Cold-induced hypertension in rats

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Cold-induced hypertension in rats
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Papanek, Paula Elizabeth, 1958-
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Hypertension   ( mesh )
Hypothermia, Induced   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1989.
Bibliography:
Bibliography: leaves 149-162.
Statement of Responsibility:
by Paula Elizabeth Papanek.
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Typescript.
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Vita.

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COLD-INDUCED HYPERTENSION IN RATS


By



PAULA ELIZABETH PAPANEK















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

















This dissertation is dedicated to my family, and
especially to my father, John Papanek Jr.
whom I miss dearly.


















ACKNOWLEDGEMENTS

I wish to express my sincere gratitude and appreciation to the chairman of my

supervisory committee, Dr. Melvin J. Fregly, for his patience, gentle guidance, and

unending confidence in me, over the years here at Florida. My sincere thanks are

extended to the members of my committee, Drs. Steven Baker, Wendell Stainsby, Colin

Sumners, and Charles Wood. In particular, I wish to thank Drs. Maureen Keller-Wood

and Charles Wood for their personal time commitment to my education which cannot

adequately be expressed. Their friendship has subtly affected by thought process. A

very special note of gratitude is extended to Mr. Tom Connor and Mr. Curt Kane for

their expert technical assistance, willingness to help just in the nick of time and, more

importantly, their friendship. Thanks go to a very special friend, Mr. Howard Clark.

Thanks must also go to my friends and colleagues for their encouragement, support and

challenging "arguments" or discussions, David O'Drobinak, David Sherry, Brian Masters,

Laura Mudd ("we made it"), Phillip French, John Burton, O.R.M. Wilking, Kevin Fortin,

Drs. Linda Myers, Susan Hathaway, Bill Henley, Kelly Standifer, Martin Steiner, Michael

Murphy, and especially Dr. Orit Shechtman for the many brain-storming sessions.

I must sincerely thank my family for the unending support. To M.A. Papanek-

Miller, who may never really know all she has done to help, thanks. Finally, a very

special note goes to Mr. Bowen P. lerna who reminded me just how beautiful life can

be, and was there to take off the edge and deal with the frustrations. Thanks go to Mr.

lerna for three wonderful years.

















TABLE OF CONTENTS


ACKNOWLEDGEMENTS .......................


ABSTRACT


CHAPTER ONE BACKGROUND ........................... 1

Cold Research .................... .......... 1
Role of the Sympathetic Nervous System
in Adaptation to Cold ........................... 5
Hypertension ..................... .......... 8
Cold Hypertension as a Model of
Essential Hypertension .......................... 14

CHAPTER TWO COLD-INDUCED HYPERTENSION IN RATS ......... 16


CHAPTER


Introduction ......................
M ethods . . . .
Results . . . .
Discussion ... ...... .... ...... ...

THREE THE EFFECT OF A LOW SODIUM DIET
ON COLD-INDUCED HYPERTENSION .


Introduction
Methods .
Results .
Discussion .


CHAPTER


FOUR THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM
IN COLD-INDUCED HYPERTENSION ..........


Introduction
Methods .
Results .
Discussion .









CHAPTER FIVE


THE ROLE OF VASOACTIVE HORMONES
IN COLD-INDUCED HYPERTENSION ......


Introduction . . . .
M ethods . . . . .
R results . . . . .
Discussion . . . . .
Sum m ary . . . . .

CHAPTER SIX SUMMARY .............................

APPENDIX A EXTRACTION OF PLASMA CATECHOLAMINES ......

APPENDIX B ARGININE VASOPRESSIN RADIOIMMUNOASSAY ....

APPENDIX C PLASMA RENIN ACTIVITY ASSAY .............

APPENDIX D CORTICOSTERONE RADIOIMMUNOASSAY .........

REFERENCES ......................................

BIOGRAPHICAL SKETCH ...............................


102

102
104
107
117
128

129

140

142

144

147

149

163














Abstract of Dissertation Presented to the
Graduate School of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy

COLD-INDUCED HYPERTENSION IN RATS

By

Paula Elizabeth Papanek

May, 1989

Chairman: Melvin J. Fregly
Major Department: Physiology

Chronic exposure of male rats to low environmental temperature (6C) results in

the development of hypertension which is characterized by an increased mean arterial

pressure (124 + 3 mm Hg) and cardiac hypertrophy. The onset occurs within 2 weeks,

is repeatable, and results in a systolic blood pressure of 150 + 3 mm Hg after 42 days

of exposure to cold whether measured in cannulated animals or indirectly by the tail-

cuff technique. The concentrations of certain vasoactive agents in the plasma of resting

animals whose femoral artery was chronically cannulated strongly suggest a large

neurogenic component in this model. Thus, the concentration of norepinephrine in

plasma was significantly increased throughout the duration of exposure, while the

concentrations of epinephrine and corticosterone in plasma increased initially, but

returned to basal levels within 2 weeks. Similarly, plasma renin activity increased

initially, but was returned to the level of controls at 20 days. The concentrations of

aldosterone, sodium, potassium, and arginine vasopressin in plasma and osmolality of

serum were unchanged. The sensitivity of the baroreceptor reflex was decreased, this

suggests an alteration in the central processing of sensory (blood pressure) information.

Food intake, urine output, and hematocrit were significantly increased, while water intake

remained unaffected, suggesting a self-imposed volume contraction. The effect of cold








vii

does not appear to be unique to the rat as there is evidence to suggest that low

environmental temperature adversely affects blood pressure in humans as well.

Although the exact mechanism for cold-induced hypertension remains

undetermined, it can be concluded that chronic exposure to cold represents a unique

method for the induction of hypertension which does not involve either surgical

intervention or the administration of pharmacological doses of a drug. The results

support the hypothesis of a neurogenically-induced, renal-mediated form of hypertension

which may involve the central and peripheral renin-angiotensin II system. Additional

work will be required to determine the exact mechanisms involved in cold-induced

hypertension.














CHAPTER ONE
BACKGROUND

Exposure of either rats or humans to low environmental temperatures results in a

variety of physiological responses aimed at maintaining body temperature either at or near

normal. The investigation of the physiological responses induced by changes in

environmental temperature is not a new area to physiologists. Indeed, physiologists have

been interested for many years in both the acute and chronic changes which affect

survival. The information relating to the regulation of body temperature is so vast that

textbooks must devote entire chapters to the subject to ensure its adequate review.

Cold Research

Among the first studies utilizing cold were those performed in the early 1870s by

Claude Bernard (13). In these classical studies, Bernard suggested the dependence of

heat production on two primary sources, muscular activity, and chemical processes that

are not involved in muscular activity. Both are ideas which continue to provoke

considerable research. According to Bard (6), another very early idea which was later
"rediscovered" is that of Ott who, in 1887, suggested a hypothalamic center for

temperature regulation. This idea was essentially ignored for over 50 years. In 1888,

Hoesslin (94) described the adaptation of small animals to a reduced temperature as a

slow process which involved the formation of a protective coat of fur and fat for

insulation. This mechanism for induction of adaptation is one displayed by many

mammalian species. Isenschmid and Krehl (98) in 1912 concluded that the diencephalon

was the region which was important in temperature regulation. Sherrington (177),

demonstrated that the essential temperature regulatory center was located prespinal and

showed that decerebrate animals effectively became poikilothermic. These very early

studies, when physiology was a young science, were involved in describing the behavioral








2

responses to cold exposure and were primarily associated with the very basic ideas of

control by the central nervous system.

It appears that the pioneering investigations of Cannon and his associates (22) in

the mid 1920s were the first series of studies which undertook a systematic investigation

of the physiological mechanisms involved in maintaining temperature by chemical means.

The major emphasis in these studies was to determine the function of the adrenal

medulla and to understand how its secretion was controlled. Cannon et al. (25)

demonstrated that the output from the adrenal gland was not fixed or tonic but rather

variable. In addition, it was controlled by the sympathetic nervous system (SNS).

Cannon et al. (24) were the first to use sympathectomized cats with respect to responses

to cold. Behaviorally, these cats were poikilothermic; much like Sherrington's (177)

decerebrate animals. They "lived" on the room heaters, leaving mainly for food. This

suggests an inability either to generate heat internally or to prevent loss of body heat.

In addition, these cats demonstrated a greater decrease in core temperature when

challenged by cold. These studies suggested a major role for the sympathetic nervous

system (SNS) and adrenal medulla in the mechanisms for defence against low temperature.

In time, Cannon et al. (25) described the role of an adrenal medullary secretion which

increased the rate of oxidation in tissues and provided substrates for the oxidative

processes. Hartman et al. (82), as well as Cannon, demonstrated an increase in the

release of a substance (later shown to be epinephrine) from the adrenal medulla when the

animals were exposed to cold. These, as well as other studies, led Cannon (23) to

conclude that the adrenal substance was the primary mediator in the chemical control of

body temperature. It was during this period of time that Cannon also demonstrated that

this adrenal substance played a significant role in the generalized response to stress.

During the 1930s a major emphasis was directed toward the localization of centers

within the central nervous system which influenced both heat production and heat loss,

and in establishing critical body temperatures for survival. The general emphasis in

physiology at this time was on the concepts of neurotransmission, chemical mediators and








3

neurocontrol centers. Ranson (161) formulated the idea of two distinct hypothalamic

centers, one which responded to heat and one which responded to cold. Ranson noted

that the neurons which made up the primary area responsible for the protection from

cooling were distributed though a large part of the hypothalamus and may be in part,

identical with those regions involved in controlling vasoconstriction, piloerection and

some other sympathetic functions (161). Thus, the center which responds to low

temperature is not a discrete nucleus, but rather a diffuse collection or series of neurons

localized within the hypothalamic region. Further, it appears that there is a significant

amount of overlap with respect to anatomical location and the control of physiological

responses. It is well known that this same region serves a variety of other key autonomic

functions including feeding, drinking, origin of circadian rhythms, and control of blood

pressure.

From 1940 to the early 1970s a tremendous amount of research was conducted in

environmental physiology and the physiological responses to cold. Perhaps some initiative

was contributed by the problems generated by World War II and the military. A great

deal of impetus was also generated by the rapid advances made in the areas of electronics

as well as in laboratory techniques, such as the development of spectroscopy,

chromatography, recording equipment, computers, and radioimmunoassays. Many studies

investigated the influence which changing environmental temperature and altitude had on

various factors including: the circulatory, pulmonary and endocrine systems, as well as

internal temperature, metabolic rate, and survival. The classical study by Herrington (89)

demonstrated an increased metabolic rate in small laboratory rats, mice, and guinea pigs

in response to a decreased environmental temperature. The author (89) measured thermal

conductances, skin and internal temperatures, and heat production over a wide range of

ambient temperatures. In accordance with Herrington's equation for calculating the

increase in heat production with decreasing environmental temperatures, an increase of

157% above basal would occur at 60C, in the case of the rat (89). The changes in

metabolic rate demonstrated by Herrington in 1940 are striking and have been duplicated








4
repeatedly as techniques for measuring metabolic rate in laboratory animals and humans

have improved. The elaborate balance involved in maintaining core temperature has been

described by DuBois (44). Heat producing factors (basal metabolic rate, specific dynamic

activity of food, moderate activity and exercise, or shivering) must be equally offset by

heat loss mechanisms (basal heat loss, increased peripheral cooling, and sweating) if

normal temperature is to be maintained. When any factor is changed, the balance is

temporarily altered until the opposing mechanisms which are capable of restoring

equilibrium are engaged.

Utilizing the basic scheme as described by DuBois (44), it can be seen that the

ability to maintain body temperature in response to cold is thus dependent on both a

decrease in heat loss and an increase in heat production. Increased heat production is

accomplished initially by shivering and an elevation of basal metabolic rate (BMR). This

increase in metabolic rate can be dramatic as indicated above. As exposure to cold

continues, the emphasis is shifted away from mechanical heat producing mechanisms, i.e.

shivering, and towards biochemical mechanisms. Although shivering ceases, body

temperature is maintained by "nonshivering" thermogenesis (NSTG), i.e. a biochemical

process of heat production. It is this NSTG which has generated many questions. What

is the stimulus for NSTG to begin functioning? Which animals possess this mechanism?

Where is the site of NSTG? How is NSTG regulated? What hormonal systems are

involved in this response?

A great deal of research has been directed toward each individual hormonal system

as it is related to acute cold exposure, NSTG, and adaptation. A thorough review of any

of these systems is beyond the scope of this dissertation, and further, each has been the

subject of published reviews. The phasic responses of the major hormones, their

interactions to provide additional substrate for heat production, and the subsequent

increase in systemic responsiveness of thermogenic mechanisms have been reviewed

elsewhere (57,59,92,141). However, the role of the sympathetic nervous system merits

a brief review.








5

Role of the Sympathetic Nervous System in Adaptation to Cold

In 1942, VonEuler (194) isolated norepinephrine (NE) and quickly developed a

bioassay. In 1946, VonEuler (194) predicted that NE was the neurotransmitter substance

of the SNS. It was nearly 10 years later that this idea was documented when

fluorescence histochemical techniques became available (194). In 1955, VonEuler and

Floding (195) developed a fluorimetric technique for measuring both NE and E in urine.

The ability to measure NE and E concentrations resulted in a major stimulus for research

in the role of the sympathetic nervous system in thermoregulation.

When animals are exposed to cold, the SNS is markedly stimulated. That this

occurs is evident by a significantly increased excretion of catecholamines (NE and E) in

a variety of species including rat (133,121,122) and man (6,183). Although a great deal

of variability exists in the actual amount of catecholamines excreted, the cold-induced

increases were large in all cases. The variations in the results can be attributed to

differences in assay techniques, temperature, and the age of the animal. In addition, the

fluorimetric technique is not sufficiently sensitive to detect the low concentration (pg/ml)

of catecholamines in the plasma at rest. The variation attributed to assay techniques is

no longer a problem due to the development of high performance liquid chromatography

(HPLC) and radioenzymatic (RE) techniques for measurement of plasma, urinary, and

tissue catecholamines. These newer techniques have both greater sensitivity and decreased

variability. The major difference between these two techniques (HPLC and RE) is not

attributed to the precision, but rather one of methodologies. The size of the sample

required is quite different (HPLC at least 0.5 ml vs 0.05 ml for radioenzymatic) as is the

cost per sample (HPLC significantly less). The size of the sample of plasma necessary

for reliable analysis becomes an important consideration when the protocol involves either

a small animal model or serial sampling. This is often the limiting factor in the design

of an experiment in which the concentration of catecholamines in the plasma is to be

determined. The concentrations of catecholamines in plasma are a more accurate

representation of temporal changes within the organism, whereas the concentrations of








6

catecholamines in urine represent both sympathetic and central nervous system activity

over an extended period., Unless sympathetic changes can be sustained for long periods

of time, the concentration of catecholamines in urine are difficult to interpret. The

concentration of catecholamines in the urine is affected not only by the concentration

of catecholamines in the blood, but also by the factors which influence their metabolism

and renal clearance. The development of high pressure liquid chromatographic technique

for measurement of catecholamines has contributed to a renewed interest in the area of

sympathetic function during exposure to cold.

As mentioned above, adaptation to cold is characterized by a markedly increased

thermogenesis. The increased nonshivering thermogenesis (NSTG) is attributed to both

changes in heat producing mechanisms (88) and a supersensitivity to the metabolic effects

of NE (41,96). Hsieh and Carlson (97) were among the first to demonstrate an enhanced

calorigenic response to administered (i.m.) catecholamines. The metabolic response to

norepinephrine was much greater than that to E given i.m. This led these authors to

conclude that NE was the primary mediator of the enhanced calorigenic response.

Depocas (41) demonstrated an enhancement of the calorigenic response to NE infused

intravenously in cold-acclimated rats. These results also lead Depocas (41) to conclude

that NE, and thus the sympathetic nervous system, is primarily responsible for the

markedly increased rate of oxygen uptake (VO2) seen with exposure to cold. However,

when infused intravenously, E results in an equally large calorigenic response (91). The

work of several investigators suggested that NE was the primary mediator of NSTG while

E served multiple functions, including supplying substrates for metabolism and acting

as a backup to NE, if needed. For instance, when adrenalectomized rats (glucocorticoids

replaced) are exposed to cold, no increase in E is detected (170). Norepinephrine

responses are unaffected by adrenalectomy (100). Various attempts have been made to

eliminate the NE response to cold by immunosympathectomy, ganglionic blockade,

adrenergic blockade, and NE depletion. Although each of these techniques has

limitations, it can be concluded that when NE responses are limited, E release is








7

enhanced when possible. Based on such studies, it would appear that NE, and thus the

SNS, is primarily responsible for the markedly increased VOz seen during exposure to

cold as concluded by Hsieh and Carlson (97), and Depocas (41). Further, E serves the

multiple functions of supply of substrate for the energy processes and as a backup to

NE, if needed.

An interesting characteristic of cold-adapted animals is an increased sensitivity to

NE and E. While an increased sensitivity to NE and E is advantageous to the animals,

it is contradictory to traditional concepts of the regulation of receptors (201); as cold-

exposed animals are characterized by increased circulating levels of the endogenous

ligands, NE and E, as already mentioned. An increased sensitivity may occur for several

reasons including a) changes in receptor affinity; b) changes post-receptor; for example,

either changes in secondary messengers or in the cascade system which follows; and c)

decrease in either reuptake or removal of the transmitter. Further, a change in

sensitivity in the heat producing systems may imply selectivity in the regulation of

receptors for their ligand based on location or function. Since the stimulatory effect of

NE on metabolism is related to its beta-adrenergic potential, it might be expected that

a similar stimulatory effect on cardiovascular responses, another beta-adrenergic

dependent response, would also be observed. In addition, NE also possesses alpha-

adrenergic potential. However, Fregly et al. (60) and Bryar et al. (19) have reported a

decrease in responsiveness to alpha-adrenergic agonists in aortic rings of cold-adapted rats

in vitro. Barney et al. (8) reported an increased metabolic and an unchanged

cardiovascular responsiveness to exogenously administered beta-adrenergic agonist,

isoproterenol, in cold-adapted rats. Thus, chronic exposure to cold increases the

concentration of NE in plasma and the metabolic (beta) responsiveness to exogenously

administered NE but actually decreases the vascular (alpha) and the cardiovascular (beta)

responsiveness is decreased or unchanged. Thus, the altered responsiveness appears to be

related more to function or location rather than to the concentration of the ligand.

Whether chronic exposure to cold is unique in this respect is undetermined.








8

With chronic exposure to cold, the urinary output of NE decreased toward control

levels and urinary E output returned to control levels within 30 days (122). This suggests

that the concentration of NE in the plasma may decrease toward control levels and E

concentrations should not be different from controls after 30 days of exposure. If this

is the case, it suggests that the animal is 1) adapted to the generalized stress of cold-

exposure, and thus decreasing the concentration of E; 2) meeting substrate demands

without additional E release; and 3) increasing its sensitivity to NE, thereby decreasing

the need for a greater output of NE. The cold-adapted animal therefore can be

characterized by an increased concentration of NE but a normal concentration of E in

the plasma, and a specifically enhanced responsiveness to the metabolic effects of these

ligands.

Clearly, chronic exposure to cold results in a variety of hormonal, receptor and

post-receptor responses, and a large sustained increase in output from the SNS. The

majority of work investigating these responses has been directed toward their role in

maintaining core temperature and thus survival. Whether these same responses result in

additional physiological changes is unknown.

Hypertension
Hypertension is a major risk factor in cardiovascular disease. Cardiovascular

disease is not only a major problem among the elderly, it also causes premature deaths

with more than 100,000 deaths annually in people under the age of 65 (191).

Epidemiological studies, such as the Framingham Study (103) have examined the

relationships between cardiovascular disease and many variables, including hypertension.

Although not providing direct evidence for cause-and-effect interactions, some important

associations are evident between the risk of cardiovascular disease and these predisposing

factors.

An elevation of arterial blood pressure is associated with a significantly increased

risk of sudden cardiac death, myocardial infarction, and atherosclerosis (71,72,69,191).

Further, pharmacological treatment of elevated arterial blood pressure decreases the total








9

cardiovascular morbidity and mortality among hypertensive patients (45). Hence, it is of

little wonder that vast amounts of research have been directed toward the mechanisms

involved in the elevation of blood pressure during the past decade. In spite of the large

amount of work conducted in this area, no single abnormality of any one depressor or

pressor system has been demonstrated as the cause of essential hypertension.

Hypertension is a complex and multifactorial syndrome. The syndrome of

hypertension is more than just an elevation of arterial pressure, although it is this aspect

with which the general public is most familiar. Hypertension when fully developed is

characterized by an increase in vascular resistance to blood flow, cardiac hypertrophy,

often an increase in cardiac output, an increased output of the SNS, changes within the

vascular smooth muscle cells, often atherosclerosis, and abnormalities in renal function.

Hypertension, like many other chronic diseases develops silently with small subtle changes

occurring in the contributing factors over many years. This, in part, explains why the

exact etiology of the hypertension remains unknown. In most cases, the hypertension

remains undetected until the most significant symptom of abnormality (i.e. an elevation

of arterial pressure) is expressed. It is the increased arterial pressure and resistance to

blood flow which are suddenly of great concern.

Increased vascular resistance is due to constriction of the vascular smooth muscle,

primarily in the high resistance vessels. Depending on the form of hypertension, various

factors are known to alter vasomotor tone, including a) increased activity of the

sympathetic nervous system innervating the vascular smooth muscle; b) increased

concentration in plasma of vasoactive agents such as NE, arginine vasopressin (AVP),

angiotensin II (All), and the eicosinoids, which stimulate the vascular smooth muscle to

constrict; c) alterations in activity within the areas of the central nervous system

responsible for regulating the discharge to the periphery via the sympathetic and

parasympathetic nervous systems; d) morphological changes in the vascular smooth muscle;

and e) reduced renal excretory function due to intrinsic alterations of kidney function

or the extrinsic factors mentioned above (a-d). The diversity of these factors, any of








10

which can increase vascular resistance, has resulted in various hypotheses and thus models

for hypertension. Due to the unknown etiology of hypertension, the use of various

research models, each of which induces the disease by a different mechanism yet with

the same end result, is advantageous.

Neurogenic Model

The neurogenic hypothesis and various models stem logically from the ability of

the SNS to stimulate vasoconstriction. Sympathetic tone, and thus peripheral

vasoconstriction, is controlled by a complex network of neurons located in the ponto-

medullary region in the brainstem. The tonic discharge of this center is under negative

feedback control (79). Lesions in the nucleus tractus solitarus (NTS) result in excess SNS

activity; either a transient or sustained elevation of blood pressure; a loss of the

baroreceptor reflex, and a decrease in the plasma concentrations of renin and aldosterone

(43). Higher brain centers are also known to influence sympathetic outflow. The

hypothalamus-thalamus-septal area has a large number of interneurons connecting it with

the NTS. Electrical stimulation of this region results in constriction of resistance vessels,

vasodilatation in skeletal muscle, an increase in stroke volume, heart rate, and blood

pressure (51). These physiological responses are very similar to those elicited in defense

of internal temperature. The NTS region is the first synapse of the baroreceptor

pathway. Thus, baroreceptor denervation is the most familiar neurogenic model of

hypertension.

In 1958, Heymans and Neil (90) denervated both the carotid sinus and aortic arch

of dogs and reported an increased blood pressure. Although the elevation of blood

pressure was dramatic in the acute preparation, the pressure was very labile. Utilizing

baroreceptor denervated dogs (long-term denervation) and chronically implanted cannulae,

Cowley et al. (32) reported no difference in the average level of blood pressure and only

an increased variability. These authors concluded that the baroreceptors act to minimize

variations in blood pressure rather than to control the absolute level. Ito and Scher (99),

as well as others (113,48), disagree. These authors (99,113,48) have demonstrated a small








11

but significant and sustained elevation of blood pressure in dogs and rats upon lesioning

of the aortic baroreceptors.

In addition to the direct effects the baroreceptors have on the circulation,

neurogenic models may also influence the circulation indirectly via changing AVP,

ACTH, or All levels as well as renal function. The major limitations of the neurogenic

model are a) requirement for extensive surgical intervention and b) the lack of evidence

demonstrating sustained hypertension rather than increased liability of blood pressure. It

is a good model, however, in the respect that central mechanisms which influence blood

pressure must certainly be altered in essential hypertension as suggested by the large class

of very effective antihypertensive drugs which act centrally on SNS function. In

addition, this model permits an investigation of the interaction, or lack of interaction,

between the CNS and the kidney.

Pharmacological Model

The most common pharmacologically induced model of hypertension in the rat is

that using deoxycorticosterone acetate plus high salt intake (DOCA/salt). This

hypertension is salt dependent in its initiation and often involves surgical reduction of

renal mass. Thus, the DOCA/salt model is often used in combination with unilateral

nephrectomy. This combination results in hypertension, cardiac hypertrophy, and

nephrosclerosis (175). The hypertension which develops is thought to be dependent on

volume expansion. An increase in fluid volume would increase central venous pressure,

cardiac output and thus reflexly lead to vasoconstriction and rarefaction to adjust flow

(77,80). In addition, there is evidence that AVP may be important in both the

development and maintenance of blood pressure in this model (36,37). The sympathetic

nervous system also appears to play a role in the development of DOCA/Salt hypertension

(104). The increased output of the SNS may act to change renal function, as denervation

of the kidneys affects both the onset and the severity of the disease (104). The major

limitations of the DOCA/Salt model are a) the pharmacological doses of drug required;

b) requirement for surgical reduction of renal mass; and c) required ingestion of large










amounts of NaCI. A significant advantage of this model is the potential to investigate

the role of sodium in the developmental stages of hypertension.

Genetic Models

Hypertension and cardiovascular disease (CVD) may have genetic predeterminants.

There is a predisposition to hypertension and CVD in those humans with a positive

family history. The notion of genetic predisposition for the development of hypertension

has been strengthened by the development of genetic or spontaneous models of

hypertension. The two most common models are the Dahl salt-sensitive, salt-resistant

hypertensive strain and the spontaneously hypertensive strain developed by Okimoto and

Aoki.

The spontaneously hypertensive strain (SHR) is used extensively. Some investigators

feel it to be the best model for human essential hypertension. Some hemodynamic

alterations which occur during the development of the disease are similar to human

essential hypertension, i.e. a high cardiac output early, and in the adult, a normal cardiac

output and an increased vascular resistance (188). In this model, there is also an increase

in the activity of the sympathetic nervous system which may contribute to the

development of the disease. Thus, electrical activity in the renal sympathetic nerve is

increased (188), while both renal blood flow and glomerular filtration rate are normal (5)

and renal vascular resistance is increased (188). The advantage of these similarities with

human essential hypertension (29) is important. A major drawback of this model is the

lack of an appropriate control, one which has been genetically altered yet is free from

the disease and the complexity of the genetic mutations which have affected not only

blood pressure but many other regulatory systems as well.

Renal-Dependent Models

One factor which appears consistently altered in all of these models is renal

function. That the kidney is involved in sodium and water conservation and, thus,

intimately involved in the regulation of blood pressure is well accepted. Since 1934,

when Goldblatt et al. (66) induced an elevation of blood pressure by partially constricting








13

the renal artery of the dog, many renal-dependent models have been developed.

Basically, this form of experimental hypertension can be divided into two general groups,

two-kidney hypertension (clip one kidney while leaving the other intact), and one-kidney

hypertension (clip one kidney and remove the other). This form of hypertension has

been induced in dogs, rats, rabbits, sheep, and cats (202).

In the two-kidney model, the renin-angiotensin II system has been implicated as

a major contributing factor in the pathogenesis of the disease (14,124). The renin-

angiotensin II response is biphasic, with an elevation lasting up to I week and thereafter

returning to normal (14,124). The sustained elevation of blood pressure may still be All-

dependent, however, as Bean et al. (9) have demonstrated the ability of All to shift its

own dose-response curve. Thus, a previously sub-pressor dose of All would now result

in an elevation of blood pressure. The basic hypothesis for the model is that the increase

in blood pressure results from the influence of the clipped kidney on the normal kidney.

Thus, error signals (changes in hormones and sympathetic nervous system activity) to a

normal kidney result in changes in sodium and water handling, and thus, changes in

blood pressure.

In the one-kidney model, the early elevation of blood pressure is also All-mediated

(53). Due to the removal of the other normal kidney, no compensatory increase in

sodium and water excretion can occur, and hence, fluid volume is retained (18). This

model is thus a sodium-fluid volume dependent model.

The hypertension which develops in these two renal-dependent models is reversible,

i.e. when the clip is removed, arterial pressure returns to normal (124,125,184). This is

a unique feature of these models which has received little attention. Further, if the

reversal of hypertension is a time-dependent phenomenon, it would suggest an important

window in which changes occur which are not reversible, perhaps structural changes.

In addition, these models provide the opportunity to investigate the changes which occur

specifically at the level of the kidney, as well as the role of the kidney in long-term

blood pressure control.










Summation

Regardless of the model or method of inducing an elevation of blood pressure,

the ultimate goal is the development of the hypertensive. Each model has limitations and

advantages while none of them clearly duplicates the essential hypertension that occurs

in humans. Perhaps the commonalities of an altered activity of the"SNS and an alteration

in renal function are clues to a unifying mechanism in hypertension. The need for new

models that may contribute additional information to an understanding of hypertension

is clear. In addition, the investigation of each of these models during the developmental

stages of hypertension is imperative in efforts to determine the mechanisms associated

with an elevation of blood pressure, and thus effective in treating hypertension.

Cold Hvyertension As A Model Of Essential Hypertension.

An interesting characteristic of adaptation to cold in rats, which was originally

mentioned in 1950 by Gilson (64), is an elevation of systemic blood pressure. Fregly in

1954 (54) as well as Heroux and Dugal (87) have also reported an increase in systolic

blood pressure in the rat, while LeBlanc (118) reported no difference in anesthetized

pentobarbitall) rats. An increase in mean arterial pressure in response to acute exposure

to cold has also been reported by Thompson et al. (185) in sheep, and in young oxen by

Bell and Thompson (10). Since these studies were primarily designed to investigate

substrate utilization, metabolism and adaptation during exposure to cold, and since blood

pressure measurements were often recorded as secondary information, these observations

have received relatively little attention.

Recently however, Papanek and Fregly (151,152) have begun to investigate the

effects of chronic exposure to cold on blood pressure in rats. Fregly et al. (61) have

measured systolic, diastolic and mean blood pressures in cold-adapted rats with indwelling

cannulae. Blood pressure, as well as heart rate and ventricular weight, was significantly

elevated in cold-adapted rats (61). The combination of an increase in arterial blood

pressure and heart weight led Fregly et al. (61) to conclude that the cold-adapted rat

is hypertensive. If this is the case, then chronic exposure to cold represents a unique








15

method for the induction of hypertension which does not involve either surgical

intervention or pharmacological dosage of drugs.

Cold-induced elevation of blood pressure is demonstrable in human beings as well

as in rats. Reed et al. (162) have reported a significant elevation of mean arterial

pressure in young men during extended residence in Antarctica. Over the course of 42

weeks, mean arterial pressure rose from baseline values of 85 mm Hg to 101 3 mm Hg.

An elevation of systolic, diastolic and mean arterial pressures in addition to an increase

in plasma NE has also been shown in response to acute cold (193,199,172). Scriven et

al. (172) showed a strong correlation between the plasma concentration of NE and both

systolic and diastolic blood pressures. Further, Brennan et al. (17) have reported seasonal

variations in arterial pressure during the British Medical Research Council's treatment

trial for mild hypertension. Their results show that for each age, sex, and treatment

group, both systolic and diastolic pressures were higher in winter than in summer (17).

Likewise, Hata et al. (83) reported a seasonal variation of blood pressure in patients with

borderline and established essential hypertension. These authors also reported seasonal

variations for plasma NE, urinary catecholamines, and urinary sodium, without variations

in plasma renin activity or the concentration of aldosterone in plasma (83). In 1961,

Rose (166) reported a seasonal influence on blood pressure during a 1-3 year study of

ischemic heart disease. However, his peak values were reported for the spring rather

than the winter. With the exception of the study by Rose (166), the above studies

suggest a negative correlation of ambient temperature on blood pressures in humans. The

study of cold-induced hypertension in rats may allow a better understanding of some of

the mechanisms involved in the cold-induced elevation of blood pressure in humans.














CHAPTER TWO
COLD-INDUCED HYPERTENSION IN RATS

Introduction
Exposure of either rats or humans to low environmental temperatures results in a

variety of physiological responses aimed at maintaining body temperature at or near

normal. The remarkable constancy of core temperature in the face of varying

environmental temperatures received attention early in physiological studies. Cannon

noted in "The Wisdom of the Body" that temperature was so well maintained from

individual to individual, that manufacturers actually stamped "normal" at 98.6*F on their

thermometers (23).

Initial responses to sudden exposure to low environmental temperature are a

reduction in heat loss (piloerection and peripheral vasoconstriction), and an increase in

heat production. Initially, the increase in heat production is accomplished

neuromuscularly, i.e. by shivering. As exposure to cold continues, the emphasis is shifted

away from neuromuscular mechanisms and toward biochemical mechanisms, i.e.

nonshivering thermogenesis. Much of the work investigating cold has centered on the

hormonal and metabolic factors involved in thermogenesis, and in adaptation to cold

(57,59,92,141). Cold-adapted animals are characterized by an increase in nonshivering

thermogenesis (NSTG) which is attributed both to changes in heat producing mechanisms

(88) and to a supersensitivity to norepinephrine (NE) (41,96). An increased sensitivity

to NE by heat producing systems is interesting because it occurs in the presence of an

increased concentration of circulating levels of the endogenous ligands, NE and

epinephrine (E) (121,122). Leduc (122), Lutherer et al. (133) and many others

(100,183,6) have measured large increases in urinary outputs of NE and E during chronic

exposure to cold. An increased metabolic sensitivity to NE is advantageous to the cold-








17

exposed animal, and suggests a change in the regulation of receptors induced by exposure

to cold. However, this suggestion is contradictory to the traditional concepts of the

regulation of receptors (201).

An important characteristic of exposure to cold in rats, which was originally

mentioned in 1950 by Gilson (64), is an elevation of systemic blood pressure. Similarly,

Fregly (54) in 1954, as well as Heroux and Dugal (87) have also reported an increase in

systolic blood pressures in the rat, while LeBlanc (118) reported no difference in blood

pressure. However, in the experiment by LeBlanc (118), the rats had been anesthetized

with pentobarbital, heparinized, and removed from the cold. The depressant effects of

pentobarbital on blood pressure are well known. An increase in mean arterial pressure

in response to acute exposure to cold has also been reported by Thompson et al. (185)

in sheep, and in young oxen by Bell and Thompson (10).

With the exception of the work of LeBlanc (118), the majority of the studies using

rats were designed to investigate either substrate utilization or other adaptive mechanisms

during exposure to cold, and blood pressure measurements were recorded for the most

part as secondary information. The observation that chronic exposure to cold resulted

in the development of a sustained elevation of blood pressure has received relatively little

attention. Recently however, Fregly et al. (61) measured systolic, diastolic, and mean

blood pressures in cold-adapted rats with indwelling cannulae. Blood pressure as well

as heart rate and heart weight were significantly elevated in rats exposed to cold (6C)

for 4 weeks. The combination of an increase in blood (systolic, diastolic, and mean)

pressure and heart weight led Fregly et al. (61) to conclude that the cold-adapted rat is

hypertensive. If this is the case, then cold-adaptation represents a unique method for

the induction of hypertension which does not involve either surgical intervention or

pharmacological dosage of drugs.

The purpose of the first series of experiments was to describe the development

of hypertension in rats chronically exposed to air at 6C.








18

Methods

Male rats of the Sprague Dawley strain initially weighing 200-250 g were used.

All animals were housed in individual cages and provided powdered Purina Laboratory

Chow (#5001) and tap water ad libitum. The vivarium was maintained at 26C and on

a 12:12 light:dark cycle. Two weeks were allowed for the animals to adapt to their new

environment.

Experiment One

Twelve male rats were divided randomly into two equal groups, i.e. either a control

(26C) or a cold-treated (6 + 2C) group. Body weights and systolic blood pressures

(SBP) were measured during the initial two week control period with all animals

maintained at 26C. At 0800 h on the first experimental day, all animals were moved

into temperature controlled environmental chambers (either 26 or 6C). Systolic blood

pressure and body weight (BW) were recorded weekly throughout the remainder of the

experiment.

Systolic blood pressures were measured from unanesthetized rats indirectly from the

tail. Animals were removed from their home environments and placed into stainless steel

restraining cages. The restrained animals were placed onto a heating pad and were

warmed by means of a heat lamp. Ambient temperature was maintained at 30 + 2C.

Approximately 15 minutes were allowed for the animals to vasodilate prior to beginning

pressure measurements. Systolic blood pressure was measured using a pneumatic pulse

transducer (MK III series), coupled to a Narco Bio (model DMP-48) physiograph

recorder. The occlusion cuff was always placed at the base of the tail to insure

reproducibility. This procedure for the measurement of SBP is essentially that previously

described by Fregly (56). The SBP data represent the mean of ten measurements per

animal. At the end of the measurements, the animals were immediately returned to their

home environments.










Experiment Two

Twenty male rats were divided equally into either a control or cold-treated group.

Each animal was provided with a spill-resistant food container (55). Fluid containers

consisted of infant nursing bottles with cast bronze spouts (117). These minimized, but

did not eliminate spillage. Reservoirs were attached under each water bottle to collect

any water that spilled. Each cage was fitted with a stainless steel funnel for the

collection of urine separate from feces. Urine was collected into volumetric tubes. Food

and water intakes were determined gravimetrically. Water intakes were corrected for

spillage. Baseline measurements were recorded for two weeks. On the first experimental

day, all animals were moved into temperature-controlled environmental chambers as

described above. All measurements were recorded daily at 0800 h throughout the

duration of the experiment.

At the end of each experiment, animals were sacrificed and the heart, kidneys,

adrenal and thyroid glands were rapidly removed. All organs were trimmed of fat and

connective tissue, blotted and weighed on a torsion balance. The heart was then

subdivided further: the atria were removed first, then the right ventricle was lifted and

cut away leaving the left ventricle and the entire septum. The atria, right and left

ventricles were weighed. In experiment one, the interscapular brown fat and epididymal

white fat were removed and weighed. After the initial weights were recorded, the organs

were dried overnight in an oven (Boekel, 65C) to remove all water and reweighed.

Results are expressed as mg tissue/100 g of body weight. Microhematocrit tubes were

filled in duplicate at the time of sacrifice in experiment two and immediately centrifuged

for four minutes in a microcapillary centrifuge (Model MB, International Equipment

Corp.).

Statistical analyses of SBP and BW were carried out by a two-way analysis of

variance repeated in time. Differences in organ weights were determined by a one-

tailed, Student's t-test for independent samples. The hypothesis tested was that the

variable being tested in cold-treated animals was less than or equal to that of controls








20

(Ho: cold < control, while Ha: cold > control). Significance was set at the 95% confidence

interval. A posteriori pairwise comparisons were performed when appropriate by a

Duncan's New Multiple Range test which maintained the significance at the a priori level

(p < 0.05) (111).

Results

Chronic exposure to cold resulted in a significant elevation of systolic blood

pressure (Figure 2-1). Throughout the initial exposure to cold (2 weeks), no change in

systolic blood pressure was detected (Figure 2-2). The first significant elevation of

systolic blood pressure occurred after 21 days of exposure to cold. The systolic blood

pressure of the cold-treated group remained significantly elevated for the duration of the

study. In each experiment, the body weights did not differ significantly between the two

groups prior to treatment (cold). However, there was a significant interaction between

environmental temperature and time. The slope of the line of body weight versus time

is depressed; i.e. cold-exposed rats gained weight at a slower rate (Figure 2-3, 2-4). In

addition, the cold-exposed group had a significantly increased amount of interscapular

brown adipose tissue (IBAT) and a decreased amount of white adipose tissue (Table 2-1).

These are characteristic changes associated with acclimation to cold. Due to differences

in body weights, data for the intakes and outputs were expressed per unit of body weight

(ml or g/100 g BW). Water and food intakes, as well as urine outputs, of the two groups

were similar during the control period (Table 2-2). Food and water consumption

increased significantly in the cold-exposed animals as demonstrated by both a significant

interaction and a main effect (Figure 2-5, 2-6). Both food and water intakes peaked

after one week. Daily urine output increased significantly as well and followed the

increase in water intake (Figure 2-7). These results are similar to those previously

reported by Fregly et al. (58).

A regression analysis of food versus water intake indicated that the cold-exposed

group drank less water for any given food intake (Figure 2-8). Regression analysis of









SControl
~ Cold


*I_


Baseline


Figure 2-1.


Day 50





The effect of 50 days of exposure to cold on systolic blood
pressure of rats.
Values are Mean + S.E.M.
* Significantly greater (p < 0.05) than control by one-tailed,
independent-sample, t-test.


75 1--


































PRE 5 10 15 20 25 30 35 40 45

Doys


Source SS DF MS F P
BETWEEN
Environment 5386.9 1 5386.9 11.62 <0.01
Error 5099.3 11 463.6
WITHIN
Time 19819.9 8 2477.5 14.91 <0.01
Env. Time 7323.5 8 915.4 5.5 <0.01
Error 14626.6 88 166.2


Figure 2-2.


The effect of 47 days of exposure to cold on systolic blood
pressure of rats.











o-o Control
*-*Cold


Pre 5 10 15 20 25 30 35 40 45


Days


Source SS DF MS F P
BETWEEN
Environment 38642.9 1 38642.9 9.9 <0.01
Error 43080.7 11 3916.4
WITHIN
Time 37291.0 7 527.3 50.8 <0.01
Env. Time 2703.7 7 86.2 3.7 <0.01
Error 8078.3 77 104.9


Figure 2-3.


The effect of chronic exposure to cold on body weight of rats.
Values are Mean S.E.M.











o-o Control
*--* Cold


410 -




385 --




360--




335--




310 --
PRE


Source SS DF MS F P
BETWEEN
Envirn. 16245 1 162456 1.47 .241
Error 188388 17 11081
WITHIN
Time 119224 19 6275 253.4 <0.01
Env.*Time 5418 19 285.2 11.51 <0.01
Error 7999.7 323 24.8


Figure 2-4.


The effect of chronic exposure to cold
from experiment two.
Values are Mean + S.E.M.


on body weight of rats


5 10 15
Days










0 -

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o-o Control
*-e Cold


PRE 5 10 15 20
Days


Source SS DF MS F P
BETWEEN
Envirn 1151.76 1 1151.76 329.26 <0.01
Error 59.47 17 3.5
WITHIN
Time 56.23 20 2.81 7.62 <0.01
Env.* Time 173.15 20 8.65 23.45 <0.01
Error 125.3 340 0.37


Figure 2-5.


The effect of chronic exposure to cold on the daily intake of
food of rats.
Values are Mean S.E.M.










o-o Control
*-e Cold


10
Days


Source SS DF MS F P
BETWEEN
Envirn 8965.8 1 8965.8 5.56 <0.05
Error 29031.1 18 1612.8
WITHIN
Time 3984.5 20 199.2 4.28 <0.01
Env.*Time 3866.6 20 193.3 4.15 <0.01
Error 16767.9 360 46.6


Figure 2-6.


The effect of chronic exposure to cold on the daily intake of
water of rats.
Values are Mean S.E.M.










o-o Control
*-* Cold


5 10 15 20
Days


Source SS DF MS F P
BETWEEN
Environment 613.9 1 613.9 23.3 <0.01
Error 474.4 18 26.4
WITHIN
Time 164.9 20 8.5 9.8 <0.01
Env.*Time 125.8 20 6.3 7.5 <0.01
Error 303.7 360 .84


Figure 2-7.


The effect of chronic exposure to cold on the daily output of
urine of rats.
Values are Mean + S.E.M.


21-
PRE








30





- Cold Y = 1.01 (X) + 3.42 r = 0.700
-- Control Y = 1.06 (X) + 4.51 r = 0.710


5 6 7 8 9 10 11 12
Food Intake (g/100g bw)


Figure 2-8.


The effect of 21 days of exposure to cold on the relationship
between the intake of food and water as determined by linear
regression analysis.








31
water intake versus urine output revealed that the cold-exposed group excreted more

urine for any given amount of water intake (Figure 2-9).

The effect of chronic exposure to cold (20 and 52 days) on organ weights is

presented in Table 2-1. Exposure to cold for 52 days significantly increased the weight

of the adrenal glands, kidneys, atria, left and right ventricles as well as IBAT. Exposure

to cold for 21 days resulted in significantly increased weight of the heart, kidneys, atria,

left and right ventricles and the adrenals. No change in the weight of the thyroid was

found in either experiment. In order to determine whether an increase in fluid retention

may have contributed to the increased weight of the heart and kidneys found in

Experiment 1, the atria, ventricles and kidneys from Experiment 2 were weighed, then

dried overnight in an oven and reweighed. The atria, left ventricle, and kidneys of the

cold-exposed group remained significantly greater than controls (Table 2-3).

Discussion
Although an earlier study by Fregly et al. (61) showed that systolic, diastolic, and

mean blood pressures of cold-exposed rats were elevated after 4 or more weeks of

exposure to cold, no information was available regarding the time required for the first

elevation of blood pressure to occur. The results from the present study confirm that

of Fregly et al. (61) that chronic exposure to cold results in a significant elevation of

systolic blood pressure. The present studies indicate that the first significant elevation

of blood pressure occurred within 21 days of exposure; i.e. about one week earlier than

previously demonstrated. Systolic blood pressure remained elevated throughout the

duration of exposure. The development of cold-induced hypertension can be divided into

three rather distinct time-periods (Figure 2-2). Period one is characterized by the many

physiological changes which permit the animal to survive in the cold. During this time,

no change in blood pressure was observed. Period two is the transition period. Within

a rather narrow interval of time (18-25 days), all of the cold-exposed animals became

hypertensive. Typically, a few animals made the transition early (15-21 days), while a

portion became hypertensive up to a week later (21-28 days). In every instance, all of














- Cold Y 0.248 (X) + 3.63 r 0.366
-- Control Y 0.322 (X) + 0.85 r 0.410


9 10 11 12 13 14 15 16 17
Water Intake (ml/100g bw)


Figure 2-9.


The effect of 21 days of exposure to cold
between the daily intake of water and the
determined by linear regression analysis.


on the relationship
output of urine as


9-


n 8-

0
0
7-

E
6-

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34
the animals were hypertensive in these two experiments by the 25th day of exposure.

Period three is the period of maintenance. The animals have fully adapted to the cold

but have become hypertensive. Although these experiments continued for only 52 days,

others have exposed rats to low environmental temperature for as much as three months

(122), or longer, (22 months) (95). This suggests that chronic exposure to low

temperature is well tolerated by the rat. In addition, it provides evidence that although

chronic exposure to cold represents an unremitting stress, it is different from other

general stressors. Holloszy and Smith (95) reported that chronic exposure to cold was not

associated with the deleterious effects on health which have been shown for other

stressors such as fear, noise, or overcrowding (86,153,167).

It is well known that one of the major mechanisms by which the cold-exposed

rat increases heat production is by an increase in nonshivering thermogenesis and a

supersensitivity to norepinephrine (NE). Exposure to cold results in a large increase in

sympathetic activity. Initially, the response may be characterized as a generalized "stress"

response similar to that described by Selye (174). This is evident by a large increase in

adrenal cortical and medullary hormones, i.e. glucocorticoids and epinephrine (E),

respectively (158,192). In addition, there is a large increase in urinary concentrations of

NE and E during acute exposure to cold (133,183,122). Leduc (122) measured urinary

catecholamines in rats exposed to 3C for up to 86 days. The concentration of NE in

the urine increased threefold, peaked within the first week of exposure, and continued

to decline thereafter until the study was concluded. After 86 days, urinary output of NE

remained two times greater than controls while the concentration of E in the urine had

returned to control levels within 30 days.

Norepinephrine is the neurotransmitter of the sympathetic nervous system (SNS).

The increased urinary levels of NE suggest that the activity of the SNS is significantly

elevated by both acute and chronic exposure to cold. In humans, acute exposure to cold

(cold-pressor test) is often used as an index of SNS reactivity. The cold-pressor response

is characterized by an increase in blood pressure, heart rate, NE and E concentrations








35

in plasma (199,193). An increase in sympathetic output to the periphery will induce

vasoconstriction, decrease blood flow, and thereby decrease peripheral heat loss. This is

a beneficial and well documented physiological response to cold. The decrease in flow

is accomplished by an increase in resistance in the peripheral vasculature. Thus, an

increase in peripheral resistance should result in an elevation of arterial pressure. This

is the case in the cold-pressor test mentioned above. The cold-pressor test increases the

concentration of catecholamines in plasma and results in an increase in blood pressure.

In the current studies, chronic exposure to cold did not result in an immediate elevation

of blood pressure (during period one). Blood pressure did not increase until nearly 21

days of exposure. In view of this, the results suggest that any initial increase in

peripheral resistance in the cold-exposed rat was compensated for elsewhere. What

compensations may actually occur is unknown, but may involve changes in either blood

volume, cardiac output, or resistance in other vascular beds.

Hypertension is induced primarily by an increase in vascular resistance to blood

flow. As a consequence, blood pressure increases to maintain the proper flow of blood

to tissues and cells. (The heart, which must pump against a higher peripheral vascular

resistance undergoes work-induced hypertrophyj In both of these studies, as in numerous

other reports, chronic exposure to cold resulted in significant hypertrophy of the heart.

Additionally, in the present studies, there was an increase in left ventricular weight of

both of the cold-treated groups. Ventricular hypertrophy was evident after 21 days of

exposure, the earliest these measurements were made. These results indicate that cardiac

hypertrophy occurred either at the same time, or in some animals, just prior to a

measurable increase in systolic blood pressure. There are several possible explanations

for the cardiac hypertrophy demonstrated in this model. The most likely is an increased

workload on the heart due to an increased total peripheral resistance associated with the

development of hypertension. Certainly this is an important factor in those animals

which demonstrated an elevation of blood pressure. An additional explanation is

suggested by the results of the intake study. The cold-exposed animals drank less water








36
for any given amount of food consumed. Further, these animals excreted more urine for

any given amount of water consumed. These results suggest that the cold-exposed animal

is in a state of self-imposed, or voluntary volume depletion or contraction. That the

cold-exposed rat is volume contracted has been suggested previously by Fregly et al. (58).

Whether this altered fluid balance is an attempt by the animal to compensate for the

increased central blood volume which would result from increased peripheral resistance

is speculative. If the animal is volume contracted, an increased hematocrit and an

increased viscosity would likely result. An increased viscosity could also impose a greater

work load on the whole heart and could contribute to the generalized cardiac atriaa, right

and left ventricle) hypertrophy seen in this model. The effect of viscosity on blood

pressure is often overlooked, but in accordance with Hagen-Poiseuille's Law, peripheral

vascular resistance is dependent on the viscosity of the blood. A significantly increased

hematocrit was found in the group exposed to cold for 21 days (49 + 0.86 and 47 + 0.4

for controls). Similarly, a significantly increased hematocrit was found in elderly

hypertensive patients by Tibblin et al. (186). An increase in serum osmolality after

exposure to cold has been reported previously by Fregly et al. (58).

A final contributing factor to cardiac hypertrophy may be the increased

sympathetic drive to the heart. Both exercise training and long term exposure to cold

result in an increased SNS activity, an increased cardiac output, and cardiac hypertrophy.

That the SNS activity is important in inducing this hypertrophy has been demonstrated

by chronic injections of NE (81), E (47), and the specific B-adrenoceptor agonist,

isoproterenol (147), all of which result in cardiac hypertrophy. Further, a sympathetic

blockade with guanethidine prevented the development of cardiac hypertrophy in both

exercise-trained and chronic isoproterenol-treated rats (147). In the case of chronic

exposure to cold, the increased activity of the SNS could explain the development of

cardiac hypertrophy without a significant elevation of blood pressure when other

compensatory mechanisms have been invoked.








37
The dependence of cardiac hypertrophy on excess adrenergic stimulation could be

demonstrated in this model by chronic treatment with a beta adrenoceptor antagonist or

even guanethidine as used above. However, nonshivering thermogenesis is likewise a

beta-adrenergic dependent system. Future studies will be needed to assess the role of the

adrenergic system in both the cardiac hypertrophy and hypertension seen in this model.

An increase in right ventricular weight is an interesting finding. It is tempting

to suggest that the cold air results in a localized vasoconstriction within the pulmonary

circulation. Indeed, pulmonary vasoconstriction does contribute to the right ventricular

hypertrophy and hypoxia-related hypertension seen commonly in cattle at high altitude,

i.e. Brisketts Disease. However, when Mather et al. (137) compared pulmonary artery,

left atria and core temperatures in dogs exposed to extreme cold (-18C), no differences

(gradient < 0.01C) were found between the pulmonary artery and the left atria. Over

time, all three temperatures fell, suggesting an overwhelmingly low temperature, but no

differences occurred between temperatures in the pulmonary artery and left atria. Thus,

it appears unlikely that pulmonary vasoconstriction in response to breathing cold air

contributes to right ventricular hypertrophy seen in the present studies. A more logical

hypothesis is an overall cardiac hypertrophy induced by the high cardiac output

necessitated by exposure to cold.

Significant hypertrophy of the kidneys and adrenal glands was also seen in response

to cold. These findings are in accord with the work of many others. No doubt the

hypertrophy seen in the kidneys is due primarily to the very large increase in food

ingested by the cold-treated group. Hypertrophied adrenal glands suggest that the

animals have not, as yet, adapted to the stress of cold air. This is in contrast to the

concentration of epinephrine in the urine which is no longer elevated at 30 days,

suggesting that adaptation has occurred. Reasons for this apparent conflict are unclear.

The present studies, in combination with the previous work of others, raise some

intriguing questions about cold-induced hypertension. When the rat is exposed to cold,

there is an immediate and apparently sustained increase in the activity of the SNS. That








38

this occurs is supported by an increased urinary NE (133,121,122,147), increased plasma

NE and an increased cardiac output (89). Initial indications would suggest cold-induced

hypertension to be the neurogenic model of hypertension which investigators have been

seeking. If this is the case, why then doesn't the rat immediately become hypertensive

and remain as such? What mechanisms are effective in initially offsetting this increased

activity of the SNS? Why are they ineffective later? In this respect, cold-induced

hypertension appears similar to other models of hypertension (DOCA/salt and renal

encapsulation) in that it takes about 3 weeks for the hypertension to develop. If

increased activity of the SNS is involved, this slow onset may suggest either structural

changes or damage at the level of the kidney or vasculature due to altered function

induced by continued high stimulation.

One possible mechanism which may result in changes in peripheral resistance over

time would be changes in the vascular reactivity to stimulation by the SNS. Fregly et

al. (61) tested the vascular responsiveness to an acute injection of both alpha- and beta-

adrenoceptor agonists in cold-induced hypertensive rats. These measurements were made

in unanesthetized, unrestrained rats which remained in the cold. The authors found a

decreased alpha-adrenergic or vasoconstrictor response and an unchanged, or possibly

reduced, beta-adrenergic (vasodilator) response. Similar results for alpha-adrenoceptor

stimulation were found by Bryar et al. (19) in aortic smooth muscle and by this author

(unpublished results) in femoral artery smooth muscle of cold-acclimated rats in vitro.

However, the response to the beta-adrenoceptor agonist, isoproterenol, suggested a greater

vasodilator sensitivity in isolated smooth muscle in vitro. These changes would appear

to be protective mechanisms induced by cold which would work against a more severe

elevation of blood pressure. Whether these changes occur early in the adaptation process

is unknown.

As demonstrated by the significantly increased (60% over controls) food intakes,

it can be expected that the intake of sodium is similarly increased. Whether an increase

in sodium, and thus a volume expansion, occurs and contributes to the development of








39
hypertension is undetermined. However, the results from the present studies as well as

those of an earlier study by Fregly et al. (58) suggest that the cold-exposed rat is actually

in a state of voluntary dehydration or volume contraction. At any given food intake the

water intake is lower; further, for any amount of water ingested, the amount of urine

excreted is greater in the cold-exposed rat. Once the rat is removed from the cold, a

large post-cold drink is demonstrated. In addition, the increased hematocrit seen in the

present study supports the idea of volume contraction rather than volume expansion in

this model. If the animal is volume contracted, it may represent a mechanism which

was called upon to compensate effectively for the initial vasoconstriction induced by the

immediate increase in the output of the SNS. Whether this occurs remains to be tested.

Further, whether the excessive sodium intake which occurs due to increased intake of

food plays a role in this model of hypertension remains to be considered.

Finally, whether changes in either the baroreceptor system or in the circulating

levels of the vasoactive hormones occur and contribute to this form of hypertension also

remains for further investigation.














CHAPTER THREE
THE EFFECT OF A LOW SODIUM DIET ON
COLD-INDUCED HYPERTENSION


Introduction
The importance of fluid volume in the regulation of arterial pressure has been

accepted for a great many years. A complex interaction or link between fluid volume

homeostasis and regulation of blood pressure is involved in the control of the excretion

of water and sodium by the kidneys. Neural, hormonal and intrinsic renal mechanisms

appear to control the excretion of water and sodium and thus, arterial pressure. The

basic idea that the kidneys act as a final and overriding regulator of blood pressure is

based on a simple feedback loop. When arterial pressure is elevated, an increase in

sodium and water excretion occurs which decreases blood volume (pressure-natriuresis),

and thus returns blood pressure to control levels. This suggests that an increase in the

excretion of sodium is a protective mechanism against a sustained elevation of blood

pressure. Conversely, this relationship suggests that an increase in the intake and/or a

retention of sodium may contribute to the development of hypertension if the sodium

were not completely removed.

Epidemiological and clinical studies suggest a relationship between the intake of

sodium and the incidence of hypertensive. For example, Dahl (38) examined this

relationship in several distinct populations. At a low intake, Eskimos from Alaska had

no incidence of hypertension. Conversely, at the other extreme, the prevalence of

hypertension was 39% of the population for men and women from Northern Japan whose

average intake of salt was 26 g/day. Further support for the role of sodium in

hypertension stems from the use of sodium restriction and/or the administration of a

diuretic as a major antihypertensive therapy. In addition, several experimental models








41

exist which are dependent on an increased consumption of salt; for example, the Dahl

salt-sensitive and DOCA/salt models. Additional evidence stems from the classical

experiment of Meneely et al. (138) in which rats were placed on high sodium chloride

intakes and hypertension developed. Further, Weinberger et al. (197) studied the effects

of altered sodium intake in normotensive young men and demonstrated a significantly

increased blood pressure with dietary sodium loading.

In the first series of experiments investigating cold-induced hypertension, a large

increase in food consumption was demonstrated (Chapter Two). An increase in food

intake is required by the animal in order to meet the increased energy demand required

for an elevation of metabolic rate. The cold-exposed animal increased its intake of food

by 50 to 60%. As a result of this increase, the animal also ingested 50 to 60% more

sodium than controls. Thus, the animal is forced by the need for increased calories used

for heat production to increase the intake of food and hence sodium. The purpose of

these experiments was to determine whether the increase in the intake of sodium by

animals chronically exposed to cold contributes to the development of cold-induced

hypertension.

Methods
Experiment One

Male rats of the Sprague Dawley strain used in this experiment were the same

animals used in Experiment 2, Chapter Two. All animals were housed and handled as

described previously. After the urine volume was recorded, an aliquot was removed for

the determination of the concentrations of sodium and potassium by flame photometry

(Technicon Autoanalyzer). In addition, an aliquot of urine was removed for the

determination of refractive index. A hand-held refractometer (American Optical,

Buffalo, NY) was used for these measurements. Baseline measurements were recorded

for two weeks. As described previously, on the first experimental day, all animals were

moved into temperature controlled environmental chambers (26 or 6C). Measurements








42

were recorded daily at 0800 h throughout the duration of the experiment. Rats were not

moved from either their home cages or environments.

Experiment Two

Twelve male rats of the Sprague-Dawley strain were divided randomly into two

groups. Upon arrival in the laboratory, all rats were given distilled, deionized water and

powdered Purina Laboratory Chow (#5001) ad libitum. One week was allowed for the

animals to adjust to their new environment. All rats were housed individually. After

one week, all animals were placed on a special Hartroft sodium test diet (United States

Biochemical Corp., Cleveland, OH) containing 0.3% sodium, i.e. the same concentration

as that in Purina Laboratory Chow.

Systolic blood pressures were measured from unanesthetized rats indirectly from the

tail as described previously (Chapter Two). Pressures were recorded twice each week and

represent the mean of ten measurements per animal. At the end of the measurements,

the animals were immediately returned to their home environments.

Body weight, food and water intakes, as well as urinary volume were determined

as described previously. Measurements were made daily at 0800 h except for the days

on which blood pressure was measured.

After baseline measurements were made (one week), animals were moved into

temperature controlled environmental chambers as described previously. At this time, the

cold-treated group was placed on a reduced sodium Hartroft diet containing 0.15%

sodium. Since cold-treated rats eat approximately twice as much food as controls, the

intake of sodium would approximately be the same. Controls remained on the Hartroft

diet containing 0.3% sodium. Measurements were continued for 21 days.

At the end of each experiment, animals were sacrificed and the organs rapidly

removed and handled as described previously. Blood was collected from the trunk into

chilled tubes containing either tetrasodium EDTA or no anticoagulant. The samples of

blood were quickly placed into a chilled centrifuge and spun at 26,000 rpm for 45

minutes. The plasma and serum were transferred into labeled polypropylene tubes and








43
stored at -800C for later determination of aldosterone, osmolality and concentration of

sodium and potassium in serum. In addition, two micro-hematocrit tubes were filled

with blood collected from the trunk for the determination of hematocrit.

The concentration of aldosterone in plasma was measured in unextracted samples

by radioimmunoassay. Samples (250 ul) were assayed in duplicate where possible. The

gamma-coat kit (Diagnostic Products Co., Los Angeles, CA) has a detection limit of 25

pg/ml. Any value which measured less than 25 pg/ml was recorded as 25 pg/ml.

Coefficient of variability for intraassay measurements was 3-8% and interassay variability

was 4-10%. Osmolality was determined by a vapor pressure osmometer (Model 5100C,

Wescor Co., Logan UT). Duplicate samples (5 ul) of serum were used. The osmometer

has a precision of + 2 mmol/Kg.

Statistical analyses of systolic blood pressure, body weight, and urinary outputs of

sodium and potassium were carried out by a two-way analysis of variance repeated in

time. Changes in organ weights, osmolalities, aldosterone concentrations of sodium and

potassium in serum as well as hematocrit, were determined by a one-tailed Student's t-

test for independent samples, when appropriate. Significance was set at the 95%

confidence level.

Results
Experiment One

Chronic exposure to cold resulted in an increased daily consumption of food by

an average of 50% (Table 3-1). Thus, the intake of sodium was 50% greater in the cold-

exposed group. This increased intake was not associated with an increase in fluid intake

of the same magnitude. Cold-exposed rats increased their average intake of water by

only 20%. In addition, the average daily output of urine increased by greater than 50%.

Associated with an increased intake of food, the 24 hour urinary output of sodium and

potassium were significantly increased, as demonstrated by a significant (p<0.01) cold X

time interaction and main effect (Figure 3-1, 3-2). The peak output occurred at the

eleventh day of exposure, declined slightly but remained significantly greater than




















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*--*Cold


5 10 15 20

Days


Source SS DF MS F P

BETWEEN
Environment 396.33 1 396.33 51.77 <0.01
Error 130.16 17 7.66
WITHIN
Time 123.78 19 6.62 5.13 <0.01
Env.*Time 144.81 19 7.62 6.00 <0.01
Error 410.06 323 1.27


Figure 3-1.


The effect of 21 days of exposure to cold on the daily output
of sodium in the urine of rats.
Values are Mean + S.E.M.


2 -
Pre










o- o Control
*-* Cold


26-

24-

22-

20-

18-

16

14-

12 ,

10-

8--
Pre


Source 88 DF MS F P
BETWEEN
Environment 2460.9 1 2460.9 62.9 <0.01
Error 665.13 17 39.13
WITHIN
Time 941.85 19 49.57 5.09 <0.01
Env.*Time 1076.66 19 56.67 5.82 <0.01
Error 3145.31 323 9.74


Figure 3-2.


The effect of 21 days of exposure to cold on the daily output
of potassium in the urine of rats.
Values are Mean + S.E.M.


5 10 15 20
Days








47

controls throughout the duration of the experiment. This peak occurred later in time

than did the peak for food and water intake which occurred by the fifth day of exposure

to cold. A regression analysis of intake of sodium in food versus output of sodium in

urine revealed no change in this relationship for the two groups (Figure 3-3). The cold-

treated animals consumed more sodium and similarly had a greater output of sodium in

the urine.

Daily refractive indices were measured throughout the period of exposure to cold

to assess the concentration of urine. No significant main effect of cold air, nor a cold

X time interaction was seen (Figure 3-4).

Experiment Two

The effect of administration of a reduced sodium diet during chronic exposure to

cold on blood pressure is shown in Figure 3-5. Systolic blood pressure of the cold-

treated group increased significantly (p<0.01) as indicated by both a significant cold X

time interaction and main effect. All animals in the cold-treated group were

hypertensive by day 21, as demonstrated previously.

The cold-exposed group significantly increased (54% over controls) their intake of

food (Figure 3-6). In contrast to the earlier study using Purina Laboratory Chow

(#5001), the cold-treated group did not significantly increase its intake of water (Figure

3-7). However, the increased urine output demonstrated in the earlier studies by the

cold-exposed group was seen here as well (Figure 3-8). The rate of gain of body weight

in the cold-treated group was significantly less than that of controls, as indicated by a

significant cold X time interaction (Figure 3-9).

The effect of chronic exposure to cold on organ weights was not influenced by the

reduced sodium diet. As seen previously, the ratios of organ weight to body weight for

the kidneys, heart, right and left ventricles, atria and adrenal glands were significantly

increased (Table 3-2). No difference in the weight of the thyroid gland was detected.

Due to differences in body weights, regression analyses of each organ weight (mg) on

body weight (g) were performed. No differences in slopes were detected for any of the








48






- Cold Y= 0.164 (X) + 1.12 r=0.434
-- Control Y= 0.126 (X) + 1.51 r=0.180


10 20 30 40 51
Food (g/day)


Figure 3-3.


The effect of 21 days of exposure to cold on the relationship
between the daily intake and output of sodium as determined
by linear regression analysis.
Values are Mean + S.E.M.


:3
0--

-'-6')~


SE




.D
















1.353-


1.352-


1.351-


1.350-


1.349-


1.348--
Baseline


Figure 3-4.


The effect of 20 days of exposure to cold on the refractive
index of the urine of rats.
Values are Mean + S.E.M.


--J Cold
m Control


Day 20












o0- Control
*-* Cold


100 1 I I I .
PRE 5


15 20


Days


Source SS DF MS F P
BETWEEN
Environment 901.78 1 901.78 13.17 <0.01
Error 616.1 9 68.45
WITHIN
Time 2240.98 7 320.14 5.58 <0.01
Env.*Time 1479.5 7 211.35 3.684 <0.01
Error 3614.3 63 57.37 3.68 <0.01


Figure 3-5.


The effect of 21 days of reduced sodium diet and exposure to
cold on systolic blood pressure of rats.
Values are Mean + S.E.M.










o-o Control
*-o Cold


PRE 5 10 15
Days


Source SS DF MS F P
BETWEEN
Environment 108.86 1 108.86 52.31 <0.01
Error 18.73 9 2.081
WITHIN
Time 9.141 11 0.831 4.95 <0.01
Env. Time 23.845 11 2.168 12.91 <0.01
Error 16.588 99 166.2


Figure 3-6.


The effect of a low sodium diet during exposure to cold on
the daily intake of food of rats.
Values are Mean + S.E.M.







52
o-o Control
*-* Cold

T
9-








S5--
-5 1/ -


4 --
PRE


5


10
Days


Source SS DF MS F P
BETWEEN
Environment 12.070 1 12.070 0.966
Error 87.420 7 12.489
WITHIN
Time 34.714 11 3.156 4.732 <0.01
Env. Time 15.544 11 1.413 2.118 <0.05
Error 51.370 77 0.667


Figure 3-7.


The effect of a low sodium diet during exposure to cold on the
daily intake of water of rats.
Values are Mean + S.E.M.


15








J o--o Control
*-. Cold

T
T T
I TT




/i/ -- iI I r -
11




I0


10
Days


Source SS DF MS F P
BETWEEN
Environment 11.239 1 11.239 1.329
Error 59.197 7 8.457
WITHIN
Time 40.920 11 3.720 11.735 <0.01
Env. Time 5.016 11 0.456 1.438
Error 24.377 77 0.317


Figure 3-8.


The effect of a low sodium diet during
daily output of urine of rats.
Values are Mean + S.E.M.


exposure to cold on the


PRE


I


1




























375 --




350 --
PRE


5 10 15
Days


Source SS DF MS F P
BETWEEN
Environment 0.967 1 0.967 1.488

Error 5.847 9 0.65

WITHIN
Time 7.052 15 0.47 117.50 <0.01

Env. Time 0.285 15 0.019 4.750 <0.01
Error 0.488 135 0.004


Figure 3-9.


The effect of a low sodium diet during exposure to cold on the
body weight of rats.
Values are Mean + S.E.M.




























































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56
relationships, while the intercept was significantly greater in the cold-treated group for

the whole heart (Table 3-3, Figure 3-10). When a regression analysis was carried out

on final systolic blood pressure (X) versus heart weight (Y), no change in the relationship

was found in the group chronically exposed to cold air. Simply, the cold-treated animals

moved to a higher position along this relationship. However, when a regression analysis

was performed on final systolic blood pressure (SBP) and the weight of the left ventricle,

a significant effect (p<0.05) of exposure to cold on the intercept was evident. Thus, at

any SBP, the weight of the left ventricle of the cold-treated group was greater (Figure

3-11).

There was no significant effect of the reduced sodium diet either on the

concentrations of sodium and potassium in serum or on serum osmolality (Table 3-4).

In addition, no difference in hematocrit was seen in cold-treated rats in this study (Table

3-4). This finding is different from that seen earlier when cold-exposed rats were given

Purina Laboratory Chow (0.3% sodium). The concentration of aldosterone in the plasma

was not affected by exposure to cold in either experiment. No significant effect of a

low sodium intake was seen on the level of aldosterone. Although the trend was toward

an increased concentration, the variability was quite large.

Discussion
The association between the intake of dietary sodium and the prevalence of

hypertension and cardiovascular disease has been suggested by epidemiological and clinical

studies. The use of sodium restriction and diuretic administration as effective

antihypertensive therapies in humans also supports the idea of a role for sodium in

hypertension. It also suggests a role for volume reduction. In addition, several

experimental models are dependent on an increase in sodium intake including the Dahl

salt-sensitive and DOCA/salt models. When animals are chronically exposed to low

environmental temperature there is an immediate and sustained increase in metabolic rate.

The increased metabolic rate, and thus caloric expenditure, necessitates an increase in the

intake of calories via food. The laboratory rat is restricted in its choice of food in that


























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Y = 2.841 (X) + 68.14
Y = 3.174 (X) + 126.7


Body Weight (g)


Figure 3-10.


The effect of chronic exposure to cold on the relationship
between body weight and the weight of the heart as determined
by linear regression analysis.


-- Control
- Cold


r =.546
r=.912


1000-


900-




800-




700-


hIJIJ-~


380


460














1000-


900-




800-


700-


- Cold Y = 10.17 (X) 594 r=0.736
-- Control Y = 6.59 (X) 78.3 r=0.603


-I


1


120


130


140


Systolic Blood Pressure (mm Hg)


Figure 3-11.


The effect of chronic
between systolic blood
ventricle as determined


exposure to cold on the relationship
pressure and the weight of the left
by linear regression analysis.


600

















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61

the chow provided is its sole source. Thus the composition of the diet is also restricted

in the content of sodium. The increased intake of food is accompanied by an increased

intake of sodium. In the first series of experiments, food and thus sodium intake

increased by over 50%. It was possible that changes in sodium intake of such magnitude

contributed to the elevation of blood pressure seen in this model. Hence, a low sodium

diet was employed to investigate this possibility.

In the first experiment, both groups (control and cold) were given identical Purina

Laboratory Chow (#5001) with a sodium content of 0.3%. The cold-treated group

consumed over twice as much sodium as controls. In addition, the cold-treated group

consumed only 20% more water while excreting 50% more urine. The shift in the

relationship between food:water intake and water intake:urine output supports the idea

of self-imposed volume contraction as originally proposed by Fregly et al. (58). It would

be difficult for the cold-treated animal to be volume contracted while consuming a large

amount of sodium, the primary regulator of extracellular fluid status, unless the excess

sodium could be eliminated. When urinary sodium and potassium outputs were measured,

the cold-treated group excreted 45% and 40% more sodium and potassium, respectively.

In addition, when a linear regression analysis compared the output of urinary sodium

(Y) against the intake of sodium or food (X), no difference in the relationship was

evident in the cold-treated animals (Figure 3-11). Thus, the increased consumption of

sodium was effectively offset by an increased excretion. It does not appear as if the

cold-treated animal retains the excess sodium it ingested. This would suggest that sodium

retention is not a mechanism which contributes to cold-induced hypertension. The results

do, however, suggest that extracellular fluid volume may be reduced in cold-treated rats.

Previous work with chronic exposure to cold suggests that a self-imposed imbalance

in fluid exchange occurs (58). In the present studies, an increase in food and water

intakes as well as an increase in urine output were demonstrated. One mechanism which

would prevent dehydration in this situation would be to increase the output of solutes

in the urine in a reduced urine output (i.e. increased urine osmolality). When the








62

refractive index of the urine was measured, no difference in the urine concentration

between groups was found. Similarly, Fregly et al. (58) found no difference in urine

osmolality in cold-treated rats. Thus, the increased osmolar load delivered to the kidney

as a result of the increase in food consumption was handled by an increase in urine

volume not by excreting a more concentrated urine. This cold-induced dehydration has

been suggested to occur in man (179,123,146,2) as well as in rats by other investigators

and has been reviewed extensively (57).

A retention of sodium, and thus an increase in fluid volume, is only one

mechanism by which sodium may contribute to hypertension. It is unclear whether the

elevated intake of sodium must be retained in order to be a contributing factor in an

elevation of blood pressure. Perhaps as a result of handling the increased sodium,

changes have occurred at the cellular level or at the kidney. To investigate this

possibility, a diet low in sodium (50% less than control) was administered to the

experimental group during exposure to cold in experiment two. The cold-treated group

consumed 54% more food than did controls. Thus, no difference in sodium intake

occurred. Interestingly, no increase in the intake of fluid was demonstrated in this

experiment suggesting that the increased water intake seen in the previous experiments

is associated with the increased sodium intake. Urine output however, was significantly

increased as seen with normal chow. These results further support a state of self-

imposed dehydration.

The systolic blood pressure of the cold-exposed group was significantly increased

in the presence of a normalized sodium intake. The blood pressure was increased by the

seventeenth day of exposure to cold and remained elevated when the experiment was

ended. The time-course, or the amount of exposure necessary for an elevation of blood

pressure, was unchanged, as was the magnitude. In addition, significant hypertrophy of

the whole heart, right and left ventricles, atria and adrenal glands was evident. When

regression analyses were performed on the organ weight to body weight relationships,

only the intercept for the whole heart was significantly increased. However, exposure








63

to cold significantly altered the relationship between systolic blood pressure and left

ventricular weight. When final systolic blood pressure (Y) was compared against left

ventricular weight (X), the intercept was increased. This suggests a work-induced

hypertrophy.

No effect of a reduced sodium diet during exposure to cold on either plasma

electrolytes or the primary sodium regulating hormone, aldosterone, was found. Although

the serum sodium concentration of the cold-treated group on a reduced sodium diet

tended to be lower, it was not significantly so. Likewise, there was a trend for the

aldosterone concentration of this group to be higher, but there was a large variability.

Recent studies investigating human essential hypertension have provided

inconclusive evidence for a role of sodium. From such studies, two observations seem

clear. First, not all individuals respond to an increase in sodium intake with an elevation

of blood pressure. Second, sodium restriction and diuretic therapy are not effective in

all hypertensive patients. In addition, not all experimental models of hypertension are

dependent on an increased sodium intake. The present studies failed to indicate that an

elevated sodium intake was associated with an elevation of systolic blood pressure.

However, the present studies do not eliminate the possibility of an indirect effect of

sodium in either this or other models of hypertension as intracellular sodium content,

sodium transport changes, and other sodium dependent processes were not investigated.















CHAPTER FOUR
THE ROLE OF SYMPATHETIC NERVOUS SYSTEM IN
COLD-INDUCED HYPERTENSION

Introduction.

It is well known that the sympathetic nervous system (SNS) controls both minute-

to-minute and long term arterial pressure. In addition, there is a great deal of evidence

which suggests that changes occur within the SNS in nearly every form of experimental

hypertension (202). Much of the difficulty in assessing the exact role for the central

nervous system in hypertension has been attributed to the inability to identify an area

or region of neurons responsible for maintaining the normal output of the preganglionic

sympathetic fibers and thus normal arterial pressure. The output of the SNS is controlled

by a negative feedback loop with input from volume or stretch sensitive

mechanoreceptors via the baroreceptor reflex. These receptors respond to local distention

with an increased activity which is transmitted, via the IX and X cranial nerves, to the

central nervous system. The first synapse of the baroreceptor system is in the nucleus

tractus solitarius (NTS) located in the ponto-medullary region of the brainstem. An

increased afferent activity in this region results in the stimulation of an inhibitory

pathway (1,164). This increased inhibitory input is targeted toward the tonic activity of

the sympathetic fibers which influence vasomotor tone and cardiac rate. It is well known

that the NTS receives input from higher brain centers, including the area postrema,

hypothalamus, thalamus, stria terminalis and cerebral cortex (1). The large amount of

input to the region of the NTS led Abrams (1) to suggest it as the center which

integrates central and peripheral information to change the cardiovascular system, and

thus to maintain circulatory homeostasis. In addition, the NTS projects to a large variety

of centers including the dorsal motor nucleus, parabrachial nucleus, hypothalamus, ventro-








65

lateral pressor area of the medulla, as well as direct projection to the spinal cord.

Several of these areas are also involved in the control of body temperature as mentioned

previously (Chapter Two). Thus, the potential for direct neural communication between

the centers which are involved in temperature regulation and those involved in the

regulation of blood pressure is an interesting possibility.

Over the last several years, Reis and his associates (164) have been identifying the

regions, pathways, and neurotransmitters which are ultimately involved in controlling

resting arterial pressure. Their work suggests that a specific zone within the rostral

ventrolateral quadrant of the medulla oblongata functions in this capacity. Further, a

subpopulation of adrenergic Cl fibers (which stain specifically for epinephrine (E))

directly innervate preganglionic neurons in the spinal cord (164). In addition, it is in

this area, where clonidine, a clinically efficacious antihypertensive agent, acts to lower

blood pressure (16,70,163). Interestingly, clonidine, a mixed alpha,- alpha2-adrenoceptor

agonist works as an imidazole and thus at an imidazole receptor to lower arterial pressure

rather than at an adrenergic receptor as originally hypothesized (190,46). With these new

findings, it may now be possible to examine whether changes occur centrally which

contribute to the elevated blood pressure in hypertensive animals. Further, these findings

provide impetus for a renewed interest in the role of the baroreceptors in hypertension.

Several lines of evidence suggest a role for the baroreceptor reflex in hypertension.

The first line of evidence stems from the basic idea that the baroreceptor reflex functions

via a negative feedback mechanism to decrease the output of the SNS. Many forms of

established experimental hypertension are characterized by a hyperactive SNS. A

decreased inhibitory influence is one mechanism by which the baroreceptors could

contribute to such a hyperactive state. Second, an attenuated baroreceptor reflex function

has been demonstrated in many experimental models of hypertension. Whether the

baroreceptor reflex is altered in cold-induced hypertension is unknown.

The role of the SNS is not simply a one mechanism hypothesis in which

excessive activation or output acts on the vasculature directly to result in vasoconstriction.








66

Indeed, as many additional possibilities exist as there are functions for the sympathetic

nervous system. For instance, an increased vascular reactivity has been reported in

hypertensive humans (39) as well as in various models of experimental hypertension

(60,110,49). The changes in vascular responsiveness may be due to changes in a) receptor

levels; b) post-receptor transduction; c) direct neuromodulation (by All, purines,

prostaglandins or local metabolites); d) local temperature (cold facilitates vasoconstriction)

and e) false transmitters. The exact mechanisms for the changes in vascular

responsiveness in the various models and significance of those findings remains for

further investigation.

It is also well known that the kidneys are highly innervated by the SNS. The

effects of the adrenergic system on renal function, and thus arterial pressure during the

development of hypertension is still under investigation. An increase in renal nerve

activity may increase renal resistance, reduce glomerular filtration rate, increase plasma

renin activity and directly mediate tubular sodium reabsorption. Additionally, acute

occlusion of the renal artery (109), intravenous infusion of E (107), and electrical

stimulation of the renal nerves (42) result in a decrease in sodium excretion. When NE

was chronically infused into the renal artery, Cowley and Lohmeier (33) demonstrated

a shift of the body fluid volume-arterial pressure function curve to the right and an

elevation of blood pressure. A shift in the renal function curve indicates that an increase

in arterial pressure would be necessary for any given level of sodium and water excretion

during chronic infusion of NE in order to achieve long-term fluid and electrolyte

balance. According to Guyton (75), the kidney (i.e. the renal function curve) is the

ultimate regulator of arterial pressure. No sustained change in arterial pressure is possible

unless a change has also occurred at the level of the kidney (78). Whether such changes

occur in cold-induced hypertension remain to be investigated. However, if the curve is

shifted to the right, this would provide a mechanism to explain how an elevated arterial

pressure could be sustained in the face of the volume contraction as hypothesized in the

cold-induced model (Chapters Two and Three).








67

Chronic exposure of rats to cold results in an elevation of blood pressure. In

addition, these animals are characterized, at least in the acute phase of adaptation, as

having a large increase in the activity of the SNS (134). Further, an elevated activity

of the SNS, and thus, an increased circulating level of NE would imply an elevation of

plasma renin activity and All, as well as a decreased sensitivity of the baroreceptor

reflex. Whether these alterations occur and are sustained, and whether they contribute

to the elevation of blood pressure seen with exposure to cold, is unknown. The purpose

of these experiments was to describe the blood pressure, baroreceptor and SNS profile

of the cannulated rat which is chronically exposed to low environmental temperature.

Methods

General Methods

Twenty male Sprague Dawley rats weighing between 150-200 g were housed

individually and provided with powdered Purina Laboratory Chow (#5001) and water ad

libitum. The vivarium was maintained as described previously (Chapter Two). After the

femoral arteries were cannulated, the rats were divided randomly into two groups (control

or cold-treated). Ten days were allowed for them to recover fully from the effects of

surgery. On the first experimental day, both groups were moved into temperature

controlled environmental chambers (either 26 or 6C).

Cannulation Procedure

Cannulae. Venous catheters were prepared from 50 cm lengths of 20 gauge clear

vinyl tubing (SV-55, 0.8 mm ID, 1.2 mm OD, Dural Plastics, Australia). A slight bevel

with a blunt tip was cut on one end. Arterial catheters were prepared from 3.5 cm

lengths of vinyl tubing (SV-31, Dural Plastics) and connected to a long (48 cm) length

of Tygon microbore tubing (S-54HL, 0.04" ID, 0.07" OD) with cyclohexanone. A small

piece of thin wall, stainless steel hypodermic tubing (HTX-24TW, Small Parts INC,

Miami, FL) was inserted at the SV31/SV55 junction, this allowed the suture to be

secured tightly without collapsing the cannula. The cannulae were gas-sterilized (ethylene

oxide) prior to use.










Cannula sheaths. The catheters were pulled through a sterilized (ethylene oxide)

steel spring coil (12 in. length, 0.26 in. OD, 0.206 in. ID, Exacto Spring Co, Grafton, WI)

with a dacron Patch (0.8 mm thick, Troy Mills, NH) attached to one end. The dacron

patch was implanted subcutaneously; sutured at two points to secure the catheter/spring

in place, and the skin sutured over it. The spring and catheter were exteriorized at the

front of the cage. The spring coils were lightly coated with quinidine sulfate (200 mg/ml

ethanol) to discourage chewing.

Surgery and maintenance of cannulae. Animals were fasted the night before

surgery. All surgery was conducted in a semi-sterile designated surgical area. Surgical

instruments were sterilized either in activated dialdehyde solution (Cidex, Surgikos,

Arlington, TX) for 24 hours or autoclaved prior to use. Animals were initially

anesthetized with Brevitol HCI (50 mg/kg, Lilly) given i.p. and maintained with Sodium

Pentothal (0.8 mg/kg, Abbott Laboratories, N. Chicago, IL) i.v. as needed. Once

anesthetized, a small area of the left inner leg and an area between the shoulders was

shaved and then cleansed with Zephiran Chloride solution (Winthrop Labs, New York,

NY). Sterile catheters were implanted first into the femoral vein and then into the

femoral artery. The femoral vein was separated from the femoral nerve and artery by

blunt dissection; preservation of nearby nervous tissue was emphasized. The cannula

(filled with sterile saline) was inserted approximately 35 mm into the femoral vein.

Local application of Lidocaine HCl (0.2%, Dexter Corp, Chagrin Falls, OH) was applied

to the isolated vessel to prevent vascular contraction. Once the catheter was inserted, it

was secured with sterile 5-0, monofilament nylon suture (Ethilon 698G, Ethicon, NJ).

A small portion of the femoral artery was dissected from the femoral nerve. The

catheter was inserted into the artery and secured proximally and distally with sterile 5-

0 suture. The free end of the catheters were brought around to the back subcutaneously

via a trocar and exteriorized through a small incision between the shoulder blades. Upon

completion of surgery, animals received 15 mg of Ampicillin (50 mg/ml, Bristol Labs,

NY) given subcutaneously. Bacitracin/Polymyxin B/Neomycin (ointment, Upjohn Co.,








69

Kalamazoo, MI) and 2% Xylocaine Jelly (lidocaine HCl, Astra Pharmaceutical Products

INC, Westborough, MA) were applied to all skin incisions which were closed with wound

clips (9 mm, Clay Adams, Parsippany, NJ). These precautions aid in prevention of

infection, minimized the discomfort to the animals and permitted long-term sampling.

Catheters were flushed with sterile heparin sodium (10 USP U/ml of 0.9% saline), filled

with sterile heparin (1000 u/ml, porcine derived, LyphoMed INC, Rosemont, IL), and

sealed with either an 18 or 20 gauge plug. All animals were closely monitored following

surgery. Any animal which appeared to experience more than the normal discomfort

associated with the surgical procedures was euthanized with an overdose of pentobarbital.

The use of a chronic cannulated preparation permitted easy access to arterial blood

samples, i.v. administration of drugs, and direct recording of blood pressure without

disturbing the animal. In addition, it allowed maximal freedom of movement for the rat.

Using a similar catheterization procedure and the carotid artery, this author has kept

cannulae patent for at least 6 weeks. The described procedure is quite similar to that

of Raff et al. (159,160) who have maintained cannulae patent in the femoral artery until

their experiment ended (30 days). As regards attrition, over 80% of the arterial and

venous cannulae were patent after 42 days in the controls, while only 50% of the cold-

treated group had both cannulae functioning at this time. Although this is the first

report of this difference between normo- and hypertensive animals with regard to

functional life of cannulae, it has been mentioned by Weeks (unpublished, personal

communication No. 1011, UpJohn) as well.

Sample Collection

Arterial blood samples (< 5 ml/kg) were collected at various times throughout the

experiment. At 0800 h, the dead space of the arterial line (0.4 ml) was removed and

discarded. An additional 0.4 ml of blood was withdrawn into a sterile 1 ml syringe and

set aside. The arterial sample was withdrawn into a 3 ml syringe containing tetrasodium

EDTA (50 ul 0.3 M/ml sample, Sigma, St. Louis, MO). Care was taken not to disturbed

the animal. The samples were immediately placed into chilled polypropylene tubes. In










addition, two micro-hematocrit tubes (ammonium heparin) were filled directly from the

cannula. The 0.4 ml of blood which was removed earlier was returned to the animal.

A volume of sterile saline equal to the sample volume taken was administered. The

saline served to maintain volume and to flush the cannula. The cannula was refilled with

heparin (1000 U/ml) and sealed. Blood samples were quickly placed into a chilled

centrifuge (IEC Model CRU-5000) and spun at 26,000 rpm for 45 minutes. The plasma

was removed and placed into labeled polypropylene tubes and stored at -80C until

analysis of plasma catecholamines by high pressure liquid chromatography with

electrochemical detection. After the hematocrit tubes were spun and the data recorded,

the plasma was removed and stored in micro-tubes for later determination of its sodium

and potassium concentrations.

Direct Blood Pressure Recording

Throughout the experiment, arterial blood pressure was measured in the respective

environment of the animal (26 or 60C). At 0800 h, the contents of the arterial cannula

were voided. The cannula was flushed with sterile saline and refilled with heparin/saline

(10 U/ml). The cannula was connected to a physiological pressure transducer (Model

RPl500, Narco-BioSystems, Houston, TX) with input into a low level DC preamp and

a Grass Model 5D, 4 channel direct-writing recorder polygraph. Systolic, diastolic and

mean blood pressures, as well as heart rates, from undisturbed animals which remained

in their home cage were recorded. After the measurements were completed, the catheters

were flushed with sterile saline and then refilled with heparin (1000 U/ml) and sealed.

Baroreflex Function

Baroreflex function was assessed at several times during the experiment by

pharmacological manipulation of blood pressure using a procedure similar to Smyth et al.

(178). Arterial cannulae was flushed with sterile saline and refilled with heparin/saline

(10 U/ml). The cannula were connected to a 3-way stopcock and then to a Statham

pressure transducer (P231D, Statham Instruments, Oxnard, CA). The transducer was

connected to a Gould recorder. Baroreflex tests were recorded directly online to an IBM










PC computer via an analog-digital converter (Keithley model 507, Keithley DAS,

Cleveland, OH). Sampling rate was 10 Hz. Blood pressure was increased by i.v.

administration of phenylephrine (2 ug/kg, Winthrop-Breon Laboratories, New York, NY)

diluted at a concentration so that the volume given was less than the dead space of the

venous catheter. The phenylephrine was flushed with sterile saline into the animal. A

baroreflex function-curve was constructed by plotting the heart period (1/heart rate)

against mean arterial pressure at any given time. The slope and intercept of the

relationship was determined by linear regression using a scientific software package

(Assystant Plus, Version 1.04, Assystant Scientific Software Co.). The slope was used

as an indication of the sensitivity of the gain of the reflex, and the intercept as an

indication of setpoint (84).

Daily Intakes

Daily food and water consumption, urinary volume, creatinine and catecholamine

excretions of individual rats were measured. Measurement of daily intakes were

described previously (Chapter Two). Urine was collected in graduated cylinders which

contained 0.25 ml of 3 N HC1. After total urine volume was recorded, 5 ml aliquots

were frozen for later analysis of norepinephrine and epinephrine by high-pressure liquid

chromatography with electrochemical detection (HPLC-EC).

Measurements

Plasma Catecholamines. Norepinephrine (NE) and epinephrine (E) concentrations

in plasma were determined by HPLC-EC. The procedure used is a simple extraction

with acid washed alumina (AAO) as originally suggested by Anton and Sayre (4) with

later modifications (12,145). Basically, the sample and internal standard were placed in

vials and adsorbed onto AAO directly from the plasma at pH 8.5. After shaking, the

alumina is washed with water and the catecholamines eluted with a small volume (200

ul) of acid (0.1 N HC104, Fisher #A229). The acid extract contained the catecholamines

and was injected directly into the HPLC. This procedure is selective for the parent

amines and allows for preconcentration to a small volume. The procedure is described








72

in LCEC Application Note No. 14 available from Bioanalytical Systems INC.; the entire

procedure appears in Appendix A. Dihydroxybenzylamine (DHBA, Sigma) was added to

each sample prior to extraction, served as the internal standard, and permitted

adjustments in recovery. A previously collected plasma pool as well as standards were

assayed with each run of 10 samples. This permitted the determination of intra- and

interassay coefficient of variance (Appendix A).

Chromatographv. A high pressure liquid chromatograph equipped with a LC-4

electrochemical detector (HPLC-EC) and a glassy carbon electrode (Bio Analytical

Systems, Lafayette IN) was used in all determinations. The electrode potential was 0.7

volts run at a sensitivity of 2 nAmps. This potential allowed for optimal recovery. The

solvent delivery system consisted of a Bio-Rad Model 1330 pump (Bio-Rad, Richmond,

CA) connected to a Bio Sil reverse-phase ODS-10 column (250 mm x 4 mm, Bio-Rad

#1250082). The mobile phase consisted of 0.1 M potassium dihydrogen phosphate (Fisher

#P285) buffer:methanol (95:5) containing 0.1 mM EDTA (Sigma #ED4SS) and 5 mM

heptane sulfonic acid (Fisher #0-3013). The pH was adjusted to 3.8 with 2 M

phosphoric acid prior to the addition of methanol. The mobile phase was filtered prior

to the addition of the ion-pairing agent and degassed prior to use. After an initial

equilibration period, the mobile phase was recycled during use. The linearity of the

detector, detection limits, recovery and intraassay coefficient of variance were established

prior to assaying samples. The results appear in Appendix A. Results are expressed as

ng/ml.

Urinary Catecholamines. The concentrations of catecholamines in 24 hour urines

were determined by HPLC-EC from samples after the catecholamines were isolated and

preconcentrated. Basically, the catecholamines were isolated on cation resin exchange

columns (Biorex 70 cation resin, Bio-Rad) eluted with 2 M ammonium sulfate. The

catecholamines were adsorbed on AAO as described above and the resultant acid extract

injected into the HPLC. The internal standard DHBA was added to each sample prior

to column extraction. Results are expressed as ng/day.








73

Creatinine. The concentration of creatinine in urine and plasma was determined

by a method described by Technicon Corporation (No.SE4-0011FH4). The results were

expressed as mg/kg body weight.

Statistics

Statistical analyses of data were carried out by a two-way randomized block design,

analysis of variance which tested for the main effects of environment and time.

A posteriori pairwise comparisons were performed when appropriate by a Duncan's New

Multiple Range test which maintained the significance at the a priori level (p < 0.05)

(111). Changes in the organ weights, and in the slopes and intercepts of regression

analyses were determined by a one-tailed Student's t-test for independent samples, when

appropriate. The hypothesis was that the variable being tested in cold-treated animals

was less than or equal to that of controls (Ho: cold < control). Significance was set at

the 95% confidence level.

Results

The effect of exposure to cold on systolic and diastolic blood pressures is shown

in Figures 4-1, 4-2. Systolic blood pressure was significantly increased above that of

controls on day four, the earliest time a measurement was made. Systolic blood pressure

remained elevated and relatively constant when measured again 10 days later. Systolic

blood pressure then increased to 146 1 mm Hg (day 28), and to 153 4 mm Hg (day

41). Diastolic blood pressure increased rapidly from a resting level of 90 3 mm Hg

to 96 3 mm Hg by day four and remained unchanged until day 41 when a large

increase (103 3 mm Hg) was demonstrated. The effect of exposure to cold on mean

arterial pressure is shown in Figure 4-3. Mean arterial pressure was significantly

increased by exposure to cold as indicated by a significant interaction and main effect.

Mean arterial pressure increased from pre-cold levels of 101 + 4 to 111 + 3.5 mm Hg

on day four and continued to increase up to 124 2 mm Hg when the experiment

ended. The positive slopes for systolic and mean arterial pressures suggest that even after

41 days of exposure to cold, the blood pressure had not reached its maximum.











0--0 Control
*-* Cold









/T[
0






1*
I I I


I I I


5 10 15 20 25 30
Days


35 40


Source SS DF MS F P

Environment 5509.9 1 5509.9 43.86 <0.01

Time 4411.9 4 1102.9 8.78 <0.01

Env. Time 1553.28 4 388.32 3.09 <0.05

Error 8040.75 64 125.64


Figure 4-1.


The effect of chronic exposure to cold on systolic blood
pressure of chronically cannulated rats.
Values are Mean + S.E.M.
* First point which is significantly greater (p < 0.05) than
control as determined by Duncan's New Multiple Range test.


165-

- 155-
E
E 145-


( 135-
n)
0t
12 25
0
0


105-
Cn


954
Pri


e













110-



100-



90



80


70 -
Pre


5 10 15 20 25 30 35 40
Days


Source SS DF MS F P

Environment 1731.8 1 1731.8 17.72 <0.01

Time 493.9 4 123.47 1.264

Env. Time 380.98 4 95.25 0.98

Error 3058.21 62 97.71


Figure 4-2.


The effect of 42 days of exposure to cold on diastolic blood
pressure of chronically cannulated rats.
Values are Mean + S.E.M.











135-


125-


115-


105-


95


R -i I-I


5 10 15 20


25 30 35 40


Days


Source SS DF MS F P


Environment 5229.48 1 5229.48 61.16 <0.01

Time 1589.67 4 397.42 4.65 <0.05

Env. Time 1126.87 4 281.72 3.3 <0.05

Error 5301.5 62 85.51


Figure 4-3.


The effect of 42 days of exposure to cold on mean arterial
pressure of chronically cannulated rats.
Values are Mean + S.E.M.
* First point which is significantly (p < 0.05) greater than
control by Duncan's New Multiple Range test.


0-o Control
*-* Cold
oT








/ 1
0______


1










Heart rate was significantly increased during exposure to cold as seen by a

significant interaction and main effect (Figure 4-4). Heart rate was the highest on day

four, with some animals oscillating between 480 to over 500 beats per minute (bpm)

during the recording period. Heart rate continued to decline toward pre-cold levels but

remained much higher than controls even after 41 days (404 + 3 vs 344 + 7 bpm).

Similar to previous findings, chronic exposure to cold resulted in significant hypertrophy

of the heart, kidneys and adrenal glands (Table 4-1). In addition, the ratio of

thyroid/100 g body weight was greater in the cold-treated group, a finding unique to this

study. However, when the relationship between body weight (X) and organ weight (Y)

was examined by linear regression analysis, only the slope of the relationship between

body weight and the weight of the heart was significantly increased (Table 4-2).

Baroreceptor function was assessed throughout the duration of exposure to cold

after pharmacological manipulation of blood pressure with the pressor agent,

phenylephrine. Baroreceptor function curves were formulated by plotting the mean

arterial pressure against the heart period (1/heart rate) at any given moment after bolus

i.v. injection of the pressor agent. Typical baroreceptor function curves for both a cold-

treated and a control animal prior to, and during exposure to cold are shown in Figure

4-5. The slopes and intercepts of these curves were determined by linear regression and

the results submitted to statistical analysis. No significant main effects, either cold or

time, nor an interaction was found for the intercept. However, the slope of the

relationship between mean arterial pressure (X) and heart period (Y) was significantly

depressed during exposure to cold (Figure 4-6). A significant main effect of cold

without an effect of time was found.

Chronic exposure to cold significantly (p<0.05) increased the concentration of

norepinephrine (NE) in plasma (Figure 4-7). The increase in NE is attributed to the low

environmental temperature, as only a main effect of cold without a significant main

effect of time nor an interaction was evident. The resting or basal concentration of NE

was 476 57 pg/ml and when measured after 24 hours of exposure to cold, the











o-o Control
*-e Cold


5 10 15 20 25 30 35 40
Days


Source SS DF MS F P

Environment 80868.83 1 80868.8 75.78 <0.01

Time 13264.35 4 3316.09 3.107 <0.05

Env. Time 27625.8 4 6906.5 6.472 <0.01

Error 58696.21 55 1067.2


Figure 4-4.


The effect of 42 days of exposure to cold on the heart rate of
chronically cannulated rats.
Values are Mean S.E.M.


300 -
Pre








79



Table 4-1. The effect of chronic exposure to cold on various organ weights of the
rat.

organ weight (mp)/(100 g) body weight

Heart Kidneys Adrenal Thyroid
Glands Gland

COLD 338.68 7.24* 966.62 22.30* 17.38 1.81* 6.16 032*

CONTROL 285.47 4.31 671.51 26.00 11.92 0.36 5.04 0.31


Values are mean S.E.M.
* Significantly greater than control (p<0.05) by independent-sample, one-tailed t-test.









80




0
u




b-
..












so
* I

















Ci II (S o i
o 0
0o J^ o0 o\ 0 o










40





o 0






o -


0 o0 0S















| < g ~%rt > .2
U, KQff
-^ a ^ -0S








*-* Pre cold
--' Day 41


3.2-

3.0-

2.8-

2.6-

2.4-

2.2-

2.0-


*-* Pre
---- QDay


cold
41


I I J
120 130 14
Pressure (mm Hg)


Figure 4-5.


The effect of 41 days of exposure to cold on baroreceptor function
curves of chronically cannulated rats.
Values are individual (X,Y) pairs of data describing the relationship
between mean arterial pressure and heart period (1/heart rate) for any
given time, in response to i.v. bolus injection of phenylephrine (2 ug/kg).
A. Baroreceptor function curve of a cold-treated rat prior to, and after
41 days of exposure to cold.
B. Baroreceptor function curve of a control rat prior to exposure to
cold and 41 days later.


i1


1.8
4.0


0
CO
E

0


-4-F


3.8-

3.6-

3.4-

3.2-

3.0-

2.8-


i1


90


100
Mean


110
Arterial


[0


150


I


-


9 R








o-o Control
*-* Cold


5 10


20
Days


25 30 35


Source SS DF MS F P

Environment 9.176 1 9.176 4.252 <0.05

Time 3.347 3 1.116 0.517

Env. Time 9.86 3 3.288 1.524 .22

Error 79.85 37 2.158


Figure 4-6.


The effect of 41 days of exposure to cold on the slope of the
baroreceptor function curve in chronically cannulated rats.
Values are Mean + S.E.M.












3.5-

3.0-

2.5-

2.0-

1.5-

1.0-

0.51


o-o Control
*-* Cold


IT


0 I
Pre 3 6 9 12 15 18
Days


Source


Environment 19125368.8 1 1912568.8 8.515 <0.01

Time 10981358.3 3 3660452.8 1.63 0.192

Env. Time 8013216.2 3 2671072.1 1.189 .323

Error 114552360.5 51 2246124.7


Figure 4-7.


The effect of exposure to cold on the concentration of
norepinephrine in the plasma of chronically cannulated rats.
Values are Mean + S.E.M.









84

concentration was 1813 + 904 pg/ml. Although the concentration of NE tended to

increase, reaching 2611 + 950 pg/ml after 19 days of exposure, there was no significant

effect of time. The concentration of NE in the plasma remained significantly increased

throughout the time it was measured (20 days). The concentration of NE in plasma from

the control group remained relatively stable, averaging 565 + 58 pg/ml over the 20 days

measured. Similarly, exposure to cold significantly increased the concentration of

epinephrine (E) in the plasma from basal levels of 596 + 58 to 1179 + 141 pg/ml (Figure

4-8). In addition to the main effect of cold, there was also a significant effect of time

and a significant cold X time interaction. The concentration of E in plasma in the cold-

treated group returned to pre-cold levels of 594 47 pg/ml when measured after 19 days

of exposure to cold and remained unchanged from control.

The effect of chronic exposure to cold on the urinary concentration of NE, E, and

dopamine (DA) is shown in Figures 4-9, 4-10, 4-11. Urinary catecholamine data were

expressed as ng/mg creatinine (Cr)/day, in an effort to adjust for changes which may

occur in renal activity. Norepinephrine, E and DA were all significantly increased by

exposure to cold as indicated by a significant cold X time interaction. However, the rate

at which each catecholamine returned toward control or basal levels differed. The

concentration of NE in the urine of the cold-treated group increased by the first day of

exposure and did not return to control levels even after 41 days of exposure to cold

(Figure 4-9). In general, a large amount of variability was evident within the cold-

treated group. In contrast, the urinary concentration of NE of the control group

remained relatively constant when expressed as ng/mg Cr. The urinary output of

epinephrine was elevated in the cold-treated group only on the second day of exposure.

Although higher than control levels at many times during the study, the intragroup

variability for both cold-treated and control was very large. As with epinephrine, the

urinary concentration of dopamine was significantly increased as indicated by a

significant cold X time interaction. In addition, intragroup variability was also very

large.












































Environment

Time

Env. Time

Error


o0-0o Control
*-* Cold


1.

'-N
E

C



c"

C
'k-



S 0,

0
E.
0_

O









Source


IYAJ .5. 1


416846.8

1094352.8

979958.3

6115219.2


416846.8

273588.2

244989.6

92654.8


4.499

2.953

2.644


<0.05

0.026

0.04


Figure 4-8.


The effect of 41 days of exposure to cold on the concentration
of epinephrine in plasma from chronically cannulated rats.
Values are Mean + S.E.M.


.40



.15



.90


Pre 3 8 13 18 23 28 33 38
Days











o-o Control
*-- Cold


440 +


240 4-


___


5 10 15 20
Days


Source


25 30 35 40






MS F P


Environment 100484274.6 1 100484274.7 130.0 <0.01

Time 27176949.1 7 3882421.3 5.02 <0.01

Env.* Time 16114161.2 7 2302023.0 2.98 <0.01

Error 85795610.3 111 772933.4


Figure 4-9.


The effect of 42 days of exposure to cold on the output of
norepinephrine in the urine.
Values are Mean + S.E.M











o-o Control
*-* Cold


5 10 15


20
Days


25 30 35 40


Source SS DF MS F P

Environment 53947.2 1 53947.2 .422

Time 4992838.9 7 713262.7 5.58 <0.01

Env. Time 2491191.8 7 355884.6 2.78 <0.05

Error 12792677.8 100 127926.8


Figure 4-10.


The effect of 42 days of exposure to cold on the output of
epinephrine in the urine.
Values are Mean + S.E.M.


90
80


n I ____________


n i


501

40


f











o-o Control
*-* Cold


5 10 15


20
Days


25 30 35 40


Source SS DF MS F P

Environment 396114.3 1 396114.3 .119

Time 127723031 7 18246147 5.48 <0.01

Env. Time 79174773 7 11310682 3.4 <0.01

Error 369761638 111 3331186


Figure 4-11.


The effect of 42 days of exposure to cold on the output of
dopamine in the urine.
Values are Mean + S.E.M.


9oo00 +


300 +


100 +
PR


E










In an effort to determine whether the concentrations of catecholamines in the urine

were a good index of the concentration of catecholamines in the plasma, linear

regressions were performed on plasma (X) versus urinary concentration (Y) of NE and

E. Data from urines collected for either 24 hours prior to, or 24 hours after the blood

sample was taken were compared against the plasma value. No significant correlation

could be found between the concentration of plasma (pg/ml) and urine catecholamines

(ng/mg Cr). Further, the correlation coefficients were poor, (r <0.25) for all

relationships. In an effort to determine whether expressing the urinary catecholamines

as a function of creatinine was contributing to the poor correlation, regression analyses

were rerun on plasma (pg/ml) versus urinary concentration expressed as total ng/day.

No difference was found between the two analyses, i.e. whether the concentration of

catecholamines in urine was expressed as ng/mg Cr or as total ng/day. The relationship

between these two measurements is shown for norepinephrine and epinephrine in Figures

4-12 and 4-13.

Discussion

The present study is the first to utilize chronic arterial cannulation to measure

blood pressure, baroreceptor function and the activity of the sympathetic nervous system

(SNS) during exposure to low environmental temperature. In previous experiments,

chronic exposure to cold resulted in a significant elevation of systolic blood pressure, as

assessed indirectly utilizing the tail cuff technique, and cardiac hypertrophy. Similarly,

in the present study, exposure to cold resulted in a significant elevation of systolic blood

pressure (Figure 4-1, Table 4-2) and cardiac hypertrophy. After 42 days of exposure

to cold, SBP increased to 152.5 + 4.0 mm Hg (Figure 4-1). The magnitude of this

increase is quite similar to that seen using the indirect tail technique for measuring blood

pressure after a similar length of exposure (Chapter Two). Additionally, diastolic and

mean arterial pressures as well as heart rate were significantly increased (Figures 4-2,

4-3, 4-4). This is an important finding as systolic blood pressure is primarily a function

of cardiac output, while diastolic blood pressure is indicative of a change in vascular



















Y -0.111 (X) + 5213 r = -0.088
Y = 0.192 (X) + 588 r = 0.176


c-
0

4-
0
c-






0




z
C

o
'5-


"r"


8000-

7000-

6000-

5000-

4000-

3000-

2000-

1000-

0
0


Plasma Norepinephrine
(pg/ml)


Figure 4-12.


Concentration


The relationship between the concentration of norepinephrine
in the plasma and urine of chronically cannulated control and
cold-exposed rats as determined by linear regression analysis.


- Cold
-- Control


I I


500 1000 1500 2000 2500 3000


















- Cold Y -0.138 (X) + 677 r = -0.113
-- Control Y = 0.058 (X) + 176 r = 0.107


500


1000


1500


2000


Plasma Epinephrine Concentration
(pg/ml)


Figure 4-13.


The relationship between the concentration of epinephrine in the
plasma and urine of chronically cannulated control and cold-
exposed rats, as determined by linear regression analysis.


C
0
.m
CO


C


C
C-1
a-c
c

LJ
0
C-
'Q"
MJ


1250-



1000-



750-



500-



250-


S1 1 1


2500








92

resistance. An increase in vascular resistance is a characteristic of hypertension. In the

present study, diastolic blood pressure increased to 103.0 + 2.8 mm Hg and was

accompanied by an increase in mean arterial pressure to 124 + 2 mm Hg. The significant

interaction demonstrated by each of these pressures indicates the slopes of the cold-

treated group are different from control. This suggests that the pressures had not peaked

at this time and may have continued to increase. It also suggests, that changes which

contributed to the elevation of blood pressure may also be incomplete and perhaps were

still occurring, i.e. a steady state had not been achieved. Whether blood pressure would

continue to increase in this manner, and for how long, is undetermined. The finding of

increased systolic, diastolic and mean blood pressures in combination with cardiac

hypertrophy support the hypothesis as initially proposed by Fregly et al. (61) that chronic

exposure to cold results in the development of hypertensive disease in rats.

It is interesting, that the 20 mm Hg increase in mean arterial pressure seen in the

present study is quite similar to the rise in mean arterial pressure reported by Reed et

al. (162) in young men (21+ 2 years) stationed in Antarctica. In this particular study,

the mean arterial pressure rose significantly from a baseline of 85 to 101 + 3 mm Hg

over a 42 week period of extended residence. It appears that the study by Reed et al.

(162) is the only published work in which the effect of long-term (although intermittent)

exposure to cold on blood pressure in humans has been measured. However, the idea

that low environmental temperature adversely influences blood pressure of humans is not

a new one, as several reports exist within the literature. Prineas et al. (157) measured

the resting blood pressure of 10,000 Minneapolis school children over a 4 month period

in effort to establish norms for this population. Even after extensive efforts to eliminate

methodological biases, these authors (157) reported a significant effect of season on blood

pressure, with winter (January and February) diastolic pressure more than 3 mm Hg

greater than the pressure recorded in the spring (April). Similarly, when blood pressures

which, were recorded during the British Medical Research Council's treatment trial for

mild hypertension, were analyzed on the basis of season, Brennan et al. (17) found a









93

significant seasonal influence. For each age, sex and treatment, these authors (17) found

greater systolic and diastolic blood pressures in the winter as compared to the summer.

The initial impetus for the analysis of a seasonal influence on blood pressure stems from

earlier reports which demonstrated a much higher mortality rate from ischemic heart

disease and stroke during the winter in England and Wales. In a later study, this same

relationship (increased mortality with decreasing temperature) was seen in New York,

England and Wales (20). The high correlation coefficients lead Bull (20) to suggest a

causal relationship between temperature and mortality. In a recent study by Hata et al.

(83), the same seasonal variation in blood pressure was seen in essential hypertensive but

not normotensive patients. The above reports suggest strongly, that cold-induced

hypertension may not be unique to the rat.

An important finding in the present study, in which blood pressure was measured

directly from an indwelling cannula, is the time-course or onset of the increased blood

pressures. Systolic blood pressure was significantly (p< 0.05) increased by the fourth day

of exposure to cold, the first time at which it was measured. Mean arterial pressure was

significantly increased after 13 days of exposure to cold. Previously, the first significant

elevation of systolic blood pressure was evident after 21 days of exposure to cold

(Chapter Two), nearly two weeks later than reported in the present study.

There are several possible explanations for the difference in the onset of the

elevation of blood pressure between the two techniques. First, in order to measure

indirect blood pressure, the animals must be restrained and warmed at 37C for

approximately 15 minutes. Restraint represents a very potent stress for rats. Visually,

the animals settle down over time, or they appear to adapt to the general stress of

restraint. The observation of adaptation to restraint prompted Popovic (156) to examine

this phenomenon. When the concentration of ACTH and corticosterone (B) were

measured in restraint-adapted animals after an acute episode of restraint, both ACTH and

B were significantly increased above prerestraint and controls levels (156). These results

refute the idea of adaptation to restraint in rats (156). In addition to ACTH and B, the




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