Extreme Resistance to Hypercapnia-Induced Pulmonary Edema of the African Naked … · 2018-12-30 · Extreme Resistance to Hypercapnia-Induced Pulmonary Edema of the African Naked

Extreme Resistance to Hypercapnia-Induced Pulmonary Edema of the African

Naked Mole-Rat

BY

Gregory Richard Charles Blass B.S., University of Oklahoma, 2005

THESIS

Submitted as partial fulfillment of the requirements of the degree of

Doctor of Philosophy in Biological Sciences in the Graduate College of The University of Illinois at Chicago, 2014

Chicago, Illinois Defense Committee: Robert P. Malchow, Chair Thomas J. Park, Advisor A. Don Murphy John P. Leonard Richard D. Minshall, Anesthesiology and Pharmacology

Acknowledgments

I would like to thank my wife Carrie for the love, encouragement, and awesome food that kept me going. My parents, Richard and Nancy Blass, I thank for offering guidance when I have needed it and help when requested. I thank my sister, Kelly Falgout, for all the assistance she has given in my academic pursuits and for being just a great sister. I thank Saleel Raut for broadening my cultural experiences and challenging me in many games of racquetball. I thank Daniel Applegate for dragging me to the gym. I would like to thank my advisor Thom Park for all the support he has provided during the course of my studies at UIC. I thank Don Murphy for his reminders that science is a part of you but does not need to be all there is. I also appreciate his mentorship in the game of darts for if not for him I would still not know how to play the game. I especially thank Rich Minshall for his assistance in learning surgical techniques involved in my research. I thank Paul Malchow for his insights of how results apply to the bigger picture. I thank Charles Laurito for testing my knowledge and providing thoughtful insights. I thank David Visintine for the training of surgical skills that have proved invaluable. Lastly I thank John Leonard for his help whenever it has been needed.

GRCB

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TABLE OF CONTENTS CHAPTER PAGE 1. INTRODUCTION ...............................................................................................1 1.1 Carbon Dioxide ......................................................................................1 1.1.1 Carbon dioxide transport ..............................................................1 1.1.2 Carbonic anhydrase .....................................................................2 1.1.3 Respiratory control .......................................................................3 1.1.4 Carbon dioxide as a possible intracellular signaling molecule .....4 1.1.5 Reactive species ..........................................................................4 1.2 Alveoli and Neighboring Cells ................................................................5 1.3 Pulmonary Edema .................................................................................6 1.4 Alveolar Epithelial Barrier ......................................................................7 1.5 Intracellular Pathways ...........................................................................8 1.6 Hypercapnia-Altered Gene Expression .................................................9 1.7 Lung Innervation ....................................................................................10 1.7.1 Peptidergic c-fibers ......................................................................11 1.7.2 Acid sensing by c-fibers ...............................................................11 1.7.3 Acid-sensing ion channels ...........................................................12 1.7.4 Transient receptor potential channels ..........................................12 1.7.5 Tachykinins ..................................................................................13 1.7.6 Neurokinin signaling .....................................................................14 1.8 Alveolar Macrophages ...........................................................................15 1.9 General Anesthetics ..............................................................................16 1.9.1 Isoflurane .....................................................................................17 1.10 Naked Mole-Rats .................................................................................18 1.11 Current Study ......................................................................................19 2. EXTREME HYPERCAPNIA RESISTANCE OF THE AFRICAN NAKED MOLE-RAT ........................................................................................................23

2.1 Abstract .................................................................................................23 2.2 Introduction ............................................................................................23 2.3 Materials and Methods ..........................................................................28 2.3.1 Animals ........................................................................................28 2.3.2 Environmental chamber ...............................................................28 2.3.3 Respiration rate ............................................................................28 2.3.4 Respiration rate recording analysis ..............................................29 2.3.5 Pulmonary edema measurement .................................................29 2.3.6 Lung lavage .................................................................................30 2.3.7 Enzyme-linked immunosorbent assay .........................................30 2.3.8 Statistical analysis ........................................................................31 2.4 Results...................................................................................................31 2.4.1 Respiration rate ............................................................................31 2.4.2 Lung edema .................................................................................33 2.4.3 Lung lavage .................................................................................35

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TABLE OF CONTENTS (continued) 2.5 Discussion .............................................................................................37 3. HYPERCAPNIA-INDUCED PULMONARY EDEMA EXACERBATION BY TAC1 KNOCKOUT MICE AND INHIBITION BY ISOFLURANE ........................41 3.1 Abstract .................................................................................................41 3.2 Introduction ............................................................................................41 3.3 Materials and Methods ..........................................................................44 3.3.1 Animals ........................................................................................44 3.3.2 Environmental chamber ...............................................................45 3.3.3 Respiration rate ............................................................................45 3.3.4 Respiration rate recording analysis ..............................................46 3.3.5 Pulmonary edema measurement .................................................46 3.3.6 Lung lavage .................................................................................47 3.3.7 Enzyme-linked immunosorbent assay .........................................47 3.3.8 Statistical analysis ........................................................................47 3.4 Results...................................................................................................48 3.4.1 Respiration rate ............................................................................48 3.4.2 Lung edema .................................................................................50 3.4.3 Lung lavage of wild-type mice ......................................................53 3.4.4 Lung lavage of tac1KO and trpV1KO mice ......................................55 3.4.5 Isoflurane attenuation of hypercapnia-induced edema ................57 3.5 Discussion .............................................................................................59 4. DISCUSSION .....................................................................................................63 4.1 Hypercapnia Induced Cell Signaling ......................................................63 4.2 Future Directions ...................................................................................68 CITED LITERATURE ..............................................................................................70 APPENDIX ..............................................................................................................93

VITA ........................................................................................................................95

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LIST OF FIGURES

FIGURE PAGE

1. Hypothesized mechanism of hypercapnia-induced pulmonary edema .........21 2. Respiration rate of naked mole-rats and mice at increasing CO2

concentrations ..............................................................................................32

3. Lung weight ratio of naked mole-rats and mice after hypercapnia treatments ....................................................................................................34 4. SP+HK-1 concentrations in lung lavage fluid of naked mole-rats and mice after CO2 treatments .....................................................................................36 5. Respiration rates of wild-type, tac1KO, TrpV1KO, and TrpV1KO/ASIC3KO mice during CO2 treatments .........................................................................49 6. Lung weight ratios of mice after hypercapnia treatments .............................51

7. Lung weight ratios of mice after hypercapnia and hypercapnia with isoflurane ...............................................................................................52

8. SP+HK-1 concentration in lung lavage fluid of wild-type mice after CO2 treatments ....................................................................................................54 9. SP+HK-1 concentration in lung lavage fluid of tac1KO and TrpV1KO mice after CO2 treatments ............................................................................56 10. SP+HK-1 concentration in lung lavage fluid with CO2 and CO2 with isoflurane ......................................................................................................58

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LIST OF ABBREVIATIONS

AEC Alveolar Epithelial Cell

AECI Alveolar Epithelial Cell type I

AECII Alveolar Epithelial Cell type II

AMP Adenosine Monophosphate-activated Protein Kinase

ASIC Acid-sensing Ion Channel

ATP Adenosine Triphosphate

BMAPK-1 Big Mitogen-activated Protein Kinase 1

BMR Blind Mole-rat

CA Carbonic Anhydrase

CAII Carbonic Anhydrase II

Ca2+ Calcium Ion

CO2 Carbon Dioxide

CNS Central Nervous System

DNA Deoxyribonucleic Acid

DRG Dorsal Root Ganglia

EKA Endokinin-A

EKB Endokinin-B

EKC Endokinin-C

EKD Endokinin-D

ERK Extracellular Signal-Regulated Kinases

H+ Hydrogen Ion

HA Hyaluronan

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LIST OF ABBREVIATIONS (continued)

HCO3- Bicarbonate

Hg Mercury

HK-1 Hemokinin-1

HO-1 Heme Oxygenase-1

H2O Water

IL-6 Interleukin 6

ISO Isoflurane

JNK Jun N-terminal Kinase

K+ Potassium Ion

KO Knockout

LPS Lipopolysaccharide

MAPK Mitogen-activated Protein Kinase

N2 Molecular Nitrogen

Na+ Sodium Ion

Na/K-ATPase Sodium/Potassium Active Transporter

NMR Naked mole-rat

Nav Voltage-gated Sodium Channel

NEB Neuroendocrine Body

NF-κB Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells

NK1r Neurokinin 1 Receptor

O2 Molecular Oxygen

PBS Phosphate-buffered Saline

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LIST OF ABBREVIATIONS (continued)

PCO2 Partial Pressure of Carbon Dioxide

pH Measure of Acidity or Basicity in an Aqueous Solution

PKC Protein Kinase C

PNS Peripheral Nervous System

RAR Rapidly Adapting Mechanoreceptor

PKA Protein Kinase A

RNS Reactive Nitrogen Species

ROS Reactive Oxygen Species

SAR Slowly Adapting Mechanoreceptor

SP Substance P

tac1 Tachykinin 1 Gene



TNF Tumor Necrosis Factor

Trp Transient Receptor Potential Channel

TrpV1 Transient Receptor Potential Vanilloid 1 Channel

WT Wild-Type

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SUMMARY

Extreme hypercapnia induces pulmonary edema in many terrestrial mammals.

Pulmonary c-fibers that innervate lung tissue are activated by hypercapnia-acidosis.

These fibers release the pro-inflammatory neuropeptide substance P (SP) and thus can

induce edema in the lungs. The aim of my study was to discover more about the

mechanism of hypercapnia-induced pulmonary edema. My working hypothesis was

that pulmonary c-fibers are necessary for hypercapnia-induced pulmonary edema.

Tissue CO2 level is an important stimulus for respiration rate control. As part of

evaluating the effects of hypercapnia-induced edema, respiration rate responses to

moderate hypercapnia were measured in all animal groups.

We tested SP deficient mice, tac1KO, to determine the extent SP contributes to

respiration rate and hypercapnia-induced edema. We found that these mice had a

significantly increased respiration rate in response to 10% CO2 compared to wild-type

(WT) mice. This demonstrated that substance P has a modulatory role in respiration

rate. These mice were then tested for hypercapnia-induced edema. Counter to our

hypothesis, SP released from c-fibers triggers edema, tac1KO mice had significant

increased edema at 15, 20, and 30% CO2. Hemokinin-1 (HK-1) is found in lung tissue

and has similar affinities as SP to the SP receptor NK1r. To date, HK-1 has not been

found expressed in neuronal cells but is abundant in immune cells such as alveolar

macrophages. We measured the combined SP+HK-1 concentration in lung lavage at

0% and 30% CO2. Since, tac1KO mice lack SP, HK-1 was the only peptide detected in

tac1KO mice. We found no difference between SP+HK-1 measurements between

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SUMMARY (continued)

tac1KO and WT mice. Therefore, this indicates that HK-1 is the predominant NK1r

ligand released in the lungs at 0 and 30% CO2.

To determine the role c-fiber acid sensitivity has in hypercapnia-induced edema,

we tested mice that lacked the TrpV1 (TrpV1KO) or TrpV1 and ASIC3

(TrpV1KO/ASIC3KO) ion channels. These channels account for a large portion of the

acid sensitivity of pulmonary c-fibers. These mice had no significant difference in

moderate hypercapnia induced respiration rate increase. These mice also had no

difference in hypercapnia-induced pulmonary edema. These results indicated that

pulmonary c-fiber acid sensitivity and subsequent release of SP does not have an effect

on hypercapnia-induced pulmonary edema. To confirm this, SP+HK-1 content of

TrpV1KO mice was measured at 0 and 30% CO2. No difference was observed between

these and WT mice.

The volatile anesthetic isoflurane (ISO) is known to have anti-inflammatory

effects on induced pulmonary inflammation. We combined ISO with 30% hypercapnia

to determine if ISO would attenuate hypercapnia-induced pulmonary edema. The ISO

significantly reduced hypercapnia-induced edema and significantly attenuated the

release of SP+HK-1 into the lung airways. Thus, ISO has an anti-inflammatory effect on

hypercapnia-induced pulmonary edema.

My last aim was to determine if African naked mole-rats, mammals that naturally

live in hypoxia and hypercapnia in their burrows, have attenuated response to

hypercapnia. We found that naked mole-rats have attenuated response to moderate

hypercapnia-induced respiration rate increase. We also found that naked mole-rats had

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SUMMARY (continued)

no hypercapnia induced edema at levels as high as 50% CO2. We then tested SP+HK-

1 level of lavage fluid at 0 and 30% CO2. It was surprising that naked mole-rats had no

detectable amount of SP+HK-1 at either of the CO2 concentrations. The absence of

edema and lack of SP+HK-1 in naked mole-rat lavage fluid demonstrates the existence

of an adaptation of naked mole-rats.

These findings demonstrate that extreme hypercapnia induces the release of

SP+HK-1. The protective effect of ISO may be part of the same pathway that protects

naked mole-rats against hypercapnia-induced edema. Overall, pulmonary c-fibers may

not have a function in hypercapnia-induced edema.

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1. INTRODUCTION

To understand the role of carbon dioxide (CO2) in normal and pathophysiological

conditions, it is important to review how CO2 interacts with lung tissues and the

molecular pathways involved. Elevated blood CO2, hypercapnia, that occurs in multiple

pathological respiratory conditions, such as chronic obstructive lung disease,

exacerbates these conditions resulting in increased lung barrier permeability and edema

[1]. The normal carbon dioxide level is considered to be within the range of 35-45 mm

Hg. Hypercapnia is considered to be anything above 45 mm Hg and mild is up to 50

mm Hg [2, 3]. Moderate hypercapnia range extends from 55 to 70 mm Hg [4] and

above 75 is considered extreme hypercapnia [5, 6]. The sections below discuss

moderate to severe hypercapnia effects as well as the protection isoflurane (ISO) offers

against hypercapnia-induced pulmonary edema. These sections also establish the

naked mole-rat as a model animal for studying hypercapnia-induced pulmonary edema

in the laboratory.

1.1 Carbon Dioxide

1.1.1 Carbon dioxide transport

Carbon dioxide is an important by-product of cellular respiration. While low in

concentration in earth’s atmosphere, 0.04%, it reaches much higher levels within

organisms. Detection of CO2 concentrations is involved in regulating several

physiological processes. It is utilized by some organisms in mating as an attractant, as

well as a repellant for avoiding predators and harmful substances [7].

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2

Classically, CO2 is thought to passively diffuse across cell membranes due to it

being a small non-polar molecule [8]. Overton’s rule dictates that the less impeded a

chemical dissolves in a lipid, the faster it will be transported into a cell. A study used

this rule to conclude that passive diffusion could explain CO2 transport into cells [9].

This study has since been disputed by a more recent study [10]. Likewise, CO2

transport by only passive diffusion has been challenged by a number of studies. If CO2

is permeable to all membranes by simple diffusion, the apical membranes of gastric

gland cells would not have been found to be impermeable to CO2 [11]. Red blood cells

would also not have reduced permeability to CO2 by the anion transport inhibitor 4,4’-

Diisothiocyanatostilbene-2,2’-disulfonate [12]. Even the discovery of evolutionarily

conserved proteins that can serve as CO2 transporters challenges simple diffusion as

the only transport mechanism of CO2 [13-17]. Despite this, the significance of how CO2

is transported across membranes is not fully understood. [9]. Once CO2 is either

produced or transported into cells by whatever means, it is quickly and reversibly

converted to bicarbonate (HCO3-) and hydrogen (protons) by carbonic anhydrase.

1.1.2 Carbonic anhydrase

Carbonic anhydrase is an essential enzyme the greatly speeds up the reaction of

CO2 and H2O to produce HCO3- and protons. The increased reaction rate by carbonic

anhydrase is estimated to be between 0.5 to 1 X 106 times. There are more than 15

isoenzymes of carbonic anhydrase that have been identified. Of these, carbonic

anhydrase II, CAII, is the most efficient and is the most studied [18]. The CAII activity in

the lung epithelium is involved in the elimination of CO2 into the alveolar space [19]. In

addition, CAII-deficient mice will develop respiratory acidosis due to CO2 retention in the

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lungs [20]. Since the enzymatic activity of CAII is the first reactive step in several

intracellular cascade reactions, carbonic anhydrase can be viewed as the first biological

sensor of CO2.

1.1.3 Respiratory control

The lungs are the site of most gas exchange in mammals with the outside

environment. As such, they are exposed to rapid fluctuations in oxygen and carbon

dioxide. In mammals, respiratory control is one of the most important physiological

processes regulated by CO2 detection. Respiration is the most efficient mechanism to

remove excess CO2. A one mm Hg increase of blood PCO2 can induce increased

respiratory frequency in many terrestrial mammals [21]. CO2 sensory cells are located

in both in the brain and the periphery. For the past century rhythmic respiration has

been believed to be regulated primarily by sensing arterial blood PCO2 / pH changes in

the CNS [22]. A complex network exists in the lower brainstem that is critical for

breathing. This network outputs to the phrenic nerve that stimulates the diaphragm in a

rhythmic pattern [23]. The cellular input of CO2 detection in the brainstem is still

plagued by controversy. Primarily this has been attributed to no identification of

molecular CO2 transducers in brain areas regulating breathing if one even exists. Mice

lacking CO2 chemosensitivity in the CNS by genetic modification can still breathe [24].

An alternative interpretation is that the carotid body, a peripheral source of detection,

can function as a backup CO2 sensor to drive respiration. During normal breathing, the

carotid chemoreceptors are critical for maintaining normal CO2 levels [25]. In response

to hypercapnia, the central chemosensitive neurons may become the dominant

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contributor to the respiration rate [26]. Even still, the carotid neurons are the first

responders to rapid transient changes in arterial CO2 levels [27].

1.1.4 Carbon dioxide as a possible intracellular signaling molecule

Physiological gasses are known to have a large impact in cellular function and

intracellular signaling pathways. Nitric oxide alters the activities of guanylate

cyclase and cytochrome c oxidase [28, 29], and molecular oxygen alters hydroxylase

activity leading to the activation of hypoxia inducible factor [30]. Hypercapnia

suppresses pro-inflammatory transcriptional pathways [31] related to the NF-κB

pathway [32]. The link of high CO2 with modulating the inflammatory pathway appears

to be evolutionarily conserved, found in both Drosophila melanogaster [33] and C.

elegans [34]. In mice, hypercapnia has been demonstrated to reduce cytokine release

and phagocytic activity of macrophages that were activated by lipopolysaccharide (LPS)

[35]. Alternatively, hypercapnia has been found to have pro-inflammatory effects on

endothelial cells and neutrophil responses [36]. Overall, the effects of carbon dioxide

on cells may not be direct. Rather, the CO2 conversion into other molecules such as

reactive species may be the signal for modulating cellular processes.

1.1.5 Reactive species

Carbon dioxide and its coupled acidosis can both undergo further reactions that

produce reactive oxygen species (ROS) and reactive nitrogen species (RNS). These

are considered to be second messengers that may act at different levels of cell

signaling cascades [37, 38]. In fact, activating a number of plasma membrane channels

causes their intracellular levels to increase, functioning as cellular signals. At high

concentrations, these have damaging effects on most if not all cellular components,

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such as proteins, carbohydrates, lipids, and DNA , that is kept in check by endogenous

antioxidant defenses [39, 40].

Due to the interconnections between CO2 and its products, the difficulties of

identifying which of these molecules that is directly responsible for inducing the

observed physiological reflexes has been noted [41]: altering the concentration of one

molecule will result in a concentration change in the others. Using buffers to substitute

for the intrinsic CO2/ HCO3- buffering system reduces the intracellular concentration of

HCO3-. This will affect the activation of soluble adenylyl cyclase and numerous

transport systems for HCO3-. Hepes and similar buffer compounds can inhibit gap

junctions and hemichannels [42, 43]. For example, the presence of CO2/HCO3- was

essential to see the involvement of ATP in chemosensory neuronal responses [44].

Therefore, determining which among CO2 and its products is responsible for the effect

requires careful experimental planning.

1.2 Alveoli and Neighboring Cells

The alveoli of the lungs are the site of gas exchange between the lung airspace

and the circulatory system. As such, the alveoli are exposed to wide fluctuations in CO2

concentrations and inhaled noxious stimuli. When damaged, the alveoli will repair itself

through proliferation of resident stem cells and recruit immune cells to fight against

inflections. This requires a complex system of signaling between cells within the lungs

and circulatory system.

The alveolus is comprised of an epithelium and endothelium monolayer. In

between these layers is a thin interstitial space. The short distance between the

6

alveolar airspace and the endothelial capillary lumen facilitates gas exchange (20-22).

The alveolar airspaces are lined by epithelial cells, alveolar type I (AECI) and alveolar

type II (AECII). AECI are flattened, covering 95% of the alveolar surface and are

involved in fluid clearance [45-48]. AECII are cuboidal and accounting for 5% of the

surface area. Surfactant is secreted by AECII cells for the purpose of surface tension

maintenance [49]. Tight junctions exist between the AECs creating a strong barrier that

prevents most molecules and pathogens from freely crossing. Residing on the apical

surface of AECI and AECII cells are alveolar macrophages. These cells are responsible

for initiating the inflammatory reaction of the lung. Under the epithelial layer there are

endothelial cells lining the pulmonary capillaries, a scattering of mast cells, dendritic

cells, and neuronal fibers.

At the bronchioalveolar duct are bronchioalveolar stem cells responsible for

repopulating the bronchioles and alveolar cells after lung injury. Just proximal to these

cells are club cells that are nonciliated secretory cells that protect the airways from

environmental insults [50]. The bronchioles are lined with ciliated cells that move the

mucosa, aiding in the elimination of hazardous particles and debris in the airways.

Outside the alveoli and embedded in the epithelium are neuroendocrine bodies (NEBs).

NEBs are clusters of cells that are highly innervated by nerve fibers and are thought to

function as oxygen detectors [51].

1.3 Pulmonary Edema

Extreme hypercapnia has been found to induce pulmonary edema in many

terrestrial mammals and this is the focus of the present dissertation. Pulmonary edema

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is the condition where the pulmonary epithelium barrier becomes leaky and fluid,

plasma and inflammatory cells seep into the alveolar airspace. This fluid coats the

respiratory membranes of the lungs, thereby hindering gas exchange. Other

complications will then arise including suffocation. Heart failure/circulatory system

impairment, and inflammation of lung tissue are known causes for pulmonary edema

[52]. While heart failure will cause edema by increasing blood pressure, inflammation

and its resulting edema can be triggered by inhalation of noxious stimuli. Examples are

ammonia and CO2. Due to their interaction with pulmonary nerve fibers, the resulting

edema has been classified as neurogenic pulmonary edema.

1.4 Alveolar Epithelial Barrier

The connections between cells within the alveoli are critical for regulating

contents between the alveolar airspace and lung tissue. For hypercapnia to induce fluid

and cells to leak into the alveolar airspace, the epithelial barrier of the alveoli must

become disrupted. The alveolar epithelial barrier comprises AECs expressing a

complex of proteins embedded in the paracellular AEC membranes. Both AECI and

AECII express well-organized paracellular adherens and tight junctions and are

polarized, with apical epithelial sodium channels and basolateral Na/K-ATPases [53-55].

The adherens junctions are comprised of epithelial cadherins. The extracellular

domain of cadherins enables interaction with neighboring cells. The intracellular

domain binds with intracellular adhesion molecules linking the junction to the cell actin

cytoskeleton [56-60]. Adheren junctions both establish strong cell-cell adhesion and are

involved in cell signaling [61].

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The paracellular tight junctions account for the low permeability of the epithelial

barrier [55, 62-64]. The tight junction composition determines its properties [65]. It is

comprised of occludens [66, 67], zonula occludens [64], and members of the claudin

family [68]. The most important component of the tight junction protein complex are

claudins. The alveolus epithelium expresses mainly claudin 3, 4, and 18 [65]. For

inflammatory cells (e.g. leukocytes) to infiltrate the alveolar space, the alveolar epithelial

barrier must become disrupted for inflammatory cells to pass from the interstitial space

into the alveoli lumen. Several stimuli such as oxidative stress can cause changes in

composition, expression, and location of tight junctions [69-71].

1.5 Intracellular Pathways

Hypercapnia causes a direct effect on alveolar epithelial cells as evident by

hypercapnia-induced endocytosis of Na/K-ATPase from AEC membranes. This

mechanism involves the CO2-induced rapid activation of AMP kinase by

Ca2+/calmodulin-dependent protein kinase-beta and PKC-ζ phosphorylation [72]. AMPK

activates energy-generating pathways while reducing energy-consuming pathway.

Therefore, endocytosis of Na/K-ATPase eliminates the functioning of this energy-

consuming protein that accounts for 40% of the AEC resting energy expenditure. A

cell’s need to increase energy-generating pathways may be related to CO2-induced

mitochondria dysfunction [73]. Hypercapnia can produce reactive nitrogen species

(RNS) that are toxic to cells and may mediate production of reactive oxygen species

(ROS) [74, 75]. Thus, hypercapnia may induce AEC damage leading to pulmonary

inflammation.

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Activation of cell receptors and ion channels will lead to the activation of

intracellular signaling. This includes activation of mitogen-activated protein kinases

(MAPKs) that relay exogenous and endogenous signals by phosphorylating intracellular

proteins. These proteins can be upstream such as growth factor receptors, g-proteins,

and tyrosine kinases; or downstream such as nuclear transcription factors [76].

Therefore, activating MAPKs can have short-term and lasting cellular responses. There

are four known MAPK families. They are extracellular-regulated kinases (ERKs), c-jun-

NH2-terminal kinase (JNKs), p38 MAPK and the big MAPK-1 (BMAPK-1) [77].

Hypercapnia has previously been demonstrated to activate some of these MAPKs to

promote cancer cell proliferation [78].

1.6 Hypercapnia-Induced Altered Gene Expression

Hypercapnia has been demonstrated to have anti-inflammatory properties in

models of inflammation. Hypercapnia at minimal and moderate levels ultimately alters

activation of Nuclear factor-kappaB (NF-kB) [79-81]. This is a family of transcription

factors that are intricately involved in the regulation of innate immunity and inflammation

[82]. NF-kB are DNA binding proteins that interact with enhancing domains of target

genes. NF-kB is arranged as a dimer of two members of the NF-κB/Rel/Dorsal family of

proteins [83]. NF-κB are sequestered next to and inhibited in the cell cytoplasm by

members of the IκB family. A wide variety of extracellular stimuli activate NF-κB. Once

activated, IκB is degraded and NF-κB translocates to the nucleus to induce transcription

of genes. RelA, the endotoxin-stimulated NF-κB, translocation in the nucleus is

inhibited by hypercapnia in pulmonary-artery endothelial cells [84]. This was not true of

10

human macrophages [35]. This demonstrates that the decreased activation of NF-κB is

cell specific. Alternatively, RelB, another member of NF-κB family, is activated by

hypercapnia and translocates to the nucleus in mouse fibroblasts and human pulmonary

epithelial cells. Together, the anti-inflammatory action of low to moderate hypercapnia

may in part be explained by its effects on NF-κB activity on gene regulation. Despite

this, the present dissertation used short 10 minute hypercapnia exposures that may rule

out altered activation of NF-κB.

1.7 Lung Innervation

Acidosis has been known to be a potent stimulus of lung airways [85]. Its effects

have been attributed to activation of a subset of neuronal afferent fibers innervating the

lungs. Overall, there are a number of innervating plexuses of the lungs. They either

belong to the sympathetic, parasympathetic pathways, or the dorsal root ganglion

(DRG). “Sympathetic nerve fibers originate from sympathetic cervical and thoracic

ganglia. A minority of sensory nerve fibers originates from dorsal root ganglia” [86, 87].

The parasympathetic, preganglionic neurons are all supplied by the vagus nerve and

the majority of sensory nerve fibers innervating the lungs [88, 89].

The vagal pulmonary pathway has two ganglia, the jugular and the nodose. The

cells originate from different pools of cells early on in development. The jugular cells

originate from neural crest cells, and the nodose cells originate from the epibranchial

placodes [90, 91]. This difference defines early on their phenotype of the vagal fibers.

The vagal airway afferents are grouped together as rapidly adapting mechanoreceptors

(RARs), slowly adapting mechanoreceptors (SARs), and C-fibers. Most SARs are

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located in the intrathoracic airways [92]. In guinea pigs, the SARs are only connected to

the jugular ganglion and the RARs are connected to the nodose ganglion of the vagal

nerve [93-95]. The central nerve endings of the airway afferents terminate in the

nucleus tractus solitarii of the medulla oblongata. The vagal efferent fibers control

vasodillation in the lungs whereas the afferent fibers (a-δ and C) convey pain and

stretch sensation to the central nervous system. Degeneration studies have

approximated that 75% of the vagal afferent fibers from the respiratory tract are

unmyelinated C-fibers [96]. The primary hypothesis of the present dissertation was that

a subset of pulmonary fibers, pulmonary c-fibers, would be stimulated by extreme

hypercapnia by hypercapnia acidosis. These fibers would then release peptides that

lead to induced pulmonary edema.

1.7.1 Peptidergic c-fibers

C-fibers are unmyelinated fibers that secrete several pro-inflammatory molecules

when stimulated. They are polymodal, responding to a variety of noxious stimuli.

These include heat, capsaicin, and acid. How they respond is determined by expressed

receptors and channels on their cytoplasm. Acid has been known to activate these

fibers to induce the cough reflex. Additionally, acid can potentiate c-fibers. In

asthmatic reactions, the pH of the airway water vapor condensate reduces from 7.65 to

5.23 [97, 98]. A pH of less than 5 has been demonstrated to induce the c-fiber release

of peptides that are known to initialize pulmonary edema [99, 100].

1.7.2 Acid sensing by c-fibers

Hypercapnia potentiates pulmonary c-fibers to inflammatory mediators by

hypercapnia acidosis [101]. Their acid sensitivity is mediated by a group of receptors

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expressed on their peripheral axons. These include the TrpV1 channel and acid-

sensing ion channels (ASICs). TrpV1 channel is attributed to the c-fiber sensitivity to a

mild decrease of pH to 7.0 and ASICs to a pH decrease to below 6.5. These two

channels are thought to make up the majority of the acid sensitivity of pulmonary c-

fibers [102].

1.7.3 Acid-sensing ion channels

Acid-sensing ion channels are trimeric channels selective for Na+ and are

predominantly expressed in neuronal cells. Three genes encode 5 major transcripts as

components of functional channels. The subunits ASIC1A and ASIC1B both assemble

into homomeric channels with high acid sensitivity. The ASIC2A assembles into

homomeric channels but has low sensitivity to acid [103]. The ASIC2B channel is silent

but the subunit does assemble with other ASIC subunits to modify the channel ion

selectivity [104]. The ASIC3 channels are unique where they are biphasic, generating

an early fast phase followed by a long prolonged phase. These are the only ASIC

subunit not expressed in the CNS. They are also most closely associated with DRG

cells [105]. The ASIC4 subunit is insensitive to acid and also modulates the properties

when combined with the other subunits [106, 107]. ASICs are expressed in brain

regions linked to hypercapnia-induced fear. ASIC1a knockout (KO) mice have a

severely blunted fear response to hypercapnia. This is the only genetic evidence for a

primary chemosensory transducer for CO2 [108].

1.7.4 Transient receptor potential channels

Transient receptor potential channels (Trp) have been well studied in the

airways. Activation of this class of receptors induces neurogenic inflammation. This

13

causes cough, airway constriction, and airway hyperreactivity [109-111]. The TrpV1

channel belongs to the family of 28 Trp channels spanning two groups. TrpV1 channels

are semiselective calcium channels. Each subunit is a 6 transmembrane protein and

they are arranged as a tetramer. The channel subunits can be comprised of 4 identical

subunits, or of a mixture of the other 3 Trp subunits (2-4). TrpV1 is polymodal,

activating in response to heat, acid, capsaicin, and several pungent substances. This

channel can also be activated, sensitized, and desensitized by intracellular signaling.

1.7.5 Tachykinins

The effect of tachykinins in response to noxious stimuli has been well

documented [112]. Tachykinins are a class of neuroamines encoded by three genes.

Tac1 has two splice variants, substance P (SP) and neurokinin A. Tac3 encodes

neurokinin B. The most recent tachykinin gene discovered is tac4 that encodes

hemokinin-1(HK-1). In humans, tac4 has four splice variants, endokinin A-D, as

opposed to one for the murine gene. While tachykinin lengths vary, their binding

domains are fairly well conserved. This enables every tachykinin to bind to all three of

the tachykinin receptors NK1, NK2, and NK3. Every tachykinin binding affinity is

influenced by their varying C-terminus binding domains and the composition of their N-

terminus. For example, SP and HK-1 are both eleven amino acid peptides. Their

affinity differences are attributed to the variant amino acid sequence occurring in the N-

terminus of 4 amino acid differences. Therefore, SP has a 100 fold higher affinity to

NK1 than HK-1.

Substance P is found throughout the CNS and PNS. In the CNS, it is

predominately found in areas that regulate emotion. The neurons in the brain

14

containing SP are also found in close proximity to those containing serotonin and

norepinephrine. These are the same areas that are targeted by antidepressant drugs.

In the PNS, SP is most known for its involvement in pain perception and inflammation.

African naked mole-rats, lack cutaneous SP expression in their c-fibers and as such

have no cutaneous inflammatory pain and itch perception from noxious stimuli applied

to their skin [113]. However, when SP expression via vector is introduced in their

DRGs, pain perception is induced by the same noxious stimuli. SP has also been

shown to cause vasodilation and endothelial leakiness, and migration of neutrophils into

the inflamed tissue. These effects occur by its binding to the NK1 receptor, where a

NK1 antagonist will reduce or abolish this effect [114]. There is also a link between SP

alveolar release and late airway response to allergenic challenge [115].

Substance P has been determined to be the peptide that mediates neurogenic

inflammation in the lung [116-118]. Lung permeability increases due to the stimulation

of pulmonary c-fibers and is inhibited by TrpV1 and NK1 antagonists [117, 119-121],

and exogenous SP mimics the increased pulmonary permeability [122]. Therefore, this

dissertation predicted that hypercapnia acidosis would stimulate the pulmonary c-fibers

to release SP. The release SP would thus induce pulmonary edema.

1.7.6 Neurokinin signaling

Neurokinin receptors are g-protein-coupled receptors and as such, the cellular

effect induced by their activation is a product of the 2nd messenger cascades they

initiate. Binding of NKs to NK receptors activates bound g-proteins that leads to PKC

activation and [Ca2+]i increases by IP3 receptor activation [123]. PKA can activate PKC

that phosphorylates other receptors, thereby desensitizing those receptors. The

15

extracellular activity of NKs is eliminated by extracellular proteases that break up NKs,

thereby making them inactive. After binding of NKs, NK receptors are endocytosed.

Neurons expressing NK1r will be completely desensitized to SP within 10 minutes after

initial stimulation. The receptors are translocated to early endosomes. This is mediated

by β-Arrestins that couples with agonist-occupied receptors and couples the receptors

to the clatherin-mediated endocytosis machinery. Once in the endosome, endothelin-

converting enzyme-1 degrades neuropeptides, thereby destabilizing the receptor from

β-Arrestin and promotes the receptor for recycling back into the cell membrane. Cell

sensitivity to SP is almost restored by 60 minutes after initial stimulation [124].

Desensitization is not fully mediated by endocytosis of the receptor but likely due to

phosphorylation of the receptor by 2nd messenger kinases. Phosphorylated receptors

can be resensitized by protein phosphatase 2A by it translocating to the plasma

membrane and dephosphorylating the desensitized receptors [125].

1.8 Alveolar Macrophages

Alveolar macrophages are the main resident immune cells in the lungs that

initiate and recruit other inflammatory cells. Therefore, alveolar macrophages are likely

to be the first immune cells activated by the pathway of hypercapnia-induced pulmonary

edema. They express the NK1r and can be activated by released SP [126]. Alveolar

macrophages are a subtype of macrophages that strictly reside in the lumen of the

bronchioles and alveoli. They have two functions, clearing of cellular debris in a steady-

state manner in healthy lungs, and initializing lung inflammation in response to lung

assault. They are kept in the stead-state mode by their interaction with the apical

16

surface of AECs by receptors and anti-inflammatory cytokines. The induction of the

inflammatory response in alveolar macrophages is thought to arise from either the

destruction of juxtapose epithelial cells, or loss of exposure to the epithelial secreted

regulatory ligands. A homeostatic balance exists between pro- and anti-inflammatory

signals that once tipped will initiate the alveolar macrophage inflammation.

Different signals to alveolar macrophages will determine whether they respond

by maintaining steady-state or pro-inflammatory behavior. In response to cell

apoptosis, alveolar macrophages must take an anti-inflammatory response to avoid

inflammatory response to self [127]. Alternatively, necrosis of AECs liberates pro-

inflammatory cellular constituents. Alveolar macrophages can also be stimulated

directly by increasing the pro-inflammatory pathways of alveolar macrophages, thereby

overcoming the inflammation suppression by interaction with AECs. Alveolar

macrophages have pattern recognizing receptors on their cell membrane. When these

receptors come into contact with a pathogen, pro-inflammatory responses will be

initialized. For example, oxidative stress enhances alveolar macrophage exocytosis

and thus increased surface expression of TLR4 [128]. In addition, peptidergic c-fibers

innervate lungs and secrete substance P that can induce inflammation by alveolar

macrophages.

1.9 General Anesthetics

General anesthetics are a broad group of substances that possess anesthetic

activity. There are many ion channels and receptors that anesthetics interact with.

Anesthetics can depress the firing of neurons and interfere with components of

17

chemosensitivity [129-131]. To date, the specific receptor targeted by anesthetics to

produce anesthesia is unknown. This is due to the lack of specificity of anesthetics.

Gamma-aminobutyric acid and N-methyl-D-asparate receptors in the CNS are indicated

as the likely targets needed to produce anesthesia [132]. General anesthetics are

known to have anti-inflammatory effects [133-138]. Therefore, the present dissertation

examined the effect of a general anesthetic, isoflurane, has on hypercapnia-induced

pulmonary edema.

1.9.1 Isoflurane

Isoflurane, a common volatile anesthetic, is known to excite pulmonary c-fibers

but also offers protection against induced inflammation. In humans, inhalation of ISO

induces tachypnea. The pulmonary c-fibers are thought to be the target cells of ISO to

induce this response in dogs [139]. Blocking pulmonary c-fiber conduction using

perineural capsaicin treatment of the cervical vagi abolished this response and

unblocked ISO increases the firing rate of pulmonary c-fibers. Isoflurane has recently

been found to block transient and delayed-rectifier K+ channels of the pulmonary c-

fibers of the nodose ganglia. This effectively makes the cells hyper excitable [140]. The

same authors in another study found that ISO inhibits apnea evoked by a 5-HT3 agonist

on pulmonary c-fibers, suggesting that ISO has an inhibitory effects of the ionotropic 5-

HT3 channels [141]. ISO also excites the TrpV1 receptor [142]. Not all subtypes of a

receptor is inhibited or excited by ISO [143]. In addition to cell membrane receptors, it

binds to intracellular components. ISO interferes with multiple sides of the electron

transport chain [144] including the mitochondrial electron transport chain complex III

18

[145] via potentiation of the electron transport chain complex I [146]. This ultimately

causes the mitochondria to produce ROS.

Isoflurane, as is the case with other volatile anesthetics, has a pro-inflammatory

action in some conditions, and an anti-inflammatory action in others. The difference of

effects of ISO has on inflammation appears to be dependent on the cell type and

method of induced inflammation. In alveolar macrophages, ISO increase the secretion

and gene expression of some cytokines. In AECII, ISO decreases the production of

pro-inflammatory cytokines [137, 138]. This is thought to be due ISO potentiating the

GABAA receptors [147]. Changes of gene expression by ISO exposure offers

protection from later injury known as preconditioning. This is thought to be due to

activation of inhibitory g-proteins via receptors [148] and PKC [149, 150]. ROS

generated by the mitochondria can also activate PKC. Ultimately, ISO exposure causes

increased heme oxygenase-1 (HO-1) activity. HO-1 activity utilizes mitochondria heme-

containing proteins in a reaction that is protective against induced cell apoptosis and

maintains mitochondrial membrane potential [151]. HO-1 is thought to be a scavenger

of heme liberated by oxidants in cellular oxidative stress [152]. Correlated with ISO-

induced increase HO-1 was a decrease in nitric oxide production that was mediated by

HO-1 activity [133]. This is to be expected as HO-1 inhibits iNOS expression and

activation. Of particular interest, ISO has been found to induce transient changes of

alveolar epithelial permeability [153].

1.10 Naked Mole-Rats

19

Previous studies in our lab and others have found naked mole-rats possess

remarkable insensitivities to noxious stimuli. In their skin, naked mole-rat cutaneous c-

fibers lack SP [154]. This absence was found to prevent pain-related behaviors in the

naked mole-rat. These behaviors were only observed once SP expression was

transfected into their c-fibers [155]. However, they demonstrated insensitivity to acid.

Naked mole-rats have been found to possess a special form of the Nav1.7 channel

[156]. This sodium channel version is only expressed in c-fibers and is inactivated by

acid. The naked mole-rat form has a special motif that causes the channel to be more

susceptible to acid inactivation.

Naked mole-rats have an extreme resistance to hypoxia. They were found to

survive 6 minutes of anoxia without any apparent lasting detrimental effects [157].

Hippocampal slices also revealed great hypoxia resistance, thereby negating any

effects the circulatory system [158]. Naked mole-rats were found to contain a subunit

composition similar to that of neonate mice that also have hypoxic resistance [159]. In

the tunnels of naked mole-rats, hypercapnia occurs coupled with hypoxia [160]. To

date, no studies have evaluated the naked mole-rat pulmonary resistances to

hypercapnia.

1.11 Current Study

The purpose of the current dissertation was to gain a better understanding of

hypercapnia effects on lung physiology and possible adaptations naked mole-rats have

that desensitize them to the hypercapnia in their burrows. We hypothesize that

pulmonary c-fiber acid sensitivity contributes and possibly induces the pulmonary

20

inflammatory reaction of hypercapnia (Figure 1). The naked mole-rat lack of SP and

acid sensitivity in their DRG c-fibers may be part of their adaptations to hypercapnia.

An alternative hypothesis was that pulmonary c-fiber acid sensitivity is not involved in

hypercapnia-induced pulmonary edema and hypercapnia may directly induce

pulmonary edema through its effects directly on alveolar macrophages.

21

Figure 1. Hypothesized mechanism of hypercapnia-induced pulmonary edema.

Hypercapnia causes tissue acidosis (1) that stimulates pulmonary c-fibers (2). The c-fibers release substance P (3) that binds with and increases permeability of the epithelial barrier (4). The increased permeability allows for blood plasma to exude out of the pulmonary blood capillaries and fill the alveolar airspace (5).

22

The following studies will determine (1) How resistant naked mole-rats are to

hypercapnia; (2) the level of SP+HK-1 in the lungs and whether it changes in response

to hypercapnia; (3) whether reducing pulmonary c-fiber acid sensitivity in mice alters the

level of inflammation induced by hypercapnia; (4) if eliminating SP expression alter

hypercapnia-induced respiration rate increase and pulmonary edema; (5) and finally if

isoflurane has a pro- or anti-inflammatory effect on hypercapnia-induced inflammation.

2. EXTREME HYPERCAPNIA RESISTANCE OF THE AFRICAN NAKED MOLE-RAT

2.1 Abstract

African naked mole-rats have several adaptations for living together in large

colonies underground. In their tunnels, oxygen content is reduced and carbon dioxide

builds up to noxious levels. Naked mole-rats have insensitivity to several noxious

inflammatory stimuli attributed as adaptations to overcome living in chronic hypercapnia.

The purpose of this study was to evaluate hypercapnia effects on naked mole-rat

respiration and CO2-induced pulmonary edema that occurs in other terrestrial mammals.

We observed naked mole-rats to have insensitivity to moderate hypercapnia-induced

respiration increase. At higher levels of hypercapnia, respiration rate increased but no

pulmonary edema ever developed. This is the first time an animal has demonstrated a

complete absence of pulmonary edema from extreme hypercapnia. We measured

SP+HK-1 levels in alveolar lavage fluid. These are neuropeptides that are released in

lungs, induce inflammation within lungs. No detectable amounts were observed at

basal or after acute hypercapnia exposure of naked mole-rats. The absence of these

two neuropeptides released into the alveolar airspace of naked mole-rats indicates a

sensory-inflammatory adaptation to overcome the physiological challenges of living in

hypercapnia.

2.2 Introduction

Hypercapnia in most mammals induces several physiological responses. At low

levels, respiration rate will increase which can compensate for the retention of too much

tissue CO2. At extreme hypercapnia, pulmonary edema will occur, resulting in impeded

23

24

gas exchange. The threshold for pulmonary edema induction is about the same

amongst most mammals tested to date, indicating a shared inflammatory pathway.

Mammals that normally live in a noxious hypercapnia environment are thought to have

adaptations to overcome their physiological challenges. However, only a few studies

have evaluated the pulmonary adaptations in these resistant mammals. African naked

mole rats, Heterocephalus glaber, chronically live in hypercapnia. This study evaluates

their physiological responses to acute hypercapnia.

The naked mole-rat is well adapted to its indigenous life in the semi-arid

landscape of north-eastern Africa. In this environment, naked mole-rats live completely

subterranean, avoiding the heat and desiccating effect of the surface. To remain

hydrated, they intake moisture from the roots and tubers they eat, and their kidneys

excrete little but highly concentrated urine [21]. Maximum urine concentration is about

1,500 mmol/kg [22]. Naked mole-rats display an unusual social structure called

eusociality. Their colonies consist of workers, soldiers, few male breeders and one to

two breeding females. Besides from breeding, no sexual bias exists for the roles

performed by each naked mole-rat. All individuals work together to aid in their survival

and child rearing in colonies as large as 300 individuals [23-26]. A consequence of so

many naked mole-rats in their narrow, enclosed tunnels is that oxygen levels decrease

(hypoxia) and carbon dioxide increases (hypercapnia). CO2 dissipation from the tunnels

varies depending on soil composition, recent rain fall, and depth of the tunnel systems

[161]. In the natural habitat of naked mole-rats, the soil has been described as being

“like cement” [21] and “dark red sandy clay loams” [1]. In such conditions, it would be

adaptive to be resistant to hypoxia and hypercapnia.

25

Naked mole-rats have several adaptations for living in their hypoxic tunnels.

Their metabolic rates are low compared to their size [162, 163]. Their blood has high

affinity for oxygen [164]. Under anoxia, naked mole-rat brain hippocampal slices

survive 3 to 4 times longer than mice [158]. Peterson et al. found that these slices have

reduced internal calcium that builds up from hypoxia compared to mice [165]. In

conjunction with this, they found that naked mole-rats retain a greater portion of GluN2D

NMDA subunits into adulthood than mice [159]. These subunits are known to be

protective during hypoxia.

Less is known of how naked mole-rats are adapted to hypercapnia. A known

physiological effect of hypercapnia is the coupled tissue acidosis caused by the

conversion of CO2 into HCO3- and protons. Naked mole-rat blood has a higher buffering

capacity of CO2 but it does not prevent acidosis in naked mole-rats from occurring in

their tissues. Naked mole-rats are insensitive to cutaneous acid application [154, 155].

The inflammatory response induced by acid is mediated by cutaneous c-fibers, which in

other animals rely on voltage-sensitive sodium channels (Nav1.7) to propagate action

potentials. The naked mole-rat Nav1.7 has increased inhibition by acid [156]. This

difference may explain the significant decreased activity of the trigeminal pain nucleus

from 50% acetic acid instead of the increase seen in mice [166].

Hibernating animals also need to cope with a hypercapnia. The same Nav1.7

amino acid motif responsible for the increased acid inhibition seen in naked mole-rats

has been found in these animals, occurring in both a parallel and divergent evolution

[167].

26

Another striking feature about naked mole-rats concerns SP and other pro-

inflammatory molecules that are usually released by c-fibers. Naked mole-rats lack SP

in their cutaneous c-fibers [154]. This was confirmed by transfecting fibers with SP,

which enabled a behavioral response to the TrpV1 channel agonist capsaicin. This also

demonstrated that naked mole-rats do contain the functional receptor for SP [168].

These physiological differences in naked mole-rats are suggested to be adaptations for

living in a chronic hypercapnia.

The acidosis resulting from hypercapnia is believed to induce pulmonary edema.

As it is in the skin, pulmonary c-fibers detect acid. For the majority of mammals tested

so far, most pulmonary c-fibers originate from the vagus nerve and the rest are from the

DRG. Once activated, these c-fibers release pro-inflammatory molecules. SP and

Neurokinin A, members of the tachykinin family, are the major contributors of

inflammation released by c-fibers. SP preferentially binds to the NK1 receptor and NKA

to the NK2 receptor. Hemokinin-1, the most recent discovered tachykinin, also

preferentially binds with the NK1 receptor [169, 170]. Unlike SP that is expressed

heavily in areas of the brain and in peripheral c-fibers [171], HK-1 is widely expressed

throughout immune and inflammatory cells [172-176]. As such, HK-1 has necessary

roles in their functions [169, 177-181]. Even though SP and HK-1 preferentially bind to

the NK1r, several studies have found different effect induced by SP and HK-1 [177,

182-184]. Recently, HK-1 was found to potentially act as a NK1 antagonist where SP is

present [185], although the existence of a proper HK-1 receptor has been suggested

[184].

27

Respiration rate is predominantly controlled by PCO2 in the blood. An increase

of 1 mm Hg of arterial PCO2 will increase the respiration rate by 30 percent in humans

[21]. This is an example of the sensitivity most terrestrial mammals have for regulating

their arterial PCO2. However, the same level of sensitivity might not be advantageous

for naked mole-rats who live in a chronic hypercapnia. If naked mole-rats have

comparable sensitivity to elevated CO2, then this would imply that they live normally in a

state of hyperventilation.

We predicted that naked mole-rats would have reduced physiological responses

to hypercapnia. We measured respiration rate in moderate hypercapnia at levels

representing those in their natural habitat. Additionally we measured respiration rate

during extreme hypercapnia exposure to determine if respiration rate is modulated in

naked mole-rats at these levels. Mice were used as a comparison species to the naked

mole-rats because they have similar physiological responses to hypercapnia with other

mammals and are closer to the naked mole-rat body size.

Due to the naked mole-rats known cutaneous adaptation of acid inhibition of their

Nav1.7 channels and reduced or absent SP, hypercapnia-induced pulmonary edema

was measured in the naked mole-rats. Part of the inflammatory response in the lungs

of most mammals is the release of pro-inflammatory molecules SP+HK-1 into the

alveolar airspace. The absence of SP in the naked mole-rat cutaneous c-fibers and the

c-fiber acid insensitivity may indicate abnormal expression or release of SP in their

lungs. Therefore, we measured the release of SP+HK-1 in their lungs at extreme

hypercapnia in vivo.

28

2.3 Materials and Methods

2.3.1 Animals

The present study used BL/6 mice that were bred from stock obtained from

Charles River Laboratories, Wilmington, MA. All mice were housed at a constant

temperature at 22oC (+/-2) in a 12-hour light-dark cycle. Food and water were available

ad libitum. The naked mole-rats were born and maintained in our colonies at the

University of Illinois at Chicago. Naked mole-rats were kept at 29oC (+/-1) and fed ad

libitum. Mice and naked mole-rats were of both sexes. The mice were at least 3

months old and no older than one year. The naked mole-rats were older than 1 year.

Animal protocols were approved by the University of Illinois at Chicago Institutional

Animal Care and Use Committee.

2.3.2 Environmental chamber

The environmental chamber for isolating each animal in a set gas mixture of

oxygen, nitrogen, and carbon dioxide was a circular clear plastic container (16 cm X 5.5

cm) with a lid that sealed and locked to the container. On one end of the container lid, a

small hole had the gas tube fed into it. The opposite end of the lid had a small open

hole, functioning as the exhaust for the container. Gas after passing through regulators

was mixed together before entering the environmental chamber. Between the oxygen

regulator and the environment chamber was an ISO vaporizer that was used to add ISO

to the oxygen stream for some experiments.

2.3.3 Respiration rate

The environmental chamber was washed with distilled water, dried, and wiped

with 70% ethanol before each animal was placed into the chamber. Gas was turned on

29

at a 20% O2/80% N2 ratio at 4 liters/minute. Four animals were used for each testing

condition and no animals were used in more than one condition. The environmental

chamber with the animal inside was placed on stilts that were 30 cm tall. A video

camera was placed underneath facing up towards the underside of the environmental

chamber. Recording was started five minutes into setting up the animal into the

chamber. After 5 minutes of recording, the nitrogen flow was reduced equal to the CO2

flow increase. CO2 concentrations used were 5%, 10%, 20%, and 40%. Recording

was carried out for an additional 15 minutes. At the end of the recording, animals were

removed and the gas was turned off. The recordings were later analyzed.

2.3.4 Respiration rate recording analysis

Each recording was played at half speed and number of breaths for the first 20

seconds of every minute was counted. These 20 seconds were used to calculate the

breaths per minute. The average of the first 5 minutes of recordings corresponded to

the 0% CO2 condition. Thus the first 5 respiration rate calculations were averaged

together to get a basal respiration rate for each animal. The next 15 respiration rate

calculations corresponding to the hypercapnia exposure period were averaged together

to determine the respiration rate in the hypercapnia condition.

2.3.5 Pulmonary edema measurement

Three animals were used for each testing condition. No animal was used more

than once. As it was for the respiration rate, the environmental chamber was washed

with distilled water, dried, and wiped with 70% ethanol before each animal was placed

into the chamber. Gas was turned on at a 20% O2/80% N2 ratio at 4 liters/minute. The

N2 flow was reduced by the amount of CO2 increase. CO2 concentrations used were 0,

30

10, 20, 30, and 50%. After 10 minutes, animals were anesthetized with ISO for 90

seconds and the lungs were removed from their chest. The lobes were individually

removed from the trachea and placed into saline. Each lobe was then patted dry using

a Kimwipe and all lobes from an animal were weighed together in an aluminum foil

weigh boat. The weigh boat with the lungs was placed into an oven set to 55 degrees

Celsius for drying. After 24 hours, the lungs were reweighed. The ratio of wet weight to

dry weights was calculated.

2.3.6 Lung lavage

Three animals were used for each condition. Animals were exposed to 10

minutes of a gas mixture followed by 20% O2/80% N2 with 2% ISO. After the animal

was anesthetized, it was transferred from the environmental chamber to a respiratory

mask that had the same 20% O2/80% N2 with 2% ISO flowing through it. A

tracheotomy was done and a 15 gauge needle was inserted into the tracheostomy.

Surgical thread was tied around the combined trachea and needle to create a seal.

One ml of cold PBS was infused into the lung and then aspirated. Peptidase inhibitors,

phenylmethylsulfonyl fluoride (PMSF, 1 mM) and DL-thiorphan(5mg/ml), were added to

the lavage fluid as was done by a previous study [115]. Samples were then centrifuged

for 10 minutes at 1600 rpm. The supernant was removed and stored at -20 degree

Celsius for later testing.

2.3.7 Enzyme-linked Immunosorbent Assay

Lung lavage fluid SP+HK-1 content was measured using a commercially

available enzyme-linked immunosorbent assays (ELISA) in accordance with the

manufacturer’s directions (R&D System, Mn, USA).

31

2.3.8 Statistical analysis

The respiration rate percent increase due to CO2 treatment was compared using

the one-way ANOVA test. Tukey post hoc test was used to determine significance

between respiratory rate groups. One-way ANOVA followed by Tukey post hoc test

was used to test for significance between wet/dry lung weight ratios at 0% CO2 and 10,

15, 20, 30, and 50% treatments for both naked mole-rats and mice. No statistical

analysis was done for SP+HK-1 lung lavage concentration between naked mole-rats

and mice as no detectable amount was found in naked mole-rat lavage fluid.

2.4 Results


Respiration rates for mice at 0 and 5% CO2 and naked mole-rats at 0, 5, 10, 20,

and 40% CO2 concentration was done (Figure 2). Consistent with known mouse

respiratory responses to CO2, mouse respiration rate significantly increased by 29.9%

(SE=5.38) at 5% CO2 and 31.5% (SE=4.01) at 10% CO2. No significant change was

observed for mice between 5% and 10% CO2 (t(6)=0.3371, p=0.7475). Naked mole-

rats had significant difference among the CO2 treatments (F(3,12)=5.846, p=0.0106).

Naked mole-rats had no significant change between 0% and 5% or 0% and 10% CO2

(at p<0.05). A significant increased respiration rate was observed for the naked mole-

rats at 20% CO2 compared to 5% CO2 (p=0.0248).

32

Figure 2. Respiration rate of naked mole-rats and mice at increasing CO2

concentrations. Naked mole-rats (NMRs) did not have a significant increased respiratory rate response at 5% and 10% CO2. Naked mole-rats significant increased their respiratory rate at 20% CO2. Error bars are standard error of means. *=p<0.05 (one-way ANOVA, Tukey post hoc test, N = 4).

33

2.4.2 Lung edema

The wet/dry weight ratio of mouse and naked mole-rat lung tissue was calculated

after each animal was exposed to one of the following CO2 percent concentrations: 0,

20, 33, and 50 (Figure 3). A significant difference existed amongst the mice treatment

groups (F(3,8)=87.76, p<0.0001). Tukey post hoc analysis revealed significant

difference between 0% CO2 (M=4.384, SD=0.2185) and 20 (M=7.738, SD=0.1936), 33

(M=7.079, SD=0.2232), and 50% CO2 (M=7.265, SD=0.4223) (p<0.0001). No change

was observed between 0% and 10% for either mouse or naked mole-rat. No significant

difference was observed for the lung weight ratio at all concentrations of CO2 exposure

for the naked mole-rats (F(3,8)=1.473, p=0.2934).

34

Figure 3. Lung weight ratio of naked mole-rats and mice after hypercapnia

treatments. Mice had significant increased wet/dry lung weight ratio at 20%, 33%, and 50% compared to 0% CO2. No significant difference was found between naked mole-rat (NMR) CO2 treatments. Error bars are standard error of means. ****=p<0.0001 (one-way ANOVA, Tukey post hoc test, N = 3).

35

2.4.3 Lung lavage

An ELISA kit was used to quantify the SP+HK-1 amount released in the lungs of

the mice and naked mole-rats (Figure 4). SP+HK-1 was measured in the naked mole-

rats and mice at 0% and 30% CO2. A substantial and significant increase was observed

for the mice at 0% CO2 (M=0.7217, SD=0.2021) and 30% CO2 (M=39.33, SD=19.29)

(t(4)=3.466, p=0.0257). There was no detectable amount of SP+HK-1 at either level of

CO2 in the naked mole-rats.

36

Figure 4. SP+HK-1 concentrations in lung lavage fluid of naked mole-rats and

mice after CO2 treatments. Mice had a significant increase of SP+HK-1 in lung lavage fluid after the 30% CO2 treatment compared to 0% CO2. No detectable amount of SP+HK-1 was found in naked mole-rat (NMR) lavage fluid after 0% or 30% CO2 treatments. Error bars are standard error of means. n.d. = not detected.

37

2.5 Discussion

Naked mole-rats demonstrate a remarkable resistance to the effects of

hypercapnia. Resistance to moderate hypercapnia-induced increase in respiration rate

may reflect their insensitivity to hypercapnia in their natural environment. Likewise,

absence of edema from extreme hypercapnia may represent this adaptation.

Undetectable hypercapnia-induced SP and HK-1 in alveolar lavage fluid could explain

the absence of edema. Extreme hypercapnia would reduce blood pH to acidic levels,

and the greater inactivation of their Nav1.7 channels could prevent the activation of their

c-fibers from releasing SP. However, undetectable basal levels may indicate a more

involved adaptation to hypercapnia than just their c-fiber insensitivity to acid. These

results set naked mole-rats as a model organism to further our understanding of CO2

detection and the mechanisms involved in pulmonary inflammation.

Respiration is controlled predominantly by a network of medulla oblongata nuclei

in the brainstem. This network is modulated by sensory input of the arterial PCO2

changes. A number of central and peripheral sensitive cells have been identified, but

the sensory transducer that detects PCO2 remains elusive. The coupled acidosis of

increased PCO2 is considered to be the stimulus that activates sensory input of the

respiratory network. Studies have induced increased respiration rate by increasing

blood acidity. However, increasing blood PCO2 while maintaining constant pH also

increases respiration. Decreasing blood pH could shift the CO2 equilibrium towards

increased free CO2. Thus, teasing out the physiological effects of CO2 from its coupled

acidosis has proven difficult. We propose that studying the respiration network in naked

mole-rats can reveal critical aspects to further our understanding of respiration control.

38

The SP found in alveolar airspaces of most mammals has been considered to be

from peptidergic c-fibers innervating the lungs. There are two fiber populations, one

originating from the vagus [186], the other from the DRG [187]. Naked mole-rats lack

SP in their DRG and it is for this reason and their insensitivity to noxious stimuli that we

examined SP release in the lungs. Substance P positive neuronal cells have been

identified within the lungs of naked mole-rats, presumably of vagal origin (unpublished

data). Despite SP presence in lung tissue, we did not detect any SP+HK-1 in naked

mole-rat lavage fluid. In respect to the naked mole-rat vagal c-fibers, this may indicate

these cells are not stimulated as they are in other mammals. Alternatively, the DRG c-

fibers could be the fibers that would normally release SP in other mammals but the

naked mole-rat DRG c-fibers do not express SP. Comparing functional differences of

the pulmonary nerve fibers between species is problematic because of different

innervation patterns between species. Several studies have tried to demonstrate that

the SP in the lavage fluid of other mammals is from these c-fibers. Over stimulating

these fibers can cause desensitization or degeneration. This results in reduction or

absence of SP in lavage fluid. However, these experimental conditions could have

effects on other pulmonary fibers beyond the c-fibers

We did not differentiate the release of SP and HK-1 in the lungs since both are

endogenous NK1r agonists. In another study (see Ch 3), we found HK-1 as the

dominant NK1 agonist released in the murine lungs. HK-1 is known to be expressed in

immune cells and alveolar macrophages but has not been found in neurons. This

eliminates HK-1 lung lavage fluid as not originating from pulmonary c-fibers. SP is

expressed to a lesser extent in alveolar macrophages than HK-1. These expression

39

differences of HK-1 and SP in alveolar macrophages may be related to the dominance

of HK-1 in alveolar lavage fluid. The absence of detectable alveolar SP or HK-1 in

naked mole-rat, even at basal levels, suggests a common source. Alveolar

macrophages are potential candidates as they express SP and HK-1 in humans and

mice.

Recently, alveolar macrophages have been implicated as the initiator of

inflammation induced by hypoxia [188]. Acute alveolar hypoxia has also been

demonstrated to induce systemic inflammation, independent of the systemic PO2.

Often, hypercapnia occurs with hypoxia as is the case for naked mole-rats.

Hypercapnia has been shown to induce gene expression changes in alveolar

macrophages [31] and independent of intracellular and extracellular pH [35]. Therefore,

a critical difference may exist in the naked mole-rat alveolar macrophages that may

make them desensitized to hypercapnia or perhaps lack expression of SP+HK-1.

Substance P is only still considered to be a neuropeptide for historical purposes.

SP is expressed by numerous immune cells. HK-1 is also expressed in immune cells

but to a much greater amount. HK-1 is necessary for the maturation of B cells and

enhances antibody production [177, 181, 189]. The naked mole-rat is considered to

have a strong immune system [190] but no cellular immune functions of the naked

mole-rat have been studied thus far. Naked mole-rats infected with a recombinant

herpes simplex virus type 1 show 100% mortality that was avirulent in mice. This was

suggested to be in part from the absence of SP expression in naked mole-rat cutaneous

c-fibers [191]. However, the present study indicates a greater deficit for the naked

mole-rat immune system. The frequency of alveolar macrophages is disputed as two

40

studies have described them as numerous [192] and rare [193]. In either case, naked

mole-rats do have alveolar macrophages. The absence of expressed SP in cutaneous

c-fibers of naked mole-rats suggests the possibility that naked mole-rat macrophages

do not express SP+HK-1, thereby explaining the absence of basal SP+HK-1 in lavage

fluid. Such difference would be remarkable as SP and HK-1 are encoded by two

separate genes.

Naked mole rats have several physiological differences from other terrestrial

animals that make them well suited for their subterranean environment. This study

adds to the known adaptations they possess. With the recent effort in sequencing the

naked mole rat genome, more detailed explanations can be discovered for these

adaptations. Future studies could look for expression of HK-1 in the naked mole-rat

lungs and other tissue. Differences of SP+HK-1 expression could have substantial

impact of the naked mole-rat immune system development and regulation. This can

further our understanding the relationship between the nervous and immune systems in

vertebrates. Additionally, research in naked mole-rat CO2 detection can lead to insights

of CO2 detection in mammals.

3. HYPERCAPNIA-INDUCED PULMONARY EDEMA EXACERBATION BY TAC1

KNOCKOUT MICE AND INHIBITION BY ISOFLURANE.

3.1 Abstract

Extreme hypercapnia induces pulmonary edema. The sensing of hypercapnia in

lungs was thought to be by pulmonary c-fibers that detect the hypercapnia-induced

tissue acidosis. To better understand the role c-fibers have in hypercapnia-induced

pulmonary edema, we exposed mice to several levels of extreme hypercapnia and

measured the SP levels of the lung airways. We reduced the acid sensitivity of

pulmonary c-fibers by using mice deficient in TrpV1 or TrpV1 and ASIC3 channels.

Isoflurane, known to attenuate pulmonary c-fibers to stimuli, was added to the

hypercapnia to determine if anesthetic can inhibit hypercapnia-induced pulmonary

edema. We found that hypercapnia at 15% CO2 and above but not 10% and below

caused increasing severity of pulmonary edema. The combined quantity of SP and

hemokinin-1 in lavage fluid matched the rise of edema from increasing CO2

concentrations. Tac1KO mice had increased level of edema than WT mice but no

difference was observed for TrpV1KO or TrpV1KO/ASIC3KO mice. Similar levels of HK-1

in lavage fluid of tac1KO mice compared to WT were observed after hypercapnia,

indicating a dominance of HK-1 alveolar release in mice. Concurrent exposure of mice

to ISO with hypercapnia attenuated the hypercapnia-induced pulmonary edema and

SP+HK-1 increase in lavage fluid. These results indicate pulmonary c-fibers may not be

the instigators of hypercapnia-induced pulmonary edema.

3.2 Introduction

41

42

Retention of CO2 in body tissue is a frequent, debilitating symptom in patients

with severe chronic pulmonary diseases. The elevated CO2 can induce several

pathophysiological changes presumably from the resulting tissue acidosis. CO2 is

quickly and reversibly converted to HCO3- and protons and the free protons make the

tissue acidic. Acid has been demonstrated by several studies to activate c-fibers [194,

195] and these fibers release peptides that promote pulmonary inflammation [196]. The

ability of c-fibers to detect acid is mediated by ion channels that respond to protons.

There are a multitude of acid transducers found on sensory neurons [197].

Evidence suggests there are two distinct ion channel populations responsible for the c-

fiber acid sensitivity. C-fibers have two acid-induced electrophysiological currents. The

first is a rapidly inactivated Na+ influx at pH slightly lower than normal homeostatic level.

The second is a mixed Ca2+/Na+ influx occurring at more acidic levels. The ASIC family

of channels seems to be responsible for the first and the second by TrpV1 receptor.

This implies that the TrpV1 and the ASIC channels are responsible for most of the c-

fiber acid sensitivity. Acid chemosensitive c-fibers are necessary for the inflammatory

response to noxious stimuli because they secrete the pro-inflammatory molecules. Of

those secreted, tachykinins have many effects.

Tachykinins have received much attention due to their critical role in pain

perception and tissue inflammation. They are encoded by three genes, tac1, tac3, and

tac4. Tac1 encodes the splice variants substance P and neurokinin A. Neurokinin B

and hemokinin-1 are encoded by tac3 and tac4 respectively. In humans, there are 4

splice variants of HK-1 whereas one conserved variant exists in rats and mice.

Tachykinins constitute one of the largest families of neuropeptides. They are expressed

43

abundantly throughout an animal, with specific cell type expressions. Substance P is

predominantly expressed in neuronal tissue and in the PNS this is in peptidergic c-

fibers. The physiological effects they exert are determined in part by three g-protein-

coupled tachykinin receptors: NK1, NK2, and NK3.

Every tachykinin binds to all three of the NK receptors. However, each has a

preferred receptor based on its higher affinity for each receptor. SP and HK-1

preferentially binds with NK1, NK A with NK2, and NK B with NK3 receptor. The

function of the tachykinins has largely been determined by observing the effects of each

tachykinin and blocking it with an antagonist to its corresponding receptor. However,

this does not work with SP and HK-1 that both bind preferentially to the NK1 receptor.

Recently, HK-1 has been demonstrated to have a competitive antagonist effect to SP

[185]. Since this is the first demonstration of competition between SP and HK-1, it is

unknown if HK-1has a competitive inhibitory effect on SP always where the two coexist.

Substance P is the most studied tachykinin and is considered to be one of the

major initiators of neurogenic inflammation. In the CNS, it is found in high

concentrations in the medulla and is involved in the vomiting pathway and respiration

[198, 199]. In the PNS, peptidergic c-fibers will release SP, acting as a potent

vasodilator, smooth muscle contractor, and a leukocyte chemoattractant. The wide

assortment of its effects is due to the NK1r expression in epithelial, endothelial,

macrophages, lymphocytes, neutrophils, dendritic cells, mast cells [200-202].

Alternatively, the other endogenous NK1r ligand HK-1 is expressed in endothelial,

inflammatory and immune, glial, and placental cells [172, 176, 177, 180]. Its role is

thought to be similar to SP as activating the NK1 receptor by either ligand should elicit

44

the same physiological response. However, several studies have found different effects

between the two. The discovery of HK-1 has produced a renewed interest in trying to

find other yet to be discovered tachykinins and their receptors.

Of the tachykinins, SP, NK A, NK B, and HK-1 are all expressed within lung

tissue. SP has been implicated in a number of neurogenic-induced pulmonary

inflammation studies. This is due to observed increase of SP release in the lungs after

noxious stimuli and the inflammation is inhibited by NK1r antagonists [203, 204]. In

asthmatic patients, lung inflammation is potentiated that corresponds with SP levels in

their airways.

The purpose of this study was to determine the effect hypercapnia on SP+HK-1

levels in alveolar airspace, and whether reducing the c-fiber acid sensitivity can

attenuate hypercapnia-induced pulmonary edema. These goals were pursued by

determining the following: (1) the dose response of hypercapnia on pulmonary edema

formation, (2) amount of hypercapnia-induced SP release into the lung airspace, (3) the

effect of tac1 gene elimination has on hypercapnia-induced edema, (4) the effect that

gene elimination of the acid-sensing ion channels TrpV1 and ASIC3 have on

hypercapnia-induced pulmonary edema, and (5) the effect of ISO has on hypercapnia-

induced pulmonary edema and SP+HK-1 release.

3.3 Materials and Methods

3.3.1 Animals

All mice were housed at a constant temperature at 22oC (+/-2) in a 12-hour light-

dark cycle. Food and water were available ad libitum. Wild-type BL/6 mice used in the

45

experiments were bred from stock obtained from Charles River Laboratories,

Wilmington, MA. The tac1KO mice were obtained from Jackson laboratories, Bar

Harbor, ME. The TrpV1KO and the TrpV1/ASIC3KO mice were obtained from

collaborators. All mice were at least 3 months old and no older than one year. Genetic

verification of knockout mice was done by Transnetyx, Cordova, TN. Animal protocols

were approved by the University of Illinois at Chicago Institutional Animal Care and Use

Committee.

3.3.2 Environmental chamber

The environmental chamber was a circular plastic container (16 cm X 5.5 cm)

with a lid that sealed and locked to the container. On one end of the container lid, a

small hole had the gas tube fed into it. The opposite end of the lid had a small open

hole, functioning as the exhaust for the container. The air tube fed from a gas mixing

chamber to the environmental chamber. Gas regulators were connected via tubing to

the gas mixing chamber on outlet end, and the gas tanks on the inlet end. The gasses

used were oxygen, nitrogen, and carbon dioxide. Between the oxygen regulator and

the environmental chamber was an ISO vaporizer that was used to add ISO to the

oxygen stream for some experiments.


The environmental chamber was washed with distilled water, dried, and wiped

with 70% ethanol before each animal placed into the chamber. Gas was turned on at a

20% O2/80% N2 ratio at 4 liters/minute. The environmental chamber with the animal

inside was placed on stilts that were 30 cm tall. A video camera was placed underneath

facing up towards the underside of the environmental chamber. Four animals were

46

used per group. Recording was started five minutes into setting up the animal into the

chamber. After 5 minutes of recording, the nitrogen flow was reduced by the amount

CO2 flow was set to. CO2 concentrations used were 5%, 10%, 20%, and 40%.

Recording was carried out for an additional 15 minutes. At the end of the recording,

animals were removed and the gas was turned off.

3.3.4 Respiration rate recording analysis

Each recording was played at half speed and scored for twenty seconds starting

at the beginning of every minute of the recording. The number of breaths was counted

and then multiplied by 3 to obtain the breaths per minute. The average of the first 5

minutes of recordings corresponded to the 0% CO2 duration. Thus the first 5 respiration

rate calculations were averaged together to get a basal respiration rate for each animal.

The next 15 respiration rate calculations corresponding to the hypercapnia exposure

period were averaged together to determine the respiration rate in each hypercapnia

condition.

3.3.5 Pulmonary edema measurement

As it was for the respiration rate, the environmental chamber was washed with

distilled water, dried, and wiped with 70% ethanol before each animal was placed into

the chamber. Gas was turned on at a 20% O2/80% N2 ratio at 4 liters/minute. The

environmental chamber was placed onto a table. The N2 flow was reduced as CO2 was

increased by the same amount, thereby maintaining a 4 liter/minute flow rate. CO2

concentrations used were 0, 10, 20, 30, and 50. After 10 minutes, animals were

anesthetized with 2% ISO for 90 seconds and the lungs were removed from their chest.

The lobes were individually removed from the trachea and placed into saline. Each

47

lobe was then patted dry using a Kimwipe and all lobes from an animal were weighed

together in an aluminum foil weigh boat. The weigh boat with the lungs was placed into

an oven set to 55 degrees Celsius for drying. After 24 hours, the lungs were reweighed.

The ratio of the wet weight to dry weight was then calculated.

3.3.6 Lung lavage

A group size of 8 animals was used for WT and each type of KO mice. Four

mice were used per group of the 30% CO2 with 2% ISO part of the study. After 10

minutes of gas exposure, the air was restored to a 20% O2/80% N2 and 2% ISO was

added to the gas mixture. After the animal was anesthetized, it was transferred from

the environmental chamber to a respiratory mask with the gas mixture attached to it. A

tracheotomy was done and a 15 gauge needle was inserted into the tracheostomy.

Surgical thread was tied around the combined trachea and needle to create a seal.

One ml cold PBS was infused into the lung and then aspirated. Peptidase inhibitors,

phenylmethylsulfonyl fluoride (PMSF, 1 mM) and DL-thiorphan(5mg/ml), were added to

the lavage fluid as was done by a previous study [115]. Samples were then centrifuged

for 10 minutes at 1600 rpm. The supernant was removed and stored at -20 degree

Celsius for later testing.

3.3.7 Enzyme-linked immunosorbent assay

Lung lavage fluid concentration of SP / HK-1 was measured using a

commercially available enzyme-linked immunosorbent assays (ELISA) in accordance

with the manufacturer’s directions (R&D System, Mn, USA).

3.3.8 Statistical analysis

48

Respiration rates were compared between tac1KO, TrpV1KO, and

TrpV1KO/ASIC3KO mice and WT mice using the one-way ANOVA with Dunnet multiple

comparisons test. Lung wet/dry weight ratios were compared at 0, 10, 15, 20, 30, and

50% CO2 using the one-way ANOVA test and 0% CO2 was compared post hoc to the

other CO2 treatments using Tukey post hoc test. Tac1KO, TrpV1KO, and

TrpV1KO/ASIC3KO mice lung wet/dry weight ratios were compared to WT mice ratios

using two-way ANOVA with Dunnet’s post hoc analysis. The SP+HK-1 lung lavage

concentration at 10, 15, 20, 30, and 50% CO2 was compared to 0% CO2 using one-way

ANOVA test with Dunnett’s multiple comparisons test. Lung lavage fluid SP+HK-1

concentrations of TrpV1KO and tac1KO mice were compared to WT mice using multiple t

test corrected with Holm-Sidak method of multiple comparisons. Both the lung wet/dry

weight ratios and the SP+HK-1 lung lavage fluid concentration of WT mice with 0%,

30% CO2, and 30% CO2 with ISO treatments were compared using the one-way

ANOVA with Tukey post hoc test.

3.4 Results


Mice were exposed to 0% CO2 followed by either 5% or 10% CO2 (Figure 5). A

significant increase in respiration rate was observed between tac1KO (M=378.2,

SD=24.36) mice compared to WT (M=304.6, SD=29.29) mice at 10% CO2;

F(3,12)=4.547, p=0.0238.

49

Figure 5. Respiration rates of wild-type, tac1KO, TrpV1KO, and TrpV1KO/ASIC3KO

mice during CO2 treatments. Only a significant difference was found between the tac1KO mice and WT mice at 10% CO2. Error bars are standard error of means. * = p<0.05 (One-way ANOVA, Dunnet post hoc test, N = 4).

50

3.4.2 Lung edema

Wild-type, TrpV1KO, and Tac1KO mice were exposed to increasing CO2

concentrations at 0, 10, 15, 20, 30, and 50% CO2 (Figure 6). As was previously

observed, WT mice had a significant increase in lung wet/dry ratios from basal levels at

15% CO2. This progressively increased from increasing CO2 concentrations up to 30%

CO2. A significant decrease was found from 30 to 50% CO2.

No significant difference (p<0.05) was observed for wet/dry ratio at each CO2

concentration between WT and TrpV1KO and TrpV1KO/ASIC3KO mice (Figure 6C and D).

A similar increase and then decrease was observed for the tac1KO mice as was

observed with the WT mice (Figure 6B). However, a significant increased wet/dry ratio

was observed between WT and tac1KO mice at 15, 20, and 30% CO2 (F(3,168)=12.88,

p<0.0001).

When ISO was added to the duration of 30% CO2 treatment for wild-mice (Figure

7), a significant difference existed amongst the wet/dry lung weight ratios

(F(2,13)=77.49, p<0.0001). Tukey post hoc analysis found a significant attenuation of

the CO2 with ISO (M=4.97, SD=0.2612) compared to CO2 (M=6.832, SD=0.4361)

treatments (p<0.0001). Isoflurane did not abolished the hypercapnia-induced edema as

a significant difference remained between 0% CO2 (M=4.483, SD=0.1101) and 30%

CO2 with ISO (p=0.0408).

51

Figure 6. Lung weight ratios of mice after hypercapnia treatments. A. Significant

increase was observed in WT mice for 15, 20, 30 and 50% CO2. B. Significant difference existed between tac1 mice and WT mice at 15, 20, and 30% CO2. C. No significant difference existed between TrpV1KO and WT mice at any CO2 level. D. No significant difference existed between TrpV1KO/ASIC3KO mice at any CO2 level. Error bars are standard error of means. * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001 (One-way ANOVA, Dunnet post hoc test, N = 8).

52

Figure 7. Lung weight ratios of mice after hypercapnia and hypercapnia with

isoflurane. The addition of ISO with the CO2 treatment significantly attenuated the induced edema but was not abolished. Error bars are standard error of means. * = p<0.05; **** = p<0.0001 (One-way ANOVA; Tukey post hoc test; N = 4).

53

3.4.3 Lung lavage of wild-type mice

Lung lavage fluid was measured for SP+HK-1 content using an ELISA. A CO2

dose response of SP/K-1 lung airway content was done in WT mice (Figure 8). This is

the first publication of a CO2 dose response for SP+HK-1 in pulmonary airways. A

significant difference existed amongst the treatment groups (F(4, 10)=10.07, p=0.0016).

A significant difference was found between mice at 0% CO2 (M=0.7217, 95% CI [0.22,

1.22]) and 50% CO2 (M=81.9, 95% CI [-15.18, 179]). Due to the small sample size, no

other significance was detected (at p<0.05). However, the mean SP+HK-1 did show a

trend of increased SP+HK-1 concentrations at 15 (M=2.973, SD=1.558) and 30%

(M=39.33, SD=19.29) CO2 but not at 10% (M=0.7370, SD=0.2183) CO2. Despite a

decrease in wet/dry ratio of mice from 30% to 50% CO2, an even greater amount of

SP+HK-1 was observed at 50% than 30% CO2.

54

Figure 8. SP+HK-1 concentration in lung lavage fluid of wild-type mice after CO2

treatments. Means are reported above each treatment group. Significant difference exists between 0% and 50% CO2. Error bars are standard error of means. * = p<0.01 (one-way ANOVA, Tukey post hoc test; N = 3).

55

3.4.4 Lung lavage of tac1KO and TrpV1KO mice

The SP+HK-1 content of lung lavage fluid was measured in tac1KO (Figure 9A)

and TrpV1KO (Figure 9B) mice exposed to 0% and 30% CO2. Due to the lack of SP

expressed by tac1KO mice, the concentration measured in these mice were determined

to be only HK-1. No significant difference was measured between tac1KO and TrpV1KO

compared to WT (F(2,12)=1.067, at p<0.05). This indicated that most SP+HK-1

measured in WT mice is actually just HK-1 and absence of TrpV1 had no significant

effect on SP+HK-1 released in the lung (at p<0.05).

56

Figure 9. SP+HK-1 concentration in lung lavage fluid of tac1KO and TrpV1KO mice

after CO2 treatments. A. No significant difference was observed between tac1KO and WT mice. B. No significant difference was observed between TrpV1KO and WT mice. Error bars are standard error of means. N = 3.

57

3.4.5 Isoflurane attenuation of hypercapnia-induced SP+HK-1 release

Isoflurane was added to the course of the 30% CO2 exposure of WT mice. A

significant decrease of SP+HK-1 concentration in lung lavage fluid (Figure 10) was also

observed amongst the groups (F(2,6)=11.66, p=0.0086). Tukey post hoc analysis

revealed significant difference between 0% (M=0.7127, SD=0.2021) and 30%

(M=39.33, SD=19.29) CO2 (p=0.0129) but not between 30% CO2 with ISO (M=1.770,

SD=1.027) (at p<0.05).

58

Figure 10. SP+HK-1 concentration in lung lavage fluid with CO2 and CO2 with

isoflurane. ISO addition to CO2 treatment significantly reduced SP+HK-1 concentration in lung lavage fluid. Error bars are standard error of means. * = p<0.05 (One-way ANOVA; Tukey post hoc test; N = 3).

59

3.5 Discussion

Pulmonary c-fibers are able to detect hypercapnia-induced tissue acidosis.

However, the present study indicates that pulmonary c-fibers may not be involved in the

hypercapnia-induced pulmonary edema. Additionally, this study demonstrates that

hypercapnia induces HK-1 and not SP release in large amounts into the alveolar

airspace. The attenuation of both pulmonary edema and release of SP+HK-1 by ISO

during hypercapnia treatment implicates that hypercapnia-induced pulmonary edema is

mediated by activation of pulmonary cellular receptors.

Acidosis is a common feature of microenvironments of local inflammatory sites.

It induces inflammatory pain through acid-sensing molecules on sensory c-fibers.

Acidosis also potentiates c-fibers to other irritants [205]. The present study utilized the

reduced c-fiber acid sensitivity of TrpV1KO and TrpV1KO/ASIC3KO mice. No difference of

lung wet/dry weight ratios were observed between these and WT mice from

hypercapnia-induced pulmonary edema. Therefore, this result suggests that acid

sensing by the pulmonary c-fibers does not mediate the hypercapnia-induced

pulmonary edema.

All tachykinins are found in the lungs as well as their corresponding receptors

NK1, NK2, and NK3. By eliminating the encoding genes of the tachykinins, their

receptors, or by adding exogenous tachykinins and selective antagonists, the functions

of the tachykinins have been ascertained. To date, SP has not been eliminated without

the elimination of the splice variant NKA. Even still, their differential preferred affinities

to NK1r and NK2r has enabled their functions to be determined by addition of selective

antagonists. Gene elimination of HK-1 has only recently been done [178]. The present

60

study for the first time demonstrates that HK-1 is the predominant tachykinin agonist

associated with hypercapnia-induced pulmonary edema for the NK-1r in the alveolar

airspace. Pulmonary c-fibers or AECs have not been found to express HK-1 but HK-1

has been found in alveolar macrophages. We predict that the HK-1 we detected in lung

lavage fluid originates from alveolar macrophages at unstimulated states. Previous

studies have induced pulmonary physiological effects by administering exogenous SP

into airways or cell cultures. We propose that such effects may instead be that of HK-1

in vivo. Even still, their results can still be interpreted as NK1r activation effects.

The mode of action of inhaled anesthetics is not specific and can both excite and

inhibit neurons [140]. Inhaled anesthetics [206], intravenous barbituates [207], and

opiods [208] are known to reduce sensitivities to CO2. Anesthetics bind and deactivate

receptors and ion channels. The present study suggests inhibition of cells due to

decreased SP+HK-1 in the alveolar space after adding ISO with the hypercapnia

exposure. We do not know what is the specific target cell ISO inhibits to prevent

hypercapnia-induced edema and the SP+HK-1 release or whether the HK-1 cells are

downstream of the target cells in the inflammatory pathway. Another inhaled

anesthetic, sevoflurane, has shown to increased pulmonary leukotriene C4, NO3-, and

NO2- in lavage fluid of pigs [209]. The present study demonstrates ISO offers

protection against the inflammatory effects of hypercapnia.

We expected attenuation of hypercapnia-induced edema in tac1KO mice.

Previous studies have found a decreased pulmonary inflammation induced by sepsis in

these mice [210, 211]. They suggested a protective modulatory role of SP/NKA and our

results support this protective role. They later observed increased pro- and anti-

61

inflammatory cytokine expression in these mice [212, 213]. Perhaps in the present

study, increased edema observed in tac1KO mice is a result of the increased cytokine

production of these mice. Likewise, selective destruction of capsaicin afferents with

resiniferatoxin pretreatment enhanced pulmonary inflammation [214]. The effects of

eliminating SP content in the lungs may represent increased alveolar macrophage

activity.

A similar increase in edema was observed after 72 hours of hyperoxia in NK1rKO

mice [215]. Hyperoxia is known to be toxic to lung tissues [216-218]. Consistent with

our results, they found no difference between TrpV1KO and WT mice. Hyperoxia and

hypercapnia may induce lung inflammation by the same mechanism. Hyperoxia and

hypercapnia can induce the formation of ROS and RNS respectively. Therefore, both

may cause cellular stress inducing pulmonary edema. If hyperoxia and hypercapnia

induces pulmonary edema by the same mechanism, eliminating SP and NK1r

influences the involved cells in the same manner. NK1rKO mice in hyperoxia had

neutrophil accumulation in the subepithelial space with impaired transversion. This is

consistent of studies where activation of NK1r enables this transversion. Perhaps these

also have increased cytokine expression seen in tac1KO mice.

The cell inducers of pulmonary inflammation by hypoxia is considered to be

alveolar macrophages [188]. Often in environmental and pathophysiological conditions,

hypoxia occurs in conjunction with hypercapnia. In alveolar macrophages, CO2 directly

suppresses genes involved in regulating inflammation and innate immunity [31]. As

such, alveolar macrophages can also be the beginning inducers of hypercapnia-induced

62

pulmonary edema. Further supporting this is our observation of HK-1 as the dominant

NK1r agonist released into the alveolar airways after hypercapnia exposure.

The differential expression of SP and HK-1 demonstrates the function of distinct

cell types despite SP and HK-1 shared preference of NK1r binding. As has previously

been considered, some of the functions of SP have falsely been applied [175].

Therefore, evaluating the release of each tachykinin from a specific cell type will identify

their respective functions. Discovering the complexities of pulmonary cell signaling can

aid in the development of treatments of lung diseases.

4. DISCUSSION

4.1 Hypercapnia-Induced Cell Signaling

The premise of hypercapnia-induced pulmonary edema was that hypercapnia

acidosis would activate pulmonary c-fibers. These fibers would release SP into the

alveolar airspace and induce pulmonary edema. Additionally naked mole-rats, which

naturally lives in hypercapnia, would have attenuated response to hypercapnia-induced

pulmonary edema. The combined studies in this dissertation indicate that the c-fibers

are not the source of SP in the alveolar airways and that their acid sensitivity does not

contribute to the hypercapnia-induced edema. The combination of our observed

inhibition of hypercapnia-induced pulmonary edema by isoflurane and its attenuated

SP+HK-1 release in lung airways during a short 10 minute period indicates that ISO is

possibly inhibiting the activation of alveolar macrophages. This is could be due to

inhibition of cellular processes of the AECs leading to inhibited communication with

alveolar macrophages. Naked mole-rat absence of induced-edema and SP+HK-1 in

their lavage fluid indicates that naked mole-rats and ISO inhibition may share a common

pathway. Hypercapnia has known anti-inflammatory effects at low to moderate levels

[84]. However, extreme concentration of CO2 induces pulmonary edema, demonstrated

by others and by this dissertation. How the two can coexist may be explained by their

effects on specific cell types and their interactions together. CO2 in tissues is part of a

bigger balance of molecular species, some of which are toxic.

High levels of CO2 can lead to increased ROS and RNS. These molecules have

numerous damaging effects to cells. In particular, they will cause mitochondrial

dysfunction in AECs. Thus O2 consumption and ATP production will decrease [73].

63

64

The AECs respond by reducing their energy expenditure by removing their Na/K-

ATPase from their plasma membrane [219]. ERK [220], JNK [221], and PKA1a [222]

have also been identified in the CO2-induced down regulation of Na/K-ATPase in AEC

membranes. AECs can also respond to oxidative stress by disassembling their tight

junctions and adherins junction protein complexes [223]. ROS can also depolymerize

epithelial F-actin, perhaps due to F-actin turnover [224]. Perhaps these effects are the

AECs means of either trying to protect itself during oxidative stress or preparation of

apoptosis. Whether hypercapnia induces directly the AEC barrier permeability has yet

to be determined.

Alveolar macrophages are the major sentinel cells of the alveolar airspace that

initiates inflammatory reactions to invading organisms and damaged tissue [225].

Alveolar macrophages are in a constant balance of signaling molecules that keeps them

in a steady-state mode. However, when activated, they can release many pro-

inflammatory cytokines that can activate epithelial permeability. This indicates alveolar

macrophages as the potential target of hypercapnia induced inflammation. However,

hypercapnia reduces alveolar macrophage inflammatory functions as demonstrated as

reduced phagocytic activity and reduced pro-inflammatory gene expression. This

coincides with the observed reduced inflammation in studies of inflammation when

hypercapnia is added. Perhaps the hypercapnia concentrations in the present

dissertation overcome any potential reduction of inflammatory reaction of alveolar

macrophages. The current dissertation did not find reduced SP+HK-1 concentrations

due to 0% CO2 and up to 10% CO2 in the lung. This indicates that 10 minutes of

hypercapnia at sub-edema levels does not affect SP+HK-1 release. The increased

65

hypercapnia-induced edema in tac1KO mice coincides with the increased cytokine

release by these mice [212, 213]. SP has been suggested to function as an autocrine

signal for macrophages [226]. Therefore, eliminating SP may disrupt this regulation and

cause macrophages to induce a greater level of inflammation in tac1KO mice. Further

investigation will be needed to determine what is altered in the alveolar macrophages of

these mice.

Naked mole-rat extreme resistance to hypercapnia-induced pulmonary edema

may uncover the mechanism by which hypercapnia induces pulmonary edema. A

simple explanation would be that naked mole-rats have greater protection from ROS

and RNS and thus are protected by hypercapnia effects. This was found to not be the

case and they even have reduced levels of antioxidants [227] and their proteins are

riddled with oxidative damage [228]. To compensate for the damaged proteins, naked

mole-rats have a much higher rate of cellular protein recycling, mediated by their

proteasome activity [229].

Recently Naked mole-rats have been found to contain a high-molecular-mass

hyaluronan (HA) that is five times larger than the human or mouse form [230]. This is a

disaccharide glucuronic acid/N-acetylglucosamine polymer and one of the main

components of the extracellular matrix [231]. This polymer is involved in cell signaling

and is dependent on its molecular length. Naked mole-rats express low levels of HA

degrading enzymes and a unique sequence of HA synthase 2 that is responsible for

their HA size.

Naked mole-rats do not have more HA than mice in their lungs [230]. However,

high-molecular-mass HA has anti-inflammatory properties and represses mitogenic

66

signaling [232]. Hyaluronan binds with the CD44 receptor [231, 233, 234]. When HA is

fragmented, it induces chemokine gene expression in alveolar macrophages [235].

Naked mole-rat cells have a twofold higher affinity for HA than mouse and human cells,

suggesting higher sensitivity of naked mole-rat cells to HA signaling [230]. Intercellular

domain of the CD44 receptor interacts with NF2 that mediates contact inhibition of cell

proliferation [236].

Hyaluronan and LPS both activate alveolar macrophages through toll-like

receptor 4. This is attributed to structural similarities HA and LPS have. LPS is

expressed by gram-negative bacteria and is part of the pathogen-associated molecular

patterns recognized by immune cells by TLR4 binding [237]. TLR4 activation induces

the generation of intracellular ROS. These ROS and cytokines secreted by alveolar

macrophages can induce epithelial membrane permeability and neutrophil recruitment.

Previous studies have found that ISO pretreatment attenuates the inflammation induced

by LPS. Our finding that ISO inhibits the pulmonary inflammation induced by

hypercapnia is supported by ISO anti-inflammatory action in macrophages. This is

mediated by ISO upregulating Ho-1 activity. In AECs, ISO was found to decrease the

transcription level of key tight junction protein ZO-1, indicating ISO having an effect on

AECs [238]. This effect may be mediated by ISO activating AECII GABAA receptors

[147].

The idea that AECs may act as the inducers of alveolar macrophage

inflammatory response from hypercapnia is supported by the anti-inflammatory

properties hypercapnia has directly on alveolar macrophages. Hypercapnia (10-20%)

reduces LPS-stimulated release of TNF by rat alveolar macrophages [239]. Following

67

this experiment, the IL-6 and TNF mRNA and protein expression was inhibited by

hypercapnia in both mouse and human cell lines. All of this was independent of

acidosis [35]. The protection hypercapnia provides in LPS stimulation may be due to

the CO2-sensitive RelB. This member of the NK-kB family is cleaved and translocates

to the nucleus in response to hypercapnia. This elevated nuclear RelB correlated with

the hypercapnia-induced protection [240].

Hypercapnia may have the same effect as hypoxia in inducing HA fragmentation.

Hypoxia can up-regulate hyaluronidase that can increase the transformation of HA into

fragments [241]. Naked mole-rats reduced expression of hyaluronidase may counteract

the hypoxia-induced up-regulation of hyaluronidase. However, whether hypercapnia

up-regulates hyaluronidase or if naked mole-rats up-regulate this enzyme in response to

hypoxia or hypercapnia is still unknown. We would predict that naked mole-rats would

not have increased hyaluronidase activity in response to hypoxia and hypercapnia,

thereby not developing pulmonary inflammation. Likewise, hypercapnia in mice would

increase hyaluronidase activity and thus activate a pro-inflammatory response in mouse

alveolar macrophages.

The increased size of HA in naked mole-rats is hypothesized to play a role in

cancer resistance of naked mole-rats and thus part of their longevity [230]. However,

we propose that naked mole-rats have evolved high-molecular-mass HA to inhibit

constant inflammatory reaction of hypercapnia in their burrows. An alternative

hypothesis is that this is an adaptation to prevent excessive inflammatory reactions to

subterranean parasites in their burrows. Naked mole-rats are constantly being exposed

to pathogens in their subterranean burrows. Naked mole-rats are known to have

68

several parasites that are present in their fecal pellets [242-245]. Reduced

inflammatory reactions to these pathogens would be advantageous for avoiding a

constant state of inflammation in naked mole-rat skin as well as their respiratory system.

Naked mole-rats dig with their teeth and could easily breathe in these parasites. They

are also coprophagic, thereby re-ingesting the parasites present in their feces. Both

hypotheses are supported by our finding that SP+HK-1 was undetectable in naked

mole-rat lavage fluid. This could either indicate a strong repression of pro-inflammatory

genes and/or their release by naked mole-rat alveolar macrophages. Naked mole-rats

may not even express SP+HK-1 in their alveolar macrophages. Whether HA is involved

in alveolar macrophage basal levels of SP+HK-1 requires further investigation.

4.2 Future Directions

Hypercapnia may be activating inflammation directly by activating alveolar

macrophages but the anti-inflammatory properties on alveolar macrophages suggests

extracellular signals perhaps from AECs. We propose to determine if hypercapnia

induces HA fragmentation as the pro-inflammatory cue. The threshold of hypercapnia-

induced pulmonary edema may represent a threshold of HA fragment concentration or

HA length before initiating inflammation. Perhaps this would be representative of

hyaluronidase activity or ROS concentration. HO-1 activity induced by ISO in AECs is

necessary to determine whether ISO only inhibits hypercapnia-induced alveolar

macrophage activation. The ISO-induced activation of HO-1may be receptor specific

and thus has no effect on AECs. Alternatively, maybe activation of GABAA receptors is

the trigger for the downstream activation of HO-1. If AECs are unaffected by ISO, then

69

hypercapnia-induced AEC permeability is likely attributed to alveolar macrophage

cytokine release.

Naked mole-rats have several adaptations that are attributed to living in their

natural environment. Therefore, their HA may not be the only difference that prevents

hypercapnia-induced pulmonary edema. The naked mole-rat HA is attributed to being

the only difference that explains their resistance to developing cancer [230]. This study

also found high-molecular-mass HA in the blind mole-rat. However, blind mole-rats

have been found to possess another mechanism to prevent cancer. They have a

mutation in their tumor suppressor protein p53 to prevent cells from undergoing

apoptosis. To counter over-proliferation, their cells secrete interferon-beta to trigger

cells to undergo necrosis [246]. This study did not find this to be the case for naked

mole-rats but they did take note of the increased contact inhibition. Blind mole-rats are

subterranean and also naturally live in hypoxia/hypercapnia. Thus these animals could

be used to confirm that high molecular mass HA protects lungs from hypercapnia-

induced inflammation.

Many pathophysiological conditions develop cellular hypoxia/hypercapnia

environments and most mammalian cells respond to these environments. As such,

these conditions can induce inflammation in a wide variety of tissues. Understanding

the complex extracellular and intracellular signaling of inflammation will contribute to

developing effective pathophysiological treatments. Naked mole-rats possess a unique

set of adaptations to cope with their natural environment. How extensive their

differences are could reveal more about the signal cascades of hypercapnia-induced

edema.

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APPENDIX

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Office of Animal Care and Institutional Biosafety Committee (OACIB) (M/C 672) Office of the Vice Chancellor for Research 206 Administrative Office Building 1737 West Polk Street Chicago, Illinois 60612

Phone (312) 996-1972 • Fax (312) 996-9088

7/17/2013 Thomas J. Park Biological Sciences M/C 067 Dear Dr. Park: The protocol indicated below was reviewed in accordance with the Animal Care Policies and Procedures of the University of Illinois at Chicago and will be renewed on 7/17/2013. Title of Application: Effects of Hypoxia, Anoxia, and Hypercapnia on the Naked Mole-Rat ACC NO: 12-120 Original Protocol Approval: 8/18/2012 (3 year approval with annual continuation required). Current Approval Period: 7/17/2013 to 7/17/2014 Funding: Portions of this protocol are supported by the funding sources indicated in the table below. Number of funding sources: 1 Funding Agency Funding Title Portion of Funding Matched NSF- National Science Fdtn.

Neurobiology of the Naked Mole-Rat Protocol is linked to form G 11-146

Funding Number Current Status UIC PAF NO. Performance Site Funding PI 0744979 Funded 2008-00469 UIC Thomas Park This institution has Animal Welfare Assurance Number A3460.01 on file with the Office of Laboratory Animal Welfare, NIH. This letter may only be provided as proof of IACUC approval for those specific funding sources listed above in which all portions of the grant are matched to this ACC protocol. Thank you for complying with the Animal Care Policies and Procedures of the UIC. Sincerely,

Bradley Merrill, PhD Chair, Animal Care Committee BM/kg cc: BRL, ACC File

VITA

Name: Gregory Richard Charles Blass

Education: B.S., Zoology, University of Oklahoma, Norman, OK, 2005

Ph.D., Biological Sciences, University of Illinois at Chicago, Chicago, IL, 2014 Publications: Smith, Ewan, Gregory RC Blass, Gary R. Lewis, Thomas J Park.

Absence of histamine-induced itch in the African naked mole-rat and “rescue” by Substance P. Molecular Pain, May 2010. 6 (29): pg. 1-6.

Blass G.R.C., Gaffin D.D. Light wavelength biases of scorpions.

Animal Behaviour, August 2008. 76 (2): pg. 365-373. Abstracts: Blass, G.R.C., E. S. J. Smith, G. R. Lewin, R. D. Minshall, and T. J.

Park: Resistance to CO2-induced pulmonary edema in naked mole-rats and free-tailed bats. 39th Annual Meeting of the Society of Neuroscience. Oct 18-21, 2009.

Blass, G.R.C., J. A. Ruttencutter, M. E. Schirementi, and T. J. Park: Histamine induced Substance P dependent pruritus in naked mole-rats. 37th Annual Meeting of the Society of Neuroscience. Nov 3-7, 2007.

Blass, G.R.C. and Gaffin D.D.: Scorpion locomotion regulation by light wavelengths. Annual Meeting of the American Arachnological Society. June 17-21, 2006.

Blass, G.R.C. and Gaffin D.D.: Light wavelength biases of the desert grassland scorpion. Annual Meeting of the American Arachnological Society. 2005.

Teaching: University of Illinois at Chicago: Teaching Assistant 2006 - 2014 Animal Physiology, Biology of the Brain, Cell Biology,

Neuroscience 1, Homeostasis, Developmental Biology, Microbiology, Intro to Biology

Guest Lecturer for Animal Physiology 2009 – 2014 Individual mentor for 4 undergraduate students University of Oklahoma:

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Teaching Assistant 2005 - 2006 Introduction to Zoology, Human Physiology Departmental Tutor 2002 – 2005 Awards and Honors: 2009: Teaching excellence award at UIC 2006: Phi Beta Kappa membership 2006: UROD presentation award 2006: OU Zoology Department Outstanding Senior Student award 2004: Undergraduate Research Opportunity Program grant 2002: Adams Summer Research scholarship Professional Memberships: American Arachnological Society Society for Neuroscience

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