Cellular Reactions and Behavioral Changes in Focal and ...617889/FULLTEXT01.pdf · Injury to the brain has devastating consequences for the affected individuals. What we define as

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2013

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 909

Cellular Reactions andBehavioral Changes in Focal andDiffuse Traumatic Brain Injury

A Study in the Rat and Mouse

SARA EKMARK LEWÉN

ISSN 1651-6206ISBN 978-91-554-8687-7urn:nbn:se:uu:diva-177083

Dissertation presented at Uppsala University to be publicly examined in Hedstrandsalen,Akademiska sjukhuset, 75185, Uppsala, Friday, June 14, 2013 at 13:15 for the degree ofDoctor of Philosophy. The examination will be conducted in Swedish.

AbstractEkmark Lewén, S. 2013. Cellular Reactions and Behavioral Changes in Focal and DiffuseTraumatic Brain Injury: A Study in the Rat and Mouse. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine909. 83 pp. Uppsala. ISBN 978-91-554-8687-7.

Traumatic brain injury (TBI) is a severe condition and a major cause of death and disability.There is no pharmacological treatment available in clinical practice today and knowledge ofbrain injury mechanisms is of importance for development of neuroprotective drugs. The aimsof the thesis were to get a better understanding of astrocyte reactions and immune responses, aswell as behavioral changes after focal unilateral cortical contusion injury and diffuse bilateralcentral fluid percussion injury in rats and mice.

In the focal injury models, the astrocyte reactions were generally restricted to the ipsilateralhemisphere. After diffuse TBI, vimentin and glial fibrillary acidic protein (GFAP) positivereactive astrocytes were bilaterally expressed in brain regions even distant from the injury site,including regions where axonal injury was seen. Early after diffuse TBI, there was a robustimmune response, including activation of macrophages/microglia (Mac-2+) and infiltration ofneutrophils (GR-1+) and T-cells (CD3+).

In order to measure functional outcome, the recently established Multivariate ConcentricSquare Field™ (MCSF) test for complex behaviors, including risk taking and explorativestrategies was used. The Morris water maze (MWM) was applied for testing learning andmemory. The MCSF test revealed alterations in risk taking, risk assessment and exploratorybehavior, in the mice subjected to focal injury whereas mice subjected to the diffuse injuryshowed a deviant stereotyped behavior. After focal injury mice showed a decreased ability toadapt to the arena in the second trial, when tested repeatedly in the MCSF test. Mice subjected todiffuse injury had an impaired memory but not learning, in the MWM test. Post-injury treatmentwith the anti-inflammatory anti-interleukin-1β (IgG2 a/k) antibody showed a positive effect onfunctional outcome in the diffuse injury model. Altogether, the results demonstrate that focaland diffuse TBI models produce differences in cellular reactions and behavioral outcome andthat the immune response plays a key role in the pathology after brain injury.

Keywords: Traumatic brain injury, Astrocytes, Inflammatory response, Multivariateconcentric square field test, Morris water maze, Exploratory behavior, Risk taking, Functionaloutcome.

Sara Ekmark Lewén, Uppsala University, Department of Neuroscience, Neurosurgery,Akademiska sjukhuset, SE-751 85 Uppsala, Sweden.

© Sara Ekmark Lewén 2013

ISSN 1651-6206ISBN 978-91-554-8687-7urn:nbn:se:uu:diva-177083 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-177083)

“And men ought to know that from nothing else but the brain come joys, delights, laughter and sports, and sorrows, grief, despondency, and lamentations. And by this, in an especial manner, we acquire wis-dom and knowledge, and see and hear, and know what are foul and what are fair, what are bad and what are good, what are sweet, and what unsavory... And by the same organ we become mad and deli-rious, and fears and terrors assail us... All these things we endure from the brain, when it is not healthy... In these ways I am of the opi-nion that the brain exercises the greatest power in the man.”

-Hippocrates

To my family

Front cover: Right side show reactive astrocytes stained for vimentin in the dentate gyrus of the hippocampus, three days after traumatic brain injury in mice. Left side show no vimentin positive cells in sham injured control ani-mals at the same time-point.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I. Sara Ekmark-Lewén, Anders Lewén, Charlotte Israelsson, Gui Lin Li, Mohammad Farooque, Yngve Olsson, Ted Ebendal and Lars Hillered. Vimentin and GFAP responses in astrocytes after contusion trauma to the murine brain. Restorative Neurology and Neuroscience. 2010; 28(3): 311-21.

II. Sara Ekmark-Lewén, Anders Lewén, Bengt J. Meyerson and Lars Hillered. The Multivariate Concentric Square Field test reveals be-havioral profiles regarding risk taking, risk assessment and explora-tion in mice subjected to traumatic brain injury. J Neurotrauma. 2010 Sep27(9): 1643-55.

III. Sara Ekmark-Lewén, Johanna Flygt, Olivia Kiwanuka, Bengt J. Meyerson, Anders Lewén, Lars Hillered and Niklas Marklund. Traumatic axonal injury in the mouse is accompanied by a dynamic inflammatory response, astroglial reactivity and complex behavioral changes. J Neuroinflammation. 2013; In Press

IV. Sara Ekmark-Lewén, Johanna Flygt*, Fredrik Clausen*, Gudrun Andrea Fridgeirsdottir, Olivia Kiwanuka, Anders Hånell, Bengt J. Meyerson, Anis K Mir, Hermann Gram, Anders Lewén, Niklas Marklund and Lars Hillered. Functional outcome after diffuse trau-matic axonal injury in mice is improved by post-injury neutralization of interleukin-1β. * Equal contribution. Manuscript.

Reprints were made with permission from the publishers.

Other related publications by the author which are not included in the thesis:

• Fredrik Clausen, Hanna Lundqvist, Sara Ekmark, Anders Lewén, Ted Ebendal and Lars Hillered. Oxygen free radical dependent acti-vation of extracellular signal-regulated kinase (ERK) mediates apop-tosis-like cell death after traumatic brain injury. J Neurotrauma. 2004 Sep;21(9):1168-82

Contents

CHAPTER 1 ................................................................................................. 13 Introduction .............................................................................................. 13 1.1 Traumatic brain injury ........................................................................ 13 1.2 Epidemiology ..................................................................................... 14 1.3 Classification of TBI .......................................................................... 14 1.4 Secondary events ................................................................................ 16 1.5 Treatment strategies ........................................................................... 17

CHAPTER 2 ................................................................................................. 19 Experimental models of traumatic brain injury ........................................ 19 2.1 Weight drop injury ............................................................................. 19 2.2 Controlled cortical impact .................................................................. 20 2.3 Central fluid percussion injury ........................................................... 20

CHAPTER 3 ................................................................................................. 23 Pathophysiology ....................................................................................... 23 3.1 General morphological changes ......................................................... 23 3.2 Axonal injury in TBI .......................................................................... 24 3.3 Blood-brain barrier pathophysiology ................................................. 25 3.4 Glial cell reactions .............................................................................. 26 3.5 Inflammatory responses ..................................................................... 26

3.5.1 Production of cytokines .............................................................. 27

CHAPTER 4 ................................................................................................. 29 Functional outcome evaluation ................................................................ 29 4.1 Ethoexperimental approaches to the study of behaviors .................... 29 4.2 Emotional reactivity and risk assessment ........................................... 29 4.3 Multivariate concentric square field test ............................................ 31 4.4 Morris water maze .............................................................................. 31

CHAPTER 5 ................................................................................................. 33 Present investigation – aims of the study ................................................. 33 General ..................................................................................................... 33 Specific ..................................................................................................... 33

CHAPTER 6 ................................................................................................. 35 Material and methods ............................................................................... 35 6.1 Animals and housing .......................................................................... 35

6.1.1 Anesthesia and production of trauma (Paper I-IV) ..................... 35 6.2 Survival times ..................................................................................... 36 6.3 Treatments (Paper IV) ........................................................................ 38 6.4 Sampling of brains (Paper I-IV) ......................................................... 38 6.5 Antibodies for histological evaluation (Paper I-IV) ........................... 38 6.6 Quantitative real-time PCR (qRT-PCR) (Paper I) .............................. 41 6.7 TUNEL-staining (Paper III) ............................................................... 41 6.8 Validation of anti-interleukin-1β antibody tissue concentration (Paper IV) ................................................................................................. 41 6.9 Functional evaluation (Paper II-IV) ................................................... 42

6.9.1 The Multivariate Concentric Square Field (MCSF) test (Paper II, III & IV) .......................................................................................... 42 6.9.2 Behavioral recordings in the MCSF ........................................... 43 6.9.3 Morris water maze (MWM) (Paper IV) ...................................... 44

6.10 Statistical analyses (Paper I-IV) ....................................................... 44 6.10.1 Principal component analysis (Paper II-IV) ............................. 45

CHAPTER 7 ................................................................................................. 46 Results ...................................................................................................... 46 7.1 Cellular reactions ................................................................................ 46

7.1.1 Vimentin and GFAP responses in astrocytes after focal brain injury in the rat and mouse (Paper I) ................................................... 46 7.1.2 Vimentin and GFAP responses in astrocytes after diffuse brain injury in the mouse (Paper III) ............................................................. 48 7.1.3 Axonal injury, cell death and BBB leakage after diffuse brain injury in the mouse (Paper III) ............................................................. 49 7.1.4 Inflammatory response after diffuse brain injury in the mouse (Paper III) ............................................................................................ 51

7.2 Behavioral changes ............................................................................ 53 7.2.1 Behavioral outcome after focal brain injury (Paper II) ............... 53 7.2.2 Behavioral outcome after diffuse axonal injury (Paper III&IV) 55 7.2.3 Principal component analysis of behavioral data (Paper III) ...... 56

7.3 Improvement of behavioral outcome after anti-inflammatory treatment (Paper IV) ................................................................................. 58

7.3.1 Principal component analysis of behavioral data (Paper IV) ...... 60 7.3.2 Treatment effects on learning and memory (Paper IV) .............. 62 7.3.3 Treatment effects on the inflammatory response (Paper IV) ...... 62

CHAPTER 8 ................................................................................................. 63 Discussion ................................................................................................ 63 8.1 Cellular reactions after focal and diffuse TBI .................................... 63 8.2 Behavioral changes after focal and diffuse TBI ................................. 64 8.3 Treatment strategies in traumatic brain injury .................................... 66 8.4 Future studies ..................................................................................... 67

Conclusion .................................................................................................... 68

Summary in Swedish .................................................................................... 69

Acknowledgement ........................................................................................ 71

References ..................................................................................................... 73

Abbreviations

ABC Avidin biotin complex

ATM Atmosphere

β-APP Amyloid precursor protein β

BBB Blood brain barrier

CBF Cerebral blood flow

CCI Controlled cortical impact

cFPI Central fluid percussion injury

CNS Central nervous system

DAB Di-amino-benzidine

DAI Diffuse axonal injury

EAAs Excitatory amino acids

EPM Elevated plus-maze

ERK Extracellular signal-regulated kinase

FPI Fluid percussion injury

GCS Glasgow coma scale

ICP Intracranial pressure

IFN-γ Interferon-γ

IgG Immunoglobulin G

IL-1β Interleukin-1β

iNOS Inducible nitric oxide synthase

MAPK Mitogen-activated protein kinases

MCSF Multivariate concentric square field

NIC Neurointensive care

NMDA N-methyl-D-aspartate

OF Open field test

PCA Principal component analysis

qRT-PCR Quantitative real-time polymerase chain reac-

tion

RLS Reaction level scale

ROS Reactive oxygen species

S-PBN 2-sulfo-phenyl-N-tert-butyl nitrone

TBI Traumatic brain injury

TNF-α Tumor necrosis factor α

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

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CHAPTER 1

Introduction Injury to the brain has devastating consequences for the affected individuals. What we define as personality and who we are is determined by this most complex organ of the body. There are about 100 billion neurons in the brain making up trillions of synapses, supported by glial cells that regulate their external environment. The brain response to injury is multifaceted including many different pathways. High energy consumption, about twenty percent of the energy used by the human body, dependence on glucose and the incapa-bility to store glucose, makes the brain especially sensitive to ischemia. The skull bone that surrounds the brain is in conflict with eventual swelling after injury, and instead raised intracranial pressure (ICP) may follow leading to cerebral ischemia by compromising cerebral perfusion. Knowledge about the brain response to damage and a greater understanding of the pathological mechanisms initiated after injury can hopefully lead to development of neu-roprotective drugs in the future.

1.1 Traumatic brain injury

The purpose of this thesis was to gain more knowledge about some of the key cellular mechanisms and behavioral effects of traumatic brain injury (TBI). TBI is a major global public health problem and one of the leading causes of mortality and morbidity.1,2 Depending on the severity of the insult, survivors of TBI often suffer from impairment of neurological motor and sensory function, information processing, perceptual function and memory loss. Additionally, other complications like depression and personality changes commonly persist. About 15 % of patients with mild TBI have dis-abling symptoms one year after the injury,3 and as long as 10 years following injury survivors with moderate and severe injuries suffers from anxiety and depression.4 More knowledge about the complex pathological changes after TBI may hopefully lead to treatment options for TBI patients.

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1.2 Epidemiology The annual incidence of head injury varies greatly around the world and there are difficulties in comparing data due to dissimilarity in classification and source data used.5 The highest number was recorded in Hualien Prov-ince, Taiwan with 333 per 105 of the population and a mortality rate of 89 per 105 of the population.6 This high incidence was considered to be due to the high usage of pedal and motor bicycles and lack of head protection by helmets. In Sweden the number of head injuries due to traffic accidents fell between 1987 and 2000, but the numbers of falls among elderly increased, and the total incidence rate was almost unchanged, with an average of 256 per 105 during this time period.7 In the Uppsala region with approximately 2 million citizens, there are about 100 severe TBI cases every year, in need of specialized neurointensive care (NIC).8

1.3 Classification of TBI Head injury demands a broad definition. The standard clinical definition of TBI can be summarized as an occurrence of injury to the head, arising from blunt or penetrating trauma or from acceleration-deceleration/rotational forces, that is associated with symptoms or signs attributable to the injury; decreased level of consciousness, amnesia, other neurological or neuropsy-chological abnormalities, skull fracture, diagnosed intracranial lesions or death.9 Adolescents, young adults, and the elderly are at highest risk of these injuries and the most common causes are attributed to motor vehicle crashes, falls and violence.9,10 The majority of human TBI are closed head injuries and these can be divided into two categories; focal brain damage and diffuse brain damage, including diffuse axonal injury (DAI). DAI often results from rotation of the head which occurs in approximately 30% of TBI and is asso-ciated with severe neurological deficits and poor outcomes.11 The severity of the injury can be measured e.g. by the Glasgow Coma Scale (GCS) or the Reaction level scale (RLS) which measures the level of consciousness.12,13 The initial neurological grading may be used to predict outcome after injury, and in severe TBI (GCS≤8) the risk of dying has been estimated to lay around 40%. However, there are other factors that have impact on outcome, like age and moreover there might be uncertainties when determining the correct GCS initially.14-16

One characteristic feature of TBI is the evolution of the injury over time. Tissue damage occurring during and after TBI results from both direct me-chanical injury (primary injury) and secondary autodestructive cascade reac-tions.17 It is commonly noticed that the size of the traumatic lesion expands over hours and days, and that new remote lesions may develop (Figure 1A).

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There is also an ongoing cell death process lasting for extended periods of time, up to a year or perhaps even longer (Figure 1B).18

A

B

Figure 1. (A) Secondary injury leading to development of contusions after 24 hours (arrows show examples of contusions). (B) Schematic picture of the secondary in-jury process that continues for long time after the impact. Due to this long lasting injury process, there may be an opportunity for reducing brain damage by neuropro-tective therapies.

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1.4 Secondary events TBI is a complex disease process and not a single pathophysiological event.19 The secondary injury events that appear after the primary damage can plainly be divided into a clinical and a cellular level where several fac-tors influence the outcome of the TBI patient (Figure 2). Very briefly, the primary injury (e.g. contusions, axonal injury, and hemorrhage) may lead to alterations in important physiological mechanisms such as cerebral blood flow (CBF), pressure autoregulation and brain compliance. Edema and raised ICP may develop. Altogether- focal and global ischemia may finally influence the outcome. Other clinically important factors are hypotension, hypoxemia, seizures, hyperglycaemia and hyperthermia that can lead to un-favorable effects. These factors are called avoidable and the mission of the NIC is to minimize their occurrence to improve outcome. On the cellular level after trauma, excitatory amino acids (EAAs) like glutamate activates the N-methyl-D-aspartate (NMDA) -receptors and this increase the Ca2+ - and Na+ levels inside the cells. Osmotic necrosis may follow. Marked eleva-tions of extracellular glutamate are regularly recorded in animal models of TBI20 and in the brains of patients with prolonged brain ischemia and focal cerebral contusions.21

Elevations in intracellular calcium22 may activate several calcium depen-dent free radical pathways, with deleterious effects for the cell.23 The in-crease in reactive oxygen species (ROS) can influence gene expression of immediate early genes, heat shock proteins, cytokines, growth factors, adhe-sion molecules, programmed cell death proteins and proteases, and have a role in events leading to neuronal death.24,25 The brain is considered to be the most sensitive organ in the body to ROS-mediated damage due to its low level of antioxidant defense, high levels of polyunsaturated fatty acids in the membranes and a high production of oxidative metabolic activity.26 ROS have shown to have great importance in the secondary injury mechanisms that follow traumatic brain injury. There is evidence for a ROS dependent switch in cell death, were high levels of ROS leads to necrotic death and whereas an insult with relatively low ROS levels may lead to apoptotic death.27-29

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Figure 2. Examples of secondary injury mechanisms after TBI, at a clinical and cellular level where several factors influence the outcome of the patient.

1.5 Treatment strategies Today no specific neuroprotective pharmacological treatment for TBI with proven efficacy exist;30-33 instead clinical effort is being made for the preven-tion of secondary brain insults by other means. The introduction of NIC has improved outcome after TBI34 and an organized secondary insults program and standardized NIC has led to further progress and a high rate of favorable outcomes.35

The fact that there is an ongoing cell death after TBI opens for possibili-ties to intervene with secondary injury mechanisms. It is tempting to specu-late that if we had a more detailed knowledge about the responsible second-ary injury mechanisms, new therapeutic targets would appear and the injury process could be stopped. For this to happen we need to better understand cellular pathological events and refine functional and behavioral outcome measurements. This is the basis for the present thesis.

The time-course after injury is very important when investigating differ-ent TBI treatment strategies. Some proteins/cascades/enzymes, like inflam-mation and caspases, have dual and not fully understood roles and might act both detrimental and beneficial at different time-points after injury. The lack of adequate analyses to determine optimal doses of potential therapies and find the therapeutic window may have led to clinical trial failures.36 Combi-nation therapies given at different time-points after injury might be needed.37 Further, a discussion about the clinical trial design that deals with the hete-rogeneity between TBI patients and centers, and better outcome measures might increase the possibility to find neuroprotecting agents.38

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CHAPTER 2

Experimental models of traumatic brain injury Experimental models of TBI are needed to understand the complex second-ary injury mechanisms initiated after brain trauma and to evaluate possible neuroprotective drugs.39-43 There is no single model that reproduces the en-tire range of the heterogeneity in human TBI, but each model represents a tool to investigate some specific aspects.44,45 The most commonly used mod-els are: fluid percussion injury (FPI), where trauma is induced by a pressure pulse delivered via a fluid column to the brain surface39,41,42, the controlled cortical impact (CCI) injury, where the impact is delivered using a pneumat-ic impactor46 and the weight drop injury (WDI) model, where a weight is dropped onto a piston that compresses the brain.47-50 Diffuse traumatic brain injury can be studied with a modified weight drop model48, central FPI (cFPI),51 or a model for rotational acceleration.52 These different experimen-tal models of TBI mimic major components of the complex human head injury occurring at impact. Secondary injury mechanisms such as hypoxia, hypotension, prolonged elevated ICP and others are more difficult to control and apply in rodent models but an increasing use of combination models is needed.44

In the present studies we have used the WDI model, the CCI model and the cFPI model, briefly described below.

2.1 Weight drop injury Animals are anesthetized and a craniotomy is made over the parietal cortex. Trauma is induced by dropping a weight onto a piston resting on the dura. By varying the compression depth of the brain cortex a severe, moderate and a mild injury can be produced, based on the extent of morphological and neurochemical changes.22,49 The weight drop model produces a focal lesion and has been shown to reproduce characteristics of human brain injury like disturbances in regional CBF.53 Additionally, cell loss in the hippocampus and impairment in neurologic motor and cognitive function are described.54 Post traumatic memory disturbances are related to the extent of hippocampal injury.55

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2.2 Controlled cortical impact After animals have been anesthetized, a craniotomy is made laterally be-tween the lambda and bregma sutures. Animals are subjected to trauma which is induced on the exposed dura, by using a pneumatically driven pis-ton. The injury is gradable by predetermined settings of velocity and impact depth, e.g. 3 m/s with 0.5 mm compression depth producing a severe injury in our hands (Figure 3). The severe CCI model is a focal injury model, cha-racterized by the presence of hemorrhagic cortical contusions below the im-pact site, at the grey –white matter interface, and in deep brain structures and development of a cortical cavity.56 This model produces morphological and cerebrovascular injury responses that resemble certain aspects of human TBI.46,57-61 It has been shown that behavioral deficits, and cortical and hippo-campal cell death are dependent on the impact depth.62

Figure 3. Picture showing the controlled cortical impact (CCI) device. The piston is attached to a linear velocity displacement transducer (LVDT). The velocity of the impacting shaft is controlled by gas pressure and measured directly by the LVDT that produces an analog signal that is recorded by a PC-based data acquisition sys-tem. The right picture is showing the movement of the piston as it is displayed on the computer screen.

2.3 Central fluid percussion injury Under anasthesia, a craniotomy is made in the midline between the lambda and bregma sutures, without destroying the underlying dura or the superior sagital sinus (Figure 4A). Trauma is induced after attachment of a plastic cup around the opening in the scull bone with a Luer-Lock (Figure 4B) allowing for tight connection to a plastic tubing attached to a fluid filled reservoir. A pendulum is released on to the reservoir giving rise to a pressure pulse into the cranium of the animal. The central fluid percussion injury (cFPI) model

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is a diffuse injury model, and was recently established in mice.63 It is characterized by hemorrhagic contusions on the brain surface and minor bleedings in the cortex and subcortical white matter, but no development of cortical contusions as seen after the CCI.

Figure 4. (A) Opening of the skull bone in central fluid percussion injury (cFPI) in the midline between the lambda and bregma sutures without destroying the underly-ing dura. (B) Fixation of the plastic cup over the craniotomy. (Photo by Gudrun Andrea Fridgeirsdottir).

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CHAPTER 3

Pathophysiology The following section briefly describes some of the morphological conse-quences and secondary pathophysiological events seen in the trauma models used in this thesis; mild WDI in rats, severe CCI and moderate cFPI in mice.

3.1 General morphological changes The mild WDI model in rat produces a cortical lesion without cavitation.22 The cortical lesion is characterized by a central region directly under the center of the piston where the brain tissue is mainly compressed, and a peri-pheral region in the perimeter of the piston where the tissue is subjected to shear forces (hence; the shear stress zone) from the edges of the piston. In the shear stress zone there is spongiosis, distorted neurons, polymorphonuc-lear phagocytes and scattered loss of neurons. There are also extravasations of serum proteins indicating increased blood brain barrier (BBB) permeabili-ty, i.e. vasogenic edema. In this region and in the subcortical white matter small hemorrhages are present.22,53

In contrast to the mild WDI, the severe CCI injury model in mouse pro-duces a distinct cortical lesion. The lateral CCI brain trauma, results in intra-cerebral hemorrhage, hippocampal and neuronal loss.56,64 Commonly ob-served cerebrovascular injury responses are disruption of the BBB as well as subdural and intraparenchymal hematoma, edema, inflammation, and altera-tions in CBF.61,65 In the mouse CCI model, the necrotic lesion has a diameter of about 2.5 mm extending to the subcortical white matter, resulting in a cortical cavitation (Figure 5A). There is also scattered cell loss in all regions of the ipsilateral hippocampus.

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Figure 5. (A) Large cavitation in the cortex, 7 days after controlled cortical impact (CCI). (B) No cavitation 7 days after central fluid percussion injury (cFPI) in mice.

Recently, a new cFPI mouse model was developed to mimic DAI injury.63 This mild cFPI model does not produce a cortical contusion and no cell death, but early induction of transcription factors known to regulate axon regeneration is seen.

3.2 Axonal injury in TBI DAI is a major consequence of acceleration/deceleration or rotation forces to the brain.66 It can lead to unconsciousness and persistent vegetative state that can be observed months to years following human TBI.63,66-71 In mild TBI the progressive damage to the white matter may explain the cognitive im-pairments that may persist for months to years following the initial injury.72 Axonal damage has also been recognized as a key predictor of outcome in other central nervous system (CNS) disorders.73 Mild TBI in the rat resulted in axonal injury and memory impairment, without evident intracerebral damage or contusions.74 Staining for axonal accumulation of β-amyloid pre-cursor protein (β-APP) can detect DAI within hours following TBI in ro-dents75,76 and head-injured patients.77,78 This glycoprotein is synthesized within the neuronal cytoplasm and carried along axons to the synapse by fast anterograde axoplasmic transport, but upon injury β-APP rapidly accumu-lates in axonal bulbs (Figure 6). It was recently shown that axonal damage after central- and lateral FPI in the rat was accompanied by myelin loss and apoptotic oligodendrocytes and increased expression of oligodendrocyte progenitor cells in injured white matter tracts.79 Axonal regeneration is ex-tremely limited in the CNS after injury and there are several proteins in CNS myelin and molecules associated with the astroglial scar that inhibits regene-ration.80

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Figure 6. Typical axonal bulbs stained with β-APP (black) in the corpus callosum one day after central fluid percussion injury in mice.

3.3 Blood-brain barrier pathophysiology The blood-brain barrier (BBB) is formed by brain endothelial cells con-nected by tight junctions that line the cerebral microvessels. This acts as a barrier for molecular traffic, except for small molecules as O2 and CO2 and small lipophilic agents, and has an important role in maintaining a well regu-lated microenvironment for neuronal signalling. However, the BBB limits drug delivery to the brain. What comes into the brain is regulated by a spe-cific transport system including a number of transporters and receptors, which permits the entry of required nutrients but excludes potentially harm-ful compounds.81 This network of neurons, glial cells and vasculature, named the neurovascular unit, has been shown to be important for regulation of cerebral blood flow and the function of the BBB. Astrocytes play an im-portant role in maintenance of barrier properties. Control of cerebral blood flow and many transporters of the BBB are under the control of astro-cytes.82,83

TBI can lead to production of a vasogenic edema, a pathological increase in the permeability of the BBB, which cause interstitial accumulation of plasma-derived osmotically active molecules and water. Formation of post-traumatic edema can raise the ICP and eventually reduce cerebral perfusion pressure and causing ischemia. As the brain is normally protected from many blood-borne factors, such as fibrinogen, thrombin and albumin, a loss in BBB function can cause great damage. These factors can bind to inflam-matory cells such as neutrophils and monocytes and cause oxidative stress. Albumin activates mitogen-activated protein kinases (MAPKs) pathways in

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both microglia and astrocytes, which leads to synthesis of the pro-inflammatory cytokine interleuin-1β.84 Edema development after experimen-tal brain contusion seems to be biphasic. The delayed phase reaches a maxi-mum 6 days post injury in the rat and correlates in time with a cellular in-flammatory reaction involving monocytes and lymphocytes.85,86

There has been much focus on the transporters of the BBB from the pharmaceutical industry. However, drugs that don’t pass over the BBB can be neuroprotective by acting at the endothelial level. We have previously shown in our laboratory a reduction of T lymphocyte and neutrophil infiltra-tion after treatment with the oxygen free radical scavenger S-PBN acting at the microvascular level.87 This is a novel concept for neuroprotection in TBI.

3.4 Glial cell reactions Activation of astrocytes and reactive gliosis are key features in many patho-logical conditions of the CNS, including neurotrauma.88,89 An increase of glial fibrillary acidic protein (GFAP) immunoreactivity is considered to be a sensitive marker of such injuries.90 Expression of vimentin immunoreactivity is considered to be another early sign of astrocyte activation in some patho-logical conditions of the CNS.91,92 In normal formalin fixed adult brain tissue the vimentin filament is not visualized; however after CNS injury astrocytes seem to increase the expression of vimentin making them visible by immu-nohistochemistry.93 The general view is that the vimentin response is con-fined to the vicinity of a brain lesion and that it is transient in nature.88,91,94

The glial scar involves many cell types, including oligodendrocytes, mi-croglia and astrocytes. It has been shown that astrocytes close to the lesion upregulate vimentin and nestin production and cell division are seen. The final structure of the formed glial scar is predominantly made up of astro-cytes, and these cells divide and slowly migrate into CNS injuries, eventual-ly to some extent fill in the vacant space.95 There are signals released during the initiated wound healing process which can both promote axonal regene-ration or suppress axonal growth and act neurotoxic and increase the forma-tion of a glial scar. The formation of a glial scar protects the surrounding tissue from inflammation but also inhibit axonal regeneration in this area e.g. through inhibition of gene expression needed for axonal guidance.96,97

3.5 Inflammatory responses Normally, the brain is protected from infiltration of molecules and immune cells by the BBB. The brain has been thought to be an “immune privileged” organ.96 However, today neuroinflammation is an accepted concept that plays an important part in the pathology of TBI. The inflammatory response

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to traumatic brain injury is highly complex and involves both local and sys-temic events. There is increasing evidence that points towards a both delete-rious, and paradoxically, beneficial role of the immune system after injury.98 Microglia/macrophages, astrocytes and leucocytes including T-cells and neutrophils, and the signals these cell types mediate are the key actors in the neuroinflammatory response following TBI.

Expression of activated microglia is shown to increase early after TBI and to persist for many years indicating that TBI triggers a long term inflamma-tory response.99 The long term microglia activation in human TBI99 has also been shown to be most persistent in sites of white matter damage, and mi-croglia activation in DAI in the rat showed association with axonal injury between 7-28 days post injury.99,100 Microglia is suggested to play an impor-tant role in cytokine release and as macrophages cleansing hazardous struc-tures and debris. They are also associated with brain repair, although the effect can become damaging when the activation is prolonged microglia can be cytotoxic and be predisposing for neurodegenerative disease.99

The recruitment of leukocytes (including neutrophils) is driven by che-mokines, small soluble signaling glycoproteins and neutrophil infiltration is observed at 24 h after focal injury in the rat and is dependent on the degree of BBB disruption.101 Further, inflammatory response of human focal TBI show reactive microglia, polymorphonuclear cells and T lymphocytes de-tected 3 to 5 days after trauma.102 T lymphocytes has also shown to be present in focal TBI in the rat87 and in the mouse.103

Neutrophils can increase vascular permeability and cause release of pro-inflammatory cytokines, proteases and reactive oxygen species and depletion of neutrophils reduces edema formation and tissue loss following focal TBI in the mouse.104,105 Both experimental and clinical research suggests that TBI activates the innate immune system (microglia and astrocytes) in the brain in a complex interplay with the systemic immune system, including cerebral production of several cytokines.106,107

3.5.1 Production of cytokines Cytokines are cellular signaling molecules acting in cascades showing both restorative, neurodegenerative, pro- and anti- inflammatory qualities. The cytokine interleukin (IL)-1β has been considered a major promoter of neu-roinflammation and increased production of IL-1β is harmful following brain injury.108-111 Early up regulation (within 1h) of IL-1β mRNA has been found after closed head injury in the rat112 and minutes after severe TBI in hu-mans.113 Compounds that directly or indirectly target IL-1β have demon-strated therapeutic efficacy in experimental TBI.114-117

Microglia and astrocytes are the major cellular sources of T-cell regulat-ing cytokines in CNS disease, including trauma. T-cells also produce proin-flammatory cytokines including interleukins and production of interferon

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(IFN)-γ occur to some extent. However, it is not clear if activated T-cells that enter over the BBB from the bloodstream are reactivated or not.118 How-ever, other types of T-cells are considered to have a protective role through anti-inflammatory cytokines.119 When targeting T-cells as a treatment option, one challenge in management design is to reduce harmful actions and en-hance protective actions of T-cells.

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CHAPTER 4

Functional outcome evaluation In order to evaluate neurobehavioral and cognitive outcome following expe-rimental TBI, several different behavioral models have been used. Usually, these tasks try to evaluate functions that are known to be impaired in human TBI patients, such as memory and motor function. Commonly used models for measurements of motor deficits in experimental TBI are the rotarod120, beam-balance and beam-walking tests121 or the Morris Water Maze (MWM) for evaluation of memory deficits.122

4.1 Ethoexperimental approaches to the study of behaviors The ethoexperimental approach is a combination of experimental psycholo-gy and etiology, which emphasizes objective measurement of behavioral variables in an experimental setting resembling natural environmental chal-lenges. Ethoexperimental means that native forms of behavior, derived from the natural environment from the species studied is taken into effort. This provides the possibility of greatly improved understanding of complex beha-vior patterns, and gives a more appropriate use of lower mammals as models for research on human behavior.123 The aim of using an ethoexperimental approach is to study meaningful behaviors using biologically relevant la-boratory test environments and include descriptions of animal behavior as part of the analysis.124 This has not been discussed extensively in the study of functional outcome after TBI.

4.2 Emotional reactivity and risk assessment Measurements of animal emotions focus primarily on the physiological and behavioral components of the emotional response. Emotions in both humans and animals are very diverse and involve many bodily and mental processes. Most definitions of emotion refer to the individual´s subjective experience of its situation.125,126 The term “emotional reactivity” is referred to as emotio-nality, anxiety or anxiety related behavior and is widely used in pharmaco-

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logical research. Assessment of emotional reactivity in exploration-based tests can be done by measurements of different kind of locomotor beha-viors.127 Several tests for research on the behavioral, pharmacological and neurochemical effects of emotional reactivity exist. The open field test (OF) and the elevated plus-maze (EPM) test are two commonly used models and the basis for these tests is exploration of an unfamiliar environment. The animal is facing an open arena (OF test) and can chose to stay close to the walls or explore the inner field, or as in the EPM test stay in the closed arms or explore the open arms. Emotional reactivity is assessed based on an emo-tional component in the choice that the animal has. There is a conventional concept that there is a conflict between exploration of the open arms and staying in the closed arms, and it has been shown that entrance to the open arms are associated with anxiety-related behaviors.128-130 Previous studies have suggested impairment in open field exploration after experimental TBI131-134 but in opposition, CCI to immature mice resulted in hyperactivity in both the OF and EPM tests.135

In the wild, risk assessment involves an animal’s trade-off between avoidance of predators and the exploration of an environment to find e.g. food, water or mate. The gain and risk are traded off so that the largest gain comes at the expense of the lowest risk.136 Risk assessment is perhaps one of the most common behavioral patterns for any higher animal and occurs in situations involving any considerable degree of unfamiliarity or unpredicta-bility. There are two major variants of risk assessment in rodents; when an escape route or place to hide is available, the animal tends to flee or hide to avoid risk but this does not give any information about the new environment. After some time has passed and no evidence of danger is present, the animal may systematically reenter the threatening area to explore it. Exploration of a novel area involves slow movements, clings to walls (thigmotaxis), avoid-ance of unnecessary activities, and stretched attention posture when explor-ing new objects. When the animal becomes more familiar with the environ-ment more normal activities take over. In situations where escape or con-cealment is impossible, freezing is the only effective method of avoiding risk and this may involve total immobility or freezing with scanning move-ments.123 Introduction of risk assessment and risk taking, and approach and avoidance behaviors, has lately complemented measures of emotional reac-tivity.137,138

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4.3 Multivariate concentric square field test Recently the Multivariate Concentric Square Field (MCSF) test was intro-duced.139 This is a relatively new behavioral model with an ethoexperimen-tal, and multivariate approach and has been used for behavioral profiling in rats and mice.140-142 It emphasizes measurement of exploration, risk taking, risk assessment and shelter seeking, which are evolutionary conserved strat-egies for survival. It has also been used to relate functional effects to the localization and extent of traumatic and ischemic brain lesions in the rat.143,144 The test situation in the MCSF involves a free choice of different environmental settings such as an open field, a dark room and other chal-lenges, i.e. the animals are not forced to certain behaviors such as finding a hidden platform or beam balancing. Furthermore, since the animals have several different behavioral options within the test box it is not predeter-mined what certain behavioral aspects or mental state that will be tested, and is not predictive in the sense of previous definition of a specific purpose of measuring a certain mental state. In a multivariate test situation, several measures can be recorded and these can provide a behavioral profile rather than focusing on any particular behavior.139 The MCSF test is suitable for measuring locomotion, exploration and cognitive functions like risk taking, risk assessment and safety seeking, and for this purpose some behavioral parameters from the test can be chosen. Furthermore, a memory effect may be measured in repeated trials and is depending on whether the animal has achieved a positive or negative association to the MCSF arena. The response in the repeated trial may reveal an altered reaction to the MCSF, denoted “transfer effect” i.e., the experience in the first trial is transferred to the next trial in the test.

4.4 Morris water maze TBI in humans leads to cognitive dysfunction and memory deficits.145-147 The degree of the symptoms often depends on the injury severity.3 The MWM is the most widely used test for cognitive evaluation after experimen-tal brain injury.148 The animal is placed in the pool at four different locations, and the use of visual cues should help the animal to locate a hidden platform under the water surface.149 The experimental design can vary and may in-clude pre-training before injury or testing post-injury only. Several parame-ters as latency to find the platform, swim speed and path length are regis-tered. In a probe trial, usually some days after the last training session, the

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platform is removed and the animal’s ability to remember the location of the platform, by recording passages over platform area, is measured. The MWM evaluates learning and memory function, and injury to the hippocampus seen after trauma can be measured.150-152 Also after mild FPI with minimal evi-dence for hippocampal damage, mice showed an impaired ability to learn the location of the hidden platform three weeks post-injury.153 The cause of cog-nitive changes in this study was explained by disruption of the neural net-work that is necessary for the MWM task, like damage in the dorsal thala-mus which may disrupt connections between the thalamus and hippocampus.

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CHAPTER 5

Present investigation – aims of the study

General The general aims for this study were to investigate cellular reactions includ-ing glial cell responses and immune cell responses, and to measure function-al changes after focal and diffuse experimental traumatic brain injury.

Specific

I To compare the temporal and spatial expression pattern of vimentin- and GFAP in a weight drop model of mild cortical contusion injury in the rat and to study the GFAP and vimentin response in severe cortical contusion injury produced by the CCI model in the mouse (Paper I).

II To investigate the effect of focal cortical trauma (CCI) in mice on behavioral profiles in the MCSF arena (Paper II).

III To characterize the neuroinflammatory response and glial cell reac-tions after diffuse axonal injury (cFPI) in mice. To investigate if the behavioral profiles generated in the MCSF test of mice subjected to diffuse injury differed compared to mice subjected to focal injury (Paper III).

IV To study the effect of the anti-inflammatory interleukin-1β antibody on functional outcome after cFPI in mice (Paper IV).

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CHAPTER 6

Material and methods All experiments were performed according to protocols approved by the Uppsala animal ethical committee for animal research in accordance with the Swedish Legislation.

6.1 Animals and housing

In Paper I male Sprague Dawley rats weighing 325-487 g and male C57BL6 mice weighing 21-24 g (both from B&K AB, Sollentuna, Sweden) were used. In Paper II, III and IV only male C57BL6 mice weighing 21-28 g were used. The animals were housed in temperature (24°C) and humidity (55±10%) controlled environment on a 12-hour light/dark cycle with lights on at 7:00 a.m. Animals had free access to food (R36 standard pellets, Lantmännen, Kimstad, Sweden) and water ad libitum. Gloves were used in all physical contact with the animals.

6.1.1 Anesthesia and production of trauma (Paper I-IV) During surgery, rats were artificially ventilated using a gas mixture of isoflu-rane (0.8-2.0%) and N2O/O2 (65%/35%). Mice were anesthetized using a mix of isoflurane (1-1. 2%) and N2O/O2 (70%/30%) delivered through a nose cone. Body temperature (37º ± 0.5) was maintained by a heating pad coupled to a rectal probe (CMA 150, CMA Microdialysis AB, Solna, Swe-den) and a heating lamp throughout the procedure. After shaving and clean-sing the skin with ethanol, local anaesthesia (Xylocain or Marcaine®, Astra-Zeneca, Sweden) was administered subcutaneously before the scalp was opened by a midline incision. Artificial tears lubricant eye ointment (Visco-tears, Novartis, Inc., Basel, Switzerland) was used for corneal protection during anesthesia. Animals were placed in a stereotaxic frame and a craniot-omy was made over the right parietal cortex (WDI and CCI) or in the mid-line between the bregma and lambda sutures (cFPI).

WDI (in Paper I) was produced by a 21-g free-falling weight that was dropped from a height of 35 cm over a piston resting on the exposed dura,

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allowing one single depression of the cortex of 1.5 mm. In sham operated animals the weight was not dropped.

For CCI (in Paper I &II) a CCI device (manufactured by VCU Biomedi-cal Engineering Facility, VA, USA) (Figure 3) was used30,44,154,155 with an impact depth of 0.5 mm and a velocity of the piston of 3.3 m/s. Sham-injured animals were treated identically with respect to anesthesia and surgery but did not receive a cortical impact.

For cFPI injury (in Paper III &IV) the surgical procedures of cFPI were modified from those previously described by Dixon et al58 in rats and by Greer et al.,63 in mice. A plastic cap was secured over the craniotomy using dental cement (Heraeus Kulzer GmbH, Hanau, Germany), and the integrity of the seal between the cap and the skull was confirmed adding normal sa-line into the cap (Figure 4B). Injury was produced by attaching the saline-filled hub to the Luer-Lock fitting on the fluid percussion device (VCU Biomedical Engineering Facility, Richmond, VA) and releasing a pendulum striking the end of a saline-filled reservoir transmitting a pressure wave into the closed cranial cavity. The pressure pulse measured by the transducer was displayed on an oscilloscope and the peak pressure was recorded in atmos-pheres (atm). Immediately after the injury, the animals were visually moni-tored for apnea and seizures. Following resumption of spontaneous breath-ing, the mouse was re- anesthetized with isoflurane and the cement and the cap was removed. Sham-injured animals were treated identically with re-spect to anesthesia and surgery.

In Paper I-IV, after trauma, the bone flap was replaced and the skin was closed with sutures. Following surgery animals were placed under a heating lamp until recovered from anesthesia and fully ambulatory. Body mass was registered once a day postoperatively until sacrificed or until the animals had regained their preoperative weight, i.e. up to one week.

6.2 Survival times Animals were allowed to recover for:

• 1h,4h,22h,1,3,7 and 21 days in Paper I; • 14 days in Paper II; • 1, 3 and 7 days for morphological evaluation in Paper III; • 21 days for behavior in Paper III and Paper IV.

See Figure 7 for an overview.

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6.3 Treatments (Paper IV) In study IV mice were randomized into groups that received the anti-interleukin-1β antibody (IgG2 a/k 20µg/mL, provided by Novartis, Inc., Basel, Switzerland) or the control antibody against cyclosporine A (anti-CsA; 20µg/mL) administered intraperitoneally 30 min after cFPI or sham-injury.

6.4 Sampling of brains (Paper I-IV) For immunohistochemical studies animals were deeply anesthetized given an overdose of isoflurane or an overdose of pentobarbital and were transcardial-ly perfused with isotonic saline solution followed with 4% paraformaldehyde or 4% formaldehyde depending on which study. Thereafter mouse brains were post fixed in 4% formaldehyde at 4ºC until used for immunohistoche-mistry, alternatively cryoprotected in 30% sucrose and snap frozen in cold isopentane. Brains from rats were kept in a fixative overnight, dehydrated and coronal blocks from the fronto-parietal region were embedded in parap-last.

6.5 Antibodies for histological evaluation (Paper I-IV) Histological outcome was analyzed by immunohistochemistry using antibo-dies directed against specific proteins located within the tissue. Sections of brain tissue were incubated with the primary antibody against the antigen of interest and then a biotinylated secondary antibody was added. An avidin-biotin complex (ABC) was coupled to the biotinylated secondary antibody and by adding di-amino-benzidine (DAB), which bind to the ABC, this re-sulted in a brown color visualizing the stained antigen. In this study several different antibodies were used, as shown in Table 1.

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Figure 7. Survival times and study protocol for studies included in this thesis. CCI, controlled cortical impact; cFPI, central fluid percussion injury; CsA, cyclosporine A; D, day; M, Morphological evaluation; MCSF, multivariate concentric square field test; MWM, Morris water maze; IL-1β, interleukin-1β.

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The sectioning procedure differed between studies, as described below;

In Paper I forty-micron thick coronal sections were cut from mice brains and stained using free floating technique, while six-micron thick sections were stained from paraffin embedded rat brains.

In Paper II forty-micron thick coronal sections were cut and mounted on a gelatin-treated glass slide.

In Paper III fourteen-micron thick brain coronal cryo-sections and sagittal brain stems sections were mounted on object glasses.

In Paper IV fourteen-micron thick coronal cryo-sections mounted on object glass were used.

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Table 1. Antibodies used

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6.6 Quantitative real-time PCR (qRT-PCR) (Paper I) To compare the immunohistochemistry findings in tissue with the pattern of vimentin mRNA expression, transcript levels of vimentin were studied. In order to examine transcript levels of vimentin and the control Cnp1 (oligo-dendrocyte marker), the entire neocortex and hippocampus were dissected from the injured hemisphere. The following two genes were selected for analysis by qRT-PCR using pairs of forward and reverse primers as specified below (GenBank accession number indicated within parenthesis): Vimentin (NM_011701) 5’-GGC TGC CAA CCG GAA CAA C-3’ AND 5’-CGC TCC AGG GAC TCG TTA G-3’, respectively. For CNPase (Cnp1, NM_009923) the primers were 5’-CAA GAT GGT GTC CGC TGA TG-3’ AND 5’-TCA TGT CCC GGC GGC AGT AG-3’. For confirmation of RNA amount, we used primers detecting 28S rRNA (X00525) and the primers were 5’-GGG AGA GGG TGT AAA TCT CGC-3’ and 5’-CTG TTC ACC TTG GAG ACC TGC-3’.

6.7 TUNEL-staining (Paper III) Cells that undergo apoptosis are usually difficult to identify with conven-tional light microscopy so for this reason methods have developed to detect apoptotic cells in situ. These methods involve the in situ extension of deox-yribonucleotide triphosphate to the free ends of the fragmented DNA using the enzyme terminal deoxynucleotidyl transferase.156 This terminal deox-ynucleotidyl transferase dUTP nick end labeling (TUNEL) was used in Pa-per III for detection of ongoing cell death. We used the TUNEL mix (Roche Diagnostics, GmbH, Mannheim, Germany) and incubated sections for 60 min in room temperature, thereafter the sections were washed and mounted with Vectashield with the nuclear stain DAPI (Vector laboratories, Burlin-game, CA, USA).

6.8 Validation of anti-interleukin-1β antibody tissue concentration (Paper IV) The anti-IL-1β antibody was administered by intraperitoneally injection 30 min after cFPI or sham-injury, and tissue samples were taken from the cortex and hippocampus bilaterally at 24 hours and 72 hours post-injury. The pene-tration of the anti-IL-1β antibody was analyzed by Western blotting using highly purified anti-idiotypic antibodies against the Fab fragment of the anti-IL-1β antibody.

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6.9 Functional evaluation (Paper II-IV) In Paper II, III and IV, the animals were kept in the animal unit for 1-4 weeks and to reduce stress involvement in the experimental procedure, the mice were handled for one week prior to behavioral testing started. The han-dling procedure included daily transfer from the home cage followed by placement on the arm of the handler for 1-2 min and back into the home cage. The mice were housed in groups of four to six animals in each cage on a 12-hour light/dark cycle with lights on at 7:00 a.m. An observer blinded to the treatment and injury status of each animal evaluated functional outcome.

6.9.1 The Multivariate Concentric Square Field (MCSF) test (Paper II, III & IV) The animals were tested in the MCSF at 2 days (Trial 1), 7 days (Trial 2, Paper II) or 9 days (Trial 2, Paper III &IV) post-injury. When tested, the animals were transferred from the animal facility in their home cage in a ventilated wagon and were allowed to adapt in the laboratory for 45 min before testing started. The test session lasted for 20 min and the animals were released in the center square facing the wall without openings. The light was dimmed except for the BRIDGE area that was illuminated. The MCSF apparatus consists of a square field (72 x72 cm) surrounded by an outer wall (28 cm high) with a smaller square field (CENTRE, 42 x 42 cm) located in the center of the box (Figure 8). There are circular openings (8 cm in diameter) in three of the walls of the center field that lead to the outer field and a corridor (divided into CORRIDOR A-C) is formed around the central field. CORRIDOR A has contact with the dark corner room (DCR) that is covered with a piece of board. CORRIDOR B and C have contact with the HURDLE with an elevated floor with a holeboard with two holes (1.5 cm in diameter) for exploration. From CORRIDOR B there is an angled plane up to the HURDLE but from CORRIDOR C the animals have to climb up 8 cm. From CORRIDOR C the animals can enter the SLOPE that lead up to a BRIDGE consisting of a stainless-steel wire-mesh construction (8 mm between the bars) that bridges over an illuminated opening in the floor. A photocell device is located under the holeboard floor of the HURDLE allow-ing recording of head dips into the holes. The entire arena was divided into zones (Figure 8) that forms the basis of the description and the variables of the animal´s performance in this test. The following zones were defined: CENTRE, the center field of the arena; CENTRAL CIRCLE, a circular zone in the middle of the center field used for measurement of activity in an open area; CORRIDOR A-C, the corridors surrounding the centre field; HUR-DLE, a corner with elevated floor containing a hole board to test the explora-tory drive; BRIDGE, the elevated and illuminated bridge considered to be an open area associated with risk; SLOPE, the slope leading up to the BRIDGE

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considered to be an area where the animal had to assess the risk of visiting the BRIDGE; SLOPE ENTRANCE before the SLOPE; DARK CORNER ROOM (DCR), a dark and shaded room which was considered to be a safe area. The sum of the frequencies of entries to CORRIDORs A-C (FRQ TOTCORR) and the sum of frequencies to all zones (TOTACT) were used for assessment of general loco-motor activity. The total time spent in COR-RIDORs A-C was given denomination (DUR TOTCORR). In addition, the number of rearing and grooming actions were recorded manually.

Figure 8. The Multivariate Concentric Square Field (MCSF) test and the defined zones. CORR A, B and C indicate corridors A-C; DCR, dark corner room with en-trance from CORR A; SLOPE ENTR, slope entrance. START indicates where the animal is placed when introduced to the arena.

6.9.2 Behavioral recordings in the MCSF The animals were monitored by a TV-video set-up (Panasonic Super Dy-namic WV-BP550/B camera, Panasonic NV-HD640 VHS recorder). Manual scoring of the behavior was performed using the software Score version 2.2 (Pär Nyström, Copyright Solids, Uppsala, Sweden). The latency (LAT, s), of first visiting a zone, frequency (FRQ) of visits and duration (DUR, s) of time spent in a certain zone and recordings of rearing and grooming, were all registered. Ethovison system version 2.3 (Noldus Information Technology, Wageningen, The Netherlands) was used for recording of velocity (cm/s) and distance (cm) moved in the MCSF arena (TOTARENA and DISTANCE CENTER, Paper IV only). The animal had to cross the defined zone with both hind legs to be scored as visiting that zone. The latency of first visiting

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a zone, frequency of visits, and duration of time spent in a certain zone, and also the number of animals visiting each zone (OCCURRENCE) were all registered. For some of the zones, the mean duration per visit (DUR/FRQ) was calculated.

6.9.3 Morris water maze (MWM) (Paper IV) The ability to learn the location of the hidden platform was assessed by sub-jecting each animal to 16 training trials over a four-day interval (4 trials/ day) in the MWM at day 14-17 post-injury. The MWM consists of a 1.4 m diameter circular tank with white bottom and walls and a 10 cm diameter white platform, placed in the southwest quadrant of the tank and submerged one cm below the surface of the 22°C water. Simple visual cues to aid navi-gation were placed on roller curtains surrounding the pool. The memory test (probe trial with the platform removed) was performed at day 21 post-injury, i.e. four days following the last learning trial and was analyzed for latency to pass the platform area, number of times crossing the platform area and the percentage of time spent in the correct quadrant. The first 15 and 30 seconds were analyzed as well as the entire 90 seconds. This was done to show the ability to change exploration strategy, since naive animals start searching for the platform in other quadrants when they do not find it in its previous posi-tion. Each swim trial was run by placing the mouse in the tank at one of four designated entry points (W, N, E and S) facing the wall, activating the video-based computer tracking system (HVS Image Ltd., Buckingham, U.K.), and the trial was terminated when the mouse located the platform. The mouse was allowed to remain undisturbed on the platform for 15 s to acquire the visual cues surrounding the pool. The animal was placed on the platform for 15s if it did not locate the platform within 90 s. Latency to find the platform, swim speed and path length was analyzed.

6.10 Statistical analyses (Paper I-IV) For statistical analysis Shapiro-Wilk´s W test was used to analyze if data showed a normal distribution. If data did not meet the assumption for normal distribution, the non-parametric Kruskall-Wallis Analysis of Variance of Ranks was used for overall comparisons between the groups. Pairwise mul-tiple comparisons were used for significant differences in mRNA in Paper I. Significant differences were analyzed for group wise comparisons with Mann-Whitney U-test (Paper II-IV). The Chi-square test was used for analy-sis of occurrence in Paper II. Friedman ANOVA followed by Wilcoxon signed rank test was used for comparisons between dependent groups in the first and second MCSF trial (Paper II). The parametric t-test for dependent samples was used for analysis of distance moved in the arena (Paper II).

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Area stained with IgG where analyzed with one-way ANOVA and Tukey post-hoc test (Paper III). MWM data was analyzed with one-way ANOVA and Fisher´s PLSD post hoc test (Paper IV). Statistica 10.0 software (Stat-Soft Inc., Tulsa, OK, USA) and SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA) were used for statistical analyses. A P-value < 0.05 was considered statistically significant.

6.10.1 Principal component analysis (Paper II-IV) The principal component analysis (PCA)157 has previously been used to de-scribe and dissociate behavioral profiles in animals observed in the MCSF test.138,139,141,142 It has also been used to analyze inflammatory response and biomarker patterns in complex datasets obtained from microdialysis in hu-man TBI.158 This statistical method is very useful for analysis of material with large numbers of variables in few number of animals.157 This method was used as a complement to traditional statistical analysis to create a score plot showing a summary of the relationship among the individuals, and a loading plot where variables important for these relationships can be identi-fied. The two plots are complementary and super imposable. To estimate the number of PCA components, cross-validation was used.159 SIMCA-P+ 12 software version 12.0 (Umetrics AB, Umeå, Sweden) was used for this pur-pose.

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CHAPTER 7

Results

7.1 Cellular reactions After focal brain injury in the rat and mouse, neither apnea nor mortality after injury was observed. After cFPI in mice, animals showed a shorter ap-nea, on average around 30 seconds. The mortality rate after cFPI was around 20%, as a result of long-lasting apnea. There was no development of a cor-tical contusion in the cFPI model but we observed some damage in the cor-tical tissue underlying the impact site the first days after trauma (Figure 5B). This section briefly describes cellular reactions observed in the included models of focal and diffuse brain injury.

7.1.1 Vimentin and GFAP responses in astrocytes after focal brain injury in the rat and mouse (Paper I) We investigated astroglial responses after mild cerebral contusion injury WDI in the rat and after severe CCI in the mouse. The temporal and spatial expression patterns of vimentin and GFAP in these two models were com-pared by immunohistochemical studies. Vimentin mRNA qRT-PCR analysis was also performed in order to verify an increased gene expression, hence protein synthesis. Increases in vimentin mRNA levels in the cortex and hip-pocampus started at 22 hours after injury. Vimentin positive cells were iden-tified as astrocytes based on their characteristic morphology of star-shape, slender processes and a clear resemblance of astrocytes stained with GFAP. In the mouse, at three days post-injury the number of vimentin positive cells had increased enormously and were present in the entire ipsilateral cortex, in the white matter beneath the ventricle and in the dentate hilar part of the hippocampus (Figure 9).

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Figure 9. Reactive astrocytes stained with vimentin in the dentate gyrus of the hip-pocampus (A) 3 days after sham-injury and (B) 3 days after controlled cortical con-tusion injury in the mice.

Further, the results showed that the mouse and rat brain had similar vimentin immunoreactivity pattern, but the expression in mice was more transient. After 21 days the number of vimentin expressing astrocytes had decreased considerably. Sham-injured mice, with very few exceptions, did not present any vimentin immunoreactive astrocytes. However, ependymal cells and the choroid plexus epithelium were intensely immunoreactive. Sham-injured rats displayed a large number of GFAP positive astrocytes in the gray and white matter. After injury the number of GFAP reactive astrocytes increased at the site of the impact (Figure 10). In the mouse, the increase in GFAP positive cells after injury was more widespread and included the injured cortex, the ipsilateral hippocampus, the corpus callosum and the white matter close to the ipsilateral ventricle.

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Figure 10. Schematic distribution of increase in GFAP (•) and vimentin (*) positive cells one day, three days and twenty-one days after weight drop injury in the rat and controlled cortical impact injury in the mouse.

7.1.2 Vimentin and GFAP responses in astrocytes after diffuse brain injury in the mouse (Paper III) Astrocyte reactions after diffuse injury were also studied with antibodies to vimentin and GFAP. Both markers were up-regulated with, to some extent, different distribution and time course post-injury. The immunoreactivity for vimentin was increased, especially after 3 days post-injury, in the cortex, the subcortical white matter, fimbria of hippocampus and the dentate gyrus of hippocampus in addition to the external and internal capsule (Figure 11A). Sham-injured animals showed some background staining of GFAP, which increased after cFPI. The increased GFAP staining was observed in the cor-tex, the subcortical white matter, fimbria of hippocampus and in the thala-mus (Figure 11B).

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Figure 11. Schematic overview of (A) vimentin and (B) GFAP staining after central fluid percussion injury (cFPI) in mice after sham-injury or 1, 3 and 7 days post-injury.

7.1.3 Axonal injury, cell death and BBB leakage after diffuse brain injury in the mouse (Paper III) By using amyloid precursor protein (β-APP) immunoreactivity, we detected axonal injuries, in cortex and subcortical white matter, one day post-injury. A few β-APP-positive axonal profiles were also observed in the dorsolateral rostral brain stem at this time-point (Figure 12).

TUNEL staining one day post-injury detected apoptotic cells in the cor-tical tissue underlying the impact, but only few cells were TUNEL positive in the subcortical white matter and the hippocampus. Increased permeability of the BBB in the cortex underlying the impact and the hippocampus are observed by staining for mouse immunoglobulin G (IgG), normally not ob-served in brain tissue. Plasma protein leakage (IgG) indicating a vasogenic edema, was increased up to seven days post-injury (Figure 13).

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Figure 12. Staining for β-APP 1, 3 and 7 days after cFPI in the mouse. (A) Schemat-ic distribution of the staining pattern. (B) Axonal profiles in the corpus callosum at one day post-injury (arrow). (C) β-APP positive axonal profiles in the brain stem at one day post-injury (right) compared to sham-injured mice with normal staining of nerve cell bodies (left).

Figure 13. (A) Representative pictures of blood-brain barrier leakage (IgG staining) in the brain regions underlying the craniotomy. (B) Quantification of the area (mm2) stained with IgG (above the arbitrary pre-set intensity level) at 1, 3 and 7 days post-injury. cFPI, central fluid percussion injury. Data presented as mean ± SD. *P< 0.05; **P<0.01; ***P<0.001 compared to sham-injured controls.

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7.1.4 Inflammatory response after diffuse brain injury in the mouse (Paper III) The neuroinflammatory response was studied at 1, 3 and 7 days after cFPI in mice. Inflammatory response was observed in the cortex underlying the im-pact site and in the subcortical white matter. Cell counts of micro-glia/macrophages (MAC-2), neutrophils (GR-1) and T-cells (CD3) were completed in the corpus callosum, external capsule and fimbria of hippo-campus. Activated microglia was increased throughout the observation pe-riod in the cortex and in the subcortical white matter, especially at one day post-injury. Infiltrating neutrophils and T-cells appeared one day post-injury in the cortex and subcortical white matter (Figure 14).

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Figure 14.Staining for different immune response markers after central fluid percus-sion injury (cFPI). (A-C) Schematic distribution of positive microglia, neutrophil and T-cell staining at 1, 3 and 7 days post-injury. The microglia staining was in-creased throughout the observation period in the cortex and in subcortical white matter, especially one day post-injury. Infiltrating neutrophils appeared one day post-injury in the cortex and cortex and subcortical white matter and remained in-creased over the observation period. T-cells increased in the cortex and subcortical white matter one day post-injury but then the number of cells declined. (D) No in-flammatory response in the external capsule after sham-injury (left column). Acti-vated microglia/macrophages (MAC-2), neutrophils (GR-1) and T-cells (CD3) at 1, 3 and 7 days post-injury. Inserts show the same area in the external capsule at higher magnification. (E) Cell counts of MAC-2, GR-1 and CD3 in the corpus callosum, external capsule and fimbria of hippocampus from 1, 3 and 7 days post-injury. Data are presented as mean ± SD. *indicates a difference compared to sham-injured con-trols (P<0.05).

7.2 Behavioral changes The behavioral changes after focal injury (CCI) and diffuse injury (cFPI) were studied using the MCSF test and the MWM.

7.2.1 Behavioral outcome after focal brain injury (Paper II) The results from this study revealed differences in risk taking, risk assess-ment and explorative behavior between the sham-injured animals and ani-mals subjected to CCI, tested in the MCSF test two and seven days post-injury. There was no difference in distance moved in the MCSF arena, which was used as a test for overall neurological function, between the sham and TBI mice. Due to some differences between the naive (normal non-anesthetized mice) and sham-injured mice, these two groups were not pooled and statistical comparisons were made towards sham animals. With the pur-pose to investigate the effect of the experience in the first trial and any even-tual transfer effect in trial 2, the differences in performance were calculated by taking the behavior of the sham group in trial 2 into account. There was a clear transfer effect between trial 1 and trial 2 in the sham group including more time spent in the dark corner room and less activity in the second trial. In the TBI group the transfer effect was almost completely absent and only minor differences were seen in the performance in trial 2 compared to the behavior of the sham group in trial 1. In order to make a functional interpre-tation of the measured parameters, which include frequencies, durations and latencies to all zones in the MCSF test, 2-4 parameters per behavioral cate-gory (locomotion, exploratory behaviors, risk taking and safety seeking ) were chosen for analysis of trends (Table 2). These parameters were ranked according to animal ID, added together, and statistical analysis was per-formed on the rank means for every behavior. The result from the analysis of

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trends showed that mice subjected to CCI were more exploratory (trial 2) and more risk taking (trial 1 and 2) compared to sham-injured mice. In trial 2 CCI-mice were less shelter seeking compared to sham-injured mice (Figure 15).

Table 2. The functional interpretations of the parameters in the MCSF test included in the analysis of trends.

DCR, dark corner room; Dur, duration; Frq, frequency; Lat, latency.

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Figure 15. Analysis of (a) locomotion, (b) exploratory behavior, (c) risk taking, (d) safety seeking, and (e) risk assessment of both trials in the Multivariate Concentric Square Field test. *P<0.05, **P<0.01 compared to sham-injured animals; TBI, traumatic brain injury (controlled cortical impact, CCI). Data is presented as mean ±SEM.

7.2.2 Behavioral outcome after diffuse axonal injury (Paper III&IV) Compared to sham-injured animals, animals subjected to cFPI showed a different behavioral pattern in the MCSF test. In both trials (two and nine days post-injury) diffuse injured mice were passing the center of the arena fewer times, were less active, made fewer visits to the corridors and spent more time running around in circles in the center of the arena compared to sham-injured mice. In Paper IV naive mice showed similar behavioral pro-files as sham-injured mice. However, there were some differences and there-fore the naive and sham groups were not pooled to one control group.

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7.2.3 Principal component analysis of behavioral data (Paper III) We wanted to test the hypothesis that the behavioral disturbances induced by cFPI were unique and therefore the behavioral data collected in Paper III, from mice subjected to cFPI, were compared to behavioral data from Paper II from mice subjected to CCI. A principal component analysis was per-formed which resulted in a model with three components, where the two first components shown describe 39.0% of the total variation in the data set. This was done in order to visualize eventual differences between the groups.

From the principal component analysis (PCA) from trial 1 and trial 2, it was evident that mice subjected to CCI or cFPI used different behavioral strategies and displayed altered behavioral patterns that placed these two groups in different part on the score plot (trial 2, Figure 16A). The PCA loading plot of the included variables showed that cFPI mice were more active, spent more time in the central circle, made more visits to the center of the arena and made more rears compared to CCI mice in the second trial (Figure 16B).

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Figure 16. The principal component analysis (PCA) from the second Multivariate concentric square field (MCSF) test 7 days (CCI) or 9 days (cFPI) post-injury. (A) PCA score plot showing the locations of the CCI and cFPI groups, indicating differ-ent behavioral patterns in the two groups. (B) PCA loading plot illustrating the MCSF variables included in the analysis. Variables that are located further away from the origin are most important for the model. (CCI, controlled cortical impact; cFPI, central fluid percussion injury; CTRCI, central circle; Dur, duration, D/V, duration per visit, Frq, frequency; Lat, Latency; SLOPE ENTR, Slope entrance; TOT ACTIVITY, total activity in arena; TOTCORR, total corridor entries.)

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7.3 Improvement of behavioral outcome after anti-inflammatory treatment (Paper IV) Paper IV was focused on anti-inflammatory treatment in animals subjected to cFPI using tests for functional outcome evaluation. First we analyzed that the anti-interlukin-1β antibody reached target brain regions in adequate the-rapeutic concentrations (30µg/g brain tissue) at 24 and 72 hours post-injury. In the analysis of trends (described in 7.2) the same variables was used as shown in Table 2, but stereotyped behavior was added including: distance moved in center (cm), total rear, duration in center and latency to start wall rearing. These behaviors form part of stereotyped behavior since the parame-ters were quantitatively influenced by a repetitive invariant performance that appeared not to be directed towards a purposeful action.160,161 The cFPI mod-el in mice resulted in deviant behavior in the MCSF test which included running around in the center of the arena and making many rears. The result showed that there were no differences between naive mice, cFPI+CsA mice and cFPI+IL-1β mice regarding locomotion, risk taking, exploratory beha-vior, shelter seeking and risk assessment in the analysis of trends (Figure 17 A-E). Significant differences were seen on stereotyped behavior, performed by cFPI mice (Figure 17 F). The anti-IL-1β antibody treatment had a positive effect on this abnormal behavior and cFPI+IL-1β mice performed this beha-vior to a smaller extent than cFPI+CsA mice. Sham-injured animals showed some behavioral effects compared to naive animals, and stereotyped beha-vior was slightly increased (Figure 17G). When analyzing the antibody con-centrations in brain tissue, it was shown that sham-injured mice had high concentrations of the anti-IL-1β antibody in the brain. Sham-IL-1β mice showed behavior profiles like naive mice in the MCSF test, compared to sham+CsA mice.

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Figure 17. Analysis of (A) locomotion, (B) risk taking, (C) exploratory behavior, (D) shelter seeking, (E) risk assessment and (F and G) stereotyped behavior in both trials in the multivariate concentric square field (MCSF) test. *P<0.05, **P<0.01, ***P<0.001. cFPI, central fluid percussion injury.; CsA, cyclosporine A; IL-1β, interleukin-1β. Mean ± SEM.

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7.3.1 Principal component analysis of behavioral data (Paper IV) A principal component analysis (PCA), including all variables in the second MCSF test, resulted in a model with three components, were the two first components shown describe 39.9% of the total variation in the data set. We detected one strong outlier (naive), situated far outside the ellipse with ab-normally high leverage on the model, and therefore the animal was removed from this analysis. The score plot shows that the cFPI+IL-1β mice were lo-cated closer to the naive mice, in the upper part of the score plot, and cFPI+CsA mice were located in the lower part, with some overlap (Figure 18A). The corresponding loading plot showed the relation between the va-riables (Figure 18B). The effect of the anti-IL-1β antibody was strengthened by conventional statistics showing a significant effect on e.g. rearing in both trials.

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Figure 18. Principal component analysis (PCA) from the second trial, day 9 post injury, in the multivariate concentric square field (MCSF) test. (A) PCA score plot showing the locations of the naive, cFPI +CsA and cFPI+IL-1β mice, indicating differences in behavioral patterns between the groups. (B) PCA loading plot illu-strating the MCSF variables that are included in the analysis. Variables located fur-ther away from the origin are most important for the model. (naive n=8;cFPI+CsA,central fluid percussion injury and the control antibody cyclosporine A n=16; cFPI+IL-1β; central fluid percussion injury and antibody against interleukin 1β n=16; CORRIDORS, total corridor entries; CTR CIRCLE, central circle; DUR, duration; D/V, duration per visit; FRQ, frequency; LAT, latency; SL-ENTR, SLOPE ENTRANCE; TOT ACTIVITY, total activity in arena.

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7.3.2 Treatment effects on learning and memory (Paper IV) In the MWM there were no effect of cFPI in the learning trials (14-17 days post-injury) but the memory was affected in brain injured mice in the probe trial 21 days post-injury. We could detect a significant treatment effect of anti-IL-1β in the probe trial indicating improved memory function.

7.3.3 Treatment effects on the inflammatory response (Paper IV) In our previous work the treatment with the same anti-IL-1β antibody atte-nuated the inflammatory response, e.g. activated microglia/macrophages (MAC-2 positive cells), at 3-7 days after CCI.162 Therefore, MAC-2 positive cells in the white matter were counted 21 days post-injury after cFPI in this study. There were more cells in brain-injured mice compared to sham-injured mice but we could not find an effect of the anti-IL-1β antibody on the MAC-2 positive cell number at this late time point. In order to measure eventual brain atrophy, the ventricle size was analyzed but was not affected in brain-injured mice compared to sham-injury.

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CHAPTER 8

Discussion Studies with models of focal and diffuse traumatic brain injury were in-cluded in this thesis. Differences between the models will be discussed re-garding cellular reactions and behavioral changes.

8.1 Cellular reactions after focal and diffuse TBI In Paper I and III astrocyte reactions were studied after focal (WDI in the rat and CCI in the mouse) and diffuse (cFPI in the mouse) TBI respectively. A striking difference between vimentin and GFAP was the absence of vimentin staining in normal adult astrocytes, making subtle changes in staining pat-terns after trauma more readily detectable. We concluded that vimentin im-munoreactivity was more sensitive than the GFAP staining method to dem-onstrate the distribution and time course of astrocyte reactions after contu-sion injury, especially in the white matter distant from the cortical lesion.

In Paper I after focal TBI we showed that the overall pattern of vimentin immunoreactivity was very similar in the rat and mouse TBI model. Vimen-tin-positive cells were observed on the ipsilateral side of the brain. The vi-mentin-staining in Paper III, after diffuse TBI, was more widespread includ-ing both brain hemispheres, illustrating differences between focal and dif-fuse TBI. Further, after diffuse injury there were some differences in astrog-lial response between astrocytes expressing vimentin or GFAP. Groups of GFAP positive cells were also observed in the thalamus but only vimentin immunoreactivity was increased in the internal capsule. These results indi-cate a different astroglial response between astrocytes expressing vimentin and GFAP.163,164

In Paper III we investigated axonal damage after cFPI. The distribution of axonal injury was less widespread than the immunoreactivity of glial cells positive for vimentin and GFAP. The widespread axonal injury observed in this model reflects many of the diffuse axonal injury features observed in TBI patients. We also studied the inflammatory response, including micro-glia/macrophage activation and neutrophils and T-cell infiltration, which was restricted to the cortex and the subcortical white matter and included areas of

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axonal injury. The inflammatory responses were seen in the same areas as axonal injury and glial cell reactivity.

Immunologic response following focal and diffuse trauma differs. So far the inflammatory response has been most extensively studied after focal TBI. In a study by Cuka et al. diffuse injury in the rat resulted in reactive astrocytes and activated microglia early after trauma, but the inflammation including brain infiltration of macrophages (ED1 positive cells) was re-stricted to meninges and the perivasculatory space.165 However, another study showed prolonged microglia activation in the same regions as axonal injury100 which is consistent with the results obtained in Paper III in this thesis. Our previous studies in a mouse CCI model showed a robust, early up-regulation of a number of inflammatory genes, including chemokines and their receptors, and a marked inflammatory response consisting of brain edema, infiltration of neutrophils and T-cells and activation of micro-glia.87,107,166,167 Similar inflammatory cell reactions were seen in focal weight drop injury in rats albeit with an earlier peak for T-cell invasion.87 Compared to the cFPI mouse model, the distribution of T-cell and neutrophil infiltra-tion differed in the CCI model, where immune cell infiltration were found mainly in the shear stress zone in the perimeter of the lesion 24 hours post injury.87,162 The conclusion from these studies is that the inflammatory re-sponse after diffuse axonal injury seems to be more widespread and includes areas of white matter distant from the injury site.

Differences between the rats and mice inflammatory gene expression after focal brain ischemia was recently reported.168 A rapid and strong induction of tumor necrosis factor (TNF)-α, followed by a delayed peak of IL-β and inducible nitric oxide synthase (iNOS) in rats was described, while inflam-matory gene induction in mice was overall weaker and more protracted. These differences in inflammatory responses need to be considered when comparing results from studies in different animal species.

8.2 Behavioral changes after focal and diffuse TBI In Paper II the behavioral outcome after focal TBI (CCI) was analyzed in the newly established MCSF test.139 During the first trial in the MCSF test, the animals are facing an environment that is new to them. Repeated exposure to the MCSF caused a general decrease in activity during the second trial, in the naive and sham group, while an increase in these behaviors were seen in injured mice. When the animals habituate to an open field the anxiety levels decreases and they are more willing to explore the center of the arena, an explorative behavior associated with less anxiety and perceived risk.127 This agrees with our results from sham and naive animals in the MCSF test. Other studies have observed a hyperactive behavior after trauma54 and increased motor activity and decreased habituation after ischemia.169,170 Furthermore,

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agitation and restlessness are frequent neurobehavioral consequences after human TBI.171 The CCI mice were characterized by taking more risks and had a higher level of exploration activity, and less shelter seeking. Further, cognitive impairment was suggested because lack of transfer effect between the first and second trial. While sham-injured mice changed their behavior when re-exposed to the MCSF arena, the CCI mice behaved as if the situa-tion in trial 2 still provoked a sense of novelty. The damage to the hippo-campus observed after CCI may explain the impairment of cognitive func-tion.

Diffuse axonal injury is very frequently seen in TBI patients. In Paper III and IV we used the newly established cFPI model in mice63 and behavioral changes were studied. cFPI mice were characterized by a deviant behavior that resembled similarities with stereotyped behavior, including running around in circles in the center of the MCSF arena and making many rears. However, no differences were seen regarding risk taking and exploration as seen in CCI mice.

In Paper III we compared the behavior from the CCI mice with cFPI mice, which showed that the focal and diffuse injury models resulted in unique behavioral profiles that separated them in the score plot in the prin-cipal component analysis. The focal and diffuse injury models used in these studies likely affects the brain in different ways, which results in unique behavioral patterns in these mice.

In Paper II and Paper III it differed slightly when the second trial in the MCSF test was performed, at seven days in Paper II and at nine days in Pa-per III. We reasoned that two days difference in the second trial would have minimal impact on the performance in the MCSF test at these late post-injury time points. Further, the set-up of the MCSF test was equal between studies and the experiments were performed by the same handler, supporting comparability between the studies. However, there might be other things that affect the results like living conditions at the breeder, transport to the lab and background sounds in the test room, to mention a few.

The mortality rate in the cFPI model was rather high, around 20%, due to brain-stem complications causing apnea. The injury level in the cFPI model used in this thesis is of moderate degree which might explain that the differ-ences in behavior seen between brain-injured and the sham-injured animals were not of full magnitude. However, an increase in atmospheric pressure to enhance the severity level of the cFPI would require ventilated animals. A limitation with the current studies is the facts that three different mechanical impact methods with differing intracranial pressure characteristics172 were used for focal (CCI and WDI) and diffuse (cFPI) injury. It would be interest-ing to make a comparison between focal unilateral and diffuse midline fluid percussion injury using the same level of injury pressure.

There were some effects on the behavior of sham-injured mice compared to naive mice, both in the CCI model and the cFPI model, which has been

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shown previously after sham-injury.173,174 We also observed some leakage of IgG in sham animals, indicating disturbed BBB (Paper III). Sham-injury with craniotomy in rats has previously shown to induce a cell loss in the hilus of the hippocampus.175 Therefore, sham-injury procedure using cra-niotomy might result in a unique brain injury in itself, including both pro-inflammatory response and behavioral damage.174 The influence of anesthe-sia should also be considered when comparing with naive animals. However, there were still some behavioral effects in trial 2 of the MCSF test, about one week after surgery, which cannot be explained as an effect of anesthesia.

Generally, behavioral testing results in great variability which requires large animal groups to detect some effect of treatment. Pre-testing to exclude animals that show abnormal stress response to the task or other deviations is used in some studies to reduce variability. Handling of animals prior to test-ing can minimize stress associated with handling in the behavioral test176 and was performed in the behavioral studies included in this thesis by the same investigator. The social status of the animals, the order in which the animals are tested as well as subtle differences in handling and motivation of the animals can also influence test results. Only to use larger animal groups are not a solution to this problem. Careful planning of the experiments, using the previously observed variability to calculate proper animal group size, might result in less variability and more significant results.

8.3 Treatment strategies in traumatic brain injury In Paper IV the anti-inflammatory antibody against interleukin-1β (IgG2 a/k) was given thirty minutes after cFPI, which resulted in improved functional outcome in the MCSF test. The cFPI did not result in learning effects in the MWM test, but instead an effect on memory was seen in the probe trial. The interleukin-1β antibody treatment improved memory function in diffuse injured mice in the MWM. Further, mice subjected to sham-injury that re-ceived the anti-interleukin-1β antibody showed a behavioral profile more like naive animals in the MCSF test, which suggest a positive effect of the treatment also in the sham-injured mice. Sham-injured mice also had high concentrations of the antibody in brain tissue detected with immunoglobulin G staining, which probably is a result of BBB leakage after sham-injury seen in Paper III.

Treatment with the anti-IL-1β antibody did not affect the number of mi-croglia/macrophages 21 days post injury but it is not ruled out that there was an effect of early microglia activation that needs further investigation. Re-duction of microglia was previously seen after anti-IL-1β treatment in CCI mice, but at earlier time-points post-injury.162 Since the anti-IL-1β antibody has shown improved outcome in two different experimental models of TBI this may be a promising drug for treatment of TBI pathology in humans.

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Although the development of animal models has improved our under-standing of the mechanisms underlying post-traumatic disability, no specific neuroprotective therapy is currently available.30,44,177 One reason for this may be that the outcomes measures in clinical trials and the behavioral tests used in preclinical research are not well matched.148 Functional evaluation of nov-el treatment strategies is therefore of great importance in order to successful-ly move preclinical therapeutic treatments into clinical practice. Simplified automated tests are still used to a high extent in both pharmacology and neu-roscience but many behaviorally oriented researchers use and advocate an ethoexperimental approach.124,129,178,179 Even though there is substantial simi-larity in the physiology of rodent and human brains it is clear that there are notable differences in terms of brain structure and function, and the response to trauma of comparable severity may differ.180 Differences between beha-vioral and morphological responses to TBI among different rat strains181,182 and mouse strains183 makes the situation even more complex.

Our results from the studies included in this thesis argue that behavioral evaluation is crucial in the evaluation of traumatic brain injury. The cFPI model in mice can be an important complement to other TBI models for the study of pathological mechanisms including neuroinflammation and treat-ment strategies for diffuse traumatic axonal injury. Understanding the innate inflammatory response may provide novel therapeutic opportunities in TBI.

8.4 Future studies In Paper IV the ventricle size after cFPI was measured in order to investigate eventual brain atrophy which is a common consequence of TBI. However, no difference in ventricle size between sham-injured or diffusely injured mice was shown. We will in a following investigation examine this further by measuring the hemispheric tissue loss. It would also be interesting to study β-APP staining after anti-IL-1β treatment, to see if there is less axonal damage after anti-inflammatory treatment. Double immunostaining for mi-croglia and β-APP would also be interesting to analyze. Further analysis including evaluation at later time-points is needed to study the long-term consequences initiated by the cFPI. Further refinement of the MCSF test in order to measure more subtle behavioral changes is also needed.

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Conclusion

This study showed: • In Paper I: Vimentin immunohistochemistry was more sensitive

than the GFAP method to demonstrate the distribution and time course of reactive astrocytes after mild and severe cerebral contu-sion in the rat and mouse, especially in the white matter.

• In Paper II: A changed behavioral profile characterized by altera-tions in exploration, risk taking and risk assessment behavior in mice subjected to CCI as compared to sham-injured mice was ob-served. The MCSF test was shown to be a useful, complementary tool for evaluation of functional outcome following experimental TBI.

• In Paper III: The cFPI model in mice resulted in increased blood-brain barrier permeability and diffuse axonal injury. Widespread astroglial cell reactivity and a robust inflammatory response in-cluding activation of microglia/macrophages, infiltration of neu-trophils and T-cells were also seen. Additionally, there were com-plex behavioral changes observed in brain-injured animals that appeared unique to this TBI model.

• In Paper IV: Mice subjected to cFPI had a changed behavioral profile compared to normal mice, including a stereotyped beha-vior. Cognitive function, tested in the MWM, showed a reduced memory function but no impairment in learning capacity. Treat-ment with an antibody against the pro-inflammatory cytokine IL-1β had a positive effect on behavior, especially on the deviant ste-reotyped behavior shown by animals subjected to cFPI and on memory dysfunction in the MWM probe trial. These findings sug-gest that the IL-1β antibody could be a promising new therapeutic strategy for patients with diffuse head injury.

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Summary in Swedish

Varje år drabbas omkring 20000 personer i Sverige av medelsvår till svår traumatisk hjärnskada, vilket är den vanligaste orsaken till dödsfall eller invaliditet i den yngre befolkningen (<40 år). I Uppsala är det omkring 100 fall varje år med allvarlig skallskada som är i behov av högspecialiserad neurointensivvård. Globalt sett är traumatisk hjärnskada ett stort problem och de högsta incidenserna finns i utvecklingsländer. Eftersom många över-levande får svåra handikapp och aldrig kan återvända till arbete får det även stora ekonomiska konsekvenser för de drabbade och för samhället i stort. Minnesstörningar och personlighetsförändringar är vanliga följder. Trauma-tisk hjärnskada orsakas främst av trafikolyckor, fallolyckor och sportrelate-rade olyckor. Under de senaste åren har ökad säkerhet i trafiken med bättre bilar och användandet av hjälm, samt förbättrad neurointensivvård minskat antalet döda och de som hamnar i vegetativt tillstånd. Traumatisk hjärnskada är trots detta ett stort problem. Under åren har flera prekliniska studier med olika läkemedel visat på lovande resultat när det gäller att skydda hjärnan efter skada, men tyvärr har man i uppföljande kliniska studier inte lyckats ta fram ett hjärnskyddande läkemedel som fungerar på patienter. Det är därför viktigt att studera de mekanismer som leder till skada och använda kliniskt relevanta modeller av skallskador.

I den här avhandlingen har tre djurmodeller använts för att studera cellu-lära mekanismer och beteendestörningar efter traumatisk hjärnskada; två modeller för fokal skada och en ny musmodell för diffus axonal skada (DAI). DAI är en fruktad komponent i hjärnskadan som tros ligga bakom många av de svåra kognitiva och psykiatriska följdverkningarna hos skall-skadade patienter. Modellen kan användas för att göra kliniskt relevanta studier av molekylära mekanismer för DAI i syfte att identifiera nya an-greppspunkter för behandling. Astrocyternas roll efter skallskada har under-sökts eftersom det har visat sig att de är viktiga för bildandet av så kallade gliaärr i hjärnan. Avhandlingen presenterar vidare en ny metod för beteende-utvärdering hos möss med traumatisk hjärnskada, ”multivariate concentric sqare field test” (MCSF) som väntas få genomslag hos forskare som studerar effekter av intervention efter experimentell skallskada.

Den här avhandlingen visar slutligen att inflammation är en viktig meka-nism för DAI efter experimentell skallskada. Antikroppsbehandling mot Inteleukin-1β är en lovande hjärnskyddande behandling och kan komma att

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bli ett nytt verktyg för att ytterligare förbättra slutresultatet efter traumatisk hjärnskada inom neurointensivvården.

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Acknowledgement

This study was carried out in the Department of Neuroscience, Section of Neurosurgery, Uppsala University, Sweden.

Jag vill särskilt tacka följande personer som på olika sätt har bidragit till den här avhandlingen: Lars Hillered, min huvudhandledare för stor vetenskaplig kompetens, ut-märkt handledning och för att du är en så varm och positiv person. Min biträdande handledare Niklas Marklund för all hjälp att komma igång med cFPI:n, inspiration och för att du tror på ”the best TBI-lab in the world”. Bengt Meyerson, min “beteendementor”. Du har satt dig in i resultaten och gjort ditt bästa för att hjälpa mig med din ovärderliga expertis inom beteen-devetenskapen, vilket jag verkligen uppskattar. Per Enblad, enhetschef på neurokirurgen, för all support under min dokto-randtid. Neurotrauma-gruppen: Fredrik Clausen som introducerade mig till labbet, för bra samarbete och hjälp på alla sätt. Johanna Flygt, min trevliga rumskamrat och medförfatta-re, för allt bra samarbete och ditt jobb med färgningar och figurer. Anna Erlandsson och Camilla Lööv, särskilt för vår trevliga resa till Skottland! Anders Hånell, särskilt tack för din ”att disputera guide” – väldigt använd-bar. Ulrika Wallenquist och Charlotte Israelsson för att ni kommer och livar upp ibland. Studenter som har arbetat på labbet, särskilt Gudrun And-rea Fridgeirsdottir och Olivia Kiwanuka. Karin Skoglund, Lena Ny-holm och Karlis Purins som bidragit med inblick i den kliniska forskning-en. Medförfattare: Ted Ebendal, Anis Mir, Hermann Gram, Gui Lin Li, Mohammad Farooque och Yngve Olsson. Maude Karlsdotter, Petra Millbert, Mona Persson och Gun Schönnings för all utomordentlig praktisk hjälp. Och tack Maude för trevliga ridturer! Sofi Forsberg för alla våra “spring-luncher” och trevligt prat om allt möj-ligt.

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Inger Pihl-Lundin för att du lärde mig hur mikroskåpet fungerar och för att du kan så mycket om immunofärgning. Olav Mäepea för hjälp med statisti-ca. Alla andra trevliga personer som äter lunch i foa3s lunchrum för en gemytlig atmosfär. Erika Roman för att jag har fått fråga dig om statistik och för att du ordnat intressanta beteendemöten för oss som använder MCSF:en. Marita Berg för hjälp när det strulade med ethovisionsprogrammet. Snickeriet på BMC för hjälp med delar till ”beteendelådan”. Mina kära vänner, som jag hoppas få många fler tjej-helger, middagar, skiddagar och tenniskvällar med! Ing-Britt and Bengt Lewén, världens bästa svärföräldrar som har ställt upp så mycket under de här åren och tagit väl hand om barnen. Det här hade varit omöjligt utan er! Min svägerska Hanna Lewén med familj för många trevliga middagar och intresse för avhandlingen. Mina älskade föräldrar Eva och Rasmus Ekmark som alltid tror på mig och för att jag vet att ni alltid finns där för mig. Ni har hjälpt till på alla sätt, jag skulle gärna ha er lite närmare Uppsala! Mina systrar Linda och Johanna för att ni är världens bästa och känner mig så bra! Din fina familj Linda, och min svåger Daniel Strandell för ovärderlig hjälp, särskilt när vi var grannar. Anders, för ditt stöd och din uppmuntran och för alla diskussioner om svår-tolkade resultat. Tack för superservice inklusive all god mat under avhand-lingsskrivandet! Du är den bästa man som finns! Mina älsklingar Carl, Hugo och August som får mig att känna mig så rik och lycklig.

Sara Ekmark-Lewén Uppsala Universitet

April 2013

I hope that posterity will judge me kindly, not only as to the things which I have explained, but also to those which I have intentionally omitted so as to

leave to others the pleasure of discovery. -Rene Descartes

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References

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Cellular Reactions and Behavioral Changes in Focal and ...617889/FULLTEXT01.pdf · Injury to the brain has devastating consequences for the affected individuals. What we define as