay using sRAGE. ..................... 83 Figure 18: In Vitro hNP1 cells relative metabolic activity MTT assay using sRAGE. ................. 84 Figure 19: In Vitro hNP1 cells relative cell viability Calcein assay using stem cells. ................. 94

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Figure 20: In Vitro hNP1 cells relative metabolic activity MTT assay using stem cells .............. 95

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ABSTRACT

Traumatic brain injury (TBI) has become an increasingly unmet clinical need due to

intense military conflicts worldwide. Directly impacted brain cells suffer massive death, with

neighboring cells succumbing to progressive neurodegeneration accompanied by inflammatory

and other secondary cell death events. Subsequent neurodegenerative events may extend to

normal areas beyond the core of injury, thereby exacerbating the central nervous system’s

inflammatory response to TBI. Recently CD-36 (cluster of differentiation 36/fatty acid

translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in

macrophages, has been implicated in lipid metabolism, atherosclerosis, oxidative stress, and

tissue injury in cerebral ischemia, and in certain neurodegenerative diseases.

Accordingly, we proposed that CD-36 has a pivotal role in the neuroinflammatory

cascade that further contributes to the pathology of TBI. First, we explored the

neuroinflammatory role of CD-36 after acute and chronic stages of TBI. Second, we employed a

neuroinflammatory model to test the therapeutic effect of the soluble receptor of advanced end-

glycation product (sRAGE); previously shown to abrogate increased CD-36 expression in

stroke. Third, we further examined ameliorating TBI related inflammation as a therapeutic

pathway by combination of stem cell therapy and sRAGE. At acute stages of TBI, we observed

brain co-localization of CD-36, monocyte chemoattractant protein 1 (MCP-1) and ionized

calcium-binding adapter molecule 1 (Iba-1) on impacted cortical areas, significant increases of

CD-36 and MCP-1 positive cells in the ipsilateral vs. contralateral hemispheres of TBI animals in

acute, but no significant increases of Iba-1 expressing cells over time. In early acute stages of

TBI immunoblotting showed overexpression of CD-36 in brain cortex when comparing ipsilateral

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and contralateral hemispheres vs. sham. Spleen CD-36 protein expression at acute post-TBI

stages showed no significant difference between TBI and sham groups. In addition,

immunohistochemistry revealed minimal CD-36 detection on the cortical area of impact on our

chronic group. Spleen immunohistochemistry also showed co-localization of CD-36 and MCP-1

in the red pulp of spleen in acute stages of TBI animals when compared to sham. Ongoing

ischemic and hyperlipidemic rodent models suggest that infiltrating monocytes/macrophages

from the periphery are the major source of CD-36 in the post-ischemic brain. Likewise, CD-36

expressing monocytes in the spleen after TBI may suggest its role in peripheral immune

response, which may exacerbates the inflammatory response after TBI. Therefore, CD-36 may

play a key role as a pathological link between inflammation and TBI.

Our results suggest an intimate involvement of CD-36 mediated inflammation in TBI,

providing novel insights into the understanding of disease neuroinflammation and as a potent

therapeutic target for TBI treatment. The critical timing (i.e., 24-48 hours) of CD-36 expression

(from downregulation to upregulation) may signal the transition of functional effects of this

immune response from pro-survival to cell death. This observed dynamic CD-36 expression

also suggests the therapeutic window for TBI. The detection of CD-36 expression in brain areas

proximal, as well as distal, to the site of impacted injury suggests its role in both acute and

progressive evolution of TBI. CD-36 neuroinflammatory role has clinical relevance for treating

patients who have suffered any TBI condition at acute and chronic stages.

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CHAPTER 1

INTRODUCTION

1.1 Traumatic Brain Injury (TBI)

Traumatic brain injury (TBI) results from a blast, an impact, or acceleration/deceleration

to the head causing cognitive, psychological, neurological, and anatomical alterations in the

brain. The brain suffers massive cell loss; neighboring cells undergo progressive

neurodegeneration, inflammatory and cell death events. If subsequent incidents are not

controlled on time, they may extend to normal areas beyond the core of injury; exacerbating the

central nervous system’s immune response. In recent years, TBI has drawn major public

attention due to intense military conflict worldwide, sport related injuries, and among the toddler

and senior populations. The CDC has also reported that about 53,000 people in the United

States die from TBI-related causes every year (Coronado, Xu et al. 2011).This number does not

include injuries seen at military or Veterans Health Administration health facilities. The National

Institute of Neurological Disorders and Stroke (NINDS) reports every year approximately 1.4

million people experience a TBI, 50,000 people die from head injury, 1 million head-injured

people are treated in hospital emergency rooms, and approximately 230,000 people are

hospitalized for TBI and survive.

According to the Defense and Veterans Brain Injury Center, in 2010 mild TBI accounted

for over 77.8% of all TBI injuries sustained during combat. Whether injury results from a blast or

a mechanical force to the head, it causes brain damage to different extents, with varying

severity mainly in frontal and temporal lobes of the brain (Suh, Kolster et al. 2006; Kraus,

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Susmaras et al. 2007; Hayashi, Kaneko et al. 2009). Equally debilitating, multiple exposures to

minor events may also lead to long-term TBI symptomatic sequelae (Elder, Mitsis et al. 2010),

such as cognitive, behavioral, and physical impairments, after TBI. It is estimated that 10% to

20% of veterans returning from war have suffered a traumatic brain injury as a result of

improvised explosive devices (Elder and Cristian 2009). Thus, urgent care and effective

treatment should be available to TBI patients in the short and long term to treat their head

injuries. More than 1.6 million soldiers have served in the wars in Iraq Operation Iraqi Freedom

(OIF) and Afghanistan Operation Enduring Freedom (OEF) (Tanielian T. 2008.). In both military

operations, TBI has been a major cause of mortality and morbidity, with blast-related injury the

most common cause (Elder and Cristian 2009).As of today there is no effective therapy for TBI

that has been approved by any health agency in the world. TBI defies conventional methods for

diagnosis and therapy development because of its heterogeneity and complexity. Immediate

conventional care and treatment to patients after TBI include: oxygen supply to the brain and

the rest of the body, maintaining adequate blood flow, controlling blood pressure, and

rehabilitation. Diagnosis and prognosis of a TBI patient with mild to moderate injuries may

undergo imaging testing: skull and neck X-rays to check for bone fractures or spinal instability.

In moderate to severe TBI cases, computed tomography (CT) scan is the signature.

Pharmacological treatment for moderate to severe TBI includes: medicines for pain, psychiatric

medications, such as antidepressants, and psychotherapy (DiTommaso, Hoffman et al. 2014;

Dobry, Novakovic et al. 2014). This treatment is based off those TBI patients who also present

conditions such as post-concussive syndrome (PCS) and posttraumatic stress disorder

(PTSD).According to the reports by the Eunice Kennedy Shriver National Institute of Child

Health and Human Development (NICHD), approximately 40% of all TBI patients develop PCS.

This syndrome is more common among people who have a pre-existing psychiatric problem,

such as depression or anxiety, before suffering a TBI (NINDS, 2012). The Substance Abuse

and Mental Health Services Administration in 2010 reports as complications: motor deficits,

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cognitive disabilities and mood disorders, such as anxiety, depression, and post-traumatic

stress disorder.

There is no effective therapy available that can cure TBI due to the complex cascade of

secondary events that lead to multiple sequelae. Yet, among the secondary injury mechanisms,

there is one which may not in itself detrimental: inflammation. It is a “necessary evil” by which

the body tries to regain equilibrium. Studies supporting this healing vision describe

inflammation, in most cases, as a well coordinated set of connections operating at an

intermediate time scale between neural and longer-term endocrine processes (Vodovotz, Csete

et al. 2008); and necessary for the removal of challenges to the organism and subsequent

restoration of homeostasis (Nathan 2002; Kadl and Leitinger 2005; Pate, Damarla et al. 2010).

Nonetheless, inflammation in many disease conditions is described as a double-edged sword

(Cole 1986; Smith 1994; Hagemann, Balkwill et al. 2007; Doyle and Buckwalter 2012;

Cerecedo-Lopez, Kim-Lee et al. 2014). In plain words, if inflammation had a zodiac sign it would

be a “Gemini”, Gemini are a mix of the yin and the yang, love and hate, peace and war, etc.

Inflammation in the brain can exert detrimental and healing effects. The inherent function of

inflammation is to recruit various immune cell types into the site of the injury to remove

damaged tissue and cellular debris allowing further creation of scar tissue (Cerecedo-Lopez,

Kim-Lee et al. 2014). The main surveillance immune cell responsible for responding to an injury

within the central nervous system (CNS) is microglia. Microglia activation shows three

profiles/phenotypes depending on the stimuli: classical activation, alternative activation, and

acquired deactivation. Classical activation is associated with pro-inflammatory mechanisms,

whereas the other two are promoters of anti-inflammatory mechanisms (Luo and Chen 2012).

The ambivalent property of inflammation is what makes it more valuable over other secondary

cell death events which do not have a two way street to reverse and shift from damage to repair.

The pathological consequences of TBI cannot be encompassed by the available

pharmacological, cognitive, and behavioral diagnostic tools. These tools are deficient because

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of the broad range of TBI conditions with divergent cell death mechanisms. In order to improve

care of patients with TBI we suggest the identification of inflammatory markers that shift and

promote inflammation towards repair, keeping in mind age and gender differences. For

example, in cell and animal models of cerebral ischemia, therapies designed to down regulate

inflammatory pathways are more effective in male models compared to female models (Chen,

Toung et al. 2005; Bushnell, Hurn et al. 2006). Therefore, it is relevant to keep in mind

preclinical and clinical research to help explain age and gender differences in disease to

optimize diagnosis, treatment and a favorable clinical outcome. Altogether, these findings

suggest the identification of novel inflammatory markers that promote inflammation towards

repair, taking into consideration important variables, such as age and gender that can impact

clinical outcomes.

Intense research in the past decade suggests that TBI is associated with conditions that

cause the breakdown of brain cells, such as Alzheimer’s disease (AD), Parkinson’s Disease

(PD), and chronic traumatic encephalopathy (Abisambra and Scheff 2014; Chauhan 2014;

Lucke-Wold, Turner et al. 2014). These neurodegenerative diseases have a strong

neuroinflammatory component. And interestingly enough, complications such as chronic

neuroinflammation and neurodegeneration have also been associated to TBI (Viscomi and

Molinari 2014). Thus, these neuroinflammatory and degenerative components link TBI to certain

neurodegenerative disorders. Further understanding of this neuroinflammatory process and cell

death pathways in TBI could explain neurodegeneration in AD and PD patients. In parallel, a

similar inflammatory and cell injury pathway in a neurodegenerative disease may also become

activated in TBI patients. Overall, studying the neuroinflammatory process is likely to elucidate

pathological mechanisms associated with the disease, as well as provide novel pathways in

treating TBI and related neurodegenerative disorders.

In addition to conventional TBI treatment and standard rehabilitation, novel therapeutic

approaches have been introduced to promote cell survival and arrest TBI-induced cell death.

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Proposed therapies attempt to reduce inflammation, promote neuronal survival, and rescue

injured neurons from cell death. example,, studies in acute spinal cord injury (SCI) in humans

and rodents have shown overlapping cytokine profiles, interleukin 6 (IL-6), interleukin 8 (IL-8),

and monocyte chemoattractant protein 1(MCP-1), released in an SCI injury dependent manner

(Stammers, Liu et al. 2012) suggesting these molecules as potential targets to attenuate

microglia activation and inflammation. In addition, experiments in TBI human and rodent models

coincide in cytokine profiles for: interleukin 1β (IL-1β) detection in human CSF; IL-6, and

interleukin 10 (IL-10) detection in plasma and serum of humans and rodents (Bell, Kochanek et

al. 1997; Kamm, Vanderkolk et al. 2006; Woodcock and Morganti-Kossmann 2013; Yan,

Satgunaseelan et al. 2014).Furthermore, other novel drug therapies attempt tackle oxidative

stress, a mechanism of secondary brain injury. This approach suggests the use of agents such

as mitochondria-targeted antioxidants (melatonin and new mitochondria-targeted antioxidants),

nicotinamide adenine dinucleotide phosphate (NADPH) inhibitors (antioxidant vitamins and

apocynin), and other compounds having mainly antioxidant properties (hydrogen-rich saline,

sulforaphane, U-83836E, omega-3, and polyphenols) (Fernandez-Gajardo, Matamala et al.

2014). These different compounds have shown positive and promising results in rodent TBI

models. Melatonin at appropriate doses reduced the amount of lesion volume, the loss of

motor function, the expression of pro-apoptotic proteins, inducible nitric oxide synthase (iNOS),

and matrix metalloproteinases (MMPs) (Campolo, Ahmad et al. 2013). One example is

mitochondria-targeted antioxidants: (i) plastoquinone derivatives, and (ii) ubiquinone derivatives

at high doses showed: (i) a reduction in reactive oxygen species (ROS), and cortical lesion

volume (Liberman, Topaly et al. 1969; Antonenko, Avetisyan et al. 2008); (ii) and

neuroprotective effects (James, Cocheme et al. 2005; Murphy and Smith 2007)). Other

alternative agents showing neuroprotective effects in TBI rat models were the intravenous

administration of vitamin C (Polidori, Mecocci et al. 2001; Riordan, Riordan et al. 2004), and

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dietary supplementation of vitamin E treatment in TBI rat models (Aiguo, Zhe et al. 2010; Yang,

Han et al. 2013), and in patients with severe TBI (Razmkon, Sadidi et al. 2011).

Overall, the pharmacological therapies are partially effective; they seem to reduce pro

inflammatory cytokines, promote neuroprotection, and slight functional recovery, but their level

of efficacy has not improved dramatically the quality of life in a patient’s road to recovery. The

challenge that TBI drug therapy faces is to facilitate these agents to cross the BBB to treat

patients quickly in the ER, and in the clinic. The pharmacokinetics and pharmacodynamics of

the drug and their method of delivery are also factors that should be taken into consideration.

Thus, to gain definitive insight of: (i) the pharmacokinetics and pharmacodynamics of a

drug/agent, (ii) its capacity to penetrate the BBB, (iii) and any delivery parameters; scientists

could pursue a collaborative effort/project that compiles/encompasses above features in unison

with help of genomics, proteomics and metabolomics to identify “ideal therapeutic

biomarkers/drugs. This study considers a pharmacologic approach in understanding TBI

pathology and treatment, by using an inhibitory drug that will exert an anti-inflammatory effect.

Using an in-vitro cell model we will attempt an upstream inhibitory approach to block the

suggested inflammatory biomarker and observe its effects in the cells.

In the last 4 years two novel therapeutic agents were introduced into human clinical trials

for their potential efficacy in TBI: progesterone and glyburide. The first agent is progesterone.

The hypothesis that progesterone has neuroprotective effects was introduced nearly 25 years

ago in a rat model of TBI (Roof, Hoffman et al. 1997). Next, a small pilot human TBI study

followed to assess progesterone’s safety, which showed neuroprotective effects as seen in

animal studies (Wright, Bauer et al. 2001; Pettus, Wright et al. 2005; Stein and Wright 2010).

The endogenous properties of progesterone made it an optimal therapeutic TBI candidate.

Progesterone is present in the brain already, besides being a hormone that supports pregnancy;

it plays a role in brain development in men (Wagner 2006) and women (Wright, Kellermann et

al. 2007); it has no problems crossing the BBB due to its steroidal nature and regenerative

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properties (He, Hoffman et al. 2004; Bali, Arimoto et al. 2012; Sitruk-Ware and El-Etr 2013).

Perhaps the most notorious feature of progesterone is its rapid mechanism of action to exert

anti-inflammatory, neuroprotective and regenerative effects in the CNS, and systemically,

creating a therapeutic window to treat patients. Recent human Phase III clinical trial, Protect,

seems very promising to elucidate progesterone’s efficacy when administered within hours of

brain injury to protect neurons and tissues from secondary damage mechanisms.

The second therapeutic agent in an undergoing clinical trial is the intravenous form (IV)

of glyburide (RP-1127). Glyburide or glibenclamide is an inhibitor of sulfonyl urea receptor

1(SUR-1) widely used to treat type 2 diabetes (Quast, Stephan et al. 2004) . SUR are molecular

targets of antidiabetic drugs which act to promote insulin release from the pancreatic beta cells.

Another interesting feature of glyburide is that it has anti-inflammatory effects (Koh, Maude et al.

2011). Therefore, the clinical trial should focus on ADMET parameters (as noted below) of the

therapeutic agent, safety and efficacy of the drug after TBI, and on identifying what inflammatory

trades decrease after swelling and bleeding in brain. Thus, we intend to study the features of a

novel biomarker with a potential inflammatory role in TBI. We are interested in finding traits that:

(i) suggest inflammation and a therapeutic window of the biomarker after mild TBI; (ii) address a

particular therapeutic agent that could penetrate the BBB and influence the biomarker’s activity;

and (iii) note if the therapeutic agent has any notorious absorption, distribution, metabolism,

excretion, toxicity (ADMET) parameters. Thus, with the proposed pharmacologic approach in

characterizing the TBI-induced neuroinflammation, this study would be able to gain insights on

potential inflammatory biomarkers, as well as begin to understand putative therapeutic agents

(as discussed below) which may influence the neuroinflammatory response towards brain

repair.

A third therapeutic platform is the use of cytokines/chemokines alone or in combination

with stem cell therapy to channel the migration of stem cells to the brain. These chemotactic

approaches propose the use of neurotrophic factors to treat CNS diseases (Liu, Fawcett et al.

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2001; Thorne and Frey 2001) and as guidance for stem cells from the periphery to the site of

brain injury, thereby allowing efficient brain bioavailability of the grafted cells’ secreted

therapeutic molecules (Borlongan 2011; Borlongan, Glover et al. 2011). Such inflammation-

mediated cell migration suggest that a modest cytokine/chemokine upregulation aids stem cells

in reaching their brain injured target areas (Hernandez-Ontiveros, Tajiri et al. 2013). Scientists

and clinicians should keep in mind the adverse effects of disturbing the delicate equilibrium

between pro-inflammatory and anti-inflammatory cytokines in microglial function.

Accumulating evidence has demonstrated the potential of stem cell therapy to treat TBI

and other neurodegenerative conditions. The principle behind this is to utilize the proliferative

capacity of stem cells for the appropriate neurological condition. Transplanted stem cells appear

to work by direct cell replacement and by neurotrophic effects, resulting in immunomodulation

and upregulation of endogenous stem cells (Sanberg, Eve et al. 2012). Yet, a contingency plan

should exist to determine the suitable cell type, whether to perform allogeneic or autologous

transplants based on the neurological disorder, and the level of stimulation of the host’s immune

system to the graft. Autologous stem cell transplants are preferable over allogeneic ones

because they have minimum risk of graft rejection since they are from the same patient from

which they were initially taken. In contrast, allogeneic transplants occur when the stem cells

come from another donor with closely matched immune markers, but the recipient’s immune

system may still cause an abrupt reaction with immediate rejection, severe consequences and

possibly death. Studies in the stem cell field support the two schools of thought in stem cell

repair: (i) direct cell replacement by exogenous stem cells into the damaged region of the brain,

and (ii) trophic factor stimulation of endogenous stem cells within neurogenic niches or recruited

from the bone marrow through peripheral circulation (Borlongan, Glover et al. 2011; Shinozuka,

Dailey et al. 2013). It is very likely that injected stem cells are able to migrate from the

bloodstream to the brain because the injured brain sends out chemical signals that recruit stem

cells. Additionally, stem cells have an easier way coming in since the injured brain has a

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compromised BBB. A third mechanism of action proposes a regenerative long-distance

migration of host cells from the neurogenic niche to the injured brain site can be achieved

through transplanted stem cells serving as “bio-bridges” for initiation of endogenous repair

mechanisms (Tajiri, Kaneko et al. 2013). All three mechanisms have a promising future, as they

could be translated in the clinical arena to treat patients who have suffered from TBI and other

neurodegenerative diseases.

Stem cell therapy needs optimization in the clinic. Due to the safety issues associated

with transplanting stem cells, testing donors and recipients to ascertain immune incompatibility,

will require quality assurance and control in handling, storing, and performing the surgical

techniques for the delivery of stem cells to minimize any mortality and morbidity risks. Equally

challenging in advancing novel therapies to the clinic include reproducibility of safe and effective

outcomes that will lead to robust and stable functional recovery in transplanted TBI patients. To

this end, there is a need to achieve prompt approval, regulatory guidelines, and accreditation by

the Food and Drug Administration (FDA) and existing health entities. Both of the proposed anti-

inflammatory and stem cell therapies aim to rescue dying neurons, ameliorate secondary injury

mechanisms, which translate to alleviate the symptoms and improve the quality of life of TBI

patients. We envision that our proposed anti-inflammatory agent will be screen on blood or

plasma of TBI patients arriving to the ER, and based on the described therapeutic window, treat

patients with therapeutic agents that abrogate neuroinflammation. Both therapeutic approaches

can be combined to potentiate synergistic effects that could decrease extent of the inflammatory

signaling pathways activated during TBI. A recent study proposes to stimulate and mobilize

endogenous stem/progenitor cells from the bone marrow in combination with an anti-

inflammatory agent granulocyte colony stimulating factor (G-CSF) (Acosta, Tajiri et al. 2014).

This team found out that transplanted stem cells influence the endogenous stem cells by

secreting neurotrophic factors, anti-inflammatory cytokines, overall regulating the milieu

associated with chronic neuroinflammation found in TBI. It appears that combinational therapy

10

of grafted stem cells with anti-inflammatory agents will be a more viable tool than monotherapy

of anti-inflammatory agents alone. Based on above study, in order to see improved behavioral

outcome it is necessary: (i) a successful grafted stem cells integration into the recipient brain

tissue; (ii) induction of an endogenous repair mechanism in the host; and (iii) a collaborative

brain repair process mutually solicited by grafted stem cells and endogenous ones.

1.1.1 Societal Burden Costs

The high rates of mild traumatic brain injury and posttraumatic stress disorder in current

operations are of significant concern for the long-term health of US veterans with associated

economic implications. From the estimated 1.7 million people who sustain a TBI in a year,

personal and social costs associated with TBI are between 9 and 10 billion dollars annually

(NIH, 1999). According to NINDS, total TBI costs in the USA exceed $56 billion a year. Thus,

development and testing of novel therapeutics and devices in the short and long term is a

priority to treat patients and help them regain functional recovery and rehabilitation. One factor

to keep into consideration is the therapeutic window to treat a patient with TBI. Using the

concept of combined therapy we increase our chances of finding an adequate drug therapy to

reduce excessive annual costs associated with TBI. We can also reduce healthcare and

hospitalization costs by educational campaigns for the prevention of TBI injuries among seniors,

soldiers and children. Advances in protective gear may also lower the risk for TBI. As more

sensitive and practical biomarkers are introduced in the clinical setting, it is likely that the

societal costs and burden for TBI will be minimized. To this end, this study desired to advance

our basic science knowledge in testing a potential biomarker for TBI.

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1.1.2 Gaps in Basic Science Knowledge of TBI

TBI is a multifaceted condition, not a single event. Current therapeutic strategies attempt

to limit primary and secondary brain damage that occurs within days of a head trauma. It is

difficult to stop the progression of neuronal loss and the cascade of secondary cell death events

after TBI. Countless years of research and experience have shown, almost in all CNS

pathologies, it is very unlikely that a single therapy may protect from degeneration (Viscomi and

Molinari 2014). Among the various secondary injury mechanisms, we found neuroinflammation.

Multiple studies suggest that neuroinflammation, in the form of microglial activation, is a

mechanism underlying chronic neurodegenerative processes after traumatic brain injury (Smith,

Gentleman et al. 2013). Our study focuses in identifying any inflammatory alterations associated

with CD-36 in TBI. The gap in basic science knowledge is the lack of understanding of the

neuroinflammatory process in TBI. There are numerous inflammatory signaling pathways that

we do not understand. One of these inflammatory signaling pathways may involve CD-36. This

biomarker has been proposed as a mediator of inflammation in certain metabolic and

degenerative disorders, such as hypercholesterolemia, atherosclerosis, stroke, AD, PD, and

dementia pugilistica. We hypothesize that CD-36 plays an inflammatory role in TBI pathology.

This study hopes to reduce the gap in knowledge of the neuroinflammatory process with other

molecular processes in TBI. We expect to reduce the gap in knowledge by addressing any

temporal profile of CD-36 activation/expression in acute and chronic stages of TBI. In addition,

we may have found a therapeutic window to test a pharmacological agent suitable for inhibiting

CD-36. Yet, we recognize that different strategies have to be explored and combined to target

neuroinflammation but other secondary mechanisms of damage after TBI.

1.1.3 Gaps in Translational and Clinical Knowledge in TBI

Despite the knowledge we have in the pathological mechanisms of TBI, current

advances in medicine has failed to translate into a single successful clinical trial treatment that

12

addresses all the complications that TBI patients face. Part of the challenge may be attributable

to the broad classification of TBI as mild, moderate and severe. In addition, there is also a

translational gap in the range of doses (concentrations) of a drug that elicits a therapeutic

response, known as therapeutic window. Diagnostic tools, such as imaging and proteomic

biomarkers are not as useful without defining a therapeutic window to treat TBI patients.

Delineating the therapeutic window for acute and chronic stages of TBI will reduce the number

of misdiagnosed TBI patients in the ER; capture more TBI patients, and provide with more

specific or elaborate tests that provide accurate cognitive and motor assessment in TBI patients.

At the same time, we are trying to identify novel biomarkers as therapeutic targets after brain

injury in acute and chronic TBI stages. Our novel biomarker may serve as an indicator of short

and, or prolonged stages of inflammation after TBI. In addition, the proposed novel biomarker

may be feasible for screening patients in the ER room immediately after TBI, or later after injury,

and it could serve as a prognosis and diagnostic tool in a patient’s rehabilitation process. The

translation gap in knowledge of TBI is a deficiency in diagnosis, especially when severity is mild

or moderate, as in the case of concussion or slight head trauma.

In the clinical arena, there are many novel and promising therapeutic agents which have

failed to demonstrate they can promote functional and neuroprotective effects. More evidence

needs to be compiled to assess its efficacy; whether in animal, and/or human TBI clinical trials.

Despite exciting pre-clinical results, more than 30 phase III prospective clinical trials have failed

to display significance for their primary outcome (Narayan, Michel et al. 2002; Schouten 2007;

Maas, Roozenbeek et al. 2010). Results from pre-clinical trials have not been translated into

successful clinical trials due to: (i) the pathophysiological heterogeneity of TBI patients, (ii) lack

of sufficient pharmacokinetic analysis for determination of optimal dose, (iii) and therapeutic

window of the target compounds. The general challenge we face, one that is addressed in this

study, is to find a novel therapeutic agent that favorably shifts the inflammatory response from

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damage to repair, thereby promoting neuronal survival and functional recovery in TBI within

clinical relevant parameters.

Finally, a gap in knowledge that also needs attention is the methodology differences

between animal, pre-clinical, and clinical studies. A great procedural disparity exists when

transitioning from animals to humans. Procedural factors such as (i) the influence of anesthetic

drugs (used in TBI models) on the primary outcomes of the study, (ii) potential drug/anesthetic

interactions (Statler, Alexander et al. 2006), (iii) methodological analysis of results, (iv) the use

of genetically identical populations in TBI models, and (v) the time course of pathophysiological

and therapeutic effects, among others (Fernandez-Gajardo, Matamala et al. 2014). In this study

we hope to provide evidence that serves to establish a time course profile of our novel

biomarker that helps establish a therapeutic window to treat TBI. Here you are alluding to a

likely disconnect between the basic science and the clinic and that animal modeling of TBI

needs to closely mimic the disease. You need to clearly state this gap. Current animal models

of TBI cannot identically replicate the human TBI model due to certain procedural factors.

Nevertheless, the CCI model is chosen mainly because it produces: (i) a pronounced cortical

contusion, (ii) morphological and cerebrovascular injury responses that resemble various

aspects (e.g. edema, inflammation, BBB disruption) of human TBI, and (iii) neurobehavioral and

cognitive impairments like those seen in human patients (Dixon and Kline 2009).

1.2 Cell Death Mechanisms Associated with Secondary Damage in TBI

This section will cover secondary mechanisms of injury: edema, inflammation, neuron

excitotoxicity, mitochondrial dysfunction, apoptosis and necrosis. Current animal models of TBI

include a biomechanical procedure known as controlled cortical impact (CCI) (LaPlaca, Simon

et al. 2007; Tehranian, Rose et al. 2008). These techniques are employed in an attempt to

reproduce some of the pathology present in human TBI. For example, in the CCI model animals

endure an initial necrotic process in the cortical tissue and white matter axonal injury, followed

14

by apoptosis in surrounding tissue due to secondary events such as edema, ischemia,

excitotoxicity, altered gene expression, and other pathways depicted below (Figure 1). (Park,

Bell et al. 2008; Hayashi, Kaneko et al. 2009; Boone, Sell et al. 2012; Johnson, Stewart et al.

2012; Rovegno, Soto et al. 2012).

Figure 1 below depicts the complexity of the “secondary injury” mechanisms that

exacerbate the initial injury. These molecular pathways may act in parallel , and or interact

among them (Park, Bell et al. 2008). This chaotic scenario leaves us with a very narrow window

of opportunity to react. Once the immune system engages in this chaotic environment, it mounts

very abrupt microglial activation, recruiting surveillance monocytes; and other immune cells from

the periphery to the site of injury. Focal damage is typically seen around hemorrhagic lesions,

such as contusions within the gray matter or at gray-white junctions (Richardson, Singh et al.

2010); at the frontal and temporal poles and in the orbital frontal cortex (Gennarelli and Graham

1998). White matter—the part of the brain that provides long connections between different

parts of the grey matter or cortex—exhibits different patterns of deterioration compared with

grey matter (Park, Bell et al. 2008). Traumatic axonal injury is a common occurrence in both

focal and diffuse brain trauma regardless of injury severity (Cordobes, Lobato et al. 1986;

Gentleman, Roberts et al. 1995). Equally compelling evidence suggests that following the acute

phase of focal TBI and brain ischemic events, hippocampal neurons may be the most

vulnerable neurons in the brain, as they show the earliest evidence of neurodegeneration in

experimental models (McIntosh, Saatman et al. 1998) with the dentate gyrus displaying reduced

neurogenesis (Acosta, Tajiri et al. 2013) coinciding with profound memory impairment in both

human and animal models of TBI (Smith, Okiyama et al. 1991; Umile, Sandel et al. 2002). The

sequence of secondary cell death events is very complex and hard to follow due to the

numerous signaling pathways associated with inflammation and cell death.

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Figure 1. Mechanisms associated with secondary damage in TBI. The major pathways associated with the progression of secondary injury after a traumatic brain injury: M icrocirculatory derangements involve stenosis (1) and loss of microvasculature, and the blood–brain barrier may break down as a result of astrocyte foot processes swelling (2). Proliferation of astrocytes (“astrogliosis”) (3) is a characteristic of injuries to the central nervous system, and their dysfunction results in a reversal of glutamate uptake (4) and neuronal depolarization through excitotoxic mechanisms. In injuries to white and grey matter, calcium influx (5) is a key initiating event in a molecular cascades resulting in delayed cell death or dysfunction as well as delayed axonal disconnection. In neurons, calcium and zinc influx though channels in the AMPA and NMDAreceptors results in excitotoxicity (6), generation of free radicals, mitochondrial dysfunction and postsynaptic receptor modifications. These mechanisms are not ubiquitous in the traumatized brain but are dependent on the sub-cellular routes of calcium influx and the degree of injury. Calcium influx into axons (7) initiates a series of protein degradation cascades that result in axonal disconnection (8).Inflammatory cells also mediate secondary injury, through the release of pro-inflammatory cytokines (9) that contribute to the activation of cell-death cascades or postsynaptic receptor modifications (Park, Bell et al. 2008). Reprinted from (E. Park, J. D. Bell, A. J., Baker) (Traumatic Brain Injury can the consequences be stopped?, Figure 1.The major pathways associated with the progression of secondary injury after a traumatic brain injury) Canadian Medical Association Journal (April 22 2008, Volume 178 No. 9, pages 1163–1170). © Canadian Medical Association (2008). This work is protected by copyright and the making of this copy was with the permission of the Canadian Medical Association Journal (www.cmaj.ca) and Access Copyright. Any alteration of its content or further copying in any form whatsoever is strictly prohibited unless otherwise permitted by law.

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Is the basic science able to capture these secondary cell death mechanisms? Basic

science is not able to grass all the elements of the puzzle in secondary cell death events. The

two mechanisms that occur in TBI are necrosis, happening immediately after injury; and

neuronal cell death occurring in a delayed fashion. Both mechanisms have been documented in

ischemia, and TBI (Liou, Clark et al. 2003; Zhang, Chen et al. 2005). Since apoptosis is a

programmed cell death process, it is a much better candidate to target for therapeutic

approaches. Experimental studies point at two promising pharmacological agents: caspase

inhibitors to target caspase dependent apoptosis and poly ADP-ribose polymerase (PARP)

inhibitors to target caspase independent cell death (Zhang, Chen et al. 2005).Yet, the gap of

knowledge remains at which step in the apoptotic pathway is convenient to intervene, and a

convenient therapeutic window. Because activation of these pathways occurs in parallel and

peaks 1–3 days after injury, such strategies should be: both additive and synergistic; and

should show a relatively wide therapeutic window (Stoica and Faden 2010). Currently, there are

NIH clinical and translational experiments testing anti-apoptotic agents in cancer, but not in TBI

patients. Interestingly enough, a safe anti-apoptotic agent could be developed that enables us to

test its effects in TBI. For example, the selective inhibitors of nuclear export (SINE) compounds

that inhibit the function of the nuclear export protein exportin 1(XPO1) have the potential to

provide a novel targeted therapy that enable tumor suppressor proteins to remain in the nucleus

and promote apoptosis of cancer cells (Cheng, Holloway et al. 2014; London, Bernabe et al.

2014). The company Karyopharm has discovered XPO1-inhibiting SINE compounds, including

Selinexor and they believe that SINE compounds may have the potential to provide therapeutic

benefit in various cancers, autoimmune and inflammatory diseases, wound healing, HIV, and

influenza (Karyopharm, 2014). With respect to necrosis, little can be done to stop it, since the

sole impact of brain injury causes neuronal damage and death within minutes; yet those

neurons in the vicinity of injury can still be rescued pharmacologically or with stem cell therapy.

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More investigations are necessary to further document necrosis and any key molecules

within the apoptotic cascade as potential therapeutic targets to treat TBI.

1.2.1 Neuroinflammation

This section will focus on reviewing the inflammatory response associated with CD-36 in

an effort to characterize the pathological cascade and therapeutic window associated with this

inflammatory pathway. In the past decade studies have focused attention on neuroinflammation

in the form of microglial activation, as a mechanism of potential relevance to neurodegeneration

both in AD and in the response to brain injury (Griffin, Sheng et al. 1998; Gentleman, Leclercq

et al. 2004). Emerging evidence suggests that neuroinflammation and microglial activation in the

white matter may also contribute to ongoing cellular damage (Loane and Byrnes 2010), and it

may persist for years after injury in humans (Gentleman, Leclercq et al. 2004; Chen, Johnson et

al. 2009). After brain injury cytokines are released activating microglia, the degree of activation

reflecting the severity of the injury (Igarashi, Potts et al. 2007; Smith, Gentleman et al. 2013).

Microglial activation will lead to further cytokine release, including interleukin (IL)-1, possibly

secondary to elevated levels of adenosine-5′-triphosphate (ATP) released from damaged cells

(Di Virgilio 1995), with activation of purinergic P2X7 receptors on microglia (Ferrari, Chiozzi et

al. 1997). IL-1 is expressed in increased quantities in the cerebral cortex within hours of TBI

(Griffin, Sheng et al. 1994; Smith, Gentleman et al. 2013). Griffin et al., 1998 have proposed a

“cytokine cycle” in which traumatic brain injury, or other forms of brain injury, can, in susceptible

individuals, initiate an over-exuberant sustained inflammatory response that can result in

neurodegeneration (Griffin, Sheng et al. 1998).

The inflammatory response after TBI results in neuronal injury and disruption of the

blood-brain barrier (Smith DH 1997; Nagamoto-Combs, McNeal et al. 2007; Namas, Ghuma et

al. 2009). Microglial cells quickly become activated, they act as antigen presenting cells (APCs)

releasing pro-inflammatory cytokines and chemokines (Town, Nikolic et al. 2005; Cao, Thomas

et al. 2012). Activated microglia also produces nitric oxide (NO) and superoxide free radicals

18

that generate reactive oxygen species (ROS) and reactive nitrogen species (RNS). In animal

models of cortical controlled impact (CCI); fluid-percussion brain injury in rats; combined

unilateral lesion of the primary motor cortex and of the lateral pre-motor cortex in rhesus

monkeys, microglial cells remain in their activated state for at least one year, especially in the

thalamic area (Smith DH 1997; Nagamoto-Combs, McNeal et al. 2007; Nagamoto-Combs and

Combs 2010; Jacobowitz, Cole et al. 2012; Jin, Ishii et al. 2012). Recent experiments with

human postmortem tissue showed microglial activation 17 years after TBI in sub-cortical brain

areas (Ramlackhansingh, Brooks et al. 2011). These accrued reports suggest a chronic

inflammatory stage sustained by microglia.

A recent study by Smith et al., 2013 reinforces the hypothesis that the microglial

response to head injury may persist and provoke chronic neurodegeneration in humans. The

team depicted the time-course and magnitude of the microglial reaction in human post mortem

cases. Their major finding was a neuroinflammatory response developing within the first week

and persisting for several months after TBI, but has returned to control levels after several years

(Smith, Gentleman et al. 2013). In addition, they also examined cases with diffuse traumatic

axonal injury and found that microglial reaction is particularly pronounced in the white matter.

Altogether, the current literature reinforces the thought that prolonged microglial activation is a

hallmark of traumatic brain injury, but may also suggests that neuroinflammatory response

returns to control levels after several years based on the severity of the injury. The proposed

study will address any CD-36 related inflammatory alterations in an animal model of TBI at

acute and chronic stages. We will gain insight in the expression patterns of CD-36 in the brain

and spleen, which will support our hypothesis that CD-36 has a pivotal role in the

neuroinflammatory cascade at early stages, and possibly in chronic stages of TBI-related

inflammation. Exploiting this CD-36 mechanism as a candidate therapeutic target may abrogate

secondary cell death in TBI and promote functional and cognitive brain recovery.

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A role for CD-36 has been implicated in the pathogenesis of various diseases. However,

its role in traumatic brain injury (TBI) has not been examined. According to the National Health

and Nutrition Examination Surveys approximately 70% of adults are overweight or obese;

roughly 78 million adult Americans are considered obese (NHANES, 2009-2010). Interestingly,

CD-36 has recently being associated with inflammatory events in lipid metabolic disorders,

atherosclerotic plaque formation, and cerebral ischemia, TBI pathology, and Alzheimer’s

disease (Coraci, Husemann et al. 2002; Kunjathoor, Febbraio et al. 2002; Cho, Szeto et al.

2007; Kim, Febbraio et al. 2012; Pietka, Schappe et al. 2014). CD-36 shows high affinity toward

lipid-based ligands such as oxLDL, or mLDL and long chain-fatty acids among other ligands,

including thrombospondins, fibrillar β-amyloid, and membranes of cells undergoing apoptosis

(Martin et al., 2011; Abumrad et al., 1993; Savill, 1997; Febbraio et al., 2001; Medeiros et al.,

2004). Although the effect of CD-36 on atherogenesis is debatable (Moore et al., 2005; Moore

and Freeman, 2006), uptake of pro-inflammatory oxLDL or mLDL by macrophage CD-36 is a

critical step that leads to foam cell formation, atheroma, and a chronic pro-inflammatory state

(Febbraio et al., 2000; Podrez et al., 2002a,b; Rahaman et al., 2006; Guy et al., 2007).

The signaling pathways of CD-36 are not fully understood, yet research strongly

suggests it has a key inflammatory role in cardiovascular conditions. In the case of

atherosclerotic lesions CD-36 is expressed by phagocytes and vascular cells, likely candidates

to mediate the in vivo oxidation of LDL and the maintenance of the inflammatory process

(Marsche, Zimmermann et al. 2003; Greaves and Gordon 2009; Kannan, Sundaram et al.

2012). For instance LDL-HOCl induces chemokines release of monocytes and chemotactic

migration of neutrophils (Woenckhaus, Kaufmann et al. 1998), initiates the respiratory burst of

macrophages and stimulates polymorphonuclear leukocytes to an enhanced production of

superoxide anion radical and hydrogen peroxide (Nguyen-Khoa, Massy et al. 1999).

CD-36 is expressed by macrophages in the brain and periphery, playing a big role in the

inflammatory response. This scavenger receptor, internalizes cholesterol-laden modified

20

lipoproteins. Uncontrolled cholesterol accumulation in macrophages may result as an adaptive

mechanism in response to excessive lipid load and lead to foam cell formation (Febbraio, Guy

et al. 2004). These foam cell formation is part of the initial lesion in atherosclerosis, known as

fatty streak (Lusis 2000). Oxidative modification of low-density lipoprotein (LDL) has been

hypothesized to be a key event in the conversion of LDL to a pro-atherogenic form recognized

by macrophage scavenger receptors (Steinberg 1997; Kunjathoor, Febbraio et al. 2002). Thus,

internalization of oxidized LDL (oxLDL) by macrophages is an early and potentially pivotal event

which, if inhibited through intervention at the level of the scavenger receptor, may delay the

atherosclerotic process.

Furthermore, in an atherosclerotic model with chimeric mice lacking CD-36

macrophages, stem cell transplantation revealed that absence of macrophage CD-36 was

protective against atherosclerosis. These mice lacking CD-36 were protected against

atherosclerosis, they had 88.1% reduction of lesion area throughout the aortic tree (Febbraio,

Guy et al. 2004). Thus, our interest in CD-36 lies in the characterization of any inflammatory

alterations in an adult animal model of CCI. In controlled cortical injury models, selective

neuronal death has been well described in the hippocampus (Morales, Marklund et al. 2005);

but a diffuse axonal injury animal model showed that despite the proximity of traumatic axonal

injury to the neuronal somata, neuronal cell bodies do not show a pathological progression to

cell death, and in fact exhibit changes suggestive of reorganization and potential repair

(Singleton, Zhu et al. 2002).

In this study we would like to: (i) identify what brain regions express CD-36; (ii) label and

identify what cells are expressing CD-36, and which glial cells are associated with CD-36 in the

inflammatory response at the acute and chronic TBI stages. Spleen has an important role after

CNS injury, spleen is a big reservoir of macrophages that play a big role in the immune

response after stroke and brain injury (Rasouli, Lekhraj et al. 2011; Seifert, Hall et al. 2012;

Seifert, Leonardo et al. 2012; Schwulst, Trahanas et al. 2013). We are also interested in finding

21

any time-dependent CD-36 expression patterns in both organs after TBI. We are concerned

with identifying what brain regions are mainly associated with CD-36 expression. We expect that

this project of CD-36 helps elucidate its inflammatory process after TBI; any time dependent

CD-36 expression/activation will contribute in building up a therapeutic window to treat acute

and chronic TBI stages; it will serve as basis to design more experimental studies that can be

translated to pre-clinical studies that consider CD-36 as a potential therapeutic target to reduce

neuroinflammation in TBI and other disorders.

1.3 CD-36, Fatty Acid Translocase

We will define CD-36, the main functions of this molecule, and inflammatory links to lipid

metabolic disorders, and degenerative diseases. CD-36 is referred to as a type B scavenger

receptor. It is a member of a family of receptors which also includes SR-B1/CLA-1, a high

density lipoprotein receptor. Medgen reports CD-36 is expressed by monocyte/macrophages,

platelets, microvascular endothelial cells, and adipose tissues. CD-36 recognizes a broad

variety of ligands including oxLDL, anionic phospholipids, apoptotic cells, thrombospondin

(TSP), collagen, Plasmodium Falciparum-infected erythrocytes, and long-chain fatty acids.

Scavenger receptors are known to be involved in: innate immune responses (Peiser and

Gordon 2001; Kennedy, Chen et al. 2012), cell adhesion (Avril, Tripathi et al. 2012) ,

phagocytosis of apoptotic cells (Ren, Silverstein et al. 1995; Leelahavanichkul, Bocharov et al.

2012), and lipid uptake (Mitchell, On et al. 2011; Moulle, Cansell et al. 2012).

Other relevant functions of CD-36: serve as a receptor for thrombospondin in platelets

and various cell lines. It binds to collagen, thrombospondin, anionic phospholipids and oxLDL. It

directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes, and it

binds long chain fatty acids and may function in the transport and/or as a regulator of fatty acid

transport (Pietka, Schappe et al. 2014). Unlike class A receptors, which recognize the oxidized

apo-protein portion of the lipoprotein particle, CD-36 also binds to the lipid moiety of oxLDL.

22

Binding of oxLDL to CD-36-transfected cells is inhibited by anionic phospholipid vesicles.

Recently, CD-36 was also identified as the major receptor for LDL modified by monocyte-

generated reactive nitrogen species (Podrez, Febbraio et al. 2000). Thus, CD-36 is a

multifaceted molecule regulating lipid homeostasis, clearing out excess oxLDL in the periphery

through macrophage action

Any disturbance or imbalance to this pathway may result in defective lipid metabolism,

which may lead to metabolic diseases such as obesity and insulin resistance; cardiovascular

complications and heart disturbances such as stroke or a heart attack. Thus, if patients with

any of the mentioned predispositions also suffer any kind of TBI, the inflammatory component

may be further exacerbated via CD-36, which would be reflected by an up-regulation of CD-36

in the brain, which is the main thesis of this study.

1.4 Role of CD-36 in Atherosclerosis

This segment will cover the prevalence, pathology, and treatment of atherosclerosis;

followed by the role of CD-36 in lipid processing, and its inflammatory links with atherogenesis.

Cardiovascular disease is the major cause of mortality and morbidity in developed countries and

its prevalence is increasing in developing countries, and atherosclerosis is responsible for many

of the severe manifestations, including myocardial ischemia, acute myocardial infarction, heart

failure, and stroke (Sun 2014). Atherosclerosis is a disease in which plaque builds up inside

your arteries. Arteries are blood vessels that carry oxygen-rich blood to your heart and other

parts of your body. The plaque forms when fat, cholesterol, calcium, and other substances

found in the blood accumulate in the arteries. Over the course of time, the plaque hardens and

narrows arteries, limiting the flow of oxygen-rich blood to your organs and other parts of the

body. Atherosclerosis can lead to serious problems, including heart attack, stroke, or even

death.

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Atherosclerosis is primarily a degenerative disorder related to aging with a chronic

inflammatory component. It is well-known that an inflammatory process occurs within the arterial

wall at the site of a developing plaque (van der Wal, Becker et al. 1994; Kadar and Glasz 2001;

van der Meer, Iglesias del Sol et al. 2003). Multiple studies in the late 1980’s and early 1990’s

identified various cellular receptors involved in binding and internalization of mLDLs known as

“scavenger receptors.” Their physiological roles are still unclear; they presumably have a

significant role in atherosclerotic foam cell formation (Gown, Tsukada et al. 1986; Fogelman,

Van Lenten et al. 1988; Steinberg 1997; Steinberg, Gotto et al. 1999).The progression of early

atherosclerotic lesions to clinically relevant advanced atherosclerotic lesions occurs with

increased frequency in persons with risk factors for atherosclerotic disease (e.g.

hypercholesterolemia, hypertension, cigarette smoking) (Ip, Fuster et al. 1991). Nevertheless,

scavenger receptors, including CD-36, are thought to be most important early in the progression

of atherosclerosis during macrophage uptake of modified LDL and foam cell formation

(Nicholson, Han et al. 2001). Proposed mechanisms describe how LDL particles suffer an

oxidative modification in the vessel wall by reactive oxygen metabolites produced by monocytes,

neutrophils, and other cells in the developing lesion (Nicholson, Frieda et al. 1995; Nicholson,

Han et al. 2001; Nicholson 2004; Nicholson and Hajjar 2004). In vivo studies in the field of

atherosclerosis have identified key mechanisms related to the pathogenesis of this disease. For

example, genetically engineered murine models have been used to elucidate the contribution of

the different scavenger receptors, to identify specific ligands related to LDL modifications (Feng,

Han et al. 2000; Nicholson, Febbraio et al. 2000; Nicholson, Han et al. 2001). There has been

steady progress in our understanding of the regulation of expression of CD-36 and have

demonstrated that oxLDL can stimulate its own uptake by induction of CD-36 gene expression.

OxLDL upregulates CD-36 through a mechanism involving the activation of the transcription

factor, PPAR‐γ.

24

Macrophages are big players of the inflammatory response. They have a scavenger

function, internalizing cholesterol modified lipoproteins. Uncontrolled cholesterol accumulation in

macrophages may result as an adaptive mechanism in response to excessive lipid load and

lead to foam cell formation (Febbraio, Guy et al. 2004). These foam cell formation is part of the

initial lesion in atherosclerosis, known as fatty streak (Lusis 2000). Oxidative modification of low-

density lipoprotein (LDL) has been hypothesized to be a key event in the conversion of LDL to a

pro-atherogenic form recognized by macrophage scavenger receptors (Steinberg 1997;

Kunjathoor, Febbraio et al. 2002). Thus, internalization of oxidized LDL (oxLDL) by

macrophages is an early and potentially pivotal event which, if inhibited through intervention at

the level of the scavenger receptor, may delay the atherosclerotic process. An atherosclerotic

model with chimeric mice lacking CD-36 macrophages, stem cell transplantation revealed that

absence of macrophage CD-36 was protective against atherosclerosis. These mice lacking CD-

36 were protected against atherosclerosis, they had 88.1% reduction of lesion area throughout

the aortic tree (Febbraio, Guy et al. 2004). Thus, CD-36 is not just a lipid biomarker in

cardiovascular problems; it has an inflammatory component that would help establish a

pathological link between atherosclerosis, stroke, and TBI. Variations in CD-36 expression may

suggest a pathological role in TBI, and its co-localization with other inflammatory markers, such

as MCP-1 advises its correlation with an inflammatory response after trauma. Obesity is a

comorbidity factor in stroke and TBI. CD-36 is a Fat/Lipid biomarker of obesity, stroke &

associated disorders. Stroke and TBI have overlapping pathological events. CD-36 could be the

pathological link between inflammation and TBI. CD-36 is a candidate therapeutic target to

abrogate secondary cell death in TBI and promote functional and cognitive brain recovery.

1.5 Role of CD-36 in Stroke

This section will now discuss stroke prevalence, pathology, treatment, but more

importantly it will highlight the inflammatory implications of CD-36 in animal stroke models. An

25

ischemic stroke occur when blood supply to part of the brain is suddenly interrupted or when a

blood vessel in the brain bursts, spilling blood into the spaces surrounding brain cells. Brain

cells die from hypoxia, lack of nutrients from the blood, from sudden bleeding into or around the

brain. According to the American Heart Association (AHA), the hallmark symptoms of a stroke

include: abrupt numbness or weakness, especially on one side of the body; momentary

confusion; trouble speaking or understanding speech; disrupted vision in one or both eyes;

problems with walking, dizziness, or loss of balance or coordination; or a severe headache with

no known cause. There are two forms of stroke: ischemic and hemorrhagic. The first one is

simply blockage of a vessel supplying blood to the brain, and the second one occurs when there

is a hemorrhage into or around the brain.

In 2008, mortality from stroke was the fourth leading cause of death in the United States,

and stroke was a leading cause of long-term severe disability (Minino, Murphy et al. 2011).

Nearly half of older stroke survivors experience moderate to severe disability (Kelly-Hayes,

Beiser et al. 2003). Care for stroke survivors cost an estimated $18.8 billion in the United States

during 2008, and lost productivity and premature mortality cost an additional $15.5 billion

(Roger, Go et al. 2012). Ischemic stroke may arise from the atherosclerotic large cerebral

arteries (e.g., carotid, middle cerebral, and basilar arteries) or atherosclerotic small cerebral

arteries (e.g., lenticulostriate, basilar penetrating, and medullary arteries); ischemic stroke may

also be cardio-embolic in origin (Reeves and Swenson 2008). In addition to thrombotic

occlusion at the site of cerebral artery atherosclerosis and ischemic infarction can be produced

by emboli arising from proximally situated atheromatous lesions to vessels located more distal

in the arterial tree (Mohr and Sacco 1992). Most investigations of atherogenesis have focused

on the coronary arteries, but, with some possible exceptions, similar processes occur in the

cerebral circulation.

In the brain, the process is better characterized in the larger arteries than in small

arteries supplying deep cerebral white matter. Some evidence suggests that the underlying

26

pathogenetic process in small arteries may differ from that described in larger arteries (DeGraba

TJ, Fisher M et al. 1992). A cascade of events known as “ischemic cascade” comprise:

anaerobic metabolism within the region of brain tissue affected by ischemia; anaerobic

metabolism produces less adenosine triphosphate (ATP) and release of lactic acid, which

disrupts the normal acid-base balance in the brain; followed by glutamate excitotoxicity;

mitochondrial failure, energy depletion and apoptosis. The American Heart Association and

CDC break down treatment into three stages: prevention, therapy immediately after the stroke,

and post-stroke rehabilitation (AHA, 2013). Prevention involves therapies that target an

individual's underlying risk factors for stroke, such as hypertension, atrial fibrillation, and

diabetes (CDC, 2014). Acute stroke therapies try to stop a stroke while it is happening by

administering a thrombolytic agent; it quickly dissolves the blood clot, tissue plasminogen

activator (TPA). TPA should be administered as soon as possible after the first stroke symptoms

have appeared, preferably within the therapeutic window of 3 hours. TPA improves the chances

of recovering from a stroke. Studies have shown that patients with ischemic strokes who

received TPA are more likely to recover fully or have less disability than patients who do not

receive the drug (NINDS 1995; Marler, Tilley et al. 2000). In cases of hemorrhagic stroke,

doctors will perform surgery to try stopping the bleeding. Post-stroke rehabilitation helps

individuals overcome disabilities that result from stroke damage.

CD-36 has been reported as an inflammatory mediator in animal models of ischemic

stroke and hyperlipidemia (Cho, Szeto et al. 2007; Kim, Tolhurst et al. 2008; Kim, Febbraio et al.

2012). There are studies showing that the use of antioxidant peptide, SS-31, attenuates CD-36

pervasive action and improves ischemia-induced injury (Cho, Szeto et al. 2007). Overall these

studies suggest that CD-36 mediated inflammation significantly contributes to the cytotoxic

effects seen in stroke. Additionally, studies in mice and human models of ischemic stroke

attempt to identify the benefits of a soluble form of receptor for advanced glycation end products

(sRAGE). Plasma levels of sRAGE and high mobility group box 1 (HMGB1) were both

27

significantly increased within 48 hours after stroke, and the sRAGE level was an independent

predictor of functional outcome at 3 months post-stroke (Tang, Wang et al. 2013). Thus, SS-31

and sRAGE may have potential therapeutic effects in stroke and related inflammatory disorders.

Because of overlapping cell death events between stroke and TBI, the molecular

pathways of secondary cell death implicated in stroke may also be involved in the progression

of TBI. The release of pro-inflammatory cytokines by microglia is a major feature occurring in

stroke and TBI. Similarly, previously mentioned therapies found effective in arresting the

secondary cell death in stroke may also be valuable therapies for TBI. Since CD-36 closely

accompanies stroke-induced neuroinflammation, and that neuroinflammation is a major cause

of secondary cell death in TBI, then it is likely that investigations of CD-36 in TBI will provide

novel insights into the understanding of TBI neuroinflammation. Moreover, targeting CD-36 may

prove effective in attenuating neuroinflammation and therefore stands as a potent therapeutic

pathway for treatment of TBI.

Furthermore, in animal models of ischemia/reperfusion injury oxLDL high levels activate

CD-36, increasing free radical production and tissue injury. In a recent murine ischemic study,

scientists tested an antioxidant peptide, SS-31 a cell permeable peptide that may reduce

intracellular free radicals and inhibits LDL oxidation/lipid peroxidation (Zhao, Zhao et al. 2004).

In addition, treating mice with SS-31 immediately after reperfusion significantly attenuated

ischemia-induced GSH depletion in the cortex and reduced infarct size; no protective effect of

SS-31 was observed in CD-36 knock-out mice, indicating that SS-31 is acting through inhibition

of CD-36; and treating C57BL/6 mice with SS-31 reduced CD-36 expression in post-ischemic

brain and mouse peritoneal macrophages (MPM) (Cho, Szeto et al. 2007) . These in vivo and in

vitro studies suggest the down-regulation of CD-36 by novel class antioxidant peptides may be

used to treat ischemia and possibly TBI, too. Thus, we will address the pharmacological effects

of sRAGE in an in-vitro model of TBI.

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1.6 Role of CD-36 in TBI

Although a neuroinflammatory connection has been established between CD-36 with

stroke and other degenerative diseases, there is very little information or experimental reports

connecting CD-36 with TBI. We will review this literature and address the series of in-vitro and

in-vivo experiments previously carried by Borlongan’s lab team supporting our notion: CD-36

could be the pathological link between inflammation and TBI. Recent publications have

documented diabetes mellitus (DM) as a co-morbidity factor in TBI (Cerecedo-Lopez, Kim-Lee

et al. 2014), and diabetes Insipidus (Diamandis, Gonzales-Portillo et al. 2013) contributing to

TBI pathology via CD-36 neuroinflammation.

A series of in-vitro experiments focused first on the ability of soluble receptor of

advanced glycation end products RAGE (sRAGE) in binding CD-36-oxLDL in cells of different

origin. They decided to test sRAGE because of its strong affinity for CD-36 (Marsche,

Zimmermann et al. 2003), its synergistic anti-inflammatory effect combined with long term use

of antihypertensive drugs like nifedipine and telmisartan (Falcone, Buzzi et al. 2012); and

sRAGE ability to act as a decoy for RAGE to slow carotid artery bloom injury, slow

atherosclerosis via blocking RAGE, and avoid interaction of RAGE with its pro-inflammatory

ligands (AGEs, HMGB1, S100 proteins) (Maillard-Lefebvre, Boulanger et al. 2009).

It is well documented the spleen immune function is known to alter stroke outcome.

Additionally, this immune response parallels with observed downstream pro-survival

(neurogenesis) and cell death events (neurovascular alterations revealed by matrix

metalloproteinaises breakdown and inflammatory response) over the same 24-48 hour period

(Hayashi, Kaneko et al. 2009; Shojo, Kaneko et al. 2010).

This evidence implies that sRAGE is a useful inhibitor of CD-36 mediated inflammation

response in brain and spleen of TBI animals. We were able to determine the inhibitory action of

soluble receptor of advanced glycation end products (sRAGE) in various cell types, suggesting

that sRAGE antagonistic effects may be a useful strategy to reduce CD-36 mediated uptake of

29

HOCl-LDL, and attenuate the immune response and tissue injury. These pilot experiments

further bolster the role of CD-36 mediated inflammation as a major secondary cell death

mechanism shared by stroke and TBI. Accordingly, we hypothesize that CD-36 has a pivotal

role in the neuroinflammatory cascade that further contributes to the pathology of TBI. Exploiting

this CD-36 mechanism as a candidate therapeutic target will abrogate secondary cell death in

TBI and promote functional and cognitive brain recovery. Furthermore, the therapeutic inhibitory

effects of sRAGE on CD-36 expression highlight a possible therapeutic window when

administered within 24 hours post-TBI.

1.7 Role of CD-36 in Other Neurodegenerative Diseases

We will address the different pathological scenarios in which CD-36 has been found to

take participate in neurodegenerative diseases. Recent studies have associated CD-36 to

inflammatory alterations in Alzheimer’s disease (AD), cerebral amyloid angiopathy (CAA), and

Parkinson’s disease (PD). We will address the inflammatory pathways associated with CD-36

among the mentioned disorders, and strengthen the notion of CD-36 as a therapeutic target.

1.7.1 Alzheimer’s Disease

AD is a neurodegenerative disease (AD) characterized by the accumulation of a protein

known as amyloid-β (Aβ) in the brain. Patients experience serious memory loss, confusion,

mood and behavior changes. According to reports by the Alzheimer's Association (AA), the

National institute of Aging, the CDC and recent statistical literature an estimated 5.2 million

Americans are suffering from AD in 2014, including approximately 200,000 individuals younger

than age 65 who have younger-onset Alzheimer's (Brookmeyer, Evans et al. 2011; Innes and

Selfe 2014; Weuve, Hebert et al. 2014). Of the 5 million people age 65 and older with AD in the

United States, 3.2 million are women and 1.8 million are men (Mielke, Vemuri et al. 2014). The

mentioned institutions estimate that by 2050, the number of people age 65 and older with AD

30

may nearly triple, from 5 million to as many as 16 million, making impossible to advance in

medical innovations to prevent, delay or stop the disease (Alzheimer's Association 2013). To

put things into perspective and draw a clearer picture on the urgency for effective care, Florida

alone reported close to 500,000 patients struggling with TBI and associated costs close to 600

thousand millions (Gilligan, Malone et al. 2013). Nationwide analysts from the AA predict costs

will climb from $214 billion in 2014 to $1.2 trillion by the midcentury.

The pathology of AD is very complex. It consists in the formation of amyloid plaques and

cerebral amyloid angiopathy, neurofibrillary tangles, and glial responses, and detrimental

lesions such as neuronal and synaptic loss (Hyman, Phelps et al. 2012). Many neuroscientists

have suggested further labeling as “positive lesions” amyloid plaques and CAA, neurofibrillary

tangles, and glial responses (Serrano-Pozo, Frosch et al. 2011). In contrast, the “negative

lesions” are characteristic losses of neurons, neuropil, and synaptic elements (DeKosky and

Scheff 1990; Scheff, DeKosky et al. 1990; Terry, Masliah et al. 1991; Masliah, Mallory et al.

1993; Scheff and Price 1993; Gomez-Isla, Price et al. 1996; Knowles, Wyart et al. 1999;

Serrano-Pozo, Frosch et al. 2011). Yet, no one has linked CD-36 to any of the negative lesions

listed above, but there are reports linking CD-36 to Aβ protein deposition (Ricciarelli, D'Abramo

et al. 2004) and CAA (Park, Zhou et al. 2013). Ricciarelli and his collaborators revealed some

interesting results: first mRNA and protein CD-36 expression was highly expressed in the

cerebral cortex of AD patients and cognitively normal aged subjects with diffuse amyloid

plaques compared with age-matched amyloid-free control brains. Thus, CD-36 expression in

human brain correlates with the presence of amyloid plaques. Second, in-vitro experiments by

the same team investigated whether Aβ1-42 may modulate the expression of CD-36; surely

enough after 24 hours of treatment the mRNA levels of CD-36 were upregulated in

neuroblastoma and human monocytes, but to a less extent on primary rat neurons. Likewise,

Borlongan’s team found human monocytes (not treated with sRAGE) expressed a strong

association with CD-36 ox-LDL. The third major finding being the more important, the induction

31

of CD-36 in these cell lines lasted after 48 hrs, denoting the first evidence of CD-36 expression

in cells of neuronal origin (Ricciarelli, D'Abramo et al. 2004).

Besides Aβ aggregation in the brain, AD patients also have a strong inflammatory

component with increased microglia activation. Wilkinson and Khoury describe how microglia

and Aβ have a dichotomous role in AD pathogenesis (Wilkinson and El Khoury 2012). On one

hand, microglia can phagocytose and clear Aβ, but binding of microglia to Aβ also increases

their ability to produce inflammatory cytokines, chemokines, and neurotoxic reactive oxygen

species (ROS) (Pan, Zhu et al. 2011; Wilkinson and El Khoury 2012). But who is responsible for

clearing Aβ? Scavenger receptors. Microglia carries receptors for Aβ, in particular: SCARA-1

(scavenger receptor A-1), CD-36, and RAGE (receptor for advanced glycation end products)

(Wilkinson and El Khoury 2012). CD-36 has been implicated in a mechanism of fibrillar Aβ

internalization along with α6β1 integrin and the integrin associated protein CD-47

(Koenigsknecht and Landreth 2004). SCARA-1 appears to be involved in the clearance of Aβ

(Herber, Mercer et al. 2007; Napoli and Neumann 2009); while CD-36 and RAGE are involved

in activation of microglia by Aβ (Scheff 1990). A second team reported that SR-A and CD-36

had similarities between interactions of microglia with fibrillary Aβ and of macrophages with

oxLDL in brains of AD patients (Coraci, Husemann et al. 2002). Altogether above experiments

are compelling evidence that CD-36 participates in Aβ protein aggregation and inflammatory

signaling pathways of AD. No cure or treatment exists that can reverse the effects of AD.

Medical treatment appeared in the 1990’s. Nevertheless, these pharmacological drugs alleviate

symptoms but cannot stop the progression of the disease. Current FDA-approved Alzheimer's

drugs, cholinesterase inhibitors and (N-methyl-D-aspartate) receptor (NMDA) antagonist do not

suffice. The positive effects of 1) cholinesterase inhibitors (donepezil, galantamine, rivastigmine

and tacrine) work by slowing down the disease activity that breaks down acetylcholine

(Kaduszkiewicz, Zimmermann et al. 2005; Hansen, Gartlehner et al. 2008; Parsons, Danysz et

al. 2013); and 2) memantine protects brain cells against excess glutamate released in large

32

amounts by cells damaged by Alzheimer's disease and other neurological disorders (Schneider,

Insel et al. 2011; Danysz and Parsons 2012). Thus, the study of inflammatory alterations of CD-

36 in AD, CAA, and TBI can help reduce the gap in knowledge of signaling molecules

influencing microglia activation via CD-36.

1.7.2 Cerebral Amyloid Angiopathy

With respect to CAA Park and his collaborators observed that Tg2576 mice lacking CD-

36 have a selective reduction in Aβ1-40 deposition and CAA, reduced vascular amyloid

deposition was associated with preservation of the Aβ vascular clearance receptor LRP-1, and

protection from the deleterious effects of Aβ on cerebral arterioles; these beneficial vascular

effects were reflected by marked improvements in neurovascular regulation and cognitive

performance (Park, Zhou et al. 2013). Once again, there is clear evidence suggesting CD-36

participates in the pathology of vascular amyloid deposition, and as a potential therapeutic

target.

1.7.3 Parkinson’s Disease

A second disease whose pathology may have a CD-36 inflammatory component is PD.

This disease is a neurodegenerative disorder characterized by the progressive degeneration of

dopaminergic neurons in the substantia nigra; leading to diminished striatal dopamine levels,

and the appearance of proteinaceous inclusions known as Lewy bodies (Abumrad and Moore

2011). There is a genetic predisposition to the disease associated with mutations in the parkin

gene. Parkinson disease (PD) affects 1%–2% of the world’s population over the age of 65 years

(Willis 2013). Common symptoms of PD include bradykinesia, resting tremor, muscular rigidity,

and postural instability; in addition, non-motor symptoms include autonomic, cognitive, and

psychiatric disturbances (Cohen, Klein et al. 2014). There are no available treatments that can

cure PD, only pharmacological agents, levodopa combined with carbidopa, that attenuate the

33

symptoms. Yet, there is a collaborative effort of 7 translational clinical trials in th US trying to

identify new indicators of disease progression, these PD candidates may be present at early

and late PD stages of the disease, and are being measured in different body fluids like CSF and

blood collected from patients with PD and healthy control subjects.

Kim and colleagues identified CD-36 as a new substrate of Parkin-mediated

ubiquitination and propose a potentially broad function of Parkin in the regulation of FA

metabolism (Kim, Stevens et al. 2011). Additionally, CD-36 bind certain ligands eliciting a range

of intracellular signaling processes involving Src and MAP kinases that integrate lipid

metabolism and inflammation and also involve CD-36 in pathways related to oxidative stress,

angiogenesis, platelet hyperactivity, phagocytosis, and cell migration (Silverstein and Febbraio

2009; Su and Abumrad 2009). Importantly, CD-36 membrane levels and turnover are abnormal

in diabetes, resulting in dysfunctional FA utilization. In addition, variants in the CD-36 gene were

shown recently to influence susceptibility for the metabolic syndrome, which greatly increases

the risk of diabetes and heart disease. In summary, these studies provide insights into the

connection between neurodegenerative disorders and lipid metabolism. Additional experiments

will strengthen the link between chronic neurological disorders and CD-36.

34

CHAPTER 2

MATERIAL AND METHODS

The experiments are designed to test our hypothesis that CD-36 has a pivotal role in the

neuroinflammatory cascade that further contributes to the pathology of TBI. Exploiting this CD-

36 mechanism as a candidate therapeutic target will abrogate secondary cell death in TBI may

promote functional and cognitive brain recovery. We postulate the following aims: (1) CD-36 is a

critical mediator of neuroinflammation following TBI in the cortex, this secondary cell death

mechanism may be reflected in the progressive expression of CD-36 in the spleen, providing a

key pathological link between peripheral regulation of brain injury. Aim 1 studied TBI at two

disease stages: (1.1) CD-36 mediated inflammation in acute TBI, without therapeutic

intervention, translates to (1.2) chronic neuroinflammation and it was recognized via

upregulation of neuroinflammation during disease progression. In order to test this hypothesis,

we characterized CD-36 expression in the brain and the spleen of TBI animals in acute and

chronic stages of injury thru histological and immunoblotting analysis described below (sections

2.1-2.12). The above in vivo testing is preferred over in vitro because it is more suitable for

observing the overall effects of CD-36 on a living organism. In addition, Aim 2 addresses

pharmacological interventions to inhibit inflammation in an in vitro TBI model. Experiments

attempted an inhibitory approach to reduce TBI related inflammation and secondary cell death

using sRAGE (section 4.2). Furthermore, Aim 3 examined the use of stem cell therapy in

combination with sRAGE to abrogate neuroinflammation in an in-vitro TBI model (section 5.2).

First, we characterized histological symptoms of TBI, with emphasis on

neuroinflammation, in acute (Aim 1.1) and chronic (Aim 1.2) stages of the injury. Procedures

35

outlined in sections 2.1 thru 2.13 tested these sub aims. At the completion of these experiments

we would have gained a better understanding on the role of brain and splenic CD-36

contributing to TBI neuroinflammation. The last section 2.14 briefly describes previous

preliminary in-vitro experiments performed by our team on CD-36.

2.1 Animals

Studies in Sprague-Dawley (SD) rats were carried in accordance with the

recommendation in the Guide for the Care and Use of Lab oratory Animals of the National

Institutes of Health. The protocol was approved by the Institutional Animal Care and Use

Committee (IACUC) at the University of South Florida, College of Medicine. All male rats were

housed under normal conditions (20°C, 50% relative humidity, and a 12-hrs light/dark cycle). A

separate cohort of animals, sham group, underwent the same experimental conditions. Only

male Sprague-Dawley rats were used in this study, and housed under normal conditions.

Necessary precautions were taken to reduce pain and suffering of animals throughout the study.

Next, we embarked on the animal surgical procedures for modeling of the disease to reproduce

many of the features observed in TBI in humans.

2.2 Surgical Procedures

The controlled cortical impact model in animals is an effective experimental tool to

replicate what happens to humans after TBI. Ten week old, male Sprague-Dawley rats (n=60)

were subjected to mild TBI using the controlled cortical impact (CCI) injury model (Pittsburgh

Precision Instruments, Inc, PA, USA). Animals were anesthetized using 1–2% isoflurane. Once

deep anesthesia was achieved, individual animals were fixed in a stereotaxic frame (David Kopf

Instruments, Tujunga, CA, USA), anesthesia was maintained via gas mask. TBI injury surgeries

consisted of animals subjected to scalp incision to expose the skull, using an electrical drill a

4.0mm craniectomy was performed over the right frontoparietal cortex (2.0 mm anteroposterior

36

(AP),+2.0 mm mediolateral (ML), and 1.0 mm dorsoventral (DV) to bregma (Paxinos G 2005;

Glover, Tajiri et al. 2012). The pneumatically operated TBI device (with a convex tip diameter of

3.0 mm for rats) impacted the brain at a velocity of 6.0 m/s reaching a depth of 1.0 mm for mild

TBI, below the dura mater layer and remained in the brain for 150 milliseconds (ms). The

impactor rod was angled 15° degrees to the vertical to maintain a perpendicular position in

reference to the tangential plane of the brain curvature at the impact surface (Yu, Kaneko et al.

2009). A linear variable displacement transducer (Macrosensors, Pennsauken, NJ) connected

to the impactor measured velocity and duration to verify consistency. This CCI approach

produced skull fracture overlying the frontal cortex. All animals received the analgesic

ketoprofen (5mg/kg of weight) post-operatively. Rats were monitored closely. Next, TBI animals

and sham animals were grouped and sacrified at various post TBI time-points for subsequent

histological and protein analysis to determine CD-36 alterations.

2.3 Histology

Under deep anesthesia, rats were sacrificed at different time-points: 24 hours (hrs), 48

hrs, 7 days, and 60 days post-TBI surgery, along with their corresponding sham groups. All

animals underwent transcardial perfusion, rats were placed on their backs, and blunt forceps

were used to cut through the body cavity. The opening was extended laterally until reaching the

rib cage. Rib cage was cut, and sternum was lifted to expose the heart. The heart was held

gently, and the needle was inserted ¼ inch into the left ventricle. The right atrium was cut using

the irridectomy scissors. Through the ascending aorta, approximately 200 ml of ice cold

phosphate buffer saline (PBS) followed by 200 mL of 4% paraformaldehyde (PFA) in phosphate

buffer (PB) were used for brain perfusion. Sham animal groups underwent the same procedure.

Brains and spleens were removed and post-fixed in the same fixative at 4°C for 24 hrs

followed by two rounds of 30% sucrose in PB for 48 hrs. Brains were frozen at −24°C, mounted

onto a 40 mm specimen disk using embedding medium. Coronal sectioning was carried out at a

37

thickness of 30 µm by cryostat on every 6th (1/6) coronal section spanning the prefrontal cortex

up to the entire dorsal hippocampus, starting at coordinates AP +2.70mm and ending at AP-

3.0mm relative to bregma. Coronal cryostat sections were stored in a cryoprotectant solution at

20°C until immunohistochemical staining to address sub-aims 1.1. and 1.2.

2.4 Tissue Collection for Protein Analysis

A second set of animals were sacrificed at different time-points under deep anesthesia:

24 hours (hrs), 48-hrs, 7 days, and 60 days post-TBI surgery, along with their corresponding

sham groups. All animals underwent transcardial perfusion, rats were placed on their backs,

and blunt forceps were used to cut through the body cavity. The opening was extended laterally

until reaching the rib cage. Rib cage was cut, and sternum was lifted to expose the heart. The

heart was held gently, and the needle was inserted ¼ inch into the left ventricle. The right atrium

was cut using the irridectomy scissors. Through the ascending aorta, approximately 300 mL

cold phosphate buffer saline (PBS) were used for brain perfusion. Sham animal groups

underwent the same procedure. Brain tissue was dissected into main brain regions: cortex,

striatum, dorsal and ventral hippocampus (Chiu, Lau et al. 2007). Immediately tissue sections

and spleens were placed in individual cryoprotectant vials in liquid nitrogen (-196°C) and

transferred to a -80°C freezer. Accordingly, histological and protein analysis experiments of CD-

36 expression in the brain, as well as in the spleen may reveal more detail about the central and

peripheral CD-36 inflammatory response after TBI. Neuroinflammation is a double-edged sword,

in that inflammatory signals (i.e., anti-inflammatory) may be upregulated at the acute phase, but

a different set of inflammatory cues (i.e., pro-inflammatory) will be elevated at the chronic

phase. Thus, we covered various immunohistochemical stainings in sections 2.5.1-2.5.5 to

capture for any other upregulated inflammatory markers in brain and spleen. After these

experiments, we analyzed brain tissue through confocal microscopy (section 2.6) to quantify the

number of CD-36 positive reactive cells; and spleen tissue through fluorescence microscopy

38

(section 2.7) to measure fluorescence intensity. And finally, statistical analysis was carried on

the brain data to compare any significant differences among the ipsilateral vs contralateral

hemispheres of the injured brain animals, and against sham brains, too (section 2.8). With

respect to the spleen data, statistical analysis was also carried for TBI vs. sham animals

(section 2.9).

2.5. Immunohistochemistry

2.5.1 CD-36/MCP-1 Immunohistochemistry in Brain

A series of one in six sections was selected that included the anatomical region of

interest (from prefrontal cortex to hippocampus) with a random start section for each subject for

this staining. On day one free-floating sections (30µm thickness) were incubated with mouse

monoclonal anti CD-36 (1:100, Abcam, USA), rabbit polyclonal anti MCP-1 (1:100, Abcam,

USA) primary antibodies overnight at 4C, followed by day two incubation with goat anti-mouse

secondary antibody (1:1000 Alexa 488, Molecular Probes, USA), and goat anti-rabbit secondary

antibody (1:1000-2000 Alexa 594, Molecular Probes, USA) for 2 hrs at RT. Finally, tissue was

incubated with Hoechst 33258 (bisBenzimideH 33258 trihydrochloride) for 30min (1:300

Molecular Probes, USA) to visualize cell nuclei. Then tissue underwent three washes in PBS,

and coverslipped with Fluoromount (Sigma).

2.5.2 CD-36/Iba-1 Immunohistochemistry in Brain

A series of one in six sections was selected that included the anatomical region of

interest (from prefrontal cortex to hippocampus) with a random start section for each subject for

this staining. On day one free-floating sections (30µm thickness) were incubated with mouse

monoclonal anti-CD-36 (1:100, Abcam, USA), and rabbit polyclonal anti Iba-1 (1:300, WAKO,

Japan) primary antibodies overnight at 4°C. The next day tissue was incubated for 2 hrs in goat

39

anti-mouse secondary antibody (1:1000 Alexa 488, Molecular Probes, USA) and goat anti-rabbit

secondary antibody (1:1000-2000 Alexa 594, Molecular Probes, USA), followed by incubation

with Hoechst (bisBenzimideH 33258 trihydrochloride) for 30 mins (1:300 Molecular Probes,

USA), then tissue underwent three washes in PBS for 30 mins, and coverslipped with

Fluoromount (Sigma).

2.5.3 CD-36/NeuN Immunohistochemistry in Brain

A series of one in six sections was selected that included the anatomical region of

interest (from prefrontal cortex to hippocampus) with a random start section for each subject for

this staining. On day one free-floating sections (30µm thickness) were incubated with mouse

monoclonal anti-CD-36 (1:100, Abcam, USA), and rabbit monoclonal anti-NeuN (1:500,

Millipore, USA) primary antibodies overnight at 4C. The next day tissue was incubated for 2 hrs

in goat anti-mouse secondary antibody (1:1000 Alexa 488, Molecular Probes, USA) and goat

anti-rabbit secondary antibody (1:1000-2000 Alexa 594, Molecular Probes, USA), followed by

incubation with Hoechst (bisBenzimideH 33258 trihydrochloride) for 30 mins (1:300 Molecular

Probes, USA), then tissue underwent three washes in PBS for 30 mins, and coverslipped with

Fluoromount (Sigma).

2.5.4 CD-36/GFAP Immunohistochemistry in Brain

A series of one in six sections was selected that included the anatomical region of

interest (from prefrontal cortex to hippocampus) with a random start section for each subject for

this staining. On day one free-floating sections (30µm thickness) were incubated with mouse

monoclonal anti CD-36 (1:100, Abcam, USA), and rabbit polyclonal anti-GFAP (1:2000, DAKO,

USA) primary antibodies overnight at 4C. The next day tissue was incubated for 2 hrs in goat

anti-mouse secondary antibody (1:1000 Alexa 488, Molecular Probes, USA) and goat anti-rabbit

secondary antibody (1:1000-2000 Alexa 594, Molecular Probes, USA), followed by incubation

40

with Hoechst (bisBenzimideH 33258 trihydrochloride) for 30 mins (1:300 Molecular Probes,

USA), then tissue underwent three washes in PBS for 30 mins, and coverslipped with

Fluoromount (Sigma).

2.5.5 CD-36 Immunohistochemistry in Spleen

Spleen sectioning was carried out at a thickness of 20 µm by cryostat at -19°C.

Fluorescent immunostaining was done on every 6th (1/6) coronal section and mounted directly

onto the slide. Day one involved incubating tissue sections with mouse monoclonal anti-CD-36

(1:100, Abcam, USA) overnight. The next day, tissue slides were incubated in goat anti-mouse

secondary antibody for 2 hrs (1:1000 Alexa 488, Molecular Probes, USA). Then, tissue was

washed 3 times for 10 mins and coverslipped with DAPI H-1200 mounting medium

(Vectashield).

2.6 Laser Scanning Confocal Microscopy

A multiphoton laser confocal scanning microscope (FV1000 MPE, Olympus, USA) was

used for observation and imaging for all immunofluorescence photomicrographs of the motor

cortex area(M1, M2) at a 40x magnification, only linear adjustments (brightness and contrast)

were made to the figures. When quantification of percentage of positive cells was determined, Z

stacks were created at 1 µm intervals throughout the 30 µm of the sections with a guard region

of 2 mm excluded from top and bottom of the Z stack. The Z stacks were rotated in all planes to

verify double labeling.

2.7 Fluorescencent Microscopy

A fluorescent microscope (Axio Imager.Z1, Zeiss, USA) was used for observation and

imaging of spleen slides at a 40x magnification. Six serial spleen coronal sections were

quantified using Image J and results are displayed as the average mean of fluorescent intensity.

41

2.8 Statistical Analysis on Indirect Immunofluorescence in Brain

CD-36, MCP-1, Iba-1 , Hoechst positive cells, adjacent to impact area (M1 motor cortex

area)were counted using Image J using 6 serial coronal sections, results showed the average

mean of the cell ratios of CD-36/DAPI, (CD-36 + MCP-1 cells)/MCP-1, MCP-1/DAPI, CD-

36/DAPI,Iba-1/DAPI, CD-36/Iba-1. Likewise, the contralateral side of injured brains, and sham

brains were analyzed. Explored coronal sections correspond to +2.7 to -0.3 mm with respect to

bregma. The data obtained were analyzed using the Graph Pad Prism 5.0 software, and were

evaluated statistically using one way analysis of variance (ANOVA) for group comparisons with

95% CI followed by subsequent pair wise comparisons using post hoc Bonferonni test. All data

are presented as mean values ±SEM. Statistical significance was set at p<0.05 for all analyses.

2.9 Statistical Analysis on Fluorescence Intensity in Spleen

The data obtained was analyzed using the Graph Pad Prism 5.0 software to run the

unpaired t-test with Welch’s correction to compare spleen from TBI animals vs. Sham animals.

Immunoblotting or western blotting is a technique helpful for the analysis of individual proteins in

a protein mixture or tissue lysate. We used this method to detect the expression levels of CD-36

protein in brain and spleen at the different acute TBI time-points. Sections 2.10 thru 2.12 cover

the process of tissue collection, homogenization and protein detection to sort the proteins by by

size, charge. Once separated, proteins are then transferred to a carrier membrane, and

accessible for antibody binding for detection.

2.10 Tissue Processing

Tissue from the cortex and spleen were homogenized using an electric tissue

homogenizer in 1:10 weight to volume ratio of ice-cold RIPA buffer (Cell Signaling Technology,

42

USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich, USA). Following

homogenization, sample lysates were centrifuged at 10,000rpm at 4ºC for two rounds of 30

mins. Pellets were discarded and the supernatant was then aliquoted in 50μl samples and

stored at -80°C.

2.11 Western Blot

BCA protein assay (BCA Protein Assay, Pierce, USA) was applied to determine total

protein concentration for each fraction sample. Pre-cast electrophoresis gels (4-15% Mini

Protean TGX, Bio-Rad, USA) were loaded with 30μg of total protein per sample per well, and

current applied at 70V for 45min, resuming at 100V for 30min. Proteins were transferred to

nitrocellulose membranes (Bio-Rad, USA) using a Criterion blotter (Bio-Rad, USA), at 100V, for

30 mins. The membranes were pre-incubated in 5% non-fat dairy milk (NFDM)/Phosphate

Buffered Saline (PBS, Pierce, USA)/0.05% Tween 20 (PBST, Fisher-Scientific, USA) for 1hr,

then incubated overnight at 4°C with one of the following primary antibodies: rabbit monoclonal

CD-36 (1:250, Abcam, USA); mouse monoclonal GAPDH (1:25,000). The next day, membranes

were washed with PBST, incubated with the corresponding secondary antibody): goat anti-

rabbit IR Dye 800 (1:3000, LICOR, USA) and goat anti mouse 680 (1:25,000, LICOR, USA),

during 2 hrs, at RT. The membranes were thoroughly washed with PBST, and placed into an

Odyssey infrared imager (LICOR, USA) for band detection. Images were analyzed through the

Image Studio software (LICOR, USA) for band density measurements. The band density ratios

from TBI vs.Sham groups were statistically compared.

2.12 Statistical Analysis on Western Blot

The data obtained were evaluated statistically using one way analysis of variance

(ANOVA) for group comparisons with 95% CI followed by subsequent pair wise comparisons

43

using post hoc Bonferonni test. All data are presented as mean values ± SEM. Statistical

significance was set at p<0.05 for all analyses.

The next set of experiments addressed Aims 2 and 3 using an in vitro cell model of TBI.

We performed a pharmacologic approach using sRAGE, an inhibitor of CD-36, to reveal

whether inhibiting this pathway produced parallel alterations in the downstream anti-

inflammatory effects of CD-36 seen in Aim 1. We replicated TBI in this in-vitro cell model by

treating human neural progenitor cells with tumor necrosis alpha (TNF-a). We expect that this

upstream inhibitory approach blocks the suggested inflammatory biomarker and observe

positive effects in the survival of human neural progenitor cells.

2.13 Cell Culture

Cultures of human neural progenitors were maintained in culture following the supplier’s

protocol (hNP1, Neuromics, Edina, MN). Briefly, immediately after thawing, cells

(46104cells/well) were seeded and grown in 96-well plate coated by poly-L lysine in Neurobasal

media (GIBCO, CA) containing 2 mM L- glutamine, 2% B27 (GIBCO, CA) and 50 U/ml penicillin

and streptomycin for 4 days at 37uC in humidified atmosphere containing 5% CO2. Cells were

grown in confluence for 72 hrs to achieve expansion with 61%viability. These cells were

exposed to TNF-a insult which closely simulates mild traumatic brain injury in humans.

2.13.1 Measurement of Cell Viability: Calcein-AM Fluorescence Dye

Measurement of cell viability was performed by both fluorescent live/ dead cell assay

(Bell, Cao et al. 2003) exclusion method. A two-color fluorescence cell viability assay was

performed by Calcein-AM (Invitrogen, L3224) to be retained within live cells, including an

intense uniform green fluorescence and ethidium homodimer (EthD-1) to bind the nuclei of

damaged cells (bright red fluorescence). After two hours reperfusion, the human Neural

Progenitor Cells (hNP1, Neuromics, Edina, MN) were incubated with 2 mM Calcein-AM and 4

44

mM EthD-1 for 45 min at room temperature in darkness according to the manufacturer’s

instructions. The green fluorescence of the live cells was measured by the Gemini EX

florescence plate reader (Molecular Device), excitation at 485 nm and emission at 538 nm.

2.13.2 Measurement of Mitochondrial Activity: MTT Assay

For the mitochondrial activity assay, reduction of 3-(4, 5-dimethyl-2-thiazoyl)-2,5-

diphenyltetrazolium bromide (MTT) by cellular dehydrogenases was used as described in our

previous report (Kaneko, Shojo et al. 2014). The purpose of the MTT assay is to assess cell

viability after treating cells with stem cells and sRAGE.

2.14 Preliminary in Vitro and In Vivo Experiments

2.14.1 In Vitro Study CD-36 and sRAGE Expression

In vitro cell culture studies included: RAW cells derived from mouse tumor, Chinese

hamster ovary (CHO) cells expressing murine scavenger receptor class B, type I (SR-BI,

termed CHO-SR-BI), or human CD-36 (termed CHO-CD-36), and human spleen-derived

monocytes/macrophages (MD cells). All treatment conditions performed

in quadruplicates.

2.14.2 CD-36, Spleen and TBI

Adult (2 months old) or neonatal (7 days old) Sprague-Dawley rats were subjected to

TBI, respectively, displayed upregulated and downregulated expression of CD-36 in the spleen

and brain at the supra-acute phase of injury (15 minutes after TBI) (Asterisk * p< 0.05 vs.

control. Sample size was n=20 per treatment group.

45

2.14.3 TBI and CD-36 Immune Response in Neonatal Rats.

Neonatal (7 days old) Sprague-Dawley rats underwent TBI and sacrificed at different

time points to measure expression of CD-36 in the spleen and brain at 15 minutes, 3 hours,12

hours,24 hours, and 48 hours post-TBI. Different cohorts of TBI rats were randomly euthanized

at each time point to reveal CD-36 expression. Sample size is n=6 treatment group. Controls

were age-matched rats that received sham surgery. Asterisk * p< 0.05 vs. control.

2.14.4 Soluble RAGE Modulates CD-36 Expression in Neonatal Spleen and Brain

after TBI.

In vivo model of neonatal TBI involved sRAGE treatment (25 ug/ 0.25 ml intraperineal

injection) at 15 mins, 3 hours, 12 hours, and 24 hours) or its equivalent volume of diluent (PBS)

was performed in neonatal Sprague-Dawley rats (7 days old). Animals were euthanized at 48

hours post-TBI for spleen and brain analysis of CD-36 expression. Asterisk * p<0.05 vs.

controls. Controls were age-matched rats that received sham surgery. Sample size was n=20

per group.

46

CHAPTER 3

INFLAMMATORY ROLE OF CD-36 IN AN ANIMAL MODEL OF TBI

3.1 Introduction

3.1.1 Why study CD-36 as a Biomarker of Inflammation?

The purpose of Aim 1 is to characterize any inflammatory alterations related to CD-36 in

an adult rat model of mild TBI. Investigations of CD-36 as a novel neuroinflammatory marker in

TBI derive from existing literature implicating this molecule in disorders such as obesity,

atherosclerosis, inflammation in cerebral ischemia, and certain neurodegenerative diseases

(Coraci, Husemann et al. 2002; Kunjathoor, Febbraio et al. 2002; Cho, Park et al. 2005; Cho,

Szeto et al. 2007; Cho and Kim 2009; Kim, Febbraio et al. 2012; Pietka, Schappe et al. 2014).

These disorders are well known risk factors for developing debilitating disorders among

humans. We propose CD-36 as a therapeutic target after TBI since it is a common denominator

implicated in the inflammatory response associated with the above disorders. Studies focusing

on CD-36 may provide insights in future pharmacological or genetic strategies to block the

deleterious expression and function of CD-36 to treat not just TBI patients, but also those

afflicted by previously mentioned risk factors.

47

3.1.2 Inflammatory Pathways Related to CD-36.

CD-36 is a glycoprotein also known as fatty acid translocase (FAT). It belongs to the

class B scavenger receptor family whose main function is to capture mLDLs. The CD-36 gene is

encoded in chromosome 7 in humans, chromosome 4 in rats, and chromosome 5 in mice

(GENE 2014). This protein is extensively glycosylated with a molecular weight of 88kDa and

also exists as a non-glycosylated version of 53kDa (Cho, Park et al. 2005). Among its

functions, CD-36 contributes to the uptake of free fatty acids facilitating their transport inside of

cells; it binds with high affinity to oxidized lipids from LDLs, advanced glycated proteins,

components of apoptotic cells, and cell-derived micro-particles (Goldberg, Eckel et al. 2009;

Silverstein and Febbraio 2009). However, functions of CD-36 go beyond lipid metabolism and

cell adhesion.

CD-36 has various ligands linked to signaling pathways associated with inflammation: (i)

CD-36 interacts with toll-like receptors and Aβ inducing intracellular signaling and release of

ROS, chemokines, and cytokines (Stewart, Stuart et al. 2010; Wilkinson and El Khoury 2012);

(ii) FAT binds to thrombospondins producing inflammatory factors causing endothelial apoptosis

(Cho 2012); (iii) CD-36 and advanced glycation-end products (RAGE) induce microglia

activation (Maillard-Lefebvre, Boulanger et al. 2009; Wilkinson and El Khoury 2012); (iv) CD-36

interacts with fibrillar β-amyloid (fAβ)/integrins, inducing the expression of pro-inflammatory

cytokines and chemokines (Coraci, Husemann et al. 2002; Moore, El Khoury et al. 2002; El

Khoury, Moore et al. 2003); and (v) CD-36 enhances macrophage and adipose tissue

inflammation and impairs insulin signaling (Kennedy, Kuchibhotla et al. 2011). These studies

suggest that CD-36 signaling pathways activated by different ligands mediate inflammatory

responses in various metabolic and neurodegenerative disorders.

Another interesting inflammatory pathway associated with CD-36 is the activation of

NLRP3 inflammasome. This pathway is activated by the uptake of oxLDL by CD-36, resulting in

the formation in the formation of intracellular cholesterol crystals that cause lysosomal

48

destabilization and NLRP3 activation (Sheedy, Grebe et al. 2013; Oury 2014). This CD36-

mediated inflammasome activation serves as evidence to link cholesterol build up to the chronic

inflammatory process of atherosclerosis. In addition, other cells engage in further activating the

NLRP3 inflammasome: activated or injured endothelial cells, leukocytes and platelets release

ATP, acting in a paracrine manner to transduce sterile inflammatory signals, such as P2X7

receptor activation further contributing to atherothrombosis (Sheedy, Grebe et al. 2013).

Furthermore, recent publications suggest the inflammatory involvement of CD-36 in TBI

(Cerecedo-Lopez, Kim-Lee et al. 2014), providing understanding of TBI neuroinflammation and

as a potent therapeutic target for TBI treatment.

3.1.3 Neuroinflammation in TBI Accompanied by CD-36 Expression in Brain and

Spleen.

We hypothesize that neuroinflammation via CD-36 has a temporal expression

contributing to the pathology of TBI at the acute and chronic stages. As it does in ischemic

injury, targeting CD-36 in the spleen and brain serves as a primary link between peripheral and

central regulation of brain injury. The in vitro data presented in Chapter 2 strongly suggest CD-

36 upregulation in the brain and spleen immediately after injury . At the completion of this Aim,

we would gain a better understanding of the role of splenic CD-36 contributing to TBI

neuroinflammation and if any corresponding alterations in inflammatory response exist in the

brain (i.e., cortex and hippocampus). Based on reported data indicating that the spleen immune

response contributes to the pathology observed in stroke animals, we envisioned a close

interaction of CD-36 in spleen and brain in TBI as well. The critical timing (i.e., 24-48 hours) of

CD-36 expression shift (from downregulation to upregulation) may signal the transition of

functional effects of this immune response from pro-survival to pro-death. This dynamic CD-36

expression also suggests the therapeutic window for TBI.

49

3.2 Specific Materials and Methods

Studies in Sprague-Dawley (SD) rats were carried out in accordance with the

recommendation in the Guide for the Care and Use of Laboratory Animals of the National

Institutes of Health. The protocol was approved by the Institutional Animal Care and Use

Committee (IACUC) at the University of South Florida, College of Medicine. All male rats were

housed under normal conditions (20°C, 50% relative humidity, and a 12-hou r light/dark cycle).

A separate cohort of animals, the sham group, underwent the same experimental conditions

and was also subjected to behavioral tests to assess motor function. Necessary precautions

were taken to reduce pain and suffering of the animals throughout the study.

3.2.1 Surgical Procedures

Ten week old, male Sprague-Dawley rats (n=60) were subjected to mild TBI using the

controlled cortical impact (CCI) injury model (Pittsburgh Precision Instruments, Inc, PA, USA).

Animals were anesthetized using 1–2% isoflurane. Once deep anesthesia was achieved,

individual animals were fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA,

USA), and anesthesia was maintained via gas mask. TBI injury surgeries consisted of animals

subjected to scalp incision to expose the skull; using an electrical drill, a 4.0mm craniectomy

was performed over the right frontoparietal cortex (2.0 mm anteroposterior (AP),+2.0 mm

mediolateral (ML), and 1.0 mm dorsoventral (DV) to bregma (Paxinos G 2005; Glover, Tajiri et

al. 2012). The pneumatically operated TBI device (with a convex tip diameter of 3.0 mm for rats)

impacted the brain at a velocity of 6.0 m/s, reaching a depth of 1.0 mm for mild TBI, below the

dura mater layer, and remained in the brain for 150 milliseconds (ms). The impactor rod was

angled 15° degrees to the vertical to maintain a perpendicular position in reference to the

tangential plane of the brain curvature at the impact surface (Yu, Kaneko et al. 2009). A linear

variable displacement transducer (Macrosensors, Pennsauken, NJ) connected to the impactor

50

measured velocity and duration to verify consistency. This CCI approach produced skull fracture

overlying the frontal cortex. All animals received the analgesic ketoprofen (5mg/kg of weight)

post-operatively. Rats were monitored closely. Next, TBI animals and sham animals were

grouped and sacrified at various post TBI time-points for future histological and protein analysis

to determine CD-36 alterations.

3.2.2 Immunohistochemistry of Brain

Under deep anesthesia, rats were sacrificed at different time-points: 24 hours (hrs), 48

hrs, 7 days, and 60 days post-TBI surgery, along with their corresponding sham groups. All

animals underwent transcardial perfusion; rats were placed on their backs and blunt forceps

were used to cut through the body cavity. The opening was extended laterally until reaching the

rib cage. The rib cage was cut, and the sternum was lifted to expose the heart. The heart was

held gently, and the needle was inserted ¼-inch into the left ventricle. The right atrium was cut

using the irridectomy scissors. Through the ascending aorta, approximately 200 mL of ice cold

phosphate buffer saline (PBS) followed by 200 mL of 4% paraformaldehyde (PFA) in phosphate

buffer (PB) were used for brain perfusion. Sham animal groups underwent the same procedure.

Brains and spleens were removed and post-fixed in the same fixative at 4°C for 24 hrs followed

by two rounds of 30% sucrose in PB for 48 hrs. Brains were frozen at −24°C and mounted onto

a 40 mm specimen disk using embedding medium. Coronal sectioning was carried out at a

thickness of 30 µm by cryostat on every 6th (1/6) coronal section spanning the prefrontal cortex

up to the entire dorsal hippocampus, starting at coordinates AP +2.70 mm and ending at AP-3.0

mm relative to bregma. Coronal cryostat sections were stored in a cryoprotectant solution at –

20°C until immunohistochemical staining.

On day one, free-floating sections (30µm thickness) were incubated with mouse

monoclonal anti-CD-36 (1:100, Abcam, USA), rabbit polyclonal anti MCP-1 (1:100, Abcam,

USA) primary antibodies overnight at 4°C, followed by day two incubation with goat anti-mouse

51

secondary antibody (1:1000 Alexa 488, Molecular Probes, USA), and goat anti-rabbit secondary

antibody 1:1000-2000 Alexa 594, Molecular Probes, USA) for 2 hrs at RT. Finally, tissue was

incubated with Hoechst 33258 (bisBenzimideH 33258 trihydrochloride) for 30 mins (1:300

Molecular Probes, USA) to visualize cell nuclei. Then tissue underwent three washes in PBS

and was coverslipped with Fluoromount (Sigma). The same immunohistochemistry protocol

described above was employed for all acute and chronic groups, with tissues incubated in

mouse monoclonal anti-CD-36 (1:100, Abcam, USA) and rabbit polyclonal anti Iba-1 (1:300,

WAKO, Japan) primary antibodies, followed by goat anti-mouse (1:1000 Alexa 488, Molecular

Probes, USA) and goat anti-rabbit secondary antibodies (1:2000 Alexa 594, Molecular Probes,

USA).

Only sections from the 24-hrs group underwent IHC; initial preliminary staining showed

CD-36 predominantly on the brain cortex of acute groups primarily and scarcely on the 60-day

chronic group. Sections were stained with mouse monoclonal anti-CD-36 (1:100, Abcam, USA)

and rabbit monoclonal anti-NeuN (1:500, Millipore, USA) primary antibodies overnight at 4°C.

The next day, tissue was incubated for 2 hrs in goat anti-mouse secondary antibody (1:1000

Alexa 488, Molecular Probes, USA) and goat anti-rabbit secondary antibody (1:1000-2000

Alexa 594, Molecular Probes, USA), followed by incubation with Hoechst (bisBenzimideH 33258

trihydrochloride) for 30 mins (1:300 Molecular Probes, USA), then tissue underwent three

washes in PBS for 30 mins and was coverslipped with Fluoromount (Sigma). Likewise, only

sections from the 24-hrs group went through the same incubation steps with mouse monoclonal

anti-CD-36 (1:100, Abcam, USA) and rabbit polyclonal anti-GFAP (1:2000,DAKO, USA) primary

antibodies, with goat anti-mouse (1:1000 Alexa 488, Molecular Probes, USA) and goat anti-

rabbit secondary antibodies (1:1000-2000 Alexa 594, Molecular Probes, USA).

52

3.2.3 Immunohistochemistry of Spleen

Spleen cryosectioning was carried out at a thickness of 20 µm by cryostat at -19°C.

Fluorescent immunostaining was done on every 6th (1/6) coronal section and mounted directly

onto the slide. Day one involved incubating tissue sections with mouse monoclonal anti-CD-36

(1:100, Abcam, USA) overnight. The next day, tissue slides were incubated in goat anti-mouse

secondary antibody for 2 hrs (1:1000 Alexa 488, Molecular Probes, USA). Then tissue was

washed 3 times for 10 mins and coverslipped with DAPI H-1200 mounting medium

(Vectashield). IHC was performed on spleen sections of acute and chronic TBI groups and their

respective controls.

A multiphoton laser confocal scanning microscope (FV1000 MPE, Olympus, USA) was

used for observation and imaging for all immunofluorescence photomicrographs of the motor

cortex area (M1, M2) at a 40x magnification; only linear adjustments (brightness and contrast)

were made to the figures. When quantification of percentage of positive cells was determined, Z

stacks were created at 1 µm intervals throughout the 30 µm of the sections with a guard region

of 2 mm excluded from top and bottom of the Z stack. The Z stacks were rotated in all planes to

verify double labeling. A fluorescent microscope (Axio Imager.Z1, Zeiss, USA) was used for

observation and imaging of spleen slides at a 40x magnification. Six serial spleen coronal

sections were quantified using Image J and results are displayed as the average mean of

fluorescent intensity.

3.2.4 Statistical analysis on indirect immunofluorescence in brain

CD-36, MCP-1, Iba-1, Hoechst positive cells adjacent to impact area (M1 motor cortex

area) were counted using Image J using 6 serial coronal sections. Results showed the average

mean of the cell ratios of CD-36/DAPI, (CD-36 + MCP-1 cells)/ total MCP-1, MCP-1/DAPI, CD-

36/DAPI, Iba-1/DAPI, CD-36/Iba-1. Likewise, the contralateral side of injured brains and sham

brains were analyzed. Explored coronal sections correspond to +2.7 to -0.3 mm with respect to

53

bregma. The data obtained were analyzed using the Graph Pad Prism 5.0 software and were

evaluated statistically using one-way analysis of variance (ANOVA) for group comparisons with

95% CI followed by subsequent pair-wise comparisons using post hoc Bonferonni test. All data

are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses.

Likewise for the 24-hrs group CD-36, NeuN, GFAP, and Hoechst positive cells adjacent to

impact area (M1 motor cortex area) were counted using Image J using 6 serial coronal sections.

Results showed the average mean of the cell ratios of CD-36/DAPI, (CD-36 + NeuN cells)/ total

NeuN cells, (CD-36 + GFAP cells)/ total GFAP cells. In this case, the contralateral side and the

control brains (non-TBI/naïve brains) were compared against the ipsilateral side. Coronal

sections correspond to +2.7 to -0.3 mm with respect to bregma. The data obtained was also

analyzed using the Graph Pad Prism 5.0 software and were evaluated statistically using one-

way analysis of variance (ANOVA) for group comparisons with 95% CI, followed by subsequent

pair-wise comparisons using post hoc Bonferonni test. All data are presented as mean values ±

SEM. Statistical significance was set at p<0.05 for all analyses.

3.2.5 Statistical analysis on fluorescence intensity in spleen

The data obtained was analyzed using the Graph Pad Prism 5.0 software to run the

unpaired t-test with Welch’s correction to compare spleen from TBI animals vs. sham animals.

3.2.6 Western Blot

A second set of animals were sacrificed at different time-points under deep anesthesia:

24-hrs, 48-hrs, and 7 days post-TBI surgery, along with their corresponding sham groups. All

TBI animals and shams underwent transcardial perfusion as previously mentioned. Brain tissue

was dissected into right and left brain regions for the cortex, striatum, dorsal and ventral

hippocampus (Chiu, Lau et al. 2007). Immediately, tissues and spleens were placed in

individual cryoprotectant vials in liquid nitrogen (-196°C) and transferred to a -80°C freezer.

54

Tissue from the cortex and spleen were homogenized using an electric tissue homogenizer in

1:10 weight to volume ratio of ice-cold RIPA buffer (Cell Signaling Technology, USA) containing

1% protease inhibitor cocktail (Sigma-Aldrich, USA). Following homogenization, sample lysates

were centrifuged at 10,000rpm at 4ºC for two rounds of 30 mins. Pellets were discarded, and

the supernatant was then aliquoted in 50μl samples and stored at -80°C. Next, a protein assay

(BCA Protein Assay, Pierce, USA) was applied to determine total protein concentration for each

fraction sample. Pre-cast electrophoresis gels (4-15% Mini Protean TGX, Bio-Rad, USA) were

loaded with 30μg of total protein per sample per well and current was applied at 70V for 45

mins, resuming at 100V for 30min. Proteins were transferred to nitrocellulose membranes (Bio-

Rad, USA) using a Criterion blotter (Bio-Rad, USA), at 100V, for 30 mins. The membranes were

pre-incubated in 5% non-fat dairy milk (NFDM)/Phosphate Buffered Saline (PBS, Pierce,

USA)/0.05% Tween 20 (PBST, Fisher-Scientific, USA) for 1 hr, then incubated overnight at 4°C

with one of the following primary antibodies: rabbit monoclonal CD-36 (1:250, Abcam, USA);

mouse monoclonal GAPDH (1:25,000). The next day, membranes were washed with PBST and

incubated with the corresponding secondary antibody): goat anti-rabbit IR Dye 800 (1:3000,

LICOR, USA) and goat anti mouse 680 (1:25,000, LICOR, USA) during 2 hrs at RT. The

membranes were thoroughly washed with PBST and placed into an Odyssey infrared imager

(LICOR, USA) for band detection. Images were analyzed through the Image Studio software

(LICOR, USA) for band density measurements. The band density ratios from TBI vs. sham

groups were statistically compared.

3.2.7 Statistical Analysis on Western Blot

The data were evaluated statistically using one way analysis of variance (ANOVA) for

group comparisons with 95% CI followed by subsequent pair wise comparisons using the post

hoc Bonferonni test. All data are presented as mean values ± SEM. Statistical significance was

set at p<0.05 for all analyses.

55

3.3 Results

3.3.1 CD-36 Expression in the TBI Brain

Histological experiments of acute time-points post-TBI revealed that at the acute stage

CD-36, expression was found predominantly in the M1 and M2 cortical regions, along the

cingulate cortex of the ipsilateral frontal lobe of the brain. CD-36 expression also extended to

the cingulate cortex of contralateral brain hemisphere (Figures 2-4). These results suggest that

at acute stages the CD-36 inflammatory response disseminates from the core of impact to the

rest of the motor cortex reaching the contralateral side of the brain. We correlated these

findings with other neuroinflammatory markers such as MCP-1 and Iba-1 for microglia.

We found MCP-1 positive expressing cells with two distinct morphologies: star-shaped

cells and other cells that also co-localized with CD-36. MCP-1 is typically expressed by

monocytes and vascular endothelial cells, among others in culture. MCP-1 is a chemokine, a

mediator of a number of processes: it regulates migration and infiltration of

monocytes/macrophages, immune response surveillance of tissues, and in the response to

inflammation, wound healing, and in general systemic reactions following tissue or organ

injuries (Deshmane, Kremlev et al. 2009). About 5% of cells show co-localization of CD-36 &

MCP-1. In the case of the sham acute groups at different time-points, they showed very few CD-

36+ expressing cells (<5%) compared to TBI. Thus, an up regulation of CD-36 expression

suggests its pathological role in TBI. Results on the animal group corresponding to the 60 day

time-point after TBI revealed scarce immunoreactivity for CD-36 in the area of impact and none

in the hippocampal or striatum areas (Figure 5). We observed brain co-localization of CD-36,

MCP-1 and Iba-1 on impact cortical area, significant increases of CD-36 and MCP-1 positive

cells in the ipsi vs. contra hemispheres of TBI vs. sham groups, but no significant increases of

Iba-1 expressing cells over time. Immunoblotting support overexpression of CD-36 in brain at

acute post-TBI time-points vs. sham. Next, we wanted to know if CD-36 imparted neuronal or

56

astrocytic-mediated inflammation in one of the acute groups, the 24-hrs post-TBI. Thus, we

tested CD-36 with well-known neuronal and astrocytic markers. We found CD-36 and NeuN co-

localization in neurons in the impact area in this acute stage of TBI (Figure 6 A-D).

Figure 2. Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 24 hours post-TBI. Fluorescence photomicrographs revealed co-localization of CD-36 & MCP-1, CD-36 and Iba-1 at the cortical core of impact at 24 hrs among the TBI (n=4) and sham groups (n=3). Direct quantification of CD-36 positive (+) cells in the vicinity also expressed MCP-1 along the cingulate cortex (CC) of the ipsilateral (ipsi) and contralateral (contra) sides. For the CD-36/MCP-1 stain one way ANOVA testing showed statistical significance when comparing the ipsi vs. contra and sham group CD-36/DAPI, (CD-36+/MCP-1)/MCP-1, and MCP-1/DAPI ratios. Their F values were (F (2,8) = 17.26, p < .005), (F(2,8) = 20.35, p < .0005), F (2,8) = 26.65, p < .0005), respectively. For the CD-36/Iba-1 stain one way ANOVA testing showed statistical significance when comparing the ipsi vs. contra and sham group CD-36/DAPI ratios (F(2,8) = 65.10, p < .0005). The comparison of the CD-36/Iba-1 ratio between the ipsi and sham groups showed significance F (2,8) =11.31, p < .005). The comparison of the Iba-1/DAPI ratio among the three groups was not significant. Fluorescence photomicrographs were taken at a 40x magnification.

57

Figure 3. Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 48 hours post-TBI. Fluorescence photomicrographs revealed co-localization of CD-36 & MCP-1, CD-36/Iba-1 at the cortical core of impact at 48 hrs among the TBI (n=4) and sham groups (n=3). Direct quantification of CD-36 positive (+) cells in the vicinity also expressed MCP-1 along the cingulate cortex (CC) of the ipsilateral (ipsi) and contralateral (contra) sides. For the CD-36/MCP-1 stain one way ANOVA testing showed statistical significance when comparing the ipsi vs. contra and sham group CD-36/DAPI, (CD-36+/MCP-1)/MCP-1, and MCP-1/DAPI ratios. Their F values were (F (2,8) = 33.55, p < .005), (F (2,8) = 9.618, p < .05). F (2,8) = 53.11, p < .0005), respectively. For the CD-36/Iba-1 stain one way ANOVA testing showed statistical significance when comparing the ipsi vs. contra and sham CD-36/DAPI ratios (F(2,8) =74.76, p < .0005). The comparison of the CD-36/Iba-1 ratio between the sham group vs. ipsi and contra showed significance F (2,8) =10.27, p < .005). The comparison of the Iba-1/DAPI ratio among the three groups was not significant. Fluorescence photomicrographs were taken at a 40x magnification.

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Figure 4. Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 7 days post-TBI. Fluorescence photomicrographs revealed co-localization of CD-36 & MCP-1, CD-36/Iba-1 at the cortical core of impact at 48 hrs among the TBI (n=4) and sham groups (n=3). Direct quantification of CD-36 positive (+) cells in the vicinity also expressed MCP-1 along the cingulate cortex (CC) of the ipsilateral (ipsi) and contralateral (contra) sides. For the CD-36/MCP-1 stain one way ANOVA testing showed statistical significance when comparing the ipsi vs. contra and sham group ratios of CD-36/DAPI, (CD-36+/MCP-1)/MCP-1, and MCP-1/DAPI. Their F values were (F (2,8) = 27.31, p < .0005), (F(2,8) = 13.01, p < .005), F(2,8) = 15.23, p < .005), respectively. For the CD-36/Iba-1 stain one way ANOVA testing showed statistical significance when comparing sham vs. ipsi and contra CD-36/DAPI ratios (F(2,8) =23.11, p < .0005). The comparison of the CD-36/Iba-1 ratio between the sham group vs. ipsi and contra showed significance F (2,8) =26, p < .0005). There was a significant difference in the Iba-1/DAPI ratio of the ipsilateral vs. contra and sham. Fluorescence photomicrographs were taken at a 40x magnification.

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Figure 5. Co-localization of CD-36/MCP-1, CD-36/Iba-1 at the cortical core of impact at 60 days post-TBI. Fluorescence photomicrographs showed immunodetection at the cortical core of impact at 60 days Post-TBI. No statistical significance was present when comparing the average cell ratios, CD-36/DAPI, (CD-36 + MCP-1/MCP-1), and MCP-1/DAPI among the ipsilateral, contralateral & sham groups. No statistical significance was present when comparing the average cell ratios of CD-36/DAPI and CD-36/Iba-1,There was a small significant difference in the Iba-1/DAPI ratio of the sham vs. contra and ipsi groups F(2,8) =198.6, p < 0.0005). Fluorescence photomicrographs were taken at a 40x magnification.

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Figure 6. A-D Co-localization of CD-36/NeuN at 24 hours after TBI. Fluorescence photomicrographs revealed co-localization of CD-36 with NeuN found only in the ipsilateral (A) hemisphere demonstrates that neuronal expression of CD-36 follows 24 hrs after TBI (n=4). No co-localization is shown in the (B) contralateral (n=4), and (C) non-TBI hemispheres (n=3). Z-stacked (D) Image shows co-labeling of both markers at a magnification of 100x. The data obtained were evaluated statistically using one way analysis of variance (ANOVA) for group comparisons with 95% CI followed by subsequent pair wise comparisons using post hoc Bonferonni test (F(2,8)=16.14, p < .005). All data are presented as mean values ±SEM. Statistical significance was set at p<0.05 for all analyses.

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These results provide evidence of CD-36 mediated inflammation in neurons. We also

found minimal co-localization of CD-36 with GFAP, which could indicate a partial CD-36

mediated inflammation in astrocytes (Figure 7 A-D). Nevertheless, we observed widespread

astrogliosis throughout the injured hemisphere, including the cortex, corpus callosum, and

hippocampus. GFAP immunoreactivity was also observed in the contralateral hemisphere but to

a lesser extent when compared to the ipsilateral hemisphere. Yet active astrocytes were found

to be widespread also in the cortex, corpus callosum, and hippocampus of the left hemisphere.

We did not perform the double stain on the chronic group since the initial single CD-36 stain

showed negative/minimal immunoreactivity. Accordingly, we limited this study to the acute

stage.

3.3.2 CD-36 in the TBI Spleen

Recent ischemic and hyperlipidemic rodent models suggest that infiltrating

monocytes/macrophages from the periphery are the major source of CD-36 in the post-ischemic

brain. Accordingly, we characterized the spleen pathological alterations in the acute and chronic

stages of TBI by indirect immunofluorescence and quantification of fluorescence intensity of CD-

36 positive monocytes. We observed that CD-36 positive monocytes localized mostly in the red

pulp as opposed to their scarce expression in the white pulp. The increased CD-36 expression

in spleen suggests that the splenic inflammatory response via the CD-36 pathway may

contribute to the general inflammatory response after TBI (Figures 8-11).

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Figure 7. A-D Minimal Co-localization of CD-36/GFAP at 24 hours after TBI. Fluorescence photomicrographs showed minimal co-localization of CD-36 with GFAP found in the ipisilateral hemisphere (A). No co-localization is shown in the contralateral (n=3) (B) and non-TBI (n=4) (C). Z-stacked (D) image shows co-localization of both markers in the injured motor cortex at a magnification of 100x. The data obtained were evaluated statistically using one way analysis of variance (ANOVA) for group comparisons with 95% CI followed by subsequent pair wise comparisons using post hoc Bonferonni test (F(2,8)=20, p<0.05). All data are presented as mean values ±SEM. Statistical significance was set at p<0.05 for all analyses.

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Figure 8. Spleen CD-36 Immunodetection at 24 hours post-TBI. Fluorescence photomicrographs demonstrate CD-36 positive (+) monocytes predominate in the vicinity/ borderline of the red and white pulp in the 24-hrs TBI spleen (1A) compared to 24-hrs sham spleen (1B). Scale bar = 50um. Fluorescence intensity of CD-36 positive (+) cells was increased in the spleen of TBI animals (n=3) vs. sham group (n=3). Photomicrographs were taken at a magnification of 40x. The data obtained showed no significance between the two groups, data was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction (t=0.3613, df=4).

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Figure 9. Spleen CD-36 immunodetection at 48 hours post TBI. Fluorescence photomicrographs revealed CD-36 positive (+) monocytes predominate in the vicinity/ borderline of the red and white pulp in the 48-hrs TBI spleen (2A) compared to the 48-hrs sham spleen (2B). Scale bar = 50um. Fluorescence intensity of CD-36 postive (+) cells showed an increased difference between TBI (n=3) and sham groups (n=4). Asterisk (*) denotes significance t =1.038, df = 5. Unpaired t-test with Welch’s correction. Photomicrographs were taken at a magnification of 40x. The data obtained was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction.

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Figure 10. Spleen CD-36 immunodetection at 7 days post TBI. Fluorescence photomicrographs showed CD-36 positive (+) monocytes do not show an larger expression in the vicinity/borderline of the red and white pulp in the 7-days TBI spleen (3A) compared to 7-days sham spleen (3B). Scale bar = 50um. Fluorescence intensity of CD-36 positive (+) cells in 7 day TBI group (n=3) does not show difference from sham groups (n=4). Photomicrographs were taken at a magnification of 40x. The data obtained was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction (t=0.3010, df=5).

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Figure 11. Spleen CD-36 immunodetection at 60 days post TBI. Fluorescence photomicrographs demonstrated CD-36 positive (+) monocytes predominate in the vicinity/ borderline of the red and white pulp in the 60-days TBI spleen (4A) compared to the 60-days sham spleen (4B). Scale bar = 50um. Fluorescence intensity of CD-36 postive (+) cells was increased in the spleen of TBI animals (n=3) vs. sham group (n=6). Asterisk (*) denotes significance ( t = 2.386, df = 4). The data obtained was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction. Photomicrographs were taken at a magnification of 40x.

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3.3.3 Western Blot

3.3.3.1 Protein Detection in the Brain Cortex

To further detect the presence of CD-36 in the area of impacted brain (i.e., cortex), we

used Western blot to measure and compare semi-quantitatively protein levels between brain

cortical extracts of sham and acute TBI groups. Protein separation is based on charge and

molecular weight size (MW). Despite being a useful technique, it has its limitations, since it does

not measure the absolute value of protein, but instead the amount of protein relative to others of

known MW. Western blot analysis of CD-36 protein expression was detected in the brain cortex

of TBI and sham animals. GAPDH was used as normalizing protein. Protein density bands were

detected at 78 kDa, the glycosylated CD-36 fragment. The ratio between the band density for

CD-36 vs. GAPDH showed a significant increase in CD-36 protein levels in the ipsilateral brain

cortex of the 24-hrs and 48-hrs post TBI groups vs. the contralateral brain cortex fractions, and

also against their corresponding sham groups (Figures 12-13). Western blot analysis of CD-36

protein expression of TBI and sham animals in the spleen showed no significant difference

among groups. Bands were detected at 53 kDa, the non-glycosylated CD-36 fragment.

3.3.3.2 Protein Detection in the Spleen

We decided to probe for protein levels in the spleen since it is implicated in the

inflammatory response after TBI, as documented by previous publications in stroke and our

recent findings from whole spleen lysates in the CD-36 neonatal TBI studies. Based on our IHC

results CD-36 positive monocytes in the spleen post-TBI suggest their role in the peripheral

immune response. Thus, we wanted to compare CD-36 splenic protein levels in TBI vs. sham

groups. Our results detected CD-36 in spleen lysates of TBI and sham groups. The relative

protein ratios of the 53 kDa fragment of CD-36 in TBI vs. sham showed no significant difference

relative to GAPDH.

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Figure 12. CD-36 brain cortex protein expression at 24 post-TBI. Two bands were detected, a band detected at 78 kDa, glycosylated CD-36 fragment, and the second band at 53 kDa, non-glycosylated fragment. We measure band density for the 78kDa glycosylated fragment and compared it against GAPDH. The data obtained were evaluated statistically using one way analysis of variance (ANOVA) for group comparisons (F(2,8)=6.477, p < 0.05), followed by subsequent pair wise comparisons using post hoc Bonferonni test . Significance (p<0.05) was observed in the indicated graph columns for the 24Hrs post TBI group GAPDH was used as normalizing protein. Sample size for the TBI group (n=4) and Sham groups (n=3).

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Figure 13. CD-36 brain protein expression 48 hours post-TBI. Two bands were detected, a band detected at 78 kDa, glycosylated CD-36 fragment, and the second band at 53 kDa, non-glycosylated fragment. We measure band density for the 78kDa glycosylated fragment and compared it against GAPDH. The data obtained were evaluated statistically using one way analysis of variance (ANOVA) for group comparisons, (F(2,8)=4.756, p < 0.05) followed by subsequent pair wise comparisons using post hoc Bonferonni test . Significance (p<0.05) was observed in the indicated graph columns for the 24Hrs post TBI group GAPDH was used as normalizing protein. Sample size for the TBI group (n=4) and Sham groups (n=3).

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Figure 14. CD-36 protein expression in spleen 24 hours post-TBI. A band detected at 53kDa, the non–glycosylated CD-36 fragment. No significance was observed in the indicated graph columns for the 24Hrs post TBI group. ). The data obtained was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction (t=0.5313, df=5) Sample size for the TBI group (n=4) and Sham groups (n=3).GAPDH was used as a normalizing protein.

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Figure 15. CD-36 protein expression in spleen of 48 hours post-TBI. A band detected at 53kDa, the non–glycosylated CD-36 fragment. No significance was observed in the indicated graph columns for the 48Hrs post TBI group. The data obtained was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction (t=0.5121, df=3) Sample size for the TBI group (n=3) and Sham groups (n=3). GAPDH was used as normalizing protein.

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Figure 16. CD-36 protein expression in spleen of 7 days post-TBI. A band was detected at 53kDa, the non–glycosylated CD-36 fragment. No significance was observed in the indicated graph columns for the 7 Days post TBI group. The data obtained was analyzed using the graph pad Prism 5.0 software to run the unpaired t-test with Welch’s correction (t=1.292, df=3) Sample size for the TBI group (n=3) and Sham groups (n=3). GAPDH was used as normalizing protein.

However, CD-36 protein ratio levels were slightly increased in the 24-hrs compared to

the 48-hrs and 7 day groups (Figures14-16). This suggests an elevated CD-36 inflammatory

splenic response early after TBI.

3.4 Discussion

In this study we examined the relevance of CD-36 to post-traumatic inflammation that

accompanies the secondary brain damage associated with TBI. We showed a novel observation

of increased CD-36 positive cell populations in the brain of TBI animals that appeared to peak

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during the early phase of injury, with CD-36 elevation maintained up to 7 days post-TBI, an

indication for CD-36’s role in acute neuroinflammation. Along with the production of other

inflammatory mediators, including MCP-1 and Iba-1, CD-36 cell immunoreactivity was robustly

upregulated in the area of impacted brain in acute time-points over that of the chronic TBI

period.

The non-detectable expression of CD-36 in brain cortical regions at the 60-day group

suggests a decline in the CD-36 involvement in the progressive inflammation seen in chronic

TBI. The observed dynamic CD-36 expression in the acute phase of TBI suggests a therapeutic

window for TBI within the first 24 hours when targeting CD-36-mediated neuroinflammation. In

addition, another interesting observation is the co-localization of CD-36 with NeuN, indicating a

direct influence of CD-36 in mediating inflammation, and likely cell death, in neurons in the

cortex. In contrast, minimal co-localization of CD-36 with GFAP suggests that CD-36 minimally

contributes to astrocytic-mediated inflammation. Interestingly, TBI-exposed MCP-1 knockout

mice showed no co-localization of MCP-1 with NeuN and GFAP in the cortex within 4 days of

TBI; instead, there was co-localization of MCP-1 with a subset of F4/80-positive amoeboid

macrophages accumulating in the lesion site (Semple, Bye et al. 2010). Further studies are

necessary to further characterize the cell phenotypes affected by CD-36 over-expression, which

should lead to a better understanding of the CD-36-mediate inflammation and its treatment.

Neuroinflammation involves a cascade of events contributing to progressive damage.

Our present data suggest a solid connection between CD-36 and MCP-1 after TBI. The

chemokine MCP-1 is implicated in macrophage recruitment into damaged parenchyma after

TBI. Consistent with CD-36 induction, previous work by our laboratory has established

increased CD-36 protein expression in the brain and spleen of TBI-exposed neo-natal rats.

Here, we further investigated CD-36 protein expression specifically in the brain cortex of acute

and chronic groups. CD-36 protein levels were abundant in the ipsilateral brain cortex relative to

contralateral and sham samples. In addition, we determined a critical timing (i.e., 24-48 hours,

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and 7 days) of CD-36 expression (from downregulation to upregulation) in the brain, which may

signal the transition of functional effects of this immune response from pro-survival to cell death.

The overall decline of CD-36 expression across the acute groups and the 60 day post TBI group

might be a delayed response of hematogenous macrophages to arrive at the site of injury. For

example, in a stroke study mononuclear phagocytes accumulated in the injured brain are initially

resident microglia at 3 to 4 days, and blood-derived macrophages at 6 to 7 days (Schroeter,

Jander et al. 1997). This explains brain levels, but does not explain CD-36 downregulation in the

spleen at 48 hours and 7 days.

Our study also examined CD-36 protein expression levels in the spleen during acute TBI

time- points. No significant differences in protein levels occurred between TBI and sham

animals at each time-point, but in the initial 24 hrs after TBI, CD-36 protein levels were slightly

higher than those in the 48-hrs and 7 days groups. Although a CD-36 peak splenic expression

occurred mainly at 24 hrs in the TBI, there was no significant difference between TBI and sham

groups, yet the relative protein expression is higher at 24 hrs when compared to the 48-hrs TBI

group. However, there was no significant difference when comparing TBI vs sham at 48 hrs and

7 days post-TBI. We assume this initial splenic response peaking within the first 24 hrs is CD-36

mediated, followed by a slow decline afterwards. In our study, we were limited by a small

number of animals to account for CD-36 expression in the spleen; this may be a reason why we

observed no difference. If we increase sample size, we may be able to detect a significant

increase in CD-36 protein expression among the acute and sham groups. Yet, there are cellular

evens and molecular interactions of CD-36 with pro-inflammatory and anti-inflammatory

cytokines/chemokines that may also be responsible for CD-36 downregulation in the spleen.

Recent studies support upregulation of CD-36 in brain and plasma of mice early after

TBI (Doyle and Buckwalter 2012; Kim, Febbraio et al. 2012), MCP-1 levels in human CSF

(Semple, Bye et al. 2010), and in serum of rats exposed to experimental TBI within hours of

injury (Rancan, Otto et al. 2001; Rhodes, Sharkey et al. 2009; Semple, Bye et al. 2010). This

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may indicate an initial CSF chemokine production as part of the initial responses to TBI,

triggering subsequent neuroinflammatory cascades. The detection of these inflammatory

mediators in serum and plasma of TBI patients and TBI rodents opens up new avenues for

clinical translation of CD-36 as a screening biomarker.

An important phenomenon is monocyte trafficking to the site of injury. Monocyte

recruitment is depicted as a tightly regulated event involving a steady paced influx of various

monocyte subsets that either express or do not express the pro-inflammatory chemokine

receptor CCR2 (Hughes, Allegrini et al. 2002; Babcock, Kuziel et al. 2003; Dimitrijevic,

Stamatovic et al. 2007). A possible explanation for the CD-36 protein downregulation in the

spleen of acute time-points after the initial 24 hrs boost may be that different subsets of

monocytes are activated by other inflammatory signaling pathways over those CD-36 mediated

monocytes subsets. A study found temporal differences in peripheral immunity after TBI in a

murine model of closed head injury at early time-points (1-7 days) and later ones (30 days and

60 days) after injury. The study’s relevant findings include a loss of thymocytes as early as 3

days after injury; a reduction in blood monocyte counts as early as 24 hrs after injury in TBI vs.

sham groups, a response that was sustained during the next 30 days after injury; and at 60

days post-TBI, the researchers detected a shift in the monocyte make-up towards an anti-

inflammatory (M2) phenotype (Schwulst, Trahanas et al. 2013).

A second specific connection between CD-36 and pro-inflammatory chemokines occurs

between CD-36 and the chemokine receptor CCR2, activated by the MCP-1 chemokine. For

instance CD-36 ligands induced CC and CXC chemokine production in stroke, and the

increases were attenuated in CD-36 deficient mice (Kim, Gautam et al. 1995; Moore, El Khoury

et al. 2002; Stewart, Stuart et al. 2010). In addition, previous reports linked the spleen to stroke

pathology; the larger the injury associated with hyperlipidemia, the greater the reduction in

spleen weight, and the higher the expression of CCR2 in the post-ischemic brain (Schober,

Zernecke et al. 2004). Overall, these studies suggest various subsets of monocytes traveling

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from the spleen to the area of injury. The range of monocyte subsets becoming activated after

injury, such as in stroke and TBI, may include other non CD-36 related inflammatory signaling

pathways.

In this project, we showed that CD-36 is elevated 24 hours after TBI, and even after 7

days post-TBI in the brain cortex. We have demonstrated that CD-36 is significantly elevated in

the brain cortex of acute TBI time points when compared to contralateral and sham animals.

Our findings support the upregulation of brain and splenic CD-36 mediated activation in the first

24 hours after TBI. Yet the mounted CD-36 brain response is sustained until 7 days post-TBI. In

contrast, splenic CD-36 expression remains unchanged even after days of injury, but it declines

after 1 day. The expression of CD-36 with the studied inflammatory markers, such as MCP-1

and Iba-1, suggests macrophage activation, certain/minimal astrocyte activation in the brain

cortex, and microglia activation associated to other inflammatory mechanisms after TBI. In the

case of the 60-Day post TBI group, very few cells express CD-36 in the cortex (and spleen),

suggesting that CD-36 has a minimal role in chronic inflammation, but is more prominent in the

acute stages of TBI.

Future directions in the study of CD-36 as an inflammatory mediator may expand on how

CD-36 regulates the mobilization of various monocyte subset populations by interacting with

other pro-inflammatory cytokines/chemokines and their receptors, such as CCR2. As described

by murine and human studies of inflammatory and cardiovascular diseases, monocytes possess

the ability to differentiate into inflammatory or anti-inflammatory subsets whenever they sense

environmental changes (Ingersoll, Platt et al. 2011; Yang, Zhang et al. 2014). This ability of

heterogeneity in monocyte subsets may serve valuable in predicting the outcome of TBI-related

inflammation. Current studies are focusing on defining the inflammatory and anti-inflammatory

functional phenotypes of human monocyte subsets. It will be interesting to identify temporal

profiles of cytokines/chemokines influencing monocyte subsets in the brain and spleen of TBI

animal models, and at the same time compare them to groups of animals subjected to

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splenectomy, as these observations may provide further insights on the inflammation-mediated

pathology and treatment of TBI. We also suggest focusing on those monocyte subsets that

infiltrate within hours after injury up to 7 days. This temporal phenomena of monocyte infiltration

has been described in stroke murine studies (Gelderblom, Leypoldt et al. 2009; Jin, Yang et al.

2010). Thus, in vitro studies may be conducted to block CD-36 and record any differences on

monocyte subsets, their ability to acquire a particular phenotype in circulation, and their

peripheral influence after injury.

There are also pharmacological approaches using specific CD-36 inhibitor, which would

contribute allow a much better understanding of CD-36 target engagement effects. We also

recognized that the major limitation in this study included small sample size, which likely

affected the statistical analyses of CD-36 protein expression. Alternatively, the lack of alteration

in CD-36 expression may be due to a rapid upregulation or decline associated with the supra-

acute period post-TBI not captured in this study. Nonetheless, these results demonstrated that

CD-36 closely accompanied the inflammation in the acute TBI period (24 hours to 7 days).

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CHAPTER 4

PHARMACOLOGICAL INTERVENTIONS DESIGNED TO ABROGATE SECONDARY

INFLAMMATION AFTER TBI INJURY

4.1. Introduction.

Stroke murine animal models have shown that reducing CD-36 mediated inflammation

may be a useful therapeutic strategy to diminish vascular injury, glial scar formation, and cell

death (Bao, Qin et al. 2012; Cho 2012; Doyle and Buckwalter 2012). Recent human studies

support sRAGE as a candidate in the prognosis and follow up after ischemic and hemorrhagic

stroke (Qian, Ding et al. 2012; Menini, Ikeda et al. 2013; Tang, Wang et al. 2013). These studies

reveal: a) ischemic and hemorrhagic stroke patients have an increase in sRAGE serum levels

days after stroke (ranging between 26%-296%, p<0.001), and this change paralleled recovery

and recurrence or aggravation of the episodes (Menini, Ikeda et al. 2013); b) acute ischemic

stroke patients and mice, and primary cortical neurons subjected to oxygen and glucose

deprivation showed increased sRAGE plasma levels within 48hours of injury; furthermore,

sRAGE recombinant administration (intravenously) was effective in reduction of infiltrating

immune cells, improving the outcome of injury in mice, and protecting cultured neurons against

hypoxia and cell death (Tang, Wang et al. 2013). In addition, in vivo and in vitro studies indicate

that the down-regulation of CD-36 by novel class antioxidant peptides may be a useful strategy

to treat ischemic stroke victims (Cho, Szeto et al. 2007). A series of pilot studies performed by

colleagues demonstrated the inhibitory action of sRAGE, soluble receptor of advanced glycation

end products, for the CD-36 receptor in various cell types (Chapter 1, Figure 2). Data revealed

sRAGE inhibitory activity on cell association of HOCl-LDL to: a) stably transfected Chinese

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hamster ovary (CHO) cells expressing murine scavenger receptor class B, type I (SR-BI, termed

CHO-SR-BI), human CD-36 (termed CHO-CD-36); b) and most importantly to human spleen-

derived monocytes/macrophages (MD cells). Additionally, this team tested in an in vivo model of

neonatal rat TBI the ability of sRAGE to alter CD-36. Neonatal rats (7 days old) after TBI

infliction received sRAGE at different time points (15 mins, 3 hours, 12 hours, and 24

hours)(Chapter 1, Figure 4). Forty-eight hours post-TBI results revealed that levels of CD-36

expression in spleen and brain were significantly downregulated in sRAGE-treated TBI animals

compared to vehicle-treated TBI animals (Chapter 1, Figure 5). Altogether these findings lead us

to suggest sRAGE antagonistic effects as a therapeutic strategy to reduce TBI related

inflammation. Thus, we hypothesized that sRAGE administration will reduce secondary

damaging mechanisms; such as cytokine induced cell death in an in vitro cell culture model.

4.2. Specific Materials and Methods

We established an in vitro cell model of secondary inflammation that occurs following

TBI by treating human neural progenitor cells with TNF-α, to mimic acute TBI. We treated and

plated Human Neural Progenitor (hNP1) cells according to the manufacturer’s protocol. We re-

suspended the cells in a total volume of 2 mL of fully supplemented AB2 Neural Medium (pre-

warmed to 37°C). Next we plated them in 2 mL cell suspension of hNP1 cells onto a Matrigel-

coated 35 mm dish and incubated the cells at 37°C in a 5% CO2 humidified incubator. We

exchanged the medium with fresh fully supplemented AB2 Neural medium at 24 hours post

plating every other day thereafter. Once the hNP1cells reach 100% confluence, they were

dissociated manually for passaging (e.g., by gentle and slow pipetting up and down to detach

the cells). The cells were maintained at a high density, the recommended passaging ratio was

1:2. After 48 hours 4x104 cells/well/200µL were incubated with 20µL of TNF-α (1.0 µg/mL/200uL

well) for three days. On the 3rd day hNP1 cells received the different reagent treatments for 3

hours followed by the calcein and MTT assays: control, TNF-α, sRAGE alone (5µg/mL), and

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TNF-α plus sRAGE (R&D Systems, 1145-RG-050). Finally we tested the upstream inhibitory

approach against CD-36 by treating the hNP1 cells with sRAGE.

4.2.1 Measurement of Cell Viability: Calcein-AM Fluorescent Dye

Measurement of cell viability was performed by both fluorescent live/ dead cell assay

(Bell, Cao et al. 2003) and trypan blue exclusion method. A two-color fluorescence cell viability

assay was performed by Calcein-AM (Invitrogen, L3224) to be retained within live cells,

including an intense uniform green fluorescence and ethidium homodimer (EthD-1) to bind the

nuclei of damaged cells (bright red fluorescence). After two hours of reperfusion, the human

Neural Progenitor Cells (hNP1, Neuromics, Edina, MN) were incubated with 2 mM Calcein-AM

and 4 mM EthD-1 for 45 min at room temperature in darkness according to the manufacturer’s

instructions. Afterwards, cells were washed once with phosphate buffered saline, and then the

green fluorescence of the live cells was measured by the Gemini EX florescence plate reader

(Molecular Device), excitation at 485 nm and emission at 538 nm. In addition, trypan blue

(0.2%) exclusion method was conducted and mean viable cell counts were calculated in four

randomly selected areas (1 mm2, n = 10) to further reveal the cell viability. To precisely calibrate

the cell viability, the values were standardized from fluorescence intensity and trypan blue data.

4.2.2 Measurement of Cell Metabolic Activity: MTT Assay

To measure metabolic activity we used the a colorimetric assay that measures the

reduction of yellow 3-(4, 5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) by

mitochondrial succinate dehydrogenases. MTT enters the cells and passes into the

mitochondria where it is reduced to an insoluble, colored (dark purple) formazan product. The

cells are then solubilized with an organic solvent (e.g., isopropanol) and the released,

solubilized formazan reagent is measured spectrophotometrically. MTT was used as described

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in our previous report and manufacturer’s instructions (Roche, Catalog Number 11465007001)

(Kaneko, Shojo et al. 2014). Before completing 2 hours of reperfusion, the cells were incubated

with 0.5 mg/ml MTT at 37°C and 5% CO2, and incubated with lysis buffer overnight in a

humidified atmosphere at 37°C and 5% CO2. The optical density of solubilized purple formazan

was measured at 570 nm on a Synergy HT plate reader (Bio-Tex). The relative viability of the

treated cells as compared to the control cells was expressed as the % viability, using the

following formula:

[A570 of treated cells]

[A570 of control cells]

The purpose of the MTT assay was to indirectly assess cell viability by measuring MTT

reduction only in metabolically active cells.

4.2.3 Data Analysis

Statistical analysis included the use of an unpaired two-sided t-test, and a one-way

analysis of variance (ANOVA) to analyze cell viability and mitochondrial function effects in each

experimental group versus its respective control group. Group comparisons were done with

95% CI followed by subsequent pair-wise comparisons using post hoc Bonferonni test. All data

are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses.

4.3. Results

Using the calcein assay to reveal cell viability, ANOVA revealed significant treatment

effects on cell survival of hNP1 cells (F(3,16)=8.709, p values<0.0015, Figure 17 ). Pairwise

comparison via Bonferroni’s multiple comparison test showed statistical significance among the

following treatment groups: control vs. TNF-α, control vs. TNF-α + sRAGE and TNF-α vs.

sRAGE (p<0.0012). However, there were no significant effects in cell viability when comparing

x 100

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TNF-α treated group against the TNF-α + sRAGE group (p >0.05), indicating sRAGE did not

rescue against TNF-α induced toxicity. Overall these data from the calcein assay demonstrate

significant cell survival of hNP1 cells under ambient condition when treated with the drug

sRAGE, but not under TNF-α mediated injury condition. Thus, under the given sRAGE dose, it

did not appear to rescue hNP1cells from cell death after TNF-α insult.

Using the MTT assay to measure mitochondrial activity, ANOVA revealed significant

treatment effects on the level of metabolic activity of hNP1 cells (F(3,16)=42.135, p<0.001, Figure

18). Bonferroni’s multiple comparison test showed statistical significance in the mitochondrial

activity of the following treatment groups: Control vs. TNF-α, control vs. TNF-α + sRAGE, TNF-α

vs sRAGE, and TNF-α + sRAGE vs. sRAGE alone (p values <0.05). However, similar to the

calcein assay, MTT assays revealed that sRAGE did not rescue against TNF-α induced toxicity

(p>0.05).

4.4. Discussion

Our findings indicate that after the TNF-α insult there was significant cell death of hNP1

cells when compared to control. Treatment with sRAGE was well tolerated by hNP1 cells grown

under normal cell culture condition, based on their relative cell viability comparable to that of

control, suggesting the given sRAGE dose was safe. However, treatment with sRAGE did not

suppress TNF-α induced cell death. These sRAGE and TNF-α interactions observed in the cell

death were reflected in both calcein and MTT assays (Figures 17and 18). sRAGE and

endogenous secretory RAGE (esRAGE) are associated with components of metabolic

syndrome or atherosclerosis in the absence of diabetes (Koyama, Shoji et al. 2005; Koyama,

Tanaka et al. 2014).A second therapeutic effect of sRAGE that may be further exploited is its

ability to reverse vascular leakage.

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Figure 17. In Vitro hNP1 cells relative cell viability Calcein assay using sRAGE. sRAGE treatment and CD-36 inhibitory action was well tolerated by hNP1 cells. The data obtained was analyzed statistically using one-way analysis of variance (ANOVA) for group comparisons with 95% CI followed by subsequent pair-wise comparisons using post hoc Bonferonni test. All data are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses. Using the calcein assay to reveal cell viability, ANOVA revealed significant treatment effects on cell survival of hNP1 cells ( F(3,16)=8.709, p < 0.0015). Pairwise comparison via Bonferroni’s multiple comparison tests showed statistical significance among the following treatment groups: control vs. TNF-α, control vs. TNF-α + sRAGE and TNF-α vs. sRAGE.

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Figure 18. In Vitro hNP1 cells relative metabolic activity MTT assay using sRAGE. The hNP1 cells treated with TNF-α +sRAGE had a slightly higher metabolic rate/percent when compared to the TNF-α insult. The data was analyzed statistically using multiple comparison test one-way analysis of variance (ANOVA) for group comparisons with 95% CI followed by subsequent pair-wise comparisons using post hoc Bonferonni test, (F(3,16)=42.135, p<0.001). All data are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses.

Various animal studies have validated the beneficial effects of sRAGE. One of these

benefits is neuroprotection. Rodents and human models of ischemic stroke and atherosclerosis

have identified neuroprotective benefits of a soluble form of receptor for advanced glycation end

products. One study reported that plasma levels of sRAGE and high mobility group box 1

(HMGB1) were significantly increased within 48 hours after stroke, and the sRAGE level was an

independent predictor of functional outcome at 3 months post-stroke (Tang, Wang et al. 2013).

Furthermore, atherosclerotic studies carried in diabetic mice showed an inhibition of

atherosclerotic plaques by the competition of RAGE with sRAGE, suggesting that plasma.

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In an accelerated atherosclerotic diabetic mice model, sRAGE ameliorated vascular

lesions in diabetic/atherosclerotic animals in the absence of changes in plasma lipids or

glycemia (Wautier, Zoukourian et al. 1996). Additionally, sRAGE shows beneficial effects in

reversing vascular leakage in the intestine, skin of rodent diabetic models (Wautier, Zoukourian

et al. 1996; Schmidt, Yan et al. 1999). Similarly, a human clinical study using long term

combined therapy of antihypertensive drugs, nifedipine-telmisartan, showed a beneficial effect

in increasing sRAGE plasma levels, thus exerting an atheroprotective and anti-inflammatory

activity (Falcone, Buzzi et al. 2012). The drug sRAGE may be a promising therapeutic drug in

reducing atherosclerotic plaque formation in patients who may have suffer stroke, in the

presence or absence of risk factor conditions, such as diabetes and high cholesterol. There are

other antioxidant agents as alternative treatment regimens to abrogate CD-36.

A recent study suggests the antioxidant agent, SS-31, as an alternative treatment to

downregulate CD-36 expression. The molecule SS-31 showed a reduction in CD-36 insidious

action, and ischemia-induced injury (Cho, Szeto et al. 2007); in addition, SS-31 and related

compounds were proposed as neuroprotective agents. These compounds target cardiolipin, a

complex lipid (diphosphatidylglycerol lipid) on the inner mitochondrial membrane (IMM) involved

in optimization of the electron transport chain (ETC), SS-31 and related compounds are capable

of restoring cellular metabolism in aging and diverse disease models without affecting the

normal health of an organism (Szeto and Birk 2014). Both SS-31 and sRAGE may have

beneficial effects in stroke and related inflammatory disorders. For the use of inhibitors of IL-6,

such as tocilizumab or a TNF-α inhibitor like etanercept, is a fusion protein produced by

recombinant DNA, an FDA approved drug to treat rheumatoid arthritis. Etanercept may prove

beneficial in the long-term treatment of chronic TBI. These inhibitors can also be administered

IV. Anti-TNF therapy has proven beneficial in the long-term treatment of rheumatoid arthritis and

it may also work to treat the chronic neuroinflammation present in traumatic brain injury.

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Additional studies are warranted to fully reveal the efficacy of sRAGE not just for in vitro

models, but also in vivo animal models to address neurobehavioral effects coupled with the

detection of sRAGE in the brain. In an in vitro cell model, it may pose as a challenge to assess

drug adverse effects. On the other hand, such in vitro model may allow examination of

sRAGE’s mechanisms of action. Our in vitro experiment showed sRAGE had no effect on TNF-

α induced cell death treatment. Following TNF-α treatment, hNP1 cells exhibited reduced cell

viability. Unfortunately, the TNFα-treated cells exposed to sRAGE showed a small, non-

significant increase in cell viability when compared to TNF-α alone. This may be indicate further

testing at higher sRAGE doses or manipulating the timing of TNF-α and sRAGE initiation in

order to capture the true effects of sRAGE against inflammation. Alternatively, sRAGE may

exert other mechanisms of action affecting parallel molecular or cellular pathways leading to

neuroprotection that may not have been captured by simple calcein and MTT assays. More

sensitive cell viability and cell death pathway tests may be needed. In addition, further in vivo

testing would be necessary to reveal behavioral and toxicity effects before and after sRAGE

administration in an in vivo TBI animal model to assess neuronal survival, functional recovery

and any adverse effects of sRAGE.

The present in vitro study provides the basis for pursuing more in-depth studies on

sRAGE and inflammatory-based therapies directed at sequestering the secondary cell death

associated with TBI. Further studies should focus on CD-36 inhibition via s-RAGE in a pre-TBI

prophylactic approach to test for any neuroprotective effects. Although sRAGE cannot cross the

healthy blood brain barrier (BBB), his barrier is compromised after TBI and the immediate

treatment with sRAGE thereafter may allow the drug to easily reach the injured brain site.

Alternatively, even if sRAGE is not able to cross the BBB after acute TBI, we envision sRAGE

can afford inhibitory action to downregulate CD-36 expression in the spleen, which we

discussed earlier as contributing to the systemic inflammation in TBI.

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To that end, it is possible to think that sRAGE will act on the periphery (i.e., spleen) and

significantly reduce infiltrating immune/inflammatory cells destined to exacerbate the brain

inflammation after TBI. In parallel, investigations on drugs that similarly target CD-36

inflammation may reveal new avenues of TBI research. Anti-inflammatory drugs like IL-6

inhibitors, such as tocilizumab, or TNF-α inhibitors like etanercept, as adjunctive therapy may

improve sRAGE therapeutic outcome in TBI. The modest effects of sRAGE against TNFα-

induced toxicity suggest that sRAGE alone may not be protective against TBI. Increasing the

dose and altering the drug initiation regimen may improve sRAGE efficacy in TBI. Similarly,

stem cell therapy in combination with sRAGE or SS-31 could potentially augment inflammation

while promoting neurogenesis. True to our long-standing interest in stem cell therapy, we

extended our hypothesis to test sRAGE in combination with stem cells (Chapter 5) to reveal the

efficacy of this combination therapy. In particular, we next investigated human neural progenitor

stem cells combined with sRAGE to assess any synergistic effects of these two treatments on

cell viability and metabolic cell activity.

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CHAPTER 5

STEM CELL THERAPY IN COMBINATION WITH SRAGE MAY AMELIORATE SECONDARY

INFLAMMATION THAT MEDIATES TBI INJURY IN-VITRO CELL MODEL OF TBI

5.1 Introduction

Head trauma and long-term cognitive decline has been addressed for years by

epidemiological and clinical studies. Follow up of patients 10-20 years after admission to

hospital with TBI provided further evidence of late stage neurodegeneration (Millar, Nicoll et al.

2003; Smith, Gentleman et al. 2012). In addition studies demonstrate that prolonged microglial

activation is a feature of traumatic brain injury, but that the neuroinflammatory response returns

to control levels after several years (Smith, Gentleman et al. 2012). Furthermore, a recent in

vivo rat study demonstrated prolonged pathological outcomes 60 days after experimental TBI

characterized by elevated inflammation and suppressed neurogenesis (Acosta, Tajiri et al.

2013).

Based on our previous results (Chapter 4), sRAGE had no effects against TNFα-induced

toxicity suggesting that sRAGE alone may not be efficacious against inflammation TBI.

Augmenting sRAGE dose and adjusting its administration in a timely manner may increase

sRAGE efficacy in TBI. An alternative therapy is to combine sRAGE with other anti-

inflammatory drugs, which may possibly improve CD-36, as well as non-CD-36, inhibitory

effects of sRAGE. Likewise, stem cell therapy in combination with sRAGE/or SS-31 could

substantially increase inflammation, yet promote neuronal cell survival/neurogenesis. We

studied the use of human neural progenitor stem cells combined with sRAGE, to determine any

concerted effects of these two treatments on cell viability and metabolic cell activity.

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Accumulating evidence in stroke studies has implicated the mobilization of bone marrow

(BM)-derived stem cells to promote regeneration in damaged brain regions (Borlongan, Glover

et al. 2011). These cells have the extraordinary ability to travel from the bone marrow (BM), thru

the peripheral circulatory system, to eventually enter the injured brain, and secrete

neuroprotective growth factors that promote cell survival/rescue neurons from cell death. Among

the repertoire of bone marrow derived stem cells (BMDSCs) we have: hematopoietic stem cells

(HSCs), mesenchymal stem cells (MSCs), and endothelial progenitor cells (EPCs) and very

small embryonic-like cells (VSELs). These have been demonstrated to exert therapeutic

benefits in animal models of stroke (Yasuhara, Matsukawa et al. 2006; Hess and Borlongan

2008; Hess and Borlongan 2008), human stroke clinical trials (Kondziolka, Wechsler et al. 2000;

Meltzer, Kondziolka et al. 2001; Nelson, Kondziolka et al. 2002), and in other models of

neurological diseases like epilepsy (Venturin, Greggio et al. 2011), Parkinson’s disease (Khoo,

Tao et al. 2011; Danielyan, Beer-Hammer et al. 2014), and Alzheimer’s disease (Nikolic, Hou et

al. 2008; Danielyan, Beer-Hammer et al. 2014). Encouraging outcomes of the latest TBI animal

studies using BMDCs’ transplantation have demonstrated efficacious delivery of human

BMDSCs to the injured brain region in rats; grafted cells were delivered using collagen delivery

matrices which seem to facilitate cell survival and neurite outgrowth post-transplantation (Guan,

Zhu et al. 2013). Another interesting outcome is increased glucose metabolism which may

rescue damaged neurons from cell death in an acute CCI animal model of TBI (Park, Yoon et al.

2013). Finally, last BM-derived endothelial progenitor cells promote axonal survival and

microvascular maintenance of the white matter after midline fluid percussion injury in adult rats

(Park, Park et al. 2014). Thus, stem cell therapy in combination with sRAGE, in particular

human bone marrow derived stem cells could decrease neuronal loss, ameliorate inflammation

and promote neuroprotection in TBI and other brain related diseases.

In accord with our hypothesis that upregulated CD-36 contributes to inflammation (Aim

1), and inhibition of inflammation via pharmacologic regimens (i.e., sRAGE) (Aim 2), in this Aim,

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we further hypothesized that inflammation-induced impairment TBI can be rescued by stem cell

therapy. Here, we additionally tested whether stem cell therapy alone or in combination with

sRAGE targeting human cells (i.e., human neural progenitor cells or hNP1), synergistically

reduced cell death and increased metabolic cell activity. Using the same dose of sRAGE

(Chapter 4), and stem cells known to afford therapeutic benefits in CNS disorders (Thorne and

Frey 2001; Lee, Lee et al. 2013; Acosta, Tajiri et al. 2014)), we tested their stand-alone and

combined efficacy in an in vitro model of inflammation, which is considered to replicate a major

secondary cell death process associated with TBI.

5.2 Specific Materials and Methods

Our in vitro cell model of inflammation cell model of TBI involved treating hNP1 cells with

TNF-α, to mimic acute TBI. We treated and plated hNP1 cells according to the manufacturer’s

protocol. We re-suspended the cells in a total volume of 2 mL of fully supplemented AB2 Neural

Medium (pre-warmed to 37°C). Next, we platted them the 2 mL cell suspension of hNP1 cells

onto a Matrigel-coated 35 mm dish and incubated the cells at 37°C in a 5% CO2 humidified

incubator. We exchanged the medium with fresh fully supplemented AB2 Neural medium 24

hours post plating. We exchanged with fresh medium every other day thereafter. Once the

hNP1 cells reach 100% confluence, they were dissociated manually for passaging (e.g., by

gentle and slow pipetting up and down to detach the cells). The cells were maintained at a high

density, the recommended passaging ratio was 1:2. After 48 hours 4x104 cells/well/200µL were

incubated with 20µL of TNF-α (1.0 µg/mL/200µL well) for three days. On the 3rd day hNP1 cells

received the various reagent treatments for 3 hours, and later underwent the calcein and MTT

assays: control, TNF-α only, TNF-α plus sRAGE (5µg/mL) (R&D Systems, 1145-RG-050) plus

human bone marrow derived stem cells (BMDSCs) at a concentration of 2x104 cells in a 20µL

volume (Progenitor Cell Therapy, California), TNF-α in combination with BMDSCs, and

BMDSCs alone.

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5.2.1 Measurement of cell viability: Calcein-AM fluorescence dye

Measurement of cell viability was performed by both fluorescent live/ dead cell assay

(Bell, Cao et al. 2003) and trypan blue exclusion method. A two-color fluorescence cell viability

assay was performed by Calcein-AM (Invitrogen, L3224) to be retained within live cells,

including an intense uniform green fluorescence and ethidium homodimer (EthD-1) to bind the

nuclei of damaged cells (bright red fluorescence). After two hours of reperfusion, the hNP1 cells

(Neuromics, Edina, MN) were incubated with 2 mM Calcein-AM and 4 mM EthD-1 for 45 min at

room temperature in darkness according to the manufacturer’s instructions. Afterwards, cells

were washed once with phosphate buffered saline, and then the green fluorescence of the live

cells was measured by the Gemini EX florescence plate reader (Molecular Device), excitation at

485 nm and emission at 538 nm. In addition, trypan blue (0.2%) exclusion method was

conducted and mean viable cell counts were calculated in four randomly selected areas (1 mm2,

n = 10) to further reveal the cell viability. To precisely calibrate the cell viability, the values were

standardized from fluorescence intensity and trypan blue data.

5.2.2 Measurement of Mitochondrial Activity: MTT Assay

To measure metabolic activity we used the mitochondrial activity assay by the reduction

of 3-(4, 5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) by cellular

dehydrogenases was used as described in our previous report and manufacturer’s instructions

(Roche, Catalog Number 11465007001) (Kaneko, Shojo et al. 2014). Before completing 2 hours

of reperfusion, the cells were incubated with 0.5 mg/ml MTT at 37°C and 5% CO2, and

incubated with lysis buffer overnight in a humidified atmosphere at 37°C and 5% CO2. The

optical density of solubilized purple formazan was measured at 570 nm on a Synergy HT plate

reader (Bio-Tex). The purpose of the MTT assay was to assess cell viability after treating cells

with sRAGE.

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5.2.3 Data Analysis

Statistical analysis assessed cell viability and mitochondrial functional effects in each

experimental group versus its respective control group. Group comparisons included the use a

one-way analysis of variance (ANOVA) with 95% CI followed by subsequent pair-wise

comparisons using post hoc Bonferonni test. All data are presented as mean values ± SEM.

Statistical significance was set at p<0.05 for all analyses.

5.3. Results

Using the calcein assay to reveal cell viability, ANOVA revealed significant treatment

effects on cell survival of hNP1 cells (F(4,20)=20.789, p values<0.0001) (Figure 19). Pairwise

comparison via Bonferroni’s multiple comparison test showed statistical significance among the

following treatment groups: control vs. TNF-α, TNF-α + sRAGE+BMDSCs, TNF-α+ BDMSCs,

and BDMSCs only groups against TNF-α (p<0.0001). There were no significant effects in cell

viability when comparing the following treated groups: TNF-α+sRAGE+BMDSCs, TNF-α+

BMDSCs, and BMDSCs only against control (p>0.05). Overall these data from the calcein

assay demonstrate significant cell survival of hNP1 cells under stem cell conditions than when

treated with the drug sRAGE alone. Moreover, combination therapy did lead to better survival

compared to TNF-α alone. Once again, the given sRAGE dose did not appear to have rescued

hNP1 cells from cell death after TNF-α insult (as in Chapter 4). Yet, stem cells conveyed

neuroprotection by rescuing hNP1cells, as seen in the increased cell viability. In summary,

these data from the calcein assay show significant cell survival of hNP1 cells when treated with

the drug sRAGE in combination with stem cells, but also by stem cells alone after TNF-α insult.

Although sRAGE did not seem to exacerbate TNF-α toxicity, neither to hNP1 cells nor to stem

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cells, the given sRAGE dose did not appear to have rescued hNP1 cells from cell death after

TNF-α treatment.

Meanwhile in the MTT assay designed to measure mitochondrial activity, ANOVA revealed

significant treatment effects on the level of metabolic activity of hNP1 cells (F(4,20)=545.1345,

p<0.0001) (Figure 20). Bonferroni’s multiple comparison test showed statistical significance in

the mitochondrial activity of the following treatment groups: Control vs. TNF-α, control vs. TNF-α

+ sRAGE+BMDSCs, control vs. TNF-α + BMDSCs, control vs. BMDSCs, TNF-α vs. TNF-α +

sRAGE+BMDSCs, TNF-α vs. TNF-α + BMDSCs, TNF-α vs. BMDSCs (one way analysis of

variance, p<0.0001). Taken as a whole, data from the MTT assay demonstrate significant

metabolic activity of hNP1 cells under stem cell conditions than when treated with the drug

sRAGE alone. Moreover, combination therapy did point to increased metabolism compared to

TNF-α alone. Once again, the given sRAGE dose did not appear to have rescued hNP1 cells

from dysfunctional metabolic activity after TNF-α insult (as in Chapter 4). Yet, stem cells

afforded neuroprotection by rescuing hNP1 cells, as seen in the increased metabolic activity. In

closing, these data from the MTT assay showed significant increase in metabolic activity of

hNP1 cells after TNF-α insult when treated with the stem cells alone, without enhancement

when combined with sRAGE. Although sRAGE did not seem to exacerbate TNF-α toxicity,

neither to hNP1 cells nor to stem cells, the given sRAGE dose did not appear to have rescued

hNP1 cells from deficient mitochondrial activity after TNF-α treatment.

5.4 Discussion

Results from our in vitro model of inflammation, a secondary cell death process of TBI,

demonstrated the therapeutic effects of stem cells against inflammation. BMDSCs are

efficacious after the TNF-α insult to hNP1 cells, in that stem cells alone or in combination with

sRAGE increased cell viability and metabolic activity compared to control and the TNF-α treated

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group. We initially envisioned that stem cells would act synergistically with sRAGE, a compound

shown to block TBI related inflammation.

Figure 19. In Vitro hNP1 cells relative cell viability Calcein assay using stem cells. sRAGE treatment and stem cell therapy increased neuronal cell survival. The hNP1 cells treated with TNF-α +sRAGE+BMDSCs had increased cell viability when compared to the TNF-α only, and the TNF-α +BMDSCs groups. The data obtained was analyzed statistically using one-way analysis of variance (ANOVA) (F(4,20)=20.789, p values<0.0001) for group comparisons with 95% CI followed by subsequent pair-wise comparisons using post hoc Bonferonni test. All data are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses.

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Figure 20. In Vitro hNP1 cells relative metabolic activity MTT assay using stem cells. The hNP1 cells were treated with TNF-α +sRAGE+BMDSC had increased metabolic rate when compared to the TNF-α only, and TNF-α +BMDCS groups. The data was analyzed statistically using multiple comparison test one-way analysis of variance (ANOVA) for group comparisons with 95% CI followed by subsequent pair-wise comparisons using post hoc Bonferonni test (F=545.1, df=4). All data are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses.

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However, there was no detectable significant increase in cell survival and mitochondrial

activity between combination therapy and stem cells alone in TNF-α treated cells, indicating

absence of synergistic effects of sRAGE and stem cells in abrogating inflammation. Moreover,

sRAGE alone did not increase cell survival and mitochondrial activity in TNF-α treated cells (as

in Chapter 4). These findings suggest that while sRAGE was not effective in affording direct

therapeutic benefits to hNP1 cells against inflammation, stem cells alone were able to reduce

inflammation to some extent.

Previous in vitro and in vivo studies reveal that low levels of sRAGE are not only

associated with conventional risk factors (diabetes, hypertension, hypercholesterolemia), non-

diabetic coronary artery disease, stroke, and AD (Yamagishi and Matsui 2010; Colhoun,

Betteridge et al. 2011), but also to acute ischemic stroke, in particular without the source of

cardioembolism (Park, Yun et al. 2009). Hence, in the clinical setting measuring plasma levels

of sRAGE after stroke, and TBI may be useful as a predictor of the neurological severity of the

condition, especially in subjects without identifiable conventional risk factors. In contrast, a

recent report suggests that high serum sRAGE levels in pregnant women may be associated

with recurrent pregnancy losses (RPL). sRAGE contributes to RPL by reducing uterine blood

flow and subsequently causing ischemia in the fetus via inflammatory and thrombotic reactions

(Ota, Yamagishi et al. 2014). That the present study revealed that sRAGE neither exert

neuroprotection against nor exacerbate TNF-α neurotoxicity suggests that optimal dose and

timing may need to be adjusted to fully capture the functional effects of sRAGE in inflammation

following TBI.

A bulk of the literature also shows multiple neuroprotective effects of stem cells against

inflammation in various diseases including TBI. These stem cells include using human

mesenchymal stem cells (hMSCs), human neural stem cells (hNSCs) and human adipose stem

cells (hASCs). These altogether possess overlapping abilities: i) hMSCs protect hyperoxia-

induced lung injury via downregulation of the RAGE-NF-KB signaling in the lung of newborn rats

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(Tian, Ji et al. 2012); ii) an intrinsic ability of MSCs for multipotent differentiation as potential

therapy option for various diseases, including those of the musculoskeletal, neuronal,

cardiovascular and pulmonary systems (Akram, Samad et al. 2013; Zou, Zhang et al. 2014); iii)

MSCs paracrine ability to secrete an array of factors implicated in the mitigation of pathological

conditions through anti-inflammatory, anti-apoptotic and immunomodulatory mechanisms (Zou,

Zhang et al. 2014); iv) human neural stem cells have improved symptomatic inflammation in

early-stage ischemic-reperfusion cerebral injury in mice (Huang, Wong et al. 2014).

The drug sRAGE had inconsequential effects against TNF-α induced toxicity suggesting

that sRAGE alone may not shield against TBI. Drug efficacy may be attained by adjusting

sRAGE dose and early drug administration when treating TBI. The use of other anti-

inflammatory drugs along with sRAGE may enhance its, inhibitory effects against TBI related

inflammation. Likewise, stem cell therapy in combination with sRAGE/or SS-31 could effectively

increase inflammation while promoting neurogenesis. Our long-standing interest in stem cell

therapy advances the present hypothesis of testing sRAGE in combination with stem cells to

unravel the efficacy of this combination therapy. This study solicits additional experiments in

demonstrating the role of sRAGE as stand-alone or adjunct therapy for enhancing cell viability

and metabolic cell activity against inflammation and TBI. Although the present data revealed

that sRAGE alone was not neuroprotective against TBI, we established that stem cells may

abrogate TBI-induced inflammation, and suggesting sRAGE as a potential pharmacological

agent for treating inflammation in TBI.

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CHAPTER 6

DISCUSSION

6.1 Relevance of CD-36

CD-36 is of interest as a therapeutic target molecule because of its inflammatory role in

lipid and metabolic conditions, cardiovascular disorders, and very recently in glucose

intolerance, cerebrovascular conditions, and neurodegenerative diseases. Inflammation is a

common factor among all these conditions, and also in head injuries, such as TBI. Our main

goal is to observe any CD-36 related changes after post-traumatic inflammation that follows the

secondary brain damage associated with TBI. CD-36 has significant roles in different stages of

previously mentioned diseases; we focus mainly on its inflammatory component, making it an

important molecule for therapeutics.

6.2 CD-36, Inflammation and TBI

Experiments detailed in this dissertation have detected increased CD-36 near the area

of impact after TBI. This expression reaches a peak in the early time periods after injury, in

particular after 24 hours post-TBI up to 7 days post-TBI. This suggests CD-36’s role is in acute

neuroinflammation. Other inflammatory mediators, such as MCP-1 and Iba-1, CD-36

immunoreactivity showed a stronger upregulation in cells in the area of brain impact at acute

time-points versus the chronic TBI period. The chronic group, 60-days post-TBI, barely showed

expression of CD-36 in cortical brain areas. Thus, this may indicate decay in CD-36 involvement

in the inflammatory process seen in chronic TBI. The early expression of CD-36 hours after TBI

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injury may also indicate a therapeutic window for TBI within the first couple of hours when

targeting CD-36 mediated neuroinflammation.

Furthermore, we detected another novel observation, the co-localization of CD-36 with

NeuN. We interpret these findings as straight action of CD-36 in mediating inflammation, and

eventually cell death, in neurons in brain cortical regions. Quite the opposite, there was minimal

co-localization of CD-36 with GFAP implying that CD-36 minimally contributes to astrocytic-

mediated inflammation. Yet, there are experiments reporting that TBI-exposed MCP-1 knockout

mice showed no co-localization of MCP-1 with NeuN and GFAP in the cortex within 4 days of

TBI; but, there was co-localization of MCP-1 with a subset of F4/80-positive amoeboid

macrophages accumulating in the lesion site (Rhodes, Sharkey et al. 2009; Semple, Bye et al.

2010). More studies are necessary to further characterize what types of cells are influenced by

CD-36 over-expression, which will help us understand CD-36-mediated inflammation and its

treatment. Thus, CD-36 may have an essential role in the neuroinflammatory cascade that

further contributes to the pathology of TBI.

The process of neuroinflammation involves a vast series of events contributing to

progressive brain degeneration. Our gathered results suggest a tangible association between

CD-36 and MCP-1 after TBI. MCP-1 is associated in macrophage recruitment into damaged

brain tissue after TBI. In addition, we observed a trend of increased CD-36 protein expression in

early acute TBI time points (i.e., 24-48 hours, and 7 days) in the brain, which may cue the

change of functional effects of this immune response from pro-survival to cell death. In contrast,

we observed a low CD-36 expression in the 60 day chronic post TBI group. This could possibly

be a delayed response of peripheral macrophages to arrive at the site of injury. This has been

observed in stroke studies of mononuclear phagocytes (resident microglia) accumulated in the

injured brain within 3 to 4 days when compared to blood-derived macrophages at 6 to 7 days

(Schroeter, Jander et al. 1997). This explains brain protein levels, yet it does not justify CD-36

downregulation in the spleen after 48 hours and 7 days post- TBI. In general, our experiments

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of CD-36 expressing monocytes in the spleen after TBI may help us clarify CD-36 role in the

peripheral immune response. We already know that the spleen’s response exacerbates the

inflammatory response post-TBI from the multiple experiments linking the spleen’s response to

cerebrovascular conditions, such as ischemic stroke. Likewise, CD-36 may also play a key role

in the inflammatory splenic response after TBI. Our results suggest an intimate involvement of

CD-36 mediated inflammation in TBI, providing novel insights into the understanding of disease

neuroinflammation and as a potent therapeutic target for TBI treatment.

Further examination of CD-36 protein expression levels in the spleen during acute TBI

time- points showed no significant differences in protein levels between TBI and sham animals

at each time-point. Although a CD-36 peak splenic expression occurred primarily at 24 hrs in the

TBI, there was no significant difference between TBI and sham groups. Likewise, there were no

significant differences when comparing TBI vs. sham at 48 hrs and 7 days post-TBI. We

presume this initial splenic response peaking within the first 24 hrs is CD-36 mediated, followed

by a slow decay at later time points. One explanation for the CD-36 splenic protein

downregulation in the spleen of acute time points after the initial 24 hrs increase may be that

different kind of monocytes get activated by other inflammatory signaling pathways over those

CD-36 mediated monocytes subsets. A recent study found temporal differences in peripheral

immunity after TBI in a murine model of closed head injury at early time points (1-7days) and

later ones (30 days and 60 days) after injury (Schwulst, Trahanas et al. 2013).

Our study had limitations, a small number of animals to account for CD-36 expression in

the spleen; this may be a reason why we observed no difference. If we were to increase sample

size, we may be able to observe a significant increase in CD-36 protein expression among the

acute and sham groups. There are also other cellular events and molecular interactions of CD-

36 with other pro-inflammatory and anti-inflammatory cytokines/chemokines that may be

responsible for CD-36 downregulation in the spleen. Prompt detection of CD-36 and related

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inflammatory mediators in serum and plasma of TBI patients and TBI rodents, will redirect new

avenues for clinical translation of CD-36 as a screening biomarker.

Our observations corroborate the upregulation of brain and splenic CD-36 mediated

activation in the first 24 hours after TBI. Nevertheless the increased CD-36 brain response is

sustained until 7 days post-TBI and then declines. In contrast, splenic CD-36 expression

remains unchanged even after days of injury, but it declines after 1 day. The expression of CD-

36 with the studied inflammatory markers indicate mainly the activation of MCP-1 and Iba-1,

suggesting macrophage activation, certain/minimal astrocyte activation in the brain cortex, and

microglia activation associated to other inflammatory mechanisms after TBI. In addition, the

chronic 60-Day post TBI group showed very low expression levels of CD-36 in the cortex (and

spleen), implying that CD-36 has a minimal role in chronic inflammation, but is more relevant in

the acute stages of TBI.

Future objectives in the study of CD-36 as an inflammatory mediator may expand on

how CD-36 regulates the mobilization of various monocyte subset populations by interacting

with other pro-inflammatory cytokines/chemokines and their receptors, such as CCR2. It will be

interesting to record temporal profiles of cytokines/chemokines influencing monocyte subsets in

the brain and spleen of TBI animal models, and at the same time compare them to groups of

animals subjected to splenectomy, as these observations may provide further insights on the

inflammation-mediated pathology and treatment of TBI. Additionally, in vitro studies may be

conducted to block CD-36 and record any differences on monocyte subsets, their ability to

acquire a particular phenotype in circulation, and their peripheral influence after injury. There

are also pharmacological approaches using specific CD-36 inhibitor, which would contribute

allow a much better understanding of CD-36 target engagement effects. We also recognized

that the major limitation in this study included small sample size, which likely affected the

statistical analyses of CD-36 protein expression. Alternatively, the lack of alteration in CD-36

expression may be due to a rapid upregulation or decline associated with the supra-acute

102

period post-TBI not captured in this study. Nonetheless, these results demonstrated that CD-36

closely accompanied the inflammation in the acute TBI period (24 hours to 7 days).

6.3 Effects of sRAGE in an in vitro cell model of Inflammation

Using an in-vitro cell model we did not observe a role for the soluble receptor of

advanced end-glycation product (sRAGE) in the inflammatory response after TBI. In our

previous in-vitro experiments we found out that inhibitory action of soluble receptor of advanced

glycation end products (sRAGE) in various cell types, suggesting sRAGE as a useful drug to

reduce TBI related inflammation. These may attenuate the immune response and tissue injury.

Our pilot experiments suggest/demonstrate the inflammatory role of CD-36 as the link shared by

stroke and TBI. This CD-36 mechanism may be a candidate therapeutic target to reduce

secondary cell death in TBI, and promote functional and cognitive brain recovery. We decided

to focus on sRAGE because of its strong affinity for CD-36 (Marsche, Zimmermann et al. 2003),

its synergistic anti-inflammatory effect combined with long term use of antihypertensive drugs

like nifedipine and telmisartan (Falcone, Buzzi et al. 2012); and sRAGE ability to act as a decoy

for RAGE to slow carotid artery bloom injury, slow atherosclerosis via blocking RAGE, and avoid

interaction of RAGE with its pro-inflammatory ligands (AGEs, HMGB1, S100 proteins) (Maillard-

Lefebvre, Boulanger et al. 2009). Recent human studies propose sRAGE as a candidate in the

prognosis and follow up after ischemic and hemorrhagic stroke (Qian, Ding et al. 2012; Menini,

Ikeda et al. 2013; Tang, Wang et al. 2013). These studies reveal: a)ischemic and hemorrhagic

stroke patients have an increase in sRAGE serum levels days after stroke (ranged between

26%-296%, p<0.001), and this change paralleled recovery and recurrence or aggravation of the

episodes (Menini, Ikeda et al. 2013); b) acute ischemic stroke patients, C57BL/6J mice, and

primary cortical neurons subjected to oxygen and glucose deprivation showed increased

sRAGE plasma levels within 48hours of injury; furthermore, sRAGE recombinant administration

(intravenously) was effective in reduction of infiltrating immune cells, improving the outcome of

103

injury in mice, and protecting cultured neurons against hypoxia and cell death (Tang, Wang et

al. 2013). In addition, in vivo and in vitro studies indicate that the down-regulation of CD-36 by

novel class antioxidant peptides may be a useful strategy to treat ischemic stroke victims (Cho,

Szeto et al. 2007).

6.4 Effects of Combination Stem Cell Therapy and sRAGE

The combination of stem cell therapy and sRAGE suggested their positive influence in

promoting cell survival and ameliorating inflammation as a therapeutic pathway against TBI.

Altogether, the current literature reinforces the thought that prolonged microglial activation is a

hallmark of traumatic brain injury, but may also suggest that neuroinflammatory responses

return to control levels after several years based on the severity of the injury.

Our findings indicate that after the TNF-α insult there was significant cell death of hNP1

cells when compared to control. Treatment with sRAGE was well tolerated by hNP1 cells grown

under normal cell culture condition, based on their relative cell viability comparable to that of

control, suggesting the given sRAGE dose was safe. However, treatment with sRAGE did not

suppress TNF-α induced cell death. Various animal studies have validated the beneficial effects

of sRAGE. One of these benefits is neuroprotection, rodents and human models of ischemic

stroke and atherosclerosis have identified neuroprotective benefits of a soluble form of receptor

for advanced glycation end products. One study reported that plasma levels of sRAGE and high

mobility group box 1 (HMGB1) were significantly increased within 48 hours after stroke, and the

sRAGE level was an independent predictor of functional outcome at 3 months post-stroke

(Tang, Wang et al. 2013). Furthermore, atherosclerotic studies carried in diabetic mice showed

an inhibition of atherosclerotic plaques by the competition of RAGE with sRAGE, suggesting

that plasma sRAGE and endogenous secretory RAGE (esRAGE) are associated with

components of metabolic syndrome or atherosclerosis in the absence of diabetes (Koyama,

104

Shoji et al. 2005; Koyama, Tanaka et al. 2014). A second therapeutic effect of sRAGE that may

be further exploited is its ability to reverse vascular leakage.

In an accelerated atherosclerotic diabetic mice model, sRAGE ameliorated vascular

lesions in diabetic/atherosclerotic animals in the absence of changes in plasma lipids or

glycemia (Wautier, Zoukourian et al. 1996). Additionally, sRAGE shows beneficial effects in

reversing vascular leakage in the intestine, skin of rodent diabetic models. (Wautier, Zoukourian

et al. 1996; Schmidt, Yan et al. 1999). Similarly, a human clinical study using long term

combined therapy of antihypertensive drugs, nifedipine-telmisartan, showed a beneficial effect

in increasing sRAGE plasma levels, thus exerting an atheroprotective and anti-inflammatory

activity (Falcone, Buzzi et al. 2012). The drug sRAGE may be a promising therapeutic drug in

reducing atherosclerotic plaque formation in patients who may have suffer stroke, in the

presence or absence of risk factor conditions, such as diabetes and high cholesterol. There are

other antioxidant agents as alternative treatment regimens to abrogate CD-36. In recent

experiments the molecule SS-31 showed a reduction in CD-36 insidious action, and ischemia-

induced injury (Cho, Szeto et al. 2007); in addition, SS-31 and related compounds were

proposed as neuroprotective agents. These compounds target cardiolipin, a complex lipid

(diphosphatidylglycerol lipid) on the inner mitochondrial membrane (IMM) involved in

optimization of the electron transport chain (ETC), SS-31 and related compounds are capable of

restoring cellular metabolism in aging and diverse disease models without affecting the normal

health of an organism. Both SS-31 and sRAGE may have beneficial effects in stroke and related

inflammatory disorders. For the use of inhibitors of IL-6, such as tocilizumab or a TNF-α inhibitor

like etanercept, is a fusion protein produced by recombinant (Szeto and Birk 2014) DNA, an

FDA approved drug to treat rheumatoid arthritis. Etanercept may prove beneficial in the long-

term treatment of chronic TBI. These inhibitors can also be administered IV. Anti-TNF therapy

has proven beneficial in the long-term treatment of rheumatoid arthritis and it may also work to

treat the chronic neuroinflammation present in traumatic brain injury.

105

Additional in vitro and in vivo TBI animal models should continue to look at the efficacy

of sRAGE, record neurobehavioral and drug adverse effects. In vitro cell models may help

decipher sRAGE’s mechanisms of action. Our in vitro experiments showed increased cell

survival under normal conditions after TNF-α insult and sRAGE treatment. Following TNF-α

treatment, hNP1 cells exhibited reduced cell viability. Unfortunately, the TNF-α treated cells

exposed to sRAGE showed a small, non-significant increase in cell viability when compared to

TNF-α alone. This may be indicate further testing at higher sRAGE doses or manipulating the

timing of TNF-α and sRAGE initiation in order to capture the true effects of sRAGE against

inflammation. Alternatively, sRAGE may exert other mechanisms of action beyond inflammation

affecting parallel molecular or cellular pathways leading to neuroprotection that may not have

been captured by simple calcein and MTT assays. More sensitive cell viability and cell death

pathway tests may be needed.

In addition, further in vivo testing would be necessary to reveal behavioral and toxicity

effects before and after sRAGE administration in an in vivo TBI animal model to assess

neuronal survival, functional recovery and any adverse effects of sRAGE. Our in vitro study

provides the basis for pursuing more detailed studies on sRAGE, and inflammatory-based

therapies directed at sequestering the secondary cell death associated with TBI. Although

sRAGE cannot cross the healthy blood brain barrier (BBB), this barrier is compromised after TBI

and the immediate treatment with sRAGE thereafter may allow the drug to easily reach the

injured brain site. Alternatively, even if sRAGE is not able to cross the BBB after acute TBI, we

envision sRAGE can afford inhibitory action to downregulate the splenic response, which we

discussed earlier as contributing to the systemic inflammation in TBI. To that end, it is possible

to think that sRAGE may act on the periphery (i.e., spleen) and significantly reduce infiltrating

immune/inflammatory cells destined to exacerbate the brain inflammation after TBI. In parallel,

investigations on drugs that similarly target CD-36 inflammation may reveal new avenues of TBI

research. Such as anti-inflammatory drugs like: the IL-6 inhibitor tocilizumab, and, or the TNF-α

106

inhibitor etanercept. Altogether may serve as adjunctive therapy to improve sRAGE therapeutic

outcome in TBI.

The modest effects of sRAGE against TNFα-induced toxicity suggest that sRAGE alone

may not be protective against TBI. Increasing the dose and altering the drug initiation regimen

may improve sRAGE efficacy in TBI. Similarly, stem cell therapy in combination with sRAGE or

SS-31 could potentially augment inflammation while promoting neurogenesis. In our study,

sRAGE alone was not robustly neuroprotective against TBI. Results from our in vitro model of

TBI showed limited therapeutic effects of stem cells against inflammation. BMDSCs are

efficacious after the TNF-α insult to hNP1 cells; when stem cells were used alone or in

combination with sRAGE cell viability increased compared to control and the TNF-α treated

group. There was not a notorious increase in cell survival and mitochondrial activity when

comparing those treated with combination therapy as opposed to stem cells alone.

These findings suggest that while sRAGE was not effective in affording direct

therapeutic benefits to hNP1 cells against inflammation, perhaps sRAGE influences stem cells

ability to mediate TBI related inflammation. Supporting in vivo studies on increased cell survival

afforded by certain stem cells against inflammation in various diseases and TBI include using

human mesenchymal stem cells (hMSCs), human neural stem cells (hNSCs) and human

adipose stem cells (hASCs) (Tian, Ji et al. 2012; Akram, Samad et al. 2013; Huang, Wong et al.

2014; Zou, Zhang et al. 2014). Our results indicated no significant difference in cell viability and

mitochondrial activity in the combined therapy group as opposed to stem cells alone. Our drug

sRAGE had inconsequential effects against TNFα-induced toxicity suggesting that sRAGE

alone may not guard against TBI.

107

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Zhang, X., Y. Chen, et al. (2005). "Bench-to-bedside review: Apoptosis/programmed cell death triggered by traumatic brain injury." Crit Care 9(1): 66-75.

Zhao, K., G. M. Zhao, et al. (2004). "Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury." J Biol Chem 279(33): 34682-34690.

Zou, X., G. Zhang, et al. (2014). "Microvesicles derived from human Wharton's Jelly mesenchymal stromal cells ameliorate renal ischemia-reperfusion injury in rats by suppressing CX3CL1." Stem Cell Res Ther 5(2): 40.

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APPENDIX A

PUBLICATIONS

• Tajiri N, Duncan K, Borlongan MC, Pabon M, Acosta S, de la Pena I, Hernandez-Ontiveros D., Lozano D, Aguirre D, Reyes S, Sanberg PR, Eve DJ, Borlongan CV, Kaneko Y. Adult Stem Cell Transplantation: Is Gender a Factor in Stemness? Int. J. Mol. Sci. 2014, 15(9), 15225-15243. Published Aug 28, 2014.

• Naoki Tajiri, Kelsey Duncan, Alesia Antoine, Mibel Pabon, Sandra A. Acosta, Ike

de la Pena, Diana G. Hernandez-Ontiveros, Kazutaka Shinozuka, Hiroto Ishikawa, Yuji Kaneko, Ernest Yankee, Michael McGrogan, Casey Case, and Cesar V. Borlongan. Stem cell-paved biobridge facilitates neural repair in traumatic brain injury. Front Syst Neurosci. 2014; 8: 116. Published Jun 24, 2014.

• Garbuzova-Davis S, Haller E, Williams SN, Haim ED, Tajiri N, Hernandez-

Ontiveros DG, Frisina-Deyo A, Boffeli SM, Sanberg PR, Borlongan CV. Compromised blood-brain barrier competence in remote brain areas in ischemic stroke rats at chronic stage. J Comp Neurol. 2014 Sep 1;522(13):3120-37. Published Mar 08, 2014.

• Tajiri N, Hernandez D, Acosta S, Shinozuka K, Ishikawa H, Ehrhart J, Diamandis

T, Gonzales-Portillo C, Borlongan MC, Tan J, Kaneko Y, Borlongan CV. Suppressed cytokine expression immediately following traumatic brain injury in neonatal rats indicates an expeditious endogenous anti-inflammatory response. Brain Res. 2014 Apr 22;1559:65-71. Published Mar 03, 2014.

• Tajiri N, Acosta SA, Shahaduzzaman M, Ishikawa H, Shinozuka K, Pabon M,

Hernandez-Ontiveros D, Kim DW, Metcalf C, Staples M, Dailey T, Vasconcellos J, Franyuti G, Gould L, Patel N, Cooper D, Kaneko Y, Borlongan CV, Bickford PC. Intravenous transplants of human adipose-derived stem cell protect the brain from traumatic brain injury-induced neurodegeneration and motor and cognitive impairments: cell graft biodistribution and soluble factors in young and aged rats. J Neurosci. 2014 Jan 1;34(1):313-26. Published Jan 01, 2014.

• Acosta SA, Diamond DM, Wolfe S, Tajiri N, Shinozuka K, Ishikawa H, Hernandez

DG, Sanberg PR, Kaneko Y, Borlongan CV. Influence of post-traumatic stress disorder on neuroinflammation and cell proliferation in a rat model of traumatic brain injury. PLoS One. 2013 Dec 9;8(12):e81585. Published Dec 09, 2013.

• Cerecedo-López CD, Kim-Lee JH, Hernandez D, Acosta SA, Borlongan CV.

Insulin-associated neuroinflammatory pathways as therapeutic targets for traumatic brain injury. Med Hypotheses. 2014 Feb; 82(2):171-4. Published Dec 01, 2013.

121

• Garbuzova-Davis S, Mirtyl S, Sallot SA, Hernandez-Ontiveros DG, Haller E, Sanberg PR. Blood-brain barrier impairment in MPS III patients. BMC Neurol. 2013 Nov 13;13:174. Published Nov 13, 2013.

• Garbuzova-Davis S, Rodrigues MC, Hernandez-Ontiveros DG, Tajiri N, Frisina-

Deyo A, Boffeli SM, Abraham JV, Pabon M, Wagner A, Ishikawa H, Shinozuka K, Haller E, Sanberg PR, Kaneko Y, Borlongan CV. Blood-brain barrier alterations provide evidence of subacute diaschisis in an ischemic stroke rat model. PLoS One. 2013 May 10;8(5):e63553. Published May 10, 2013.

• Hernandez-Ontiveros DG, Tajiri, N, Acosta S, Giunta B, Tan J, Borlongan CV.

Microglia activation as a biomarker for traumatic brain injury. Front Neurol.2013;4:30. Published Mar 26, 2013.

• Garbuzova-Davis S, Hernandez-Ontiveros DG, Rodrigues MC, Haller E, Frisina-

Deyo A, Mirtyl S, Sallot S, Saporta S, Borlongan CV, Sanberg PR. Impaired blood-brain/spinal cord barrier in ALS patients. Brain Research, 1469, pp.114-28. Published Jun 27, 2012.

• Svitlana Garbuzova-Davis, Maria C. O. Rodrigues, Diana G. Hernandez-

Ontiveros, Michael K. Louis, Cesario V. Borlongan, Paul R. Sanberg, 2011, “ Amyotrophic Lateral Sclerosis: a Neurovascular Disease”, Brain Research, 1398, pp.113-125. Published May 12, 2011.

• Svitlana Garbuzova-Davis, Robert L. Woods, III, Michael K. Louis, Theresa A.

Zesiewicz, Nicole, Kuzmin-Nichols, Kelly L. Sullivan, Amber M. Miller, Diana G. Hernandez-Ontiveros, Paul R. Sanberg, 2010, “Reduction of circulating endothelial cells in peripheral blood of ALS patients”, PLoS One, 5, (5) e10614. Published May 12, 2010.

• Rodrigues MC, Hernandez-Ontiveros DG, Louis MK, Willing AE, Borlongan CV,

Sanberg PR, Voltarelli JC, Garbuzova-Davis S. Neurovascular aspects of amyotrophic lateral sclerosis. Int Rev Neurobiol. 102:91-106. 2012.

122

APPENDIX B

PUBLISHER’S PERMISSION TO USE FIGURE 1

RE: Permissions Request: Proquest-USF system for dissertations Access Copyright Permissions Group <LicensingAdmin@accesscopyright.ca> Tue 8/4/2015 9:34 AM To: Hernandez, Diana dhernan1@health.usf.edu; Hi Diana, Students wishing to use CMA material in a thesis/dissertation is available at no charge, except where the thesis/dissertation is to be made commercially available for purchase through organizations like ProQuest (this is then Republishing).Please use the following copyright statement: Reprinted from (E. Park, J. D. Bell, A. J., Baker) (Traumatic Brain Injury can the consequences be stopped?, Figure 1.The major pathways associated with the progression of secondary injury after a traumatic brain injury) Canadian Medical Association Journal (April 22 2008, Volume 178 No. 9, pages 1163–1170). © Canadian Medical Association (2008). This work is protected by copyright and the making of this copy was with the permission of the Canadian Medical Association Journal (www.cmaj.ca) and Access Copyright. Any alteration of its content or further copying in any form whatsoever is strictly prohibited unless otherwise permitted by law. Kind regards, Jessica We’re moving! Access Copyright is moving as of August 24. Our new mailing address will be 320 – 56 Wellesley Street West; Toronto, ON M5S 2S3. Please update your records accordingly. Our phone and fax numbers remain the same. Jessica Luet, MLIS Research Specialist Access Copyright, The Canadian Copyright Licensing Agency One Yonge Street, Suite 800 Toronto, ON, M5E 1E5 T: (416) 868-1620 (416) 868-1620 x224 TF: 1-800-893-5777 1-800-893-5777 FREE x224 jluet@accesscopyright.ca www.accesscopyright.ca

ABOUT THE AUTHOR

Diana G. Hernandez-Ontiveros was born and raised in Mexico. She moved to the U.S.A

in 1999 to pursue her B.A. degree in Chemistry with a Biochemistry emphasis and a M.S. in

Biomedical Sciences from the University of South Florida. She collaborated with various lab

teams in multiple scientific projects, as reflected by the list of publications, and a book chapter.

Diana also attended scientific conferences, where she presented her research in Amyotrophic

Lateral Sclerosis and Traumatic Brain Injury. Diana also dedicated her time to train incoming

graduate students at the master and undergraduate level in basic neuroscience lab skills. She

would like to continue working in the field of neurosciences, motivate younger generations on

this subject, and create more awareness of neurodegenerative diseases in her community.