Corrélation entre l'expression de HIF tronc cérébral

et la réponse ventilatoire à l'hypoxie chez les rats et les

souris

Mémoire

Manju Shahare

Maîtrise en Neurobiologie

Maître ès sciences (M.Sc.)

Québec, Canada

© Manju Shahare, 2016

Corrélation entre l'expression de HIF tronc cérébral

et la réponse ventilatoire à l'hypoxie chez les rats et les

souris

Mémoire

Manju Shahare

Sous la direction de

Dr. Jorge Soliz

Département de Médecine,

Université Laval,

Québec, Canada.

iii

RÉSUMÉ Compte tenu de la faible disponibilité de l’oxygène (hypoxie) en haute altitude, l’adaptation à ce

milieu constitue un vrai défi pour les espèces adaptées au niveau de la mer. Aussi, le rat et la

souris constituent un modèle pertinent pour la compréhension des facteurs qui contribuent à une

bonne adaptation en haute altitude. En effet, les rats et les souris de laboratoire élevées à haute

altitude durant plusieurs générations possèdent un phénotype différent: les souris présentent une

plus importante ventilation, des valeurs d’hématocrite/ hémoglobine diminuées et une

hypertension pulmonaire réduite. Ces différences indiquent une mauvaise adaptation des rats qui

montrent également une importante mortalité en haute altitude. Néanmoins, les mécanismes

impliqués dans cette différence entre ces deux espèces ne sont pas connus.

Nous avons donc recherché dans un premier temps si les différences observées entre rats et

souris sont également présentent au niveau de la mer, puis dans un second temps si cela avait un

lien avec l'expression du senseur moléculaire d'oxygène HIF (Hypoxia Inducible Factor).

Nous avons mené une étude au niveau de la mer (Québec, Canada - 98m) pour comparer les

réponses ventilatoire et moléculaire entre les rats et les souris. Pour se faire, les animaux sont

exposés pendant 6 heures à différents gradients d’oxygène :

21%, 15%, et 12% O2. La ventilation est mesurée par pléthysmographie à corps entier. La

consommation d’oxygène (VO2) et la production de CO2 (VCO2) sont évaluées durant la même

période d’exposition. Après 6 heures d’exposition, les animaux sont anesthésiés et le tronc

cérébral rapidement prélevé pour effectuer une mesure de l’expression de HIF-1α à l’aide de la

technique ELISA (Enzyme LinkImmunosorbent Assay).

Comparé aux rats, les souris présentent une élévation du débit ventilatoire, une diminution de la

VO2 et de la VCO2 et une augmentation de l’équivalant ventilatoire à l’O2 (Ve/VO2) et au CO2

(Ve/VCO2) durant l’exposition à 15 et 12% d’O2. De plus, l’expression de HIF-1α au niveau du

tronc cérébral est plus élevée chez les souris en comparaison à celui des rats.

En conclusion, la différence de la réponse ventilatoire à l’hypoxie peut être liée à la différence

d’expression de HIF-1α au niveau du tronc cérébral. Ces resultantssuggèrent que les souris

possèdent une prédisposition génétique permettant une réponse adaptée en milieu hypoxique et

pouvant aisément expliquer la facilité que possède cette espèce à survivre et à établir sa colonie

en haute altitude.

Mot clés : Hypoxie, haute altitude, réponse ventilatoire à l’hypoxie, tronc cérébral,HIF.

iv

ABSTRACT

Successful adaptation at high altitude is very challenging for sea level natives due to the

low level of available oxygen (hypoxia). Rats and mice offer an interesting model to understand

the factors that contribute to efficient adaptation to high altitude. Indeed, laboratory rats and mice

that have been raised at high altitude for several generations have a different phenotype with

mice showing higher ventilation, lower hematocrit/hemoglobin values, and lower pulmonary

hypertension. These differences are clearly a failure of adaptation to high altitude in rats, as

underlined by data showing high mortality in the colony of high altitude rats. However the

underlying mechanisms behind these differences are poorly understood. We sought to address

whether these differences are also apparent in mice and rats living at Sea level, and if they are

related to different responses of the O2 molecular sensor HIF (Hypoxia Inducible Factor).

To test these hypotheses, we chose to perform the study at sea level i.e. at Quebec City,

Canada (98m) to compare the ventilatory and molecular responses in male rats and mice. The

animals were exposed to different oxygen gradients 21%O2, 15 % O2 and 12% O2 for 6 hours.

Ventilation was measured by whole bodyplethysmography, oxygen consumption (VO2) and CO2

production rate (VCO2) were also measured during the exposure. After the 6 hour’s exposure, the

animals were anesthetised, and the brainstem quickly dissected, Brainstem HIF-1α expression

was measured by Enzyme Link Immunosorbent Assay (ELISA).

Compared to rats, mice had higher minute ventilation, lower VO2, VCO2, and higher

ventilatory equivalent to oxygen and carbon dioxide, (Ve/VO2,Ve/VCO2) at 15% and 12% O2. In

addition, mice also had higher brainstem HIF-1α expression compared to rats.

We conclude that the differences in ventilatory responses to hypoxia at sea level might be

due to differences in expression of HIF-1α in the brainstem. This suggests that mice have a

genetic pre-disposition that ensure adequate response to hypoxia. This trait helps to explain that

mice are able to survive and successfully establish natural colonies at high altitude.

Key Words: Hypoxia, high altitude, hypoxic ventilatory response, Brainstem, HIF.

v

TABLE OF CONTENTS

CONTENTS

Pg. No.

Résumé iii

Abstract iv

Table of Contents v

List of tables viii

List of figures ix

Abbreviations x

Acknowledgement xii

Presentation and Participations xiii

I- INTRODUCTION 1

1. Hypoxia 2

1. 1 High altitude Physiology 3

1.2 The ventilatory response to hypoxia 6

1.2.1 The peripheral chemoreceptors: the carotid bodies 7

1.2.2 Time domains of the hypoxic ventilatory response 9

1.3 The metabolic response to hypoxia 11

1.4 Molecular response to hypoxia 12

1.4.1 Structure and composition of HIF 12

1.4.2 Regulation of oxygen homeostasis by HIF-1 α 13

1.4.3 HIF-1α target genes 15

1.4.3.a Epo is HIF-1 target genes involved in erythropoiesis and ventilatory response

to hypoxia

18

1.4.3.b HIF-1 target genes involved in glucose metabolism 18

1.4.3.c HIF-1 target genes involved in Angiogenesis 19

1.4.3.d HIF-1 target genes involved in Cell Proliferation/Survival 20

vi

1.5 Induction of Brainstem HIF-1α expression in response to hypoxia 21

II- OBJECTIVE AND HYPOTHESIS OF THE STUDY 22

III- MATERIALS AND METHODS 24

3.1Animals 25

3.2 Experimental Groups 25

3.3 Measurement of Respiratory and metabolic parameters 25

3.3.1 Whole body Plethysmography 25

i) Experimental setup 25

ii) Plethysmoraphy recordings 26

3.3.2 Analysis of respiratory and metabolic parameters 28

3.4 Allometric scaling 29

3.5 Tissue Sampling 30

3.6 Measurement of molecular parameters 30

3.6.1 Nuclear Protein extraction (HIF-1 α from brainstem) 30

3.6.2 Enzyme Link Immunosorbent Assay 31

3.7 Statistical Analysis 32

IV- RESULTS 33

4.1.Adult mice had had higher minute ventilation than rats during hypoxic exposure 34

4.1.1 Mass-specific values 35

4.1.2 Mass-corrected values 36

4.2. Adult mice had lower O2 consumption (V.O2), CO2 production rate (V.CO2),

(V.CO2/V.O2) and higher ventilatory equivalent for oxygen (V.e/ V.O2) and for carbon

dioxide exchange ( V.e/V.CO2) than rats

38

4.3 Adult mice had higher brainstem HIF-1 alpha expression than rats with decreasing

O2 concentration/hypoxic exposure

40

V- DISCUSSION 41

5.1 Limitation of the experimental approach 42

5.2 Higher ventilation and reduced metabolic rate allows hyperventilation in mice

exposed to hypoxia

43

5.3 Higher Brainstem HIF-1 alpha expression link to better ventilatory acclimatization

in mice

45

5.4 Contribution of peripheral chemoreceptor to modulate pulmonary ventilation in

mice

46

vii

VI- CONCLUSION AND FUTURE PROSPECTIVE

48

VII-REFERENCES

50

viii

LIST OF TABLES

TABLES

Pg. No.

Table.1: Selected HIF-1 target genes 16

Table. 2: Mass specific values for respiratory parameter in rats and mice 34

Table. 3: Mass specific data for metabolic variables in rats and mice 35

ix

LIST OF FIGURES

FIGURES

Pg.No.

Fig.1: The "cascade of oxygen" at sea level and high altitude 4

Fig.2: Comparative physiology in rats and mice at high altitude 6

Fig.3: Control of breathing 8

Fig.4: Time domains of hypoxic ventilatory response 9

Fig.5: Structure and composition of HIF and their isomers 13

Fig.6: Schematic illustration of HIF-1α regulation 15

Fig.7: Schematic representation of whole-body plethysmography 26

Fig.8: Ventilatory variables at different oxygen gradients in rats and mice 37

Fig. 9: Metabolic variables in adult rats and mice (2-3 months old) 39

Fig.10: Expression of brainstem HIF-1 alpha in adult rats and mice 40

Fig.11: Bidimensional plots for ˙Ve vs. ˙VO2 showing the effect of hypoxia in rats and

mice

44

Fig.12: Bidimensional plots for ˙Ve vs. ˙VCO2 showing the effect of hypoxia in mice

and rats

45

x

ABBREVIATIONS

O2= Dioxygen

CO2= Carbon dioxide

PO2= Partial pressure of oxygen

PaO2= Arterial partial pressure of oxygen

PaCO2= Arterial partial pressure of Carbon dioxide

pH2O= Vapor pressure of water

V.e = Minute Ventilation

Vt= Tidal volume

Fr= Respiratory frequency

V.O2= Volume of O2Consumed

V.CO2= Volume of CO2 produced

V.CO2/V.O2=Respiratoryexchangeratio

V.e/V.O2= Ventilatory equivalent to O2

V.e/V.CO2= Ventilatory equivalent to CO2

HIF= Hypoxia Inducible Factor EPO= Erythropoietin

VEGF= Vascular endothelial growth factor

GLUT-1= Glucose transporter -1 CRLR= Calcitonin Receptor Like Receptor ANGPT2= Angiopoietin 2 NOS2a= Inducible nitric-oxyde synthase pfkfb3= 6 phosphofructo-2-kinase

PDK1= Pyruvate dehydrogenase kinase 1 PRKAA1= AMP-activated alpha 1 catalytic subunit. IGF-1= Insulin-like growth factor 1

xi

ATP= Adenosine triphosphate PHD= Prolyl hydroxylases bHLH= basic helix–loop–helix

ODDD= O2-dependent degradation domain NAD= N-terminal activation domain CAD= C-terminal activation domain HRE= Hypoxia-responsive element 2OG= 2-oxoglutarate pVHL= Von Hippel–Lindau protein

xii

ACKNOWLEDGEMENT

I would never have been able to finish my dissertation without the guidance of my Supervisor,

committee members, friends, and my family. I would like to first thank my family for all their love and support. I owe the fact that their faith

and confidence allows me this far to pursue my studies. They were always encouraging me with

their best wishes.

I would like to express my sincere gratitude to my advisor, Dr. Jorge Soliz, for his encouraging

guidance, persistence, patience, and providing me with an excellent atmosphere for doing

research. Without your motivation, enthusiasm, immense knowledge and financial support

during my research I wouldn’t have been able to successfully take this step towards finishing my

education.

In addition, I owe deepest gratitude to Dr. Vincent Joseph for providing excellent hands on

training, advices and useful suggestions during study, without which I could not have succeeded.

I would like to also thank Dr. Richard Kinkead and Dr. Aida Bairam for providing the beautiful

educational environment during my stay in the lab. Last but not the least my friends Sandeep, Praveena, Shahid, Louana, Alexandra, who help me

professionally and emotionally by cheering me up and stood by me through the good times and

bad.

Finally, I would like to thank my all the lab members for their constant support and help.

xiii

PARTICIPATIONS AND PRESENTATIONS

This research thesis was performed as part of my ‘Masters in Neurobiology’. This offered me the

pinnacle of experiences and training reward. In addition to learning in the wonderful world of

research and to provide a solid theoretical and practical training, my masters also allowed me to

participate in various presentations, includes:

HIF: Molecular determinants of adaptation at high altitude in rats and mice. ManjuShahare,

Jorge Soliz and Vincent Joseph at DIXIEME COLLOQUE SCIENTIFICETUDIANT DE LA

SOCIETE LEGALLOIS POUR L’ETUDE DE CONTROLERESPIRATOIRE (SLECR). La

Jouvence, Orford, Quebec, Canada. 6-9th February 2014.

Correlation between brainstem HIF expression and ventilatory response to hypoxia in rats and

mice.Manju Shahare, Jorge Soliz and Vincent Joseph at ONZIEME COLLOQUE SCIENTIFIC

ETUDIANT DE LA SOCIETE LEGALLOIS POURL’ETUDE DE CONTROLE

RESPIRATOIRE (SLECR). La Jouvence, Orford, Quebec, Canada. 7-9th February 2015.

1

I - INTRODUCTION

2

1. Hypoxia

Oxygen (O2) is essential for the survival of almost all forms of life on earth. A balanced

oxygen environment is required for normal cellular and metabolic functions since either an

increase or a decrease in the oxygen levels can be detrimental to the cells by disrupting the

oxygen homeostasis. (Semenza, 2012a; Semenza, 2012b). Hypoxia may be defined as a relative

deficiency in oxygen availability/delivery for maintaining adequate physiological oxygen

tensions, and thus resulting in an imbalance between demand and supply of oxygen (Hopfl et al.,

2004; Lu and Kang 2010). A change in the oxygen environment will trigger a cascade of

physiologic and biochemical events to compensate the reduced O2 pressure. If these events are

not able to adequately compensate the reduced O2 pressure, pathological processes may develop

and affect enzyme activities, mitochondrial function, cytoskeletal structure, membrane transport,

and antioxidant defenses. In all cases, limited oxygen availability decreases oxidative

phosphorylation resulting in a decreased synthesis of energy-rich phosphates eg. Adenosine

triphosphate (ATP), and limitation of physical and mental activities (Maltepe and Saugstad,

2009).

In mammals, many factors can contribute to this oxygen imbalance, such as a decreased

concentration of functional hemoglobin or a reduced number of erythrocytes that impaired the

ability of blood to carry oxygen to the tissue (anemic hypoxia), an inability of cells to take up or

utilize oxygen from the blood stream (histotoxic hypoxia), reduced tissue perfusion (ischemic

hypoxia) and an insufficient oxygen availability to the lungs (hypoxemia/hypoxic hypoxia).

(Hockel and Vaupel 2001). Hypoxic hypoxia occurs when the partial pressure of O2 (PO2) in

arterial blood falls. This could be caused by several factors such as respiratory problems e.g.,

hypoventilation, diffusion impairment, caused by pulmonary edema, ventilation– perfusion

mismatch or anatomic shunt of blood past the gas exchange region, blocked airways, drowning

or reduction of the oxygen partial pressure in the environment, as occurring at high altitude.

3

In response to hypoxia, most animal species are able to alter their physiology to increase

the uptake of oxygen from the lungs (by increasing their ventilation), and to optimize its cellular

utilization through the activation of specialized cellular and molecular O2 sensors distributed

throughout the body.

In the present study we compared the respiratory and molecular responses to acute

exposure to hypoxic hypoxia in rats and mice. Interestingly, these two species have a different

tolerance to high altitude (see below) and we sought to address whether these differences in

responses are also apparent in mice and rats living at sea level, and if they are related to different

responses of the O2 molecular sensor. Accordingly, we will first describe how the central and

peripheral chemoreceptors in nervous system control ventilation under hypoxic condition, and

how the Hypoxia-Inducible-Factor 1 (HIF-1 – the main molecular O2 sensor) is regulated by O2

level.

1. 1 High altitude Physiology

Altitude refers to the terrestrial elevation over 1500 m and is commonly divided into high

altitude: 1500 to 3500 m, very high altitude: 3500 to 5500 m and extreme altitude: 5500 to 8850

m (Gallagher, 2004). At altitude, the fraction of O2 in the air remains the same, but as barometric

pressure decreases, the partial pressure of O2 decreases with altitude. Consequently, there is less

O2 available to breath at high altitude. Because the atmospheric PO2 is lower at high altitude, the

gradient driving O2 transport from the atmosphere to the cells is considerably less than at sea

level, and the fall of partial pressure of O2 at each consecutive step in the O2 cascade is less at

high altitude than at sea level (Fig.1).

4

(Torrance et al., 1970)

Fig.1: The "cascade of oxygen" at sea level and high altitude.

The y-axis represents the partial pressure of oxygen in mm Hg (left) or kilopascal

(‘Kpa’ right) in different compartments of the respiratory system (ambient air,

alveolar gas, arterial blood, mixed venous blood). The blue line indicates the drop in

partial pressure of oxygen at sea level and at high altitude (4500m).

Exposure to hypoxia induces different responses such as increased pulmonary ventilation and

hematocrit values, or induction of pulmonary hypertension among others. In species living at

high altitude, a good adaptation is generally indicated by high hemoglobin–oxygen affinity, high

pulmonary ventilation, low hematocrit and low pulmonary hypertension. In human, Tibetan have

been living in the Tibetan plateau (~4000m) for at least 25,000 years and Andeans (3000-4000

m) for 12,000 years, also showing successful adaptation at high altitude. These two populations

are adapted to life in a hypoxic environment to support their maintenance, growth, development

and reproduction with different traits (Petousi et al., 2013). Tibetans have lived on the Qinghai–

Tibetan plateau for many years and have developed unique phenotypes, such as protection from

polycythemia which has been linked further to prolyl hydroxylases 2 (PHD2) mutation, resulting

in the down regulation of the hypoxia inducible factor (HIF) pathway. (Tashi et al.,2014).

Different species of animals have been living at high altitude under hypoxic condition for an

extended period of time such as pika from the Tibetan plateau, Ilama in the Andes and

Himalayas‟ yak. These animals are endemic to the high altitude regions, and are

5

thereforeconsidered adapted to high altitude. It is believed that high altitude exerts selective

evolutionary pressure primarily due to its hypoxic environment, resulting in multiple adaptive

responses over the course of generations. Among animals that are not endemic to high altitude,

but have been brought by human migrations mice and rats are an interesting case. According to

ecological reports of South America, mice can be found at 4000-5000m of altitude but rats are

noticeably absent above 2000m-2500m (Sydney, 1997).

A recent comparative study at high altitude using rats and mice at the Bolivian institute

for altitude biology (IBBA - La Paz, altitude 3600 m), which have been originally imported from

France (IFFA-CREDO) in 1992 (23 years ago), and constantly bred at 3600 m from almost 30

generations, reported lower hemoglobin and hematocrit values, higher ventilation and lung

volume with lower pulmonary hypertension in mice compared to rats (Lemoine et al., 2015). In

rats, poor adaptation to altitude leads to high hematocrit and hemoglobin values, higher

pulmonary hypertension, lower metabolic rate and high mortality (Fig.2) (Lumbroso et al., 2012;

Lemoine etal., 2015).

Mice also had a higher ventilation and tidal volume compared to rats,which are likely

linked to differences in the oxygen sensing abilities of the respiratory control system to maintain

pulmonary ventilation under hypoxic condition. One of the intriguing questions that emerged

from these studies was to know whether this “resistance” to hypoxia is a pre-determined trait

characterizing rats and mice. Given that these 2 species are widely used worldwide to study

hypoxic responses for bio-medical research, this question does not appear trivial. As a first

approach, we sought to determine whether rats and mice have different responses to hypoxia at

the physiological (ventilatory response to hypoxia), and molecular level (expression of HIF-1).

6

(Lemoine et al., 2015)

Fig. 2: Comparative physiology in rats and mice at high altitude.

This figure describes the physiological differences in rats and mice living at high

altitude. Compared to mice, rats had high level of hematocrit/hemoglobin values,

very high pulmonary hypertension and lower metabolic rate. Mice had higher

ventilation and higher lung volume than rats.

1.2 The ventilatory response to hypoxia

The mammalian lungs have an impressing capacity to increase ventilation about 20 times higher

than the resting ventilation, therefore being able to fulfill O2 needs under extreme conditions

(Ganong, 1997). Hyperventilation is the first response to the reduction of environmental oxygen,

and is the most important feature of acclimatization to high altitude (Joseph and Pequignot,

2009). The hypoxic ventilatory response (HVR) is important in respiratory physiology since it

reflects the global output of a neurological system that integrates the hypoxic stimulation of

peripheral sensors (carotid bodies), the central translation of peripheral inputs to the phrenic

nerve and the metabolic response of the organism.

7

1.2.1 The peripheral chemoreceptors: the carotid bodies

The main peripheral chemoreceptors identified in mammals are the aortic and carotid

bodies. However, only carotid bodies have a significant influence in small mammals such as

mice and rats (Gonzalez et al., 1994). Carotid bodies are small bilateral organs situated near the

carotid bifurcation that permanently measure oxygen arterial pressure. While the main stimulus

of carotid bodies is the decline of PaO2, carotid bodies are also stimulated by other physical or

chemical components in blood such as PaCO2, pH, temperature, arterial flow and pressure or

osmolarity (Kumar and Prabhakaran, 2012; Gonzalez et al., 1994).

The carotid bodies are the most vascularized organs in the body (five times higher than

brain (Gonzalez et al. 1994), and are highly innervated by efferent sensory fibers and afferent

fibers from the autonomic nervous system. The main innervation comes from the carotid sinus

nerve, a branch of the glossopharyngeal nerve that contains fibers proceeding from the petrosal

ganglion. The carotid sinus nerve conveys the sensory information generated by the activation of

the carotid body, and sends this information to the nucleus tractus solitarius (NTS), located in the

dorsomedial medulla (in the brainstem) (Fig. 3). The NTS is the entry point of a variety of

visceral sensory afferent inputs, including pulmonary mechanoreceptors, peripheral

chemoreceptors, and others (Finley et al., 1992). The NTS is connected to the major respiratory

centers of the ventrolateral medulla, also in the brainstem, and the activation of NTS neurons by

carotid sinus nerve afferents contributes to increase the activity of the neuronal network that

control ventilation, also named the central respiratory command (CRC). The carotid bodies also

receive parasympathetic and sympathetic efferent innervation, the last one through small

ganglio-glomerular nerves coming from the superior cervical ganglion (Kumar and Prabhakaran,

2012; Gonzalez et al., 1994).

8

(Campbell and Reece, 2005)

Fig.3: Control of breathing.

This figure describes the role of peripheralchemoreceptors in control of breathing.

The sensors in the aorta and carotid arteries detect changes in oxygen level in the

blood and signal the pons and medulla of the brainstem to increase the breathing

rate when oxygen level becomes low.

Structurally, carotid bodies contain two types of cells, type I and type II, surrounded by

fenestrated sinusoidal capillaries and conjunctive tissue (McDonald etal., 1981; Gonzalez et al.,

1994). The type I or glomus cells are chemosensitive andhave a neural origin (Kondo et al.,

1982). These cells look like classical neurosecretory cells and their morphology resembles that of

the chromaffine adrenal medullar cells. The glomus cells have dense-core granules containing

neurotransmitters and neuromodulators that are released upon exposure to hypoxia. The principal

transmitters between chemosensitive cells and nerve endings in the carotid bodies appear to be

acetylcholine and ATP (Zhang et al., 2000; Rong et al., 2003), which depolarize the Hering‟s

nerve ending though post-synaptic purinergic and nicotinic receptors. Dopamine appears to be a

potent modulator, mainly inhibitor, of the response of the chemosensitive cells to hypoxic

exposure ( Gonzalezet al., 1995). Surrounding the glomus cells are the type II cells. These cells

do not have vesicles and morphologically resemble glial cells of theperipheral nervous system.

These cells are also called sustentacular cells (Kondo et al., 1982).

9

1.2.2 Time domains of the hypoxic ventilatory response

HVR involves complex interplay between several physiological distinct mechanisms

which differ by various features, there are: (1) the specific stimuli that elicit them (e.g. pattern

and intensity of hypoxic exposure); (2) the time course ofthe response (seconds to years);

(Fig.4). (3) The effects on the components of ventilation (tidal volume vs. frequency); (4) the

direction of their effects (facilitation vs. depression); and (5) the neurochemicals necessary for

their manifestation. (Powell et al., 1998).

A

B

(Powell et al., 1998)

Fig.4: Time domains of hypoxic ventilatory response.

Figure A describes ventilatory responses after a brief (seceonds to minutes) hypoxic

exposure (Acute response (AR), short-term potentiation (STP), short-term depression

(STD)).Figure B describes the ventilatory responses during prolonged hypoxic exposures

include hypoxic ventilatory decline (HVD), ventilatory acclimatization to hypoxia (VAH)

and hypoxic desensitization (HD) followed by extended hypoxic response across the

generations. Of note, after several generations of life at high altitude, mice had higher

ventilationthan mice.

10

Following acute hypoxia, the organism reacts by a reflex loop initiated in the carotid body

that produces a quick augmentation of ventilation in order to compensate the drop of oxygen

supply. Hypoxic hyperventilation is necessary to limit the drop of arterial oxygenation and is a

vital response during severe hypoxic exposure. During this period short-term potentiation (STP)

is initiated (Eldridge and Millhorn et al., 1986). STP is characterized by a second augmentation

phase of ventilation that appears after some minutes upon hypoxic exposure. This mechanism of

respiratory neuron hyperexcitation can be explained in part by the presynaptic calcium

accumulation that increases the release of several neurotransmitters (Wagner et al., 1991). The

electric stimulation of the carotid body fibers is able to induce STP, which indicate that glomus

cells are directly implicated in the initiation of this phenomenon (Hayashi et al., 1993). When the

hypoxic stimulation persists (some minutes), a ventilatory depression appears which is

characterized by a decrease of the respiratory rate. This phenomenon is termed short-term

depression and it is thought to depend on the noradrenergic A5 group located in the rostral part

of the ventrolateral medulla. In fact, it was demonstrated that a lesion of A5 abolishes the short-

term depression (Coles and Dick, 1996).

In the case of sustained hypoxic stimulation (mins to hrs) a new phase of ventilatory

response appears which is termed hypoxic ventilatory depression (HVD) or “roll off” (Vizek et

al., 1987) and principally involves the decrease of the tidal volume. It was hypothesized for a

long time that respiratory alkalosis which follows sustained hypoxia was mainly responsible for

the HVD. In fact, central chemoreceptors sensitive to CO2 are able to induce an augmentation of

the ventilation (Nattie, 1999). During alkalosis, the lower CO2 concentration decreases the

activation of these receptors producing a diminution of the ventilation. However it was

demonstrated that the HVD is also produced under isocapnic conditions (Bisgard, 1995). Thus,

the HVD seems to be a reflex of a central inhibition, or due to decreased activity of the

peripheral chemoreceptors (Neubauer et al., 1990).

Ventilatory acclimatization to hypoxia (VAH) appears days or weeks after hypoxic

exposure. VAH is characterized by a progressive augmentation of ventilation until reaching a

plateau. The time at which VAH is completed for a given hypoxic stimulus is species dependent.

11

While in humans and rats it is established after 10 days (Bisgard, 1995), in mice it occurs after

three days (Malik et al., 2005). VAH is absolutely dependent on the peripheral chemoreceptors

(Busch et al., 1985; Smith etal., 1986), and it was demonstrated that mice with a partial knockout

for Hypoxia inducible factor -1 α (HIF-1α) exhibited reduced carotid body sensitivity to hypoxia

and consequent impaired VAH (Bisgard, 1995; Kline et al., 2002). Finally, exposition to hypoxia

across several generations as occurring in animal species or human population permanently

living at high altitude might be accompanied by a blunting of hypoxic sensitivity, resulting in

relative hypoventilation and a phenotype of mal-adaptation to altitude. As an example, an

important fraction of high altitude natives develop a high altitude disease (Chronic mountain

sickness) mainly characterized by a pathological erythrocytosis and alveolar hypoventilation

(Monge et al., 1992). Interestingly, chronic mountain sickness occurs almost exclusively in adult

male subjects and postmenopausal women living in the Andes (Leon-Velarde et al., 1997).

1.3 The metabolic response to hypoxia

To cope with hypoxic hypoxia, mammals are provided by two physiological strategies:

Increasing the rate of O2 uptake by increasing ventilation, or decreasing the rate of O2

consumption. The decrease of the O2 consumption rate leads to a decrease of the production of

energy (ATP), and a decreased body temperature. This response is highly efficient, is conserved

across evolution, and under extreme conditions of hypoxia it ensures survival in species that

show stunningly long-lasting resistance to anoxia such as aquatic turtles (Hochachka et al., 1996;

Hochachka and Lutz, 2001). While in mammals this extreme response is not possible, a certain

degree of metabolic rate reduction is observed upon exposure to acute hypoxia, particularly in

newborn or in small adults, in which metabolic rate is high relatively to their body mass

(Mortola, 1999; Singer, 2004). The decrease of metabolic rate might be accompanied by a

reduction of core body temperature, due to a reduction of the thermoregulatory set point. Current

models are consistent with the hypothesis that hypoxia activates cAMP dependent pathways in

the hypothalamic pre-optic area, causing an elevation of the thermal sensitivity of pre-optic

warm-sensitive neurons, in turn leading to an inhibition of thermogenesis and activation of heat

loss (Steiner and Branco 2002; Branco et al., 2006; Bicego et al., 2007): in other words, this

response is a decrease of the thermoregulatory set point initiated in the pre-optic hypothalamic

area. Gasotramsmitters such as Nitric Oxide, Carbon monoxide and hydrogen sulfide appear as

12

critical mediators of this response (Branco et al., 1987; Paro et al., 2001; Steiner and Branco

2002; Branco et al., 2014). Interestingly, blockade of NMDA receptors amplifies the hypoxic-

induced fall of body temperature and this effect is present in developing rats between the 4th and

the 20th post-natal day (Baig and Joseph 2006). These data clearly illustrates that this process is

tightly regulated by the nervous system rather than a passive response to O2 limitation (Gordon

and Fogelson 1991; Barros et al., 2001; Steiner and Branco 2002; Branco et al., 2014).

To take into account the ventilatory and metabolic responses to hypoxia, it is possible to report

the ratio of ventilation to O2 consumption or to CO2 production rate (Ve/VO2 or Ve/VCO2 -

Morgan, 2014).

1.4 Molecular response to hypoxia

Mammalian cells are able to sense a decrease in oxygen tension and exposure to a low

oxygen environment triggers several immediate and long-term adaptive mechanisms both at

physiological and molecular level. Hypoxia induces various systemic cardiorespiratory

responses. However, these compensatory mechanisms are not sufficient to meet oxygen demand

of the central nervous system, especially during prolonged exposure to hypoxia. Therefore at

molecular level these responses during hypoxia are mediated by expression of HIF.

1.4.1 Structure and composition of HIF

HIF is a heterodimer that consists of the inducible HIF-α subunit and the constitutively

expressed HIF-1β. Both α and β subunits belong to the family of the basic helix–loop–helix

(bHLH) and PER-ARNT-SIM (PAS) domain-containing transcription factors (Fig.5 - Wang et

al., 1995). bHLH and PAS domains mediate

DNA binding and dimerization; the other domains in the α subunit include a unique O2-

dependent degradation domain (ODDD) and two transactivation domains: the N-terminal

activation domain (NAD) and C-terminal activation domain (CAD). Three structurally closely

related α subunits (HIF-1α, HIF-2α, and HIF-3α) have been identified to date (Huang and Bunn,

2003; Masoud and Lin, 2015). Among this HIF- 1α and HIF-2α are the best characterized. HIF-

13

1α is expressed ubiquitously, whereas HIF-2α displays tissue-specific expression (Wiesener et

al., 2003). HIF-3α has multiple splice variants, including the best known, inhibitory PAS domain

protein, which is a short protein that functions as a dominant-negative inhibitor of HIF-1

(Makino et al., 2002). The HIF-1 heterodimer binds to a conserved HIF-binding sequence within

the hypoxia-responsive element (HRE) in the promoter or enhancer regions of target genes,

thereby eliciting their transactivation and an adaptive hypoxic response (Semenza, 2003).

(Masoud and Lin, 2015).

Fig.5: Structure and composition of HIF and their isomers.

This figure describes the functional domains (bHLH, PAS and TAD) for proteins

related to bHLH-PAS family. HIF-1α and HIF-2α share highdegree of amino acid

sequence similarities and both of them have two distinct TADs (C-TAD and ODDD

N-TAD). In contrast, HIF-3α only has N-TAD.

1.4.2 Regulation of oxygen homeostasis by HIF-1 α

Regulation of HIF-1 alpha is oxygen dependent. Under normoxic conditions, HIF-1α is

hydroxylated primarily by specific prolyl hydroxylases (PHD1, PHD2 and PHD3) at two

conserved proline residues (Pro 402 and Pro564) situated within its ODDD. PHD1, PHD2, and

PHD3 have closely related catalytic domains and belong to the superfamily of 2-oxoglutarate

(2OG)-dependent oxygenases.

14

In order to be active, PHDs require O2, the citric acid cycle intermediate 2-oxoglutarate

2OG as a co-substrate, plus Fe (II) and ascorbate as cofactors. (Bruick and McKnight, 2001;

Epstein et al., 2001; Jaakkola et al., 2001). HIF-1α hydroxylation facilitates binding of tumor

suppressor gene: von Hippel–Lindau protein (pVHL) to the HIF-1a ODD (Ohh et al., 2000; Ivan

et al., 2001). pVHL forms the substrate-recognition module of an E3 ubiquitin ligase complex,

(Kondo and Kaelin, 2001) which directs HIF-1α poly-ubiquitylation and in the final step,

polyubiquitylated HIF- α is degraded in the 26S proteasome (Maxwell, 1999). Moreover an

additional hydroxylation event in the CAD domain ensures that any HIF-1α that escapes

degradation is rendered inactive. This process involves the hydroxylation of an asparagine at

Asn803 residue instead of a proline by asparaginyl hydroxylase and suppresses the recruitment

of CBP/p300 co-activators (Lando et al., 2002a). This activates FIH-1 (factor inhibiting HIF-1),

leads to steric hindrance of the interaction between α subunits and the coactivator proteins

p300/CBP. This hindrance prohibits the transactivation of target genes under high pO2 (Lando et

al., 2002b).

Upon exposure to hypoxia, hydroxylation of prolyl and asparaginyl residues is inhibited.

This inhibition enables the α subunit to escape proteolytic degradation and allow efficient

translocation into the nucleus where the phosphorylated form of HIF-1 alpha dimerizes with

HIF-1 β through intermolecular interactions between the HLH and PAS domains. The α:β

heterodimer associated with the transcriptional coactivators p300/CBP to regulate transcription

of various target gene via HRE binding sites (Fig.6).(Brahimi-Horn et al., 2005; Shimoda, 2012).

15

(Shimoda, 2012).

Fig.6: Schematic illustration of HIF-1α regulation.

Under normoxic conditions, prolyl hydroxylase domain (PHD) proteins use

molecular oxygen as a substrate to hydroxylate HIF-1α. Once hydroxylated, HIF-1α

binds von Hippel-Lindau (VHL) protein and becomes polyubiquitylated (Ub) and

targeted for proteosomal degradation. Under hypoxic conditions, PHD activity is

reduced and HIF-1α escapes hydroxylation, accumulating and translocating to the

nucleus where it binds with HIF-1β and CBP/p300 at the hypoxia response element

(HRE).

1.4.3 HIF-1α target genes

Activated HIF-1α plays a crucial role in adaptive responses to changes in O2 in organisms

through transcriptional activation of over 100 downstream genes which regulate vital biological

processes required for survival under hypoxia. HIF-1 activity leads to the upregulation of genes

that are involved in erythropoiesis,iron metabolism, apoptosis, glucose metabolism,

angiogenesis/vascular tone and cell proliferation/survival etc. (Semenza, 2001a; Semenza,

2003b).The following table summarizes the HIF-1α target genes information.

16

Table.1: Selected HIF-1 target genes.

Gene Product Functions References

α1B-adrenergic receptor Vascular tone (Eckhart et al., 1997)

Adenylate cyclase Nucleotide metabolism (Wood et al., 1998)

Adrenomudulin Vascular tone, cell survival (Cormier-Regard et

al.,1998)

Aldolase A Glucose metabolism (Iyer et al., 1998; Ryan

etal., 1998)

Aldolase C Glucose metabolism (Iyer et al., 1998)

Carbonic anhydrase 9 pH regulation (Wykoff et al., 2000)

Ceruloplasm Iron metabolism (Mukhopadhay et al.,

2000)

Endothelin-1 Vascular tone (Hu et al., 1998)

Enolase 1 Glucose metabolism (Iyer et al., 1998)

Erythropoietin Erythropoiesis, cell survival (Jiang et al., 1996)

Glucose transporter 1 Glucose metabolism (Iyer et al., 1998; Ryan

etal., 1998)

Glucose transporter 3 Glucose metabolism (Hogenesch et al.,1998)

Glyceraldehyde-3-P-

dehydrogenase Glucose metabolism

(Iyer et al., 1998; Ryan

etal., 1998)

Heme oxygenase Vascular tone, cell survival (Lee et al., 1997)

Hexokinase 1 Glucose metabolism (Iyer et al., 1998)

Hexokinase 2 Glucose metabolism (Iyer et al., 1998)

IGF-binding protein 1 Cell proliferation and survival (Tazuke et al., 1998)

IGF-binding protein 2 Cell proliferation and survival (Feldser et al., 1999)

IGF-binding protein 3 Cell proliferation and survival (Feldser et al., 1999)

Insulin-like growth factor

(IGF-2) Cell proliferation and survival (Feldser et al., 1999)

Lactate dehydrogenase A

NIP3 Cell proliferation and survival

(Iyer et al., 1998; Ryan

etal., 1998)

17

Nitric oxide synthase 2 Cell proliferation and survival (Bruick, 2000)

p21 p35srj Cell proliferation and survival

(Palmer et al.,

1998Carmeliet et

al.,1998)

Phosphofructokinase L Glucose metabolism (Bhattacharya et

al.,1999)

2-kinase/fructose-2,6-

biphosphatase Apoptosis (Iyer et al., 1998)

Phosphoglycerate kinase

1

Vascular tone, cell survival, Cell

proliferation (Minchenko et al., 2002)

Plasminogen activator

inhibitor 1 Regulation of HIF-1 activity

(Iyer et al., 1998; Ryan

etal., 1998)

Prolyl-4-hydroxilase α (I) Glucose metabolism (Kietzmann et al., 1999)

Pyruvate kinase M Glucose metabolism (Takahashi et al., 2000)

RTP801 Glucose metabolism (Shoshani et al., 2002)

Transferin Glucose metabolism (Rolfs et al., 1997)

Transferin receptor Angiogenesis, (Lok et al., 1999)

Transforming growth

factor β3

Collegen metabolism, Glucose

metabolism (Tacchini et al., 1999)

Triosephosphate

isomerase Apoptosis, cell survival (Iyer et al., 1998)

Vascular endothelial

growth factor Iron metabolism

(Iyer et al., 1998; Ryan

et

VEGF receptor 1 Angiogenesis, cell proliferation, Glucose

metabolism, Angiogenesis/cell survival

angiogenesis

(Gerber et al., 1998)

Among hundreds of HIF target genes, some important HIF target genes are described as follows.

18

1.4.3.a Epo is HIF-1 target genes involved in erythropoiesis and ventilatory response to

hypoxia.

In response to hypoxia, the capacity of red blood cells to transport oxygen isup-regulated

by the expression of EPO gene involved in erythropoiesis. HIF-1α beingan important

transcription factor for EPO, releases from the kidneys and increases the O2 carrying capacity of

blood by stimulating erythropoiesis in bone marrow (Semenza, 1999).

Studies on transgenic mouse (Tg6), which over-expresses EPO in the brain and in the

blood suggested that EPO exerts effects on the hypoxic ventilatory response both centrally and

peripherally. In Tg6 mice, there is an increased level of norepinepherine in the A5 cell group of

the pons, which could contribute to increase the respiratory frequency (Soliz et al., 2007).

However, systemic EPO also increases frequency by acting on the carotid body (Soliz et al.,

2005). In addition, the Tg21 line of transgenic mice reported 3-fold higher EPO levels in the

brainstem than wild type mice. With chronic hypoxia, Tg21 mice increase ventilation more than

wild type mice (Soliz et al., 2005). Although HIF-1α is widely expressed, HIF-2α has now

emerged as the main regulator of the hypoxic induction of EPO in vivo. Recently histological

studies, demonstrated that the location of HIF-2α expressing renal cells coincided with the

location of EPO-producing renal interstitial fibroblast-like cells (Paliege et al., 2010). Moreover,

genetic studies in mice have demonstrated that renal and liver EPO synthesis is HIF-2 dependent

and not HIF-1 dependent (Chavez et al., 2006; Rankin et al., 2007).

1.4.3.b HIF-1 target genes involved in glucose metabolism

Depletion of O2 changes the energy metabolism of cells, the cells don't use the oxygen

dependent metabolic pathway such as the tri carboxylic acid (TCA) cycle to generate energy

through the oxidation of acetate derived from carbohydrates, fats and proteins into carbon

dioxide and chemical energy in the form of ATP. Instead of the TCA cycle, the cells switch to

the O2-independent metabolic pathway, and they start using glycolysis as the primary mechanism

of ATP production (Dang and Semenza, 1999; Seagroves et al., 2001). The TCA cycle provides

38 ATPs from glucose, but glycolysis provides only two. Therefore, the hypoxic condition

requires more glycolysis than normoxic condition. HIF-1 regulates the expression of all enzymes

19

in the glycolytic pathway as well as expression of the glucose transporters gene GLUT1 and

GLUT3 that mediate cellular glucose uptake (Chen et al., 2001). Studies on glucose transport

and metabolism in hypoxia-ischemic rat brain causes upregulation of GLUT1 and GLUT3 gene

expression via HIF-1α to increase glucose transport and glycolytic rate (Vannucci et al., 1998).

Enhanced lactate production and lower intracellular pH results from the increase in anaerobic

glycolysis, potentially limiting this source of ATP despite sufficient glucose supply (Swietach et

al., 2000). Regulation of pH has its own importance in cell death under hypoxia, thus

mechanisms of pH regulation via carbonic unhydrase is likely to be vital pathways for survival.

Thus, transmembrane carbonic anhydrases were reported to regulate the pH by converting

protons and bicarbonate to carbon dioxide, so that it could taken up by erythrocytes for

transportation to the lung (Wykoff et al., 2000). Whereas increased levels of GLUT1 could

satisfy the elevated glucose requirement for anaerobic glycolysis under hypoxia. In addition,

GLUT1 may support the adaptive response of the brain during hypoxia which facilitates glucose

transport from the blood into brain parenchyma through the blood–brain barrier (Wenger, 2002).

1.4.3.c HIF-1 target genes involved in Angiogenesis

Angiogenesis is the development of new blood vessels and is subjected to a complex

control system with proangiogenic and antiangiogenic factors. One of the most important

proangiogenic factors is the vascular endothelial growth factor (VEGF). It is one of the major

target genes of HIF-1 and participates in the angiogenesis by recruiting endothelial cells into

hypoxic and a vascular area and stimulates their proliferation (Neufeld et al., 1999; Josko et al.,

2000; Conway etal., 2001). It has also been shown that hypoxia induces the expression of

VEGFmRNA and protein, suggesting that hypoxia stimulates angiogenesis through the up-

regulation of VEGF expression (Neufeld et al., 1999; Harris, 2000).

Among seven members of VEGF family, VEGF-A plays a central role in angiogenesis

and neovascularization, by increasing delivery of both oxygen and energy substrates. VEGF-A

expression can be induced when cells are subjected to hypoxia or hypoglycemia. This response

seems to depend on Hypoxia Regulated/ Responsive Element/Enhancer sequences in the 5’ and

3’ regions of the VEGF-A gene (Tsuzuki et al., 2000). HIF-1 directly activates the expression of

20

vascular endothelial growth factor which promotes the formation of new blood vessels, thus

restoring the supply of O2 and nutrients during hypoxia.

VEGF interacts with its receptor, VEGFR, which is specifically expressed in endothelial

cells, and this stimulates endothelial cell proliferation (Josko et al., 2000; Conway et al., 2001).

Disruption of the genes in Flk-1-deficient mice encoding the VEGF tyrosine-kinase receptors

VEGFR-2 (Shalaby et al., 1995) and VEGFR- 1 (Fong et al., 1995) results in severe

abnormalities of blood vessel formation in homozygous animals. In addition, mice lacking one of

the two VEGF alleles die before birth because of defects in the development of the

cardiovascular system indicating that the development of the cardiovascular system depends on

the generation of precise VEGF concentration gradients, and decrease in the amounts of the

VEGF produced during the development of the embryo may lead to decreased angiogenesis with

fatal consequences (Ferrara et al., 1996).

1.4.3.d HIF-1 target genes involved in Cell Proliferation/Survival

Hypoxia-induced growth factors most notably insulin-like growth factor-2 (IGF2) and

transforming growth factor-alpha (TGF-α) are known to promote cell proliferation and survival.

(Feldser et al., 1999; Krishnamachary et al., 2003). Binding of these factors to their cognate

receptors the insulin-like growth factor 1 receptor (IGFIR) and epidermal growth factor receptor

(EGFR) respectively, activates signal transduction pathways mitogen-activated protein kinase

(MAPK) and Phosphatidylinositol 3-OH kinase (PI3K), that lead to cell proliferation and

survival by activating HIF-1 activity. Activation of HIF-1 system leads to increased HIF-1

transcriptional activity of target genes, encoding IGF2 and TGF-α, thereby contributing to

autocrine-signaling pathways that are crucial for cancer progression (Semenza, 2003). In

addition PI3K activity is also increased in hypoxic conditions (Chen, 2001). PI3K is one of the

key downstream mediators of many tyrosine kinase signaling pathways, and is involved in

regulating cell proliferation and suppression of apoptosis. The PI3K pathway is inhibited by the

phosphoinositide phosphatase (PTEN), and mutations in PTEN enhance HIF-1 activated

responses (Zundel et al., 2000).

21

1.5 Induction of Brainstem HIF-1α expression in response to hypoxia

Physiological responses to hypoxia occur at the systemic and cellular level. The systemic

response is mediated by chemoreceptor stimulation and activation of the central and peripheral

nervous system whereas, the cellular response is mediated by HIF. Understanding the adaptive

capacity of the brain to deal with oxygen deficiency is important because the molecular

mechanisms responsible for this appear to be activated under hypoxia and other

pathophysiological conditions. (Bergeron et al., 1999; Jin et al., 2000).

Immunohistochemical study revealed that HIF-1α displays an organ-specific expression

in mice under normoxic condition and increases in response to systemic hypoxia. The maximum

HIF-1α expression was found after 4–5 hrs of hypoxic exposure in brain and other organs. The

achievement of maximal HIF-1α expression depends on the degree and duration of the hypoxic

exposure and is different between the different organs (Stroka et al., 2001).

Interestingly, some studies suggested that HIF-1α induction is the primary regulatory response

that triggers the cascade of different events leading to physiological acclimatization. Several

study suggested that HIF deletion in knock out (KO) mice impairs normal responses and

ventilatory acclimatization to hypoxia (Klein et al., 2002; Powell et al., 2008). HIF-1α increases

in the Central Nervous Systen (CNS) and brainstem is probably a major central structure for

HIF-1α expression during hypoxia as respiratory nuclei of brainstem are responsible to modulate

ventilation under hypoxic condition (Pascual et al., 2001; Lindsey et al., 2013). Therefore, it

might be predicted that an understanding of the patterns of HIF expression in the brainstem

would provide important insights into processes mediated by these molecules during hypoxia.

While the literature is rich describing the ventilatory response to hypoxia in mice and rats

separately, literature directly comparing ventilatory responses between these species is poor.

Moreover, the interspecies comparison of hypoxic ventilation from the perspective of the HIF

expression in the brainstem was not yet performed. Based on the in line evidences we

hypothesized that the differences in ventilatory response in adult rats and mice are linked to

differences in the brainstem HIF expression in response to hypoxia.

22

II- OBJECTIVE AND HYPOTHESIS OF THE

STUDY

23

The fact that rats and mice raised at high altitude have different resistance to hypoxia is

intriguing. Because these animals are not endemic at high altitude, but have been imported by

humans, we postulated that the higher “resistance” to hypoxia in mice compared to rats is a pre-

determined trait characterizing these species. As a first approach, we sought to determine

whether rats and mice have different responses to hypoxia at the physiological (ventilatory

response to hypoxia), followed by molecular level (expression of HIF-1).

24

III- MATERIALS AND METHODS

25

3.1 Animals

We used 20 adult male Sprague-Dawley rats and 22 male FVB mice of age 2-3 months

old from Charles River, St. Constant, Quebec, Canada. The animals had access to water and food

ad libitum and are maintained under a 12:12-h light-dark cycle, with controlled humidity and

temperature. The animals were allowed to adapt for one week at animal house prior to

experiment. All the experimental protocol is approved by local animal care committee of

‘University Laval’ in accordance with guidelines of the Canadian Council of Animal Care

(CCAC).

3.2 Experimental Groups

Adult rats and mice were divided in three groups. First, normoxic group of 20.9% O2

and remaining two were hypoxic group of 15% O2 and 12% O2 obtained by mixing a

predetermined flow of nitrogen gas to room air. Each group has minimum 6 and maximum 8

animals which were exposed for 6 hrs.

3.3 Measurement of Respiratory and Metabolic Parameters

3.3.1 Whole body Plethysmography

i) Experimental setup

Whole body Plethysmography (Emka Technologies, Paris, France) was used to measure

the ventilatory and metabolic parameters in adult rats and mice (Seaborn et al., 2013).The

respiratory flow trace was recorded using a differential pressure transducer. The flow of air

through the chamber was set and continuously monitored at 0.35 l/min for mice and 1.5 l/min for

rat using a pump and gas flow restrictor/monitor (Emka Technologies, France). Inlet and outlet

gases were alternatively subsampled, directed toward a water pressure analyzer (RH-300), and

then the air was dried and directed to an oxygen/carbon dioxide analyzer (AEI technology, USA)

for respiratory gases analysis. All signals (plethysmograph, gas analyzers, and flow meter) were

directed towards computer running the software (Spike2-7.06, CED-Cambridge Electronic

Designs.UK) for online storage and calculation of respiratory and metabolic values (Fig.7).

26

Fig.7: Schematic representation of whole-body plethysmography (Seaborn.et al.,2013)

ii) Plethysmoraphy recordings

First, calibration of oxygen and CO2 analyser (AEI Technologies) was done using 20.9 %

O2 and 5.09% CO2 gas followed by calibration of chamber by using known volume of air (0.5ml

for mice/3ml for rat). Later level of O2 is maintained to 20.9% for normoxic and 12%, 15 % for

hypoxic condition using nitrogen gas. Once the O2level get stabilized, animal was placed inside

the chamber for 6 hours to measure the respiratory and metabolic recordings. Respiratory traces

were recorded to determine the frequency (fR, breaths/min), tidal volume (VT, ml), and minute

ventilation (VE = fR x VT). During each measurement, a small, accuratelymeasured volume of

air was injected rapidly into the animal chamber for calibration purposes. The pressure change

caused by injection was used in the calculation of tidal volume by the equation (Drorbauh and

Fenn, 1955). VT was obtained via integration of the negative downward deviations of the flow

trace and corrected using a standard equation expressed as BTPS (Bartlett, 1970).

27

VT (BTPS) = PT/PK x VK x [TR (PB-PC)/ (TR [PB-PC] – TC [PB-PR])] 1

The symbols in the equation 1 are defined as follows:

VT = tidal volume

BTPS = volume expressed in terms of vapor pressure of water and corrected for body

temperature of the animal/organism

PT = amplitude of the respiration pressure change

PK = amplitude of the calibration of pressure variation

VK = calibration volume (ml)

TR = internal temperature of the animal (K)

TC = temperature in the plethysmography chamber (K)

PB = barometric pressure (mmHg)

PC = saturated vapor pressure in the plethysmography chamber (mmHg)

PR = saturated vapor pressure at the internal temperature of the animal (mmHg)

Oxygen consumption (VO2) and CO2 production (VCO2) rates were calculated using the

following equations (Lighton, 2008).

28

O2 consumption = Flow x [(O2in –O2out) – O2out x (CO2 out – CO2in)]

(1–O2out)

2

CO2 production = Flow x [(CO2out –CO2in)-CO2out x (O2 in – O2out)]

(1–CO2out)

3

Where, ‘Flow’ is the flow of air measured before entry into the chamber, ‘O2, in’ and

‘CO2, in’ are the gas fractions in the inflowing air (considered at 20.9% and 0.038%,

respectively), and O2, out and CO2, out are the gas fractions measured in the outflowing line. The

respiratory exchange ratio was calculated as CO2 production/O2 consumption.

In these equations, O2 and CO2 concentrations were corrected with the following term:

PB/ (PB- PH2O), where, PB is barometric pressure, and PH2O is the partial pressure of water in the

inflowing or outflowing air. This correction compensates for the diluting effect of water pressure

on measured O2 and CO2 levels (Melanson et al., 2010).

3.3.2 Analysis of respiratory and metabolic parameters

Ventilatory and metabolic recordings were averaged every hour over the 6 hours after the

onset of normoxic and hypoxic exposure by selecting periods of stable breathing patterns without

movements. Periods of recording showing error due to body movements were excluded.

Analysis of respiratory parameters, respiratory frequency (fR), tidal volume (VT),VO2 and VCO2

were analyzed from the plethysmograph. All metabolic volumes are expressed in conditions of

BTPS and expressed per 100 grams of body weight (Bartlett, 1970).

29

The respiratory quotient (RQ) or respiratory coefficient, is a dimensionless number

obtained by dividing the volume of carbon dioxide (VCO2) produced by an organism to the

volume of oxygen consumed (VO2). This quotient is useful because the volumes of CO2 and O2

produced depends on which fuel source is being metabolized for the production of ATP.

Measuring RQ is a convenient way to gain information about the source of energy an animal is

using. Eg.Glucose oxidation or fatty acid oxidation. We can then compare the metabolism of

animals under different environmental conditions. The respiratory quotient (RQ) was calculated

as CO2 production/O2 consumption (VCO2/VO2). The ventilatory equivalent for oxygen and

carbon dioxide exchange, was calculated as (VE/ VO2: ‘ml’ of air need to ventilate to consume

‘ml’ of oxygen (O2) and VE/ VCO2: ‘ml’ of air need to ventilate to produce ‘ml’ of carbon

dioxide (CO2) (Lemoine et al., 2015).

3.4 Allometric scaling

Allometry, is also called biological scaling, which describes the change in organisms in

relation to proportional changes in body size. Allometric scaling is the standard approach to

compare animals of the different sizes. (Maina et al., 1989; Stahl, 1967).

Allometric equations in general represented as ‘X = aMb ’

Where, X is biological variable, M is a measure of body size, and b is scaling exponent.

The most common example of allometry is geometric scaling, in which surface area is a function

of body mass. The scaling exponent ‘b’ were obtained by calculating the slope of a regression

line fitted through a log–log plot of a parameter (X) as a function of body mass (M).

In general, for organisms that preserve their basic shape as they vary in size, the

organisms linear dimensions vary as the 1/3 and their surface area as the 2/3 powers of their

body mass. Another important example of scaling is based on Kleibers law. Which describes the

relationship of energy consumption (or metabolic rate) and body mass in mammals: metabolic

rate scales as the 3/4 power of body mass.

30

We used allometric scaling to compare physiological and morphological values between rats and

mice, from the above equation,we reported mass-specific variables with Mb. For the respiratory

variables, we used the scaling variable calculated by Stahl (1967), which are: fR, b=−0.25; VT,

b=1.04; V˙E, b=0.8; VO2 consumption and VCO2 production, b=0.75. Data corrected for the

allometric scaling variables are referred as mass corrected values in the text description

(Lemoine et al., 2015).

3.5 Tissue Sampling

Once the plethysmography recordings were finished, anesthesia with 2-3 % isoflurane

was circulated inside the plethysmography chamber under hypoxic condition. Body temperature

was evaluated immediately after the animals were perturbed by the anesthesia, in the way that

body temperature was not altered by the isoflurane (Albrecht M. et al., 2014), followed by

cardiac puncture to collect the blood sample under hypoxic condition. Then immediately animal

was sacrificed and brainstem was collected.

3.6 Measurement of molecular parameter

3.6.1 Nuclear Protein extraction (HIF-1 α from brainstem)

We used nuclear protein extraction kit (Item No: 10009277) from Cayman Chemical

Company USA. Rat and mice brainstems were used for extraction of transcription factor i.e.

HIF-1 alpha. The summarised protocol was as follows:

Whole Brainstem was taken into prechilled vial containing known volume of ice- cold 1x

hypotonic buffer supplemented with DTT and Nonidet P-40 per gram of tissue. The sample vial

was homogenized with homogeniser on ice for 15 min. Then prechilled micro centrifuge tubes

were centrifuged at 300 x g for 10 min at 4 O C to separate first cytosolic fraction. Pellet obtained

from centrifugation was gently resuspended in additional volume of 1x hypotonic buffer into

each vial for complete lysis of cells followed by additional 15 min incubation on ice. After

31

incubation, Nonidet P-40 was added, mixed well and centrifuged at 14,000 x g for 30 seconds

(pulse spin) at 4O C in a micro centrifuge to separate second fraction of cytosolic fraction. The

pellet was re-suspended in known volume of ice –cold 1x Extraction buffer (with protease and

phosphatase inhibitors). Each vial was vortexed for 15 seconds at the highest setting and then

rocked gently on ice for 15 minutes using shaking platform. Sample was vortexed for additional

30 seconds at the highest setting and rocked gently for an additional 15 minutes. The sample was

then centrifuged at 14,000 x g for 10 minutes at 4 O C to obtained nuclear fraction as a

supernatant. Aliquoted fraction were stored at -80 O C and used further for transcription factor

assay.

3.6.2 Enzyme Linked Immunosorbent Assay (ELISA)

We used HIF-1 α transcription factor assay kit (Item No: 10006910) fromCayman

Chemical Company, USA. Rat and mice brainstem nuclear fraction were used to detect HIF-1 α.

The summarised protocol was as follows:

Ready to use 96 well ELISA plate was used. Known volume of complete transcription

factor binding assay buffer (CFTB), - competitor dsDNA, positive control and sample containing

HIF-1 α was added in appropriate wells. The ELISA plate was incubated at 4 OC for overnight.

After incubation all the wells were washed five times with known volume of 1x wash buffer and

then known volume of HIF-1 α antibody was added except blank well followed by 1 hr

incubation at room temperature. After incubation wells were washed properly with 1x wash

buffer and secondary antibody was added in each well except blank well. Again incubated was

done for additional 1 hr at room temperature. Each well were washed again with 1x wash buffer,

then developing solution was added in each well and incubated for another 15 – 45 min. After

final incubation, stop solution was added in each well and absorbance was measured at 450 nm.

32

3.7 Statistical Analysis

We used Graph Pad Prism software (version 6.04 for windows) for all analyses. All

values are reported as the means ± SEM and the significant P value was set as 0.05. P values are

reported in the figures with following general pattern: *, **, *** and **** for P, 0.05, 0.01,

0.001 and 0.001, respectively. We used two way ANOVA to compare hypoxic ventilatory

response (moderate hypoxia; 15% O2 and severe hypoxia; 12 % O2) and expression of HIF-1

alpha between rats and mice.

33

IV- RESULTS

34

4.1. Adult mice had higher minute ventilation than rats during hypoxic exposure

4.1.1 Mass-specific values

Mass-specific values for respiratory and metabolic parameters are presented in tables 2

and 3. In rats and mice, respiratory frequency (Fr) and minute ventilation (V.e) recorded in

hypoxia were higher than in normoxia, and values for metabolic rate decreased. Significant

interaction between species and hypoxia appeared for respiratory frequency, minute ventilation,

V.O2, V.CO2, and V.e/V.O2, showing different responses to hypoxia in mice compared to rats.

The mass-specific values indicate that compared to rats, mice have a higher respiratory

frequency, tidal volume, and minute ventilation under hypoxia, and higher values of V.e/V.O2

and V.e/V.CO2, indicating effective hyperventilation.

Table. 2: Mass-specific values for respiratory parameters in rats and mice

Parameters O2 level Rats Mice P values

(%)

No. of 21% 6 6 -

animals (n) 15% 5 7 -

12% 6 7 -

Body 21% 333 ± 21 26.7 ± 0.6 O2 = 0.1

Weight (g) 15% 367 ± 13 29.1 ± 0.7 Species < 0.0001

12% 320 ± 17 26.9 ± 0.2 x =0.15

Rectal 21% 36.8 ± 0.2 35.4 ± 0.3 O2 = 0.004

temperature 15% 36.3 ± 0.2 35.5 ± 0.3 Species < 0.0001

at end of

experiment 12% 35.6 ± 0.3 34.6 ± 0.1 x = 0.4

(Tr OC)

35

Fr (bpm/bw) 21% 103±3 136±9 O2< 0.0001

15% 134±6** 217±6**** Species < 0.0001

12% 162±6**** 269±7**** x < 0.0001

Vt (ml/bw) 21%

0.48±0.05 0.62±0.05 O2 = 0.002

15%

0.43±0.03 0.61±0.06 Species = 0.002

12%

0.63±0.060.058 0.89±0.11** x = 0.65

21% 49.5 ± 4.3 86.2 ± 8.3 O2< 0.0001

V.e 15% 57.7 ± 6.6 131± 14* Species < 0.0001

(ml/min/bw)

12% 102 ± 10* 236± 29**** x = 0.02

This table represents mass specific values for respiratory variables in rats and mice. n- no. of

animals; O2 level (%)– oxygen level in percentage; species- Rat vs mice; x- significant

interaction between species and hypoxia; All values are reported as the means ± SEM; *, **,

*** and **** for P, 0.05, 0.01, 0.001 and 0.001, respectively.

Table. 3: Mass specific data for metabolic variables in rats and mice

O2

level

Parameters (%) Rat Mice P values

V.O2

(ml/min/bw0.75) 21% 3.38 ± 0.07 7.03 ± 0.58 O2 < 0.0001

15% 1.90 ± 0.3* 3.86 ± 0.61**** Species < 0.0001

12% 1.93 ± 0.08* 2.56 ± 0.15**** x = 0.02

36

V.CO2 21% 1.76 ± 0.09 3.31 ± 0.25 O2 < 0.0001

(ml/min/bw0.75) 15% 1.49 ± 0.06 2.81 ± 0.39 Species < 0.0001

12% 1.33 ± 0.08 1.49 ± 0.09**** x = 0.007

V.e/V.O2

21% 14.7± 1.3 11.7± 1.1 O2< 0.0001

15% 34.1± 7.1 45.2± 12.4 Species = 0.04

12% 52.5± 4.0** 88.9± 14.0**** x = 0.07

V.e/V.CO2

21% 28.2± 2.4 24.2 ± 1.2 O2< 0.0001

15% 38.9 ± 4.6 46.3 ± 2.7* Species = 0.0002

12% 76.5 ± 5.7**** 149 ± 14**** x < 0.0001

This table represents mass specific data for metabolic variables in rats and mice. n-no.of

animals; O2 level (%)– oxygen level in percentage; species- Rat vs mice; x-significant

interaction between species and hypoxia; All values are reported as the means ± SEM; *, **,

*** and **** for P, 0.05, 0.01, 0.001 and 0.001, respectively.

4.1.2 Mass-corrected values

Mass-corrected values for respiratory and metabolic parameters are presented in figures 8 and 9

respectively. In normoxic condition (21% O2), we observed a lower mass corrected respiratory

frequency in mice compared to rats. During hypoxia, at 15 % O2 hypoxic exposure, respiratory

frequency was higher as expected for both rats (P<0.0001) and mice (P<0.0001) compared to

normoxia. It was also higher at 12 % O2 hypoxic exposure in rats (P<0.01) and mice (P<0.01).

At both level of hypoxic exposure adult mice had lower mass-corrected respiratory frequency

than rats (Fig. 8A).

In case of tidal volume, in normoxia, there was a trend towards higher tidal volume in

mice (p <0.07) than rats. In rats, tidal volume does not increase in response to hypoxia. In mice,

tidal volume was similar at 15 % O2 and higher at 12 % O2 compared to normoxia and at both

levels of hypoxia, tidal volume was higher in mice compared to rats. (Fig. 8B).

37

For minute ventilation (Ve), we observed similar values between rats and mice in

normoxia. In rats, at 15 % O2 hypoxic exposure, minute ventilation was not different from 21%

O2 but it was higher at 12 % O2. Mice had a stronger increase of minute ventilation in hypoxia

than rats, and at 12 % O2, mice had a higher level of minute ventilation than rats. (Fig. 8C).

Fig.8: Ventilatory variables at different

oxygen gradients in rats and mice. (A-

C).

A. respiratory frequency (fr). B. tidal

volume (Vt) and C. Minute ventilation

(Ve), in adult (2-3 months old) rats and

mice. Means ± SEM. *P<0.05,

**P<0.01,***P<0.001and****P<0.000

1 for hypoxic exposure and ‘O’ for rats

vs mice.

38

4.2. Adult mice had lower O2 consumption (V.O2), CO2 production rate (V.CO2),

(V.CO2/V.O2) and higher ventilatory equivalent for oxygen (V.e/ V.O2) and for carbon

dioxide exchange (V.e/ V.CO2) than rats.

For oxygen consumption (V.O2), we observed that there was no significant difference

between rats and mice in normoxia. During hypoxia, at 15 % O2 hypoxic exposure, oxygen

consumption (V.O2) was lower than normoxia in rats (P<0.001) and mice (P<0.0001) and there

was no difference between the level of oxygen consumption (V.O2) in rats and mice. At 12% O2

hypoxic exposure, in rats, there was no difference in oxygen consumption (V.O2) but in mice, at

12 % O2 hypoxic exposure, oxygen consumption (V.O2) was lower (P<0.05) than 15% O2

hypoxic exposure. (Fig. 9A).

In case of CO2 production rate (V.CO2), there was no significant difference in normoxia

between rats and mice. Also, there was no decrease in V.CO2 in rats and mice at 15% O2 hypoxic

exposure. At 12 % O2 hypoxic exposure, there was significant decrease (P<0.0001) in V.CO2 in

mice but not observed in rats, consequently mice had a lower V.CO2 compared to rats. (Fig. 9B).

The respiratory exchange ratio (V.CO2/V.O2) was similar between rats and mice in normoxia. At

15% O2, rats had a significant increase (P<0.01) in (V.CO2/V.O2) compared to normoxia.

(Fig.9C), whereas mice maintained the similar (V.CO2/V.O2).

For ventilatory equivalent for oxygen (V.e/ V.O2) and carbon dioxide exchange (V.e/

V.CO2) in normoxic condition, there was no significant difference between rats and mice. (Fig.9

D-E). Under hypoxic exposure, (V.e/ V.O2) and (V.CO2/V.O2) increased in mice. (Fig.9 D-E).

But the effect was only significant (P<0.01) in rats at 12 % O2 compared to normoxia for (V.e/

V.O2) and (V.CO2/V.O2). At 12 % O2 mice had a higher (P<0.0001) (V.e/ V.O2) and

(V.CO2/V.O2) than rats.

39

Fig. 9: Metabolic variables in adult rats

andmice (2-3 months old).

(A) O2consumptionrate (V.O2 ) and (B)

CO2 production rate (V.CO2) in adult rats and

mice. A and B are mass-corrected values. (C)

Respiratory exchange ratio (V.CO 2/V.O2).

(D)Ventilatory equivalent for oxygen (V.e/ V.O2)

and (E) for carbon dioxide exchange

(V.e/V.CO2). Means ± SEM. *P<0.05,

**P<0.01,***P<0.001 and****P<0.0001 for

hypoxic exposure and ‘O’ for rats vs mice.

40

4.3 Adult mice had higher brainstem HIF-1 alpha expression than rats with decreasing O2

concentration/hypoxic exposure

To avoid potential confounding effect of species specific reactivity of the HIF-1 assay, the

expression level of HIF-1 alpha has been normalise to the normoxic level for each species.

(Fig.10). In rats, there was no significant effect of hypoxia for brainstem HIf-1 alpha expression

but, mice had a significant (P<0.001) increase in brainstem HIF-1 alpha expression both at 12 %

O2 and 15 % O2 hypoxic exposure compared to normoxic level.

Fig.10: Expression of brainstem HIF-1 alpha in adult rats and mice.

Means ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 for hypoxic exposure and

‘O’ for rats vs mice.

41

V- DISCUSSION

42

We compared the physiological and molecular responses in laboratory rats and mice at

sea level (98m above sea level) at Quebec, Canada.

Recently a comparative study from our laboratory between rats and mice at high altitude

(3600m at IBBA Bolivia) had reported the divergent adaptation successes between these species

who had differences in the physiological response with respect to pulmonary ventilation,

hematocrit/hemoglobin values and pulmonary hypertension. In comparison to rats, mice had

higher pulmonary ventilation, low hematocrit values and lower pulmonary hypertension,

indicating mice are well adapted at high altitude but not the rats (Lemoine et al., 2015).

The rational of performing this comparative study between rats and mice at sea level is to test the

hypothesis that whether rats and mice have different responses to hypoxia at the physiological

(ventilatory response to hypoxia), and molecular level (expression of HIF-1).

This study is useful for sea level natives who might have different adaptation success for

their survival at high altitude.

Our results show that compared to rats, mice had higher minute ventilation, lower VO2,

VCO2, and higher ventilatory equivalent to oxygen and carbon dioxide, (Ve/VO2, Ve/VCO2) at

15% and 12% O2. In addition, mice had higher brainstem HIF-1 alpha expression compared to

rats.

5.1 Limitation of the experimental approach

We used whole brainstem for the said study, as brainstem had a different nuclei which are

responsible for the control of breathing which involves pontine respiratory regions,

retrotrapezoid nucleus/parafacial respiratory group, Bötzinger complex, pre-Bötzinger complex,

rostral ventral respiratory group, caudal ventral respiratory group, nucleus tractus solitaries

(NTS) and brainstem raphé nuclei (Lindsey et al., 2013; Smith et al., 2013). As immune

43

histochemical study with rat brainstem revealed that the neuronal expression of HIF-1a protein

was located in two regions: (i) an extensive dorsomedial brainstem region including the NTS, the

dorsal motor vagal nucleus, the hypoglossal nucleus and (ii) a more restricted region of

ventrolateral brainstem (Pascual et al., 2001). Brainstem is very heterogeneous in terms of

neuronal population and their control of breathing. For the said study we dont know which

brainstem nuclei has more impact on the HIF expression in rats and mice. We did not perform

the immune histochemical study to determine the HIF expression in each nuclei. This is the big

limitation of the study. The intension behind using whole brainstem was to have overexpression

of HIF and to have a maximum expression of different proteins eg. EPO, VEGF etc. In order to

characterise these proteins we need to have a cytosolic and nuclear fractions of brainstem, and

thats why we choose whole brainstem and not the particular nuclei for our experiments (Nuclear

protein extraction, ELISA).

5.2 Higher ventilation and reduced metabolic rate allows hyperventilation in mice exposed

to hypoxia

To cope up with hypoxia, many animals are able to compensate for a

reduced O2 supply by reducing the rate of O2 uptake and increasing ventilation (Ramirez et al.,

2007).

Compared to rats, mice have lower the O2 consumption (VO2) and CO2 production rate

(VCO2) in response to hypoxia (Fig.9 A-B). The decrease of metabolic rate in mice is seen as

being protective and contributes to the preservation of arterial oxygen pressure in hypoxia. It

might be accompanied by the active process resulted in reduction of the thermoregulatory set

point by reducing core body temperature initiated in the pre-optic hypothalamic area. Mice had

reduced rectal temperature of 34.6 ± 0.1 OC than rat which had 35.6 ± 0.3 OC after 12 % O2

hypoxic exposure (Table. 2). It was reported that hypoxia activates cAMP dependent pathways

in the hypothalamic pre-optic area, causing an elevation of the thermal sensitivity of pre-optic

warm-sensitive neurons, in turn leading to an inhibition of thermogenesis and activation of heat

loss (Stainer and branco, 2002; Branco et al., 2006; Bicego et al., 2006).

44

For respiratory exchange ratio (VCO2/VO2), rats showed a significantly higher VCO2/VO2 at 15

% O2 compared to normoxia (Fig 9C), but there is no difference in mice. VCO2/VO2 is useful as

the volume of O2 consumed and CO2 produced depends on which fuel source is being

metabolized for the production of ATP. The oxidation of glucose produces the highest ratio of

ATP synthesized for each molecule of O2 consumed compared with other metabolites. For

glucose this VCO2/VO2 ratio is 1 and for lipids it is 0.76. The higher value of VCO2/VO2 at 15

% O2 in rats represents energy production is mostly accomplished by oxidation of glucose

molecules, which is an effective way to optimize synthesis of ATP to meet their energy demand

under hypoxic condition (Hochachka and Somero, 2002; Lemoine et al., 2015). In case of

ventilatory equivalent for O2 (Ve/VO2) and CO2 (Ve/VCO2), mice had a very strong increase in

Ve/VO2 and Ve/VCO2, which was not seen in case of rats (Fig 9 D-E). ˙VE/˙VO2 and

˙VE/˙VCO2 plots represent the interaction between metabolic needs and minute ventiltation,

metabolic and respiratory data for males were plotted on bidimensional graphs, one for ˙Ve

versus ˙VO2 (Fig.11 ) and the other for ˙Ve versus ˙VCO2 (Fig.12 ).

Fig.11: Bidimensional plots for ˙Ve vs. ˙VO2 showing the effect of hypoxia in rats

and mice.The dotted line represents different oxygengradient 21% O2, 15% O2 and

12% O2 respectively from bottom to top.

45

Fig.12: Bidimensional plots for ˙Ve vs. ˙VCO2 showing the effect of hypoxia in

mice and rats.The dotted line represents different oxygengradient 21% O2, 15% O2

and 12% O2 respectively from bottom to top.

This presentation of the data emphasizes the effect of hypoxia in rats and mice fortheir

ventilatory and metabolic interaction. Below 15 % O2 rats are no longer able to decrease their

metabolic rate and they had only a slight increase in their ventilation. On the contrary, in case of

mice, between 15 % O2 and 12% O2, they are still able to decrease their metabolic rate and to

increase the minute ventilation.

This leads to much higher Ve/VO2 and Ve/VCO2 in mice, indicating stronger hyperventilation

compared to rats.

5.3 Higher Brainstem HIF-1 alpha expression link to better ventilatory acclimatization in

mice

Several studies reported that O2 chemo sensitive network exists in brainstem which

modulate ventilation under hypoxic condition (Smith et al., 2009a; Smith etal., 2013b). HIF is a

master regulator of hypoxic condition which helps to restore O2 homeostasis at cellular, local and

46

systemic level by regulating many target genes which are involved in glucose metabolism,

angiogenesis, erythropoiesis, cell survival etc. (Stroka, 2001). In our study, compared to rats

mice had a higher expression of HIF-1 alpha in the brainstem in response to hyoxia.

The striking difference in brainstem HIF-1 alpha expression between rats and mice could

be due to differences in the genetic background between species. In case of laboratory animal, in

vivo hypoxic study in rats reported that the chemosensory inputs are integrated in brainstem

which control cardiorespiratory response by HIF-1 alpha expression (Pascual et al., 2001).

Suggesting that the preadaptation of the laboratory animals who were never exposed to altitude is

characterized by their innate response and their predisposition for better survival under hypoxia.

As a consequence of difference in the brainstem HIF-1 alpha expression there is a difference in

the HIF target gene expression. Rats and mice could be varying in their expression of HIF target

gene for survival under hypoxia. The variety of genes regulated by HIF-1 alpha could be the

early determinant inducing the cascade of events leading to the functional plasticity which is

characteristically associated with acclimatization to hypoxia (Dumas et al., 1996; Powell et al.,

2000, Powell and Fu, 2008).

5.4 Contribution of peripheral chemoreceptor to modulate pulmonary ventilation in mice

An essential adaptation to acute and chronic hypoxia is an increase in ventilation that

depends on the activity of peripheral chemoreceptors, particularly the carotid body, which detect

changes in arterial O2 concentration and relay sensory information to the brain stem neurons that

regulate breathing (Prabhakar, 2000).

Rats and mice showed a different hypoxic ventilatory response at sea level. Compared to

rats, mice had lower mass corrected respiratory frequency, higher tidal volume and higher

minute ventilation. (Fig 8 A-C). These differences between rats and mice are likely linked to

differences in the oxygen sensing ability of the peripheral chemoreceptors to maintain pulmonary

ventilation under hypoxic condition. The HVR is the reflex response to hypoxic stimulation of

carotid body chemoreceptors. Along with HVR, carotid bodies are also critical for VAH

(Bisgard, 2000). In experimental animals, bilateral denervation of carotid bodies attenuates or

47

abolishes VAH (Forster, 1981; Bisgard, 2000). Also, direct recording of carotid body sensory

activity showed progressive increase in afferent discharge in response to prolonged hypoxia

(Nielsen, 1988). The difference in the hypoxic response could be first due to change in the

sensitivity of the carotid body glomus cells to O2 during hypoxia. It is reported that carotid

bodies in rats were enlarged and showed pronounced neovascularization after prolonged

exposure to hypoxia (Laidler and kay 1978; Dhillon et al., 1984; Kusakabe et al., 2004). In

addition the glomus cells showed hypertrophy and hyperplasia in rats leading to reduced

sensitivity of CB (Pequignot et al., 1984; Pallot et al., 1990). Second there is an increase in the

CNS responsiveness to afferent input from the carotid body (Wolff, 2000). Moreover increase

ventilation owing to plasticity in the O2 sensing function of the carotid body chemoreceptors and

in central integration sites within the neural feedback loop that regulates breathing (Black and

Tenney, 1980; Powell, 2007).

48

VI- CONCLUSION AND FUTURE

PROSPECTIVE

49

Mice raised at sea level showed higher pulmonary ventilation and brainstem HIF-1α

expression compared to rats after 6 hrs of hypoxic exposure. The differences in the ventilatory

response to hypoxia at sea level might be due to a different HIF expression pattern in the

brainstem between species. Suggesting that preadaptation of the mice who had never been

exposed to altitude is characterized by an innate response that would predispose them for a better

survival under hypoxia. These differences help to explain why mice and rats had different

adaptation success in the high altitude colonization history.

In the future we want to measure the expression of the proteins of HIF target genes in

adult rats and mice. To do so, we will compare the brainstem expression of HIF target genes

proteins in control animals or animals that were exposed 6 hrs at 12% O2. Proteomics analysis

will include the following proteins: Vascular Endothelium Growth Factor (VEGF), Calcitonin

Receptor Like Receptor (CRLR), angiopoietin 2 (ANGPT2), inducible nitric-oxide synthase

(NOS2a), glucose transporter 1 (GLUT1), 6 phosphofructo-2-kinase (pfkfb3), pyruvate

dehydrogenase kinase 1 (PDK1), protein kinase AMP-activated, alpha 1 catalytic subunit

(PRKAA1), erythropoietin (EPO), and insulin-like growth factor 1 (IGF-1). This work will be

conducted in collaboration with the CHUL hospital, Quebec, Canada that will held the

systematic identification and quantification of the complete complement of protein expressed in

the brainstem tissue of rats and mice. In addition, we want to do the time domain hypoxic

responses and HIF expression between rats and mice to understand the differences in hypoxic

responses and respective HIF expression between rats and mice.

50

VII – REFERENCES

51

Albrecht M., Henke J., Tacke S., Markert M. and Guth B.(2014).Effects of isoflurane, ketamine-

xylazine and a combination of medetomidine, midazolam and fentanyl on physiological variables

continuously measured by telemetry in Wistar rats.BMC Veterinary Research,10:198, 1-14.

Baig M.S. and Joseph V. (2006). Activation of NMDA receptors prevents excessive metabolic

decrease in hypoxic rat pups. Respiratory Physiology &Neurobiology, 152, 61–71.

Bartlett D. and Tenney S.M. (1970).Control of breathing in experimental anemia.Respir Physiol,

10, 384–395.

Barros R.C., Zimmer M.E., Branco L.G., and Milsom W.K. (2001).Hypoxic metabolic response

of the golden-mantled ground squirrel.Journal of appliedphysiology, 91, 603-612.

Bergeron, M., Yu, A.Y., Solway, K.E., Semenza, G.L. & Sharp, F.R. (1999).Induction of

hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat

brain.Eur. J. Neurosci, 11, 4159-4170.

Bicego K.C., Barros R.C., and Branco L.G. (2007). Physiology of temperature regulation:

Comparative aspects. Comparative biochemistry and physiology Part A,Molecular& integrative

physiology, 147, 3, 616-39.

Bisgard G.E. (2000). Carotid body mechanisms in acclimatization to hypoxia.Respir Physiol,

121, 237–246.

Bisgard G.E. and Neubauer J.A. (1995).Peripheral and central effects of hypoxia. In: Dempsey,

J.A., Pack, A.I. (Eds.), Regulation of Breathing, Marcel Dekker, New York, 617–618.

Black, C. P. and Tenney, S. M. (1980).Oxygen transport during progressive hypoxia in high

altitude and sea level waterfowl.Respir. Physiol, 39, 217-239.

52

Brahimi-Horn C., Mazure N., Pouyssegur J. (2005). Signalling via the hypoxia inducible factor-

1α requires multiple posttranslational modifications. Cell Signal, 17, 1–9.

Branco L.G., Carnio E.C., and Barros R.C. (1987).Role of the nitric oxide pathway in hypoxia-

induced hypothermia of rats.The American journal of physiology, 273, R967-971.

Branco L.G., Gargaglioni L.H., and Barros RC.(2006). Anapyrexia during

hypoxia.Journal of thermal biology, 31, 82-89.

Branco L.G., Soriano R. N., and Steiner A. A. (2014).Gaseous mediators in temperature

regulation.Comprehensive Physiology, 4, 1301-1338.

Bruick R K, McKnight S L. (2001). A conserved family of prolyl-4-hydroxylases that modify

HIF.Science, 294, 1337–40.

Busch M.A., Bisgard G.E., and Forster H.V. (1985). Ventilatory acclimatization to hypoxia is

not dependant on arterial hypoxemia. J Appl Physiol, 58, 1874-1880.

Chavez J.C., Baranova O., Lin J., Pichiule P. (2006).The transcriptional activator hypoxia

inducible factor 2 (HIF-2/EPAS-1) regulates the oxygen-dependent expression of erythropoietin

in cortical astrocytes. J Neurosci, 26, 9471–81.

Chen C., Pore N., Behrooz A., Ismail-Beigi F., and Maity A. (2001).Regulation of glut1 mRNA

by hypoxia-inducible factor-1.Interaction between H-ras and hypoxia.J Biol Chem, 276, 9519–

9525.

Coles S.K. and Dick T.E. (1996). Neurones in the ventrolateral pons are required for post-

hypoxic frequency decline in rats. J. Physiol, (Lond.) 497, 79–94.

Conway E.M., Collen D., and Carmeliet P. (2001).Molecular mechanisms of blood vessel

growth.Cardiovasc Res, 49, 507–521.

53

Dang C.V. and Semenza G.L (1999).Oncogenic alterations of metabolism. Trends, Biochem Sci.

24, 68–72.

Dhillon D.P., Barer G.R., and Walsh M. (1984). The enlarged carotid body of the chronically

hypoxic and chronically hypoxic and hypercapnic rat: A morphometric analysis. Quart. J. Exp.

Physiol, 69, 301-317.

Eldridge F.L. and Millhorn D.E. (1986).Oscillation, gating, and memory in the respiratory

control system. In: Cherniack, N.S., Widdicombe, J.G. (Eds.), Handbook of Physiology, section

3: The Respiratory System: Control of Breathing, part 1, vol. II. American Physiological Society,

Washington, DC, pp. 93–114.

Epstein A.C., Gleadle J.M., McNeil L.A., et al. (2001). C. elegans EGL-9 and mammalian

homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell, 107,

43–54.

Feldser D., Agani F., Iyer N.V., Pak B., Ferreira G., and Semenza G.L. (1999).Reciprocal

positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2.Cancer

Res, 59, 3915–3918.

Ferrara N., Carvermoore K., Chen H., Dowd M., Lu L., Oshea K. S., Powell braxton L., Hillan

K. J., and Moore M. W. (1996). Heterozygous embryonic lethality induced by targeted

inactivation of the VEGF gene. Nature (London), 380, 439–442.

Finley J. and Katz D. (1992).The central organization of carotid-body afferents-projections to the

brainstem of the rat.Brain Res, 572, 108-116.

Forster H.V., Bisgard G.E., Klein J.P. (1981).Effect of peripheral chemoreceptor denervation on

acclimatization of goats during hypoxia.J Appl Physiol, 50, 392– 398.

54

Fong G. H., Rossant J., Gertsenstein M., and Breitman M. L. (1995).Role of the Flt-1 receptor

tyrosine kinase in regulating the assembly of vascular endothelium.Nature (London), 376, 66–

70.

Ganong, W. (1997).Review of medical physiology. USA.

Gallagher S. A and Hackett P H. (2004).High-altitude illness.Emerg Med Clin NAm, 22, 329–

355.

Gonzalez C., Almaraz L., Obeso A., and Rigual R. (1994). Carotid body chemoreceptors: from

natural stimuli to sensory discharges. Physiol Rev, 74, 829-898.

Gonzalez C., Lopez-Lopez J.R., Obeso A., Perez-Garcia M.T., and Rocher A. (1995).Cellular

mechanisms of chemoreception in the carotid body.RespirPhysiol, 102, 137-147.

Gordon C. J. and Fogelson L. (1991).Comparative effects of hypoxia on behavioral

thermoregulation in rats, hamsters, and mice.The American journal ofphysiology, 260, R120-125.

Harris A.L. (2002). Hypoxia--a key regulatory factor in tumour growth. Nat RevCancer, 2, 38–

47.

Hayashi F., Coles, S.K., Bach, K.B., Mitchell, G.S., Mc-Crimmon, D.R. (1993). Time dependent

phrenic nerve responses to carotid afferent activation: Intact vs. decerebellate rats. Am. J.

Physiol, 265, 811–819.

Hochachka P.W., Buck L.T., Doll C.J., and Land S.C. (1996).Unifying theory of hypoxia

tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen

lack.Proceedings of the National Academy of Sciences of theUnited States of America, 93, 9493-

9498.

55

Hochachka P.W. and Lutz P.L. (2001).Mechanism, origin, and evolution of anoxia tolerance in

animals.Comparative biochemistry and physiology, Part B, Biochemistry & molecular biology,

130, 435-459.

Hockel, M. and Vaupel P. (2001). Tumor hypoxia: definitions and current clinical, biologic, and

molecular aspects. J Natl Cancer Inst, 93, 4, 266-276.

Hopfl G., Gassmann M., and Desbaillets I. (2004).Differentiating embryonic stem cells into

embryoid bodies.Methods Mol. Biol, 254, 79-98.

Huang L.E, Bunn H.F. (2003). Hypoxia-inducible factor and its biomedical relevance.J Biol

Chem, 278, 19575–8.

Ivan M, Kondo K, Yang H, et al. (2001). HIF α targeted for VHL- mediated destruction by

proline hydroxylation: implications for O2 sensing. Science, 292,464-8.

Jaakkola P., Mole D.R., Tian Y.M., et al. (2001).Targeting of HIF-α to the von Hippel–Lindau

ubiquitylation complex by O2-regulated prolyl hydroxylation. Science, 292, 468–72.

Jin K.L., Mao X.O., Nagayama T., Goldsmith P.C., and Greenberg D.A. (2000).Induction of

vascular endothelial growth factor and hypoxia inducible factor HIF-1a by global ischemia in

rat.Brain Neuroscience, 99, 577-585.

Joseph V. and Pequignot J.M. (2009).Breathing at high altitude.Cell Mol Life Sci, 66, 22, 3565-

73.

Josko J., Gwozdz B., Jedrzejowska-Szypulka H., and Hendryk S. (2000).Vascular endothelial

growth factor (VEGF) and its effect on angiogenesis.Med Sci Monit, 6,1047–1052.

56

Kline D.D., Peng Y.J., Manalo D.J., Semenza G.L., Prabhakar N.R. (2002).Defective carotid

body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient

for hypoxia-inducible factor 1 α. Proc NatlAcad Sci, (U S A), 99, 821–826.

Kumar P. and Prabhakar N. R. (2012). Peripheral chemoreceptors: function and plasticity of the

carotid body. Compr Physiol, 2, 1, 141-219.

Kusakabe T., Hirakawa H., Oikawa S., Matsuda H., Kawakami T., Takenaka T., Hayashida Y.

(2004). Morphological changes in the rat carotid body 1, 2, 4, and 8 weeks after the termination

of chronically hypocapnic hypoxia. Histol Histopathol, 19, 1133–1140.

Krishnamachary B., Berg-Dixon S., Kelly B., Agani F., Feldser D., Ferreira G., Iyer N., LaRusch

J., Pak B., Taghavi P., et al. (2003).Regulation of colon carcinoma cell invasion by hypoxia-

inducible factor 1.Cancer Res, 63, 1138– 1143.

Kondo K. and Kaelin W. G. (2001).The von Hippel–Lindau tumor suppressor gene.Exp Cell Res,

264, 117–25.

Kondo H., Iwanaga T., and Nakajima T. (1982).Immunicytochemical study on the localization of

neuron-specific enolase and S-100 protein in the carotid body of rats.Cell Tissue Res, 227, 291-

295.

Laidler P. and Kay J.M. (1978). A quantitative study of some ultrastructural features of the type I

cells in carotid bodies of rats living at a simulated altitude of 4300 meters. J. Neurocytol, 7, 183-

192.

Lando D., Peet D. J., Whelan D.A., Gorman J.J., Whitelaw M.L. (2002). Asparagine

hydroxylation of the HIF transactivation domain a hypoxic switch.Science, 295, 858–61.

57

Lando D., Peet D. J., Gorman J.J., Whelan D.A., Whitelaw M.L., Bruick R.K. (2002). FIH-1 is

an asparaginyl hydroxylase enzyme that regulates thetranscriptional activity of hypoxia-

inducible factor. Genes Dev, 16, 1466–71.

Lemoine A. J., Villalpando G., Gonzales M., Valverde I., Soria R and Joseph V.

(2015).Divergent physiological responses in laboratory rats and mice raised athigh altitude. The

Journal of Experimental Biology, 218, 1035-1043. doi:10.1242/jeb.112862

Lighton J. R. B. (2008). Flow-through respirometry using incurrent flow measurments.

Measuring Metabolic Rate: A manual for scientists. New York: Oxford University Press; Inc.

105–131.

Lindsey B G., Rybak I. A., and Smith J. C. (2013). Computational Models and Emergent

Properties of Respiratory Neural Networks, Compr Physiol, 2, 3, 1619–

1670. doi:10.1002/cphy.c110016.

León-Velarde F., Ramos M.A., Hernández J.A., De Idiáquez D., Muñoz L.S., Gaffo A., Córdova

S., Durand D., and Monge-C C. (1997). The role of menopause in the development of chronic

mountain sickness.Am J Physiol, 272, R90-R94.

Lu, X. and Y. Kang (2010). Hypoxia and hypoxia-inducible factors: master regulators of

metastasis. Clin Cancer Res, 16, 24, 5928-5935.

Lumbroso D., Lemoine A., Gonzales M., Villalpando G., Seaborn T., and Joseph V. (2012).

Life-long consequences of postnatal normoxia exposure in rats raised at high altitude. J. Appl.

Physiol, 112, 33-41.

McDonald D. (1981).Peripheral chemoreceptors.Structure-function relationships of the carotid

body.In Regulation of Breathing, ed. Hornbein T, 105-119. Dekker, New York.

58

Melanson E.L., Ingebrigtsen J.P., Bergouignan A., Ohkawara K., Kohrt W.M., et al. (2010).A

new approach for flow-through respirometry measurements in humans.Am J Physiol Regul

Integr Comp Physiol, 298, R1571–1579.

Maltepe E. and Saugstad O. D. (2009). The Role of Oxygen in Health and Disease - A Series of

Reviews, Padiatric Research, 65, 3, 261-268.

Malik M.T., Peng Y.J., Kline D.D., Adhikary G., and Prabhakar N.R (2005). Impaired

ventilatory acclimatization to hypoxia in mice lacking the immediate early gene fos B. Respir

Physiol Neurobiol, 145, 23–31.

Maina J. N., King A. S., and Settle G. (1989).An allometric study of pulmonary morphometric

parameters in birds, with mammalian comparisons.Philos. Trans. R.Soc. Lond. B Biol. Sci, 326,

1-57.

Masoud G. N. and Lin W. (2015). HIF-1α pathway: role, regulation and intervention for cancer

therapy, Pharmaceutica Sinica, B,1-12.

Makino Y, Kanopka A, Wilson W.J, Tanaka H, and Poellinger L (2002). Inhibitory PAS domain

protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia inducible factor-3α locus. J

Biol Chem, 277, 32405–32408.

Morgan B.J., Adrian R., Bates M. L., Dopp J.M., and Dempsey J.A. (2014).Quantifying

hypoxia-induced chemoreceptor sensitivity in the awake rodent.Journal of applied physiology,

117, 816-824.

Maxwell P.H., Wiesener M., Chang G.W., et al. (1999).The tumor suppressor protein VHL

targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature, (Lond), 399, 271–5.

59

Monge-C.C., Arregui A., and León-Velarde F. (1992).Pathophysiology and epidemiology of

chronic mountain sickness.Int J Sports Med, 13, S79-S81.

Mortola J.P. (1999). How newborn mammals cope with hypoxia. Respirationphysiology, 116,

95-103.

Nattie E. (1999).CO2, brainstem chemoreceptors and breathing.Prog Neurobiol, 59, 299–331.

Nielsen A.M., Bisgard G.E., Vidruk E.H. (1988).Carotid chemoreceptor activity during acute

and sustained hypoxia in goats.J. Appl Physiol, 65, 1796–1802.

Neufeld G., Cohen T., Gengrinovitch S., and Poltorak Z. (1999).Vascular endothelial growth

factor (VEGF) and its receptors.FASEB J, 13, 9–22.

Neubauer J.A., Melton J.E., and Edelman N.H. (1990).Modulation of respiration during brain

hypoxia.J Appl Physiol, 68, 441-451.

Ohh M., et al. (2000).Ubiquitination of hypoxia-inducible factor requires direct binding to the b-

domain of the von Hippel-Lindau protein. Nat. Cell Biol, 2, 423– 427.

Pallot D.J., Bee D., Barer G.R., and Jacob S. (1990).Some effects of chronic stimulation on the

rat carotid body. In: Arterial chemoreception. Eyzaguirre C., Fidone S.J., Fitzgerald R.S., Lahiri

S. and McDonald D.M. (eds). Springer-Verlag. New York. 293- 301.

Paliege A., Rosenberger C., Bondke A., et al. (2010). Hypoxia-inducible factor-2alpha-

expressing interstitial fibroblasts are the only renal cells that express erythropoietin under

hypoxia-inducible factor stabilization. Kidney Int, 77, 312–8.

60

Pascual O., Denavit-Saubie M., Dumas S., Kietzmann T., Ghilini G., Mallet J., and Pequignot

J-M.(2001). Selective cardiorespiratory and catecholaminergic areas express the hypoxia-

inducible factor-1a (HIF-1a) under in vivo hypoxia in rat brainstem, European Journal of

Neuroscience, 14, 1981-1991.

Paro F.M, Steiner A.A., and Branco L.G.S. (2001).Thermoregulatory response to hypoxia after

inhibition of the central carbon monoxide-heme oxygenase pathway.J Thermal Biol, 26, 339-

343.

Petousi N., Croft Q. P. P., Cavalleri G. L., Cheng H-Y., Formenti F., Ishida K., Lunn D.,

McCormack M., Shianna K. V., Talbot N. P., Ratcliffe P. J.. and Robbins P. A. (2013). Tibetans

living at sea level have a hyporesponsive hypoxia-inducible factor. J Appl Physiol,

doi:10.1152/japplphysiol.00535.2013.

Pequignot J.M., Hellström S., and Johansson C. (1984). Intact and sympathectomized study of

the carotid bodies of long term hypoxic rats: A morphometric ultrastructural study. J.

Neurocytol, 13, 481- 493.

Powell F.L., Milsom W.K., Mitchell G.S. (1998). Time domains of the hypoxic ventilatory

response.Respiration Physiology, 112, 123–134.

Powell F. L. (2007).The influence of chronic hypoxia upon chemoreception.Respir.

Physiol. Neurobiol. 157, 154-161.

Powell F. L. and Fu Z. (2008).HIF-1 and ventilatory acclimatization to chronic hypoxia, Respir

Physiol Neurobiol, 164, 1-2, 282–287.

Prabhakar N.R. (2000). Oxygen sensing by the carotid body chemoreceptors.JAppl Physiol, 88,

2287–2295.

61

Rankin E.B., Biju M.P., Liu Q., et al. (2007). Hypoxia-inducible factor-2 (HIF-2) regulates

hepatic erythropoietin in vivo. J Clin Invest, 117, 1068–77.

Ramirez J.-M., Folkhow L. P., and Blix A. S. (2007). Hypoxia tolerance in mammals and birds:

from the wilderness to the clinic. Annu. Rev. Physiol, 69, 113-143.

Rong W., Gourine A.V., Cockayne D.A., Xiang. Z., Ford A.P., Spyer K.M., and Burnstock G.

(2003). Pivotal role of nucleotide P2X2 receptor subunit of the ATP-gated ion channel mediating

ventilatory responses to hypoxia. J Neurosci, 23, 11315-11321.

Seagroves T.N., Ryan H.E., Lu H., Wouters B.G., Knapp M., Thibault P., Laderoute K., and

Johnson R.S (2001). Transcription factor HIF-1 is a necessary mediator of the pasteur effect in

mammalian cells. Mol Cell Biol, 21, 3436–3444.

Seaborn T., Gassmann M., and Soliz J. (2013).Using Plethysmography toDetermine

Erythropoietin’s Impact on Neural Control of Ventilation. Tissue-Protective Cytokines: Methods

and Protocols, Methods in Molecular Biology, 982, 4-18. DOI 10.1007/978-1-62703-308.

Semenza G.L., Nejfelt M.K., Chi S.M., and Antonarakis S.E (1991). Hypoxia-inducible nuclear

factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad

Sci, USA. 88, 5680–5684.

Semenza G. L. (2012). Hypoxia-inducible factors in physiology and medicine.Cell, 148, 3, 399-

408.

Semenza G. L. (2012). Hypoxia-inducible factors: mediators of cancer progression and targets

for cancer therapy. Trends Pharmacol Sci, 33, 4, 207-214.

Semenza G. L (2003). Targeting HIF-1 for cancer therapy.Nat Rev Cancer, 3,721– 732.

62

Shalaby F., Rossant J., Yamaguchi T. P., Gertsenstein M., Wu X. F., Breitman M. L., and Schuh,

A. C. (1995). Failure of blood is land formation and vasculogenesis in Flk-1-deficient mice.

Nature, (London), 376, 62–66.

Shimoda L. A. (2012). 55th Bowditch Lecture: Effects of chronic hypoxia on the pulmonary

circulation: Role of HIF-1, J Appl Physiol, 113, 1343–1352.

Singer D. (2004). Metabolic adaptation to hypoxia: cost and benefit of being small.

Respiratory physiology & neurobiology, 141, 215-228.

Smith J. C., Abdala A. P.L., Borgmann A., Rybak I A., and Paton J. F.R. (2013). Brainstem

respiratory networks: building blocks and microcircuits, Trends inNeurosciences, 36, 3, 152-162.

Smith C., Bisgard G., Nielsen A., Daristotle L., Kressin N., Forster H., and Dempsey J. (1986).

Carotid bodies are requiered for ventilatory acclimatization to chronic hypoxia. J Appl Physiol,

60, 1003-1010.

Soliz J., Soulage C., Hermann D.M., Gassmann M. (2007). Acute and chronic exposure to

hypoxia alters ventilatory pattern but not minute ventilation of mice overexpressing

erythropoietin. Am J Physiol Regul Integr Comp Physiol, 293, R1702–R1710.

Soliz J., Joseph V., Soulage C., Becskei C., Vogel J., Pequignot J.M., Ogunshola O., Gassmann

M. (2005). Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem

and carotid bodies. J Physiol, 568, 559–571.

Stahl, W. R. (1967).Scaling of respiratory variables in mammals.J. Appl. Physiol,22, 453-460.

Steiner A. A. and Branco L.G. (2002). Hypoxia-induced anapyrexia: implications and putative

mediators. Annual review of physiology, 64, 263-288.

Stroka D. M., Burkhardt T., Desbaillets I., Wenger R. H., Neil D. A. H., Bauer C., Gassmann M.,

63

Candinas D. (2001). HIF-1 is expressed in normoxic tissue and displays an organ specific

regulation under systemic hypoxia. FASEB J, 15, 2445– 2453.

Swietach P., Vaughan-Jones R.D., Harris A.L. (2007). Regulation of tumor pH and the role of

carbonic anhydrase 9. Cancer Metastasis Rev, 26, 299–310.

Sydney. A. (1997). Mammals of Bolivia: Taxonomy and distribution. Bulletin ofthe American

Museum of Natural History. 231. New York: AMNH. http://hdl.handle.net/2246/1620.

Tashi T., Fengb T., Koul P., Amaru R., Hussey D., Lorenzo F. R., RiLi G., and Prchal J T.

(2014). High altitude genetic adaptation in Tibetans: No role of increased hemoglobin–oxygen

affinity, Blood Cells, Molecules and Diseases, 53, 27–29.

Torrance J. D., Lenfant C., Cruz J., and Marticorena E. (1970). Oxygen transport mechanisms in

residents at high altitude. Respir. Physiol, 11, 1–15.

Tsuzuki Y., Fukumura D., Oosthuyse B., Koike C., Carmeliet P., and Jain R.K. (2000). Vascular

endothelial growth factor (VEGF) modulation by targetinghypoxia inducible factor-1alpha

hypoxia response element VEGF cascade differentially regulates vascular response and growth

rate in tumors. Cancer Res. 60, 6248–6252.

Vannucci S.J., Reinhart R., Maher F., Bondy C.A., Lee W.H., Vannucci R.C., and Simpson I.A.

(1998).Alterations in GLUT1 and GLUT3 glucose transporter gene expression following

unilateral hypoxia ischemia in the immature rat brain.BrainRes. Dev. Brain Res, 107, 255-264.

Vizek M., Pickett C.K., and Weil J.V. (1987). Interindividual variation in hypoxic ventilatory

response: potential role of carotid body. J Appl Physiol, 63, 1884-1889.

Wang G. L., Jiang B. H., Rue E. A., and Semenza G. L. (1995). Hypoxia inducible factor 1 is a

basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci, U

S A, 92, 12, 5510-4.

64

Wagner P.G. and Eldridge F.L. (1991).Development of short term potentiation of

respiration.Respir. Physiol, 83, 129–140.

Wenger R.H (2002). Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-

inducible transcription factors, and O2-regulated gene expression. FASEBJ. 16, 1151–1162.

Wiesener M. S., Jurgensen J. S., Rosenberger C., Scholze C. K., Horstrup J. H., Warnecke C.,

Mandriota S., Bechmann I., Frei U. A., Pugh C. W., Ratcliffe P. J., Bachmann S., Maxwell P. H.,

and Eckardt K. U. (2003).Widespread hypoxia-inducible expression of HIF-2alpha in distinct

cell populations of different organs.FASEB J, 17, 2, 271-3.

Wolff C. B. (2000).Cerebral blood flow and oxygen delivery at high altitude.HighAltitude

Medicine and Biology, 1, 1, 33–38.

Wykoff C. C., Beasley N. J., Watson P. H., Turner K. J., Pastorek J., Sibtain A., Wilson G. D.,

Turley H., Talks K. L., Maxwell P. H., Pugh C. W., Ratcliffe P. J., and Harris A. L.

(2000).Hypoxia-inducible expression of tumor-associated carbonic anhydrases.Cancer Res, 60,

7075–7083.

Zhang M., Zhong H., Vollmer C., and Nurse C. A. (2000). Co-release of ATP and ACh mediates

hypoxic signalling at rat carotid body chemoreceptors. J Physiol, 525 Pt 1, 143-158.

Zundel W, Schindler C, Haas-Kogan D, Koong A, Kaper F, Chen E, et al. (2000). Loss of PTEN

facilitates HIF-1-mediated gene expression. Genes Dev, 14, 391–96.