Date ’ 0-7539 I ‘ EMERABY i Michiganflate University ' A r; ————— This is to certify that the thesis entitled HORMONAL RESPONSES OF SPONTANEDUSLY HYPERTENSIVE AND NORMOI'ENSIVE RATS TO STRESS presented by Jahan Shah EFI‘EIGIAR has been accepted towards fulfillment of the requirements for MASTER degree in Physiology R.A. BERNARD Adj/B4 “1454 Major professor /#fll(’{flc )7 A) 5?)! MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES MW Blflfi‘fl‘x’ififlflfl‘fl--- Place in book drop to remove this checkout from your record. Elfl§§_will be charged if book is returned after the date stamped below. x... 9‘"? ~T"‘a“"‘r€:‘:‘a 3. 00 A 2b 1 'l # HORMONAL RESPONSES OF SPONTANEOUSLY HYPERTENSIVE AND NORHOTENSIVE RATS TO STRESS BY Jahan Shah Eftekhar A THESIS Submitted to . Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology I 985 ii He taught me all the necessary survival skills. He raised my stress threshold to a level that no other man could have. And to him i owe my health, my success my accomplishments, and above all--my existence. To my father with respect, love and passion. IICKNOIIILEIIGMENTS I wish to express thanks to many people who collectively made it possible to execute these studies and prepare this thesis. Dr. Rudy A. Bernard, my major professor and advisor from the Physiology department, deserves utmost gratitude for his inspiration, conceptual support, enthusiastic encouragement, constructive criticisms, guidance and provison of supplies and facilities during the two years that I worked In his laboratory. Dr. John Chimoskey and Dr. Richard J. Hall deserve recognition for their critical role and counsel as the members of the Guidance Committee for this thesis. Timothy w. Priehs and Kenneth J. Price, my friends and colleagues, deserve special credit for their support, cooperation, kind assistance and the crucial part that they played in all the experimental phases of this work. Karen J. Money and Dana Pfeifer have my appreciation for their technical assistance in hemodynamic measurements. Dr. Ali Rassuli deserves a special note of thanks for his friendship and statistical assistance. Further, I wish to express my gratitude to Dr. Raymond Nachreiner and his staff from Animal Health Diagnostic Laboratory, for their collaboration In carrying out the radioimmunoassays. I also would like to express my gratitute to Douglas J. Ereg, Sister I'lary Honora Kroger, and Patricia Soutas-Little since their iii iv encouragement and support have enhanced the breadth of my graduate experience. Finally I wish to acknowledge and extend my deep appreciation to my wife and partner, Glsso, for her material and moral support, enduring patience and understanding; and to thank the unborn child of ours, whose declaration decorated the final draft of this thesis work with happiness and contentment. TflBlE OF CONTENTS PAGE LIST OF TABLES ....................................................................................... vii LIST OF FIGURES ....................................................................................... viii INTRODUCTION .......................................................................................... 0i REVIEW OF THE LITERATURE -- -- ....... - 04 Hypertension, Pituitary-adrenocortical and Sympa- thetic Nervous System -- 04 Stress, PItuitary-adrenocortical and Sympathetic Nervous System I4 Stress, Salt Intake and Hypertension ............................. 20 Stress, Hypertension and Spontaneously Hyperten- sive (SHR) rats ---- -... 24 Chronic Catheterizaion in Rat ........................................... 26 MATERIALS, METHODS AND PROCEDURES ................................. 29 Protocol of the Experiment .............................................. 29 Experimental animals ......................................................... 30 Housing and Feeding Condition of the Rats ............... 30 Salt Preference Test .......................................................... 32 Chronic Catheterization ..................................................... 32 Surgical Procedure ................................................... 33 Post-surgical Procedures ..................................... 35 Heart Rate, Blood Pressure and Hematocrit Measurements - -- --.-- . ..................... 3S Blood Sampling Procedures .................................................... Stress Tests - .- ........... -- - ............................................ Ether Stress ....................................................................... Vibration Stress _ - .................................. Manual Restraint Stress .............................................. Radioimmunoassay ................................................................... Catecholamines -- ................ ....... Cortisol and Corticosterone ...................................... ACTH Assay ....................................................................... Statistical Analysis Calibrating ACT H/Corticosterone Concentration ...... RESULTS .......................... _ - ................... Growth Rate - - - _ _ -.--__ Blood Pressure -- -- - - Ether Stress - - _ _ ...... ACTH and Corticosterone _ Epinephrine and Norepinephrine ............................... Vibration Stress - - ....... ACTH and Corticosterone .............. Epinephrine and Norepinephrine ............................. Manual Restraint Stress ACTH and Corticosterone Epinephrine and Norepinephrine Evening and Morning Resting Level of Hormones ....... Salt Preference Tests --- - _ ....... - -- DISCUSSION - -- ..................... LIST OF REFERENCES .................. - APPENDIX (PLATES) ....... -- - ......... PAGE 36 37 37 38 38 38 39 39 40 40 4I 42 42 42 49 49 S3 53 53 6O 64 64 68 72 79 83 89 LIST OF TflBLES TABLE PAGE 01. Growth Rate of WKY and SHR rats .................................................. 45 02. Mean Arterial Blood Pressure in Rat Groups ............................... 48 03. ACTH and Corticosterone in Response to Ether Stress .......... 52 04. Epinephrine and Norepinephrine Response to Ether Stress S6 05. ACTH and Corticosterone Response to Vibration Stress ....... S9 06. Epinephrine and Norepinephrine Response to Vibration Stress -- -- ....... 63 07. ACTH and Corticosterone Response to Manual Restraint 67 08. Epinephrine and Norepinephrine Response to Manual Restraint -- --_ - 71 09. Evening (1 1:00 PM) Resting Level of Stress Hormones ......... 75 lo. Pooled Morning (I I 0.0 AM) Resting Level of Stress Hormones - -- -- - 78 I I. Salt Preference of 24 Week Old, Group A Rats ....................... 80 I2. Salt Preference of I6 Week Old, Group B Rats ....................... 81 13. Salt Preference of 18 Week Old, Group B Rats ...................... . 82 vii llST 0F FIGURES FIGURE PAGE OI. The growth rate of SHR and WKY rats for a period of I30 days, fromday86upto day 2I6 ................................................... 44 02. Morning (1 1:00 AM) and evening (1 1:00 PM) resting mean arterial pressure of the SHR, WKY and 80 rats --- ------ . 47 03. Plasma level of ACTH and Corticosterone before and after 30 minutes ether stress in WKY and SHR rats SI 04. Plasma level of epinephrine and norepinephrine before and after 30 minutes ether stress in WKY and SHR rats ..... SS 05. Plasma level of ACTH and corticosterone before and after 30 minutes vibration stress in WKY and SHR rats ................ $8 06. Plasma level of epinephrine and norepinephrine before and after 30 minutes vibration stress in WKY and SHR rats ...... 62 07. Plasma ACTH and corticosterone before and IS minutes after a 5-minute manual restraint stress in WKY, SHR andSDrats _- _ - - _ 66 08. Plasma level of epinephrine and norepinephrine before and I5 minutes after a 5-minute manual restraint stress in WKY, SHR and SD rats ..... - _ _- -- - 70 09. Evening (I l :00 PM) resting plasma level of ACTH, corticosterone, epinephrine and norepinephrine in WKY, SHR and 50 rats __ - - 74 viii FIGURE PAGE I0. Pooled morning (1 mo AM) resting Plasma level of ACTH, corticosterone, epinephrine and norepinephrine in SHR and WKY rats ........................................................................................... 77 RBSTBRCT W RESPONSES OF SPONTANEOUSLY HYPERTENSI VE All) WTENSIVE RATS TO STRESS BY Jahan Shah Eftekhar Plasma concentration of ACTH, corticosterone , epinephrine and norepinephrine was measured by radioimmunoassay in 22-34 week old male spontaneously hypertensive (SI-R) and normotensive Wistar-Kyoto (WKY) rats before and after exposxre to various forms of stress. Blood samples were obtained througi catheters chronically Implanted in the left ca‘otid a‘tery aid attached to a swivel wove each cage, such that the animals were completely undistirbed except when exposed to experimental stress Resting morning blood pressure (mean 1 standard deviation) was ISO 2 24 mmHg In the SIR rats (ti-l4) and I07 3 27 In the WKY (h-I6) [p<0.0i1 In one experiment blood was taken before and after the rats were exposed to 30 minutes of ether ( n-4 SHR, I0 WKY) and/or 30 minutes _ vibration (n-S SIR, BWKY) stress In a randomly alternate sequence that allowed 4-7 days between each test In a second experiment, samples were taken IS minutes after the rats were manually restrained for 5 minutes (n-9 SI-R, 8 WKY). Resting values were obtained both individually for each Jahan Shah Eftekhar test and also were pooled across all experiments. Results of pooled morning resting level of the hormones were as follows: ACTH, I28199 vs. 97166 pg/ml; corticosterone, 961IOI vs. 96177 ng/ml; epinephrine, 79194 vs. 251I8 pg/ml (p<0.0l); and norepinephrine, 2621l37 vs. M9158 pg/ml (p< 0.005) for SHR and WKY rats respectively. The post etherstress level of the hormones were as follows: ACTH, 5i01I44 vs. 5221I66 pg/ml; corticosterone, 368147 vs. 396195 ng/ml; epinephrine, 743316757 vs. 3621273 pg/ml (p< 0.00; and norepinepi'rine, 229812375 vs 5%70 pg/ml (p< 0.05). Post vibration-stress findings were ACTH, 4001120 vs 4501i I2 pg/ml; corticosterone, 4861l29 vs 4Il1l08 ng/ml; epinephrine, 7l31378 vs I85192 pg/ml (p< 0.025); and norepinephrine, 6801l56 vs. 4i31l35 pg/ml (p<0.025). Finally, the post manual-restraint results were: ACTH, 35I1l49 vs. 400190 pg/ml ; corticosterone, 395154 vs 336158 ng/ml (p<0. 05); epinephrine, 5241453 vs. 114156 pg/ml (p<0.0l); aid norepinephrine, 5581336 vs. 200184 pg/ml (p<0.005). The higher concentration of catecholamines found in SHR both at rest and In response to a variety of stresses Indicates that these hypertensive rats have a higher level of sympathetic activity than their normotenslve , controls However, comparable levels of ACTH and corticosterone suggest that SHR and WKY do not differ simificantly In pituitary-awenocortical function INTRIIIIIICTIBN The interrelationships among 'hypertension', 'stress’ and 'salt make are complex and controversial. On one hand they seem to be causally related--that is, the occurrence of any of them In a person , is followed by the occu‘rence of at least one if not both of the other conditions; on the other hand, one sees evidence in the literature that tends to refute the causal association of these conditions. There are also discrepancies In the literature over the issues of: finding the one condition among the three which is the final cause of the other two; or finding the material order of causation among them-~that Is, which one comes first, which one next and which one last; or determining the organ/system of the body that can best serve to link these conditions; or to decide which level of organization--from subcellular to organismal or even societal, can best serve to advance our knowledge of these states. Finally, and perhaps the most Important of all Is the lack of a universal ag'eement over the exact boundaries which delineate the concepts of 'stress ' aid 'hypertension'. This problem seems to be the major intensifier of the complexity of the Issue--since not all the scientists who refer to the concept of (for example) 'stress', really refer to the same thing This thesis we in part with the simplified, mechanistic aid prag'natlc definition of stress proposed by Yates eta,” I974). These 1 2 authors state that " a stress Is any stimulus, Internal or external, chemical, physical or emotional, that excites neurons of the hypothalamus to release corticotropIn-releasing hormone (CRH) at rates greater than would occur at that time of the day In the absence of the stimulus. The CRH drives the pituitary to release ACTH, and ACTH stimulates adrenal cortex to secrete corticosteroids". However this thesis also includes activation of the sympathetic nervous system, either dependent or independent of the ACTH release mechanism as part of the comprehensive definition of stress. The underlying hypothesis of this thesis is that stress activates humoral and neural mechanisms in the body that lead to elevated blood pressire (hypertension) and Increased salt intake. More specifically the hypothesis states that stress stimulates the pituitay-ad‘enocortical axis, and the released ACTH molecule, then, either alone and/or through the adrenal steroids causes elevated salt appetite ( Blaine etaL, I 975; Denton e131,, I980) and heightened blood pressure (Scoggins stat, I974; Kawasaki e121,, I976; Genesteta], I978; McCaa, I978; Rauh stat, I979; Freeman, e111,, I980). Spontaneously hypertensive rats (SHR), widely studied as an animal model of essential hypertension (Okamoto and Aoki, I963; Okamoto e131,, I966; Okamoto, I972) were chosen for these experiments because they also have a naturally elevated salt appetite (Catalanotto at at. I972; Bernardstat. I980). The specific hypothesis tested in these experiments is that the higi blood pressure and high salt Intake of the SHR rats are due to an abnormally active pituitay-ad‘enocortical system manifested either by a him resting level of ACTH, or a heigitened ACTH response to stress or 3 both . Although a role for catecholamines is not proposed In the hypothesis, their plasma levels are also measured for two reasons. First, to perform amore complete assessment of the stressresponse of the SHR rats; and second, because of the possible synergistic effect of the catecholamines on ACTH fmction (Ramey et aL, I95I; Lohmeir and Guyton, I98I). Finally, as part of the methodological strategy, the experiment employs three types of stress, namely; ether, vibration and manual-restraint--in order to provide a variety of stress options--from physical (manual-restraint) to neural (ether) and to psycho-physical (vibration). BEIIIEIIJ 0F TIIE lITEIIflTIIIIE A- HYPERTENSION, PITUITARY-ADRENOCORTICAL AND SYMPATHETIC IERVOUS SYSTEM Selye and his peers (I942) were among the early pioneers who demonstrated that the adrenal cortex plays a role in the physiology of hypertension They showed that hypertension Is easily inducible In baby chicks by a higi dose of desoxycorticosterone acetate. A ya: later, consistent with this finding, Sarason(l943) published theresults of m autopsy study on patients with essential hypertension . The report Indicated presence of mm aa‘enals In most of these cadav -- implying a state of hyperactivity. Another ea‘ly line of support for the association of hypertension aid ad‘eno-cortical activity is evident In the work of Plotz 91a], 0952), who stated that a connection between him secretion of cortisol aid higi diastolic blood pf‘esares has been known for quite a long time in most of the patients with various forms of Cushing's syncrome. From the 5th decade of the 20th century onward, one sees more experimental work on the interrelationship of hypertension and the pituitai'y-ad‘enocortical system . Knowlton ata], (I952), were able to induce moderate to severe arterial hypertension in normal and adenalectomized rats by administering cortisone acetate to them. The same glucocorticoid was also demonstrated to Induce hypertension In 4 mice, one to two days after the first injection (Clark etaL, I968). Also In a study on methylprednlsone, a potent cortisol analog, Krakoff eta], (I975) were able to bring forward evidence In favor of the role of cortisol in hypertension. They demonstrated that this substance Increased the plasma renin activity and the pressor response to angiotensin II in the rat. They further showed that treatment of rats with angiotensin II inhibitor (Sar' -Ala8-Angiotensln Il)caused a drop in blood pressue. Based on these findings, they proposed a role for glucocorticoids in general and cortisol In particular In the elevation of arterial pleasure, and ascribed a precipitating function to the renIn-angiotensin system. The idea of searching for precipitating elements In explaining the role of the glucocorticoids in the etiology of hypertension is also not a new Idea Early In the I950’s, Ramey and his collaiorators were able to demonstrate that the pressor response to norepinephrine could not be maintained In adenalectomized dogs, and bloodpressire fell back to shock level despite la‘ge doses of norepinephrine. However, when they added cortisol to the post sirgical dug regimen of the dogs, the blood pressu‘e responded favoraily to the previously ineffective norepinephrine doses. Their conclusion was that cortisol may have a permissive role in the maintenance and elevation of blood pressre (Ramey eta], I95I ). There is also evidence In the literature on the Interaction between stressful stimuli and aa‘enocortical activity in the maintenaice of blood meme. For example, Goldstein ata], (I950) were able to observe that glucocorticoids (cortisol and cortisone) and not mineralocorticoids (deoxycorticosterone and aldosterone) were Involved in the maintenance of blood pressure. They showed that airenalectomized 6 dogs under severe muscular exercise failed to maintain blood pressure-whether they were injected with deoxycorticosterone or not. Literature also shows controversial evidence regarding the role of corticosterone In the maintenance of blood pressu‘e. Thornat a], (I953) indicated that corticosterone raised blood pressure to hypertensive levels In patients that were bilaterally ad‘enalectomized for hypertension treatment 0n the contrary, Peterson and Pierce (I960) based on their findings on the plasma level of corticosterone in man, have suggested that this hormone, although a weak Na“ retainer and a modest glucocorticoid and pressor stbstanceuls unlikely to have a major role In the maintenance of blood presetre. Their reasoning was based on the fact that this hormone Is produced In a very small anoint; I.5-40 mg/day, by the ad‘enal cortex in mm The experiments aid findings cited move all have one thing In common-they all ascribe a role to the acrenal cortex aid the concomitaitly released glucocorticoids In the maintenance and/or origin of blood pressire. Nevertheless, the fact that the WI cortex Is controlled by the anterior pituitary gland and the latter Is in tun under the influence of the hypothalanus raises the question of what role or roles is the hypothlanus-pituitaiy-mml axis playing In hypertension, and more specifically, to what extent and how Is the ACTH molecule Involved in the etiology and/or maintenance of hypertension In what follows, the focus of the reports and experiments will center a‘ouid answeringthese questions. Walser and colleagues (I955), based on experiments wherein they studied the effects of salt loading in conjtnction with ACTH and corticosterone administration In Sprague-Dawley rats, suggested that Increase In blood volune Is not a necessary sequel to salt and water retention, nor an inevitable consequence of adrenocortical steroid adnlnistration but an outcome of the Interaction of both of them. Raul and associates (i979) who studied the effects of continuous (5 days) Infusion of ACTH on aa‘enocortical function, electrolyte metabolism and blood pressure of normotenslve and hypertensive children, reported a continuous rise In the plasma cortisol and deoxycorticosterone, a transient kalilresis and a continuous fall in serum K’ , retention of Na’, and weight gain in all the patients. They observed that plasma aldosterone rose only transiently In both normo- and hypertensive gulps They also showed that the cunulative retention of Na‘ had a signirlcant correlation with the rise of systolic blood piessue in both g‘otps Ftrthermore, they found evidence that ACTH on a low salt diet cal cause a small rise In blood pressu‘e. However, they were not dale to show my simificalt correlation between the level of plasma cortisol, deoxycorticosterone, aldosterone and the rise in blood bresstre. Overthree decades ago, Levitt eta], (I95I) were able to show that cortisone and ACTH could induce a transient shift of water, sodium and chloride into the measurable extracellular fluid compartment In man They observed that the maximum value was attained 8 to 9 days after the beginning of the therapy. Based on their experiment, they proposed that the rise In blood presetre could be related to the role of ACTH In volume expansion and salt retention One should note that this suggestion Is in contrast with Walser atal‘s recommendation cited previously. Vazirata], (I981), based on their experiments on Sprague-Oawley rats, showed that ACTH cal Induce fulctional tralsformatlon of glunerolusa cells to fasciculata-llke cells In the «finals, and that the 8 induced hypertension is related to the Increased secretion of corticosterone and l8-hyd‘oxy-deoxycorticosterone, and not to aldosterone or to le-hyaoxycorticosterone. They further observed a decrease In body weight and an increase In atrenal weight In all the experimental rats. Haack at a]. (I978) studied the effect of chronic ACTH treatment on blood pressure and trinary excretion of steroids in male Wistar rats. They observed that chronic ACTH amlinistration caused a loss of body weiglt and Increased blood pressure, while It had no effect on aldosterone prediction. This work Is firther smported by the research of Freeman at a], (I980) who studied the effects of continuous ACTH ackninistration on blood pressure and water metabolism In male Sprague-Dawley rats They were able to show an ACTH induced hypertension without prior sensitizing maneuvers such as unilateral nephrectomy or salt-loading Firthermore, they observed that the Induced hypertension was entirely reversible if the ACTH infusion was stopped Also they explicitly affirmed that the volune expansion was not critically Involved In the development of ACT H-induced hypertension-m both the experimental and control gems in the study were on positive liia‘~ balmce. W Concomitant to the lines of the aforementioned works, the role of the autonomic nervous system in the regulation aid malntenmce of the essential hypertension has also been the swject of Intensive study by other scientists. Guyton and his associates (I974), writing on the role of the autonomic nervous system In the regulation of arterial blood pressure, pointed out that althougl the long term control of blood pressu‘e ls accomplished by the kitxleys through modulation of salt aid water excretion, the baroreceptor reflexes, the chemoreceptors In the walls of the goat vessels , and the receptors of vasomotor centers are responsible for the short term control of blood pressure. Centrally active antihypertensive drugs have been used for quite a long time as clinical evidence In favor of the role of the sympathetic nervous system In the maintenance and/or etiology of him blood pressure . A recent study by W109 £1 at (I977) has shown that the antihypertensive dug, clonidine, which lowers blood pressure by a central nervous action, reduces the sympathetic tone and causes a drop In plasma norepinephrine level. This study affirms the role of norepinephrine In the maintenance of the hlql blood pressre. Further support for the role of norepinephrine comes from the work of Louis at a], (I974). These authors demonstrated a higl correlation between plasma norepinephrine level and blood pressu‘e on one hand, and the level of dopamine B-hydroxylase--an enzyme that converts dopamine to norepinephrine, on the other. in another study on the heart of young and old spontaneously hypertensive rats, Louis ata], (I969) showed that the cardiac epinephrine tunover time of the older hypertensive rats Is much lower than the older control groups. This suggested to them that hypertension mlmt be due to the concentration of epinephrine which stays longer In the crdiac vasculattre. However, In contrast to this suggestion, Yarntri (I974) who studied the activity of autonomic nervous system In young Spontaneously hypertensive rats (l-2 months age), observed an Increased cardiac ttrnover of epinephrine compared to the normotenslve controls 10 A further Investigation was reported by Hallbach (I976) who studied the Interaction of centeral autonomic hyperactivity and environmental stimuli In relation to their roles in the devolpment of hypertension in rat The report supports the idea that genetic hypertension Is mediated through central adrenergic hyperactivity-~since mental stress Imposed on yomg normotenslve Okamoto rats resulted In a d‘amatic elevation of a~terial presstre and In an ether onset of hypertension. Additional support for Hallbach's study comes from the work of Weiss and colleagues (I974). They studied the effectiveness of early B-ad‘energic blockade treatment in reducing the Increase in blood pressu‘e that occurs with age In the spontaneously hypertensive rats, aid suggested that Increased sympathetic activity in early age may be the cause of spontaneous hypertension p39° £1 a]. (I958), In an effort to summarize the factors that regulate blood presstre and tissue perfusion, named cardiac output, blood volune, blood viscosity, vascular caliber, vascular elasticity, vascula‘ reactivity, chemical stbstances and netral activity as the major precipitating factors They further Indicated that netrogenlc sympathetic constrictor outflow Is certainly a simificant factor in essential hypertension even If not a necessay cause of it Finally, In a more recent review on the relationship between blood pressu‘e, heart rate and plasma norad‘enaline, Reid gt 3], (I978) wrote: “there are many possible explmations for the controversial and contradictory reports on the role of autonomic mechanisms In hypertension Host of these directly or Indirectly are the consequence of the use of plasma levels of noradrenaline as an Index of sympathetic 11 activity“. The authors highlighted the major reasons and causes of these controversies as the following: methodological problems of sensitivity, specificity, and reproducibility; site and conditions of blood sampling; variability In race, sex and age of the subjects; and the differences In clearance and metabolism of noradrenaline and the sensitivity of receptors to neurotransmitters. The work of these authors casts some light upon the comprehensibility of the reports of other reseachers like Hoobler gt a], 0954), who indicated that plasma concentration of catecholamines in patients with essential hypertension was not markedly different from concentrations In normal subjects. Or the contrasting work of Franco-Morselli gt a], (l978) who measured plasma cathecholamines in human essential hypertension and in DOCA salt hypertension of rat, and reported that there Is a slight Increase In plasma norepinephrine and a marked rise In the level of epinephrine In the hypertensive patients compared to control normotenslves . These authors further were able to show a positive correlation between plasma epinephrine level and blood pressure-with plasma epinephrine appearing more elevated than norepinephrine. They remarked that epinephrine represents a more accurate Index of sympathetic activity than norepinephrine, which is subjected to a more complex diffusion from nerve endings to the lumen of blood vessels. To obtain a reliable estimate of the plasma norepinephrine level under various conditions of rest and exercise, Watson gt :1. (I978) used a forearm indwelling cannula in patients with mild to moderate essential hypertension. They observed that plasma norepinephrine was lowest during sleep (02730.03 ug/l) and progressively increased through sitting, standing and walking, reaching the highest value during bicycle exercise 12 (23210.47 ug/l). They firther found a significant correlation between the log of plasma norepinephrine and blood pressure under different physical conditions of the experiment The literattre also shows experiments and reports that propose to mveil the complex cellular and subcellular mechanisms for the Interaction of hypertension and hypophysio-ack‘enocortical or autonomic nervous system. Kalsner (I969), who studied the mechanism of hyd‘ocortisone potentiation of responses to epinephrine and norepinephrine in rabbit aorta, concluded that corticosteroids are required for the vasoconstrictor action of norepinephrine. This study showed that In the absence of glucocorticoids, the vasopressor action of catecholanine hormones is diminished or lost aid blood pressure Is decreased Kalsner » proposed the following detailed mechaiism: glucosteroids Inhibit the enzyme catecholanine-o-methyltrmsferase (CG'IT) which Is responsible for Inactivating the catecholanine hormones at their action sites. if this enzyme is not inhibited by corticosteroids, catecholamines a‘e metabolized so midly that they cannot accumulate in sufficient anomts to cause vasoconstrictlon. Tobian gt :1, (I956) studied the effect of norepinephrine on the electrolyte composition of arterial smooth muscle in rats and concluded that regulation of Ion distribution on the two sides of the cell membrane may be the basic determinant of the steady tension produced by these muscles They showed that infusion of norepinephrine sufficient to case a1 increased blood presslre Is accompanied by a decrease in extracellular sodlun and an Increase In the sodium content of arterial smooth muscle. On a closer look at the cellular mechalisms which account for the 13 correlation between sodium metabolism and peripheral vascular resistance, Blaustein (I977) proposed that the sodium electrochemical g‘adient across the vascular smooth muscle cell plasma membrane (sarcolemma) plays an Important role In the regulation of cell calciun. The author argued that since there Is a sigiificant resting tension In most resistance vessels, the Ionized calcium must be maintained above the threshold level for their contractlllty. Accordingly, Ca“ transport in the sacrcolemma and, presunably, Na’-Ca“ exchange mechanisms must be held In such a way as to hold the Ca“ inside at the required high level. Any change in the Na” gradient will then be reflected by a change In Ca“ Inside m In ttrn In the tension of the vessels aid peripheral resistance. The author flrther suggested the possibility of a circulating agent, such as natritretic hormone, affecting the Na+ g‘adient across the sacrcolemma, the Ca“ Inside, and the tension of the vasculattre as a consequence. Consistent with this report and In the same year, De Wadener(I977), In a review on natrllretic hormone, wrote that an increased concentration of natriuretic substaices that are Na’ transport inhibitors is demonstrated in both mm aid animals dlring a high intdte of Na“ and after acute volume expansion These substances, while retirning the sodiun balance toward normal, might also cause hypertension Postnov gt at (I976) noticed an altered permeability in the erythrocyte membrane of SHR rats for sodium and potassium Ions compared to Wistar-Kyoto aid Sprague-Dawley rats They fomd a higher permeability of the SHR membrane for these two Ions The Increased permeability, according to the authors, reflected a widespread cell membrale defect which would serve as a general cause for activating the mechanism for maintaining hig'i blood pressure. 14 Finally, Luft gt a]. (I979) studied the plasma and trinary norepinephrine values at the extremes of sodium intake from l0-l500 mEq/24 hrs to determine the plasma and irinary norepinephrine values in man They offered to each of the I4 sibjects In the study a graded Intake of sodiun for three days--starting with l0 and proceeding to 300, 800 and I500 mEq/24 hows. The results showed that mean arterial pressure of the stbjects Increased from 83.8:l to l00.3:3 mml-lg Venous plasma norepinephrine decreased from 467:63 to 67:24 pg/ml, while irinaly norepinephrine excretion decreased from 54323.4 to 234:2.9 liq/24 hr. The calculations showed that trinary excretion of sodiun was inversely correlated with the trinay norepinephrine values. This suggested to the authors that sympathetic nervous system activity may decrease with sodiun loading In normal slbjects aid facilitate the excretion of massive salt loads which in tin will modulate the Increase in blood pressire. Accordingly, It Is possible that the higi blood pressire of hypertensive patients and Sin rats is due to a defect In their ability to excrete hlgi Illar loads mder higi norepinephrine concentration of plasma 8- STRESS, PIWITARY-ADRENCORTICAL All) SYHPAflETlC KRVOUS SYSTEM Cannon (I929) Identified the fight/fligit response to stress and described the accompa'iying Increase In sympathetic and adenocortical activity. Selye in the I930's described the general adaptation syntrome (GAS) In response to stress and demonstrated that this can be beneficial or hamful depending on the length of time that the emergency bodily responses are sustained According to his view, animals are In the state of stress when they maiifest a synlrome of three changes. hypertrophied 15 mnals, atrophled lymphatic organs, and bleeding gastrointestinal ulcers. This syndrome of changes, he called ”GAS“. Stressors, according to his hypothesis, produce a generalized state of stress in the body. The state of stress in turn Inaugates a series of “alarm signals" which he postulated can act througi the floor of the brain (presumbly the hypothalamus) to stimulate the sympathetic nervous system and the pituita'y gland Stimulation of the sympathetic nervous system stimulates the aa‘enal medulla which In tlrn Increases the secretion of epinephrine. Concomitantly, stimulation of the anterior pituitary Increases the secretion of ACTH, which acts on the acrenal cortex and enhaices glucocorticoid secretion The latter produces a hyperglycemic condition in the body--that Is, it mobilizes the tissue fats aid proteins, aid Increases the llver's gluconeogenesis. 0n the thymus glad, the glucocoticoids cause an atrophy, followed by a decreased number of lymphocytes and eosinophils. The former causes a condition of decreased Immmity, while the latter decreases the allergic reactions of the body. Consequently, the corticosteroids enable the animals to resist or adapt to the stressors or the stresses (Selye, I936). Following the work of the above two authors, the wound was paved for father research by many Interested authors on the link between stress and hypertension on one hand, and the role of the nervous aid endocrine system in this relationship on the other. Friednann and Paul (I952) influenced by the Idea of dehyd‘ation as a stress condition fomd that vasopressin In large doses could elevate blood pressure In rats and rabbits. lngle (I954) studied the role of the adrenal cortex In stress aid fomd that corticosteroids have a permissive action In 16 stress-although their presence at a basal level is necessary to permit animals to cope with the stresses, their Increase beyond the normal level does not add to the ability of the animals to adapt to the stress challenge. Anderson 0966) studied the hormones of peripheral blood In male Fischer rats after suijecting them to severe physiological stress. It was fomd that their blood contained ACTH-releasing and antidiuretic activity and that the hormones associated with this dual activity enter the general circulation by way of the portal vessels of the anterior pituita'y gland According to the author, the most severe stress, lapa‘otomy alder ether anaesthesia followed by rapid removal of blood from the aorta , provoked the himest ACTH-releasing and mtidiu‘etlc response compared to control hypophysectornized rats Riegle (l973) studied the effect of chronic stress on the ad‘enocortical responsiveness of yomg and aged male and female rats. He sibjected the rats, twice daily, for two hairs to restraint or ether stress for a period of 20 days He observed that the acenocortical responsiveness to restraint or ether vapor stress was decreased In all the stressed mar-being geater in the yomger than the older rats This suggested that the corticosteroid feediack from chronic stress activation of the aa‘enal cortex may result In incomplete Inhibition of the adrenocortical control mechanisms A year later, Buckingiam and Hodges, being Interested In the effects of ether anaesthesia as a stress factor, studied the interrelationships of the anterior pituitary and plasma corticosterone in ad‘enalectomized aid stressed alrenalectomized rats. They correlated the changes in the pituitary and plasma ACTH, with the chaiges In plasma corticosterone before and after exposire of the rats to stress They observed that adenalectomy Increased the ACTH level 17 of the plasrna--presunbly due to removal of the feecback Input from the arterial. They also found that ether stress caused a small rise In the plasma ACTH level of the intact, and a profound Increase in the plasma level of the alrenalectomized rats. However this exaggerated response was shown to be reduced to normal when physiological doses of corticosterone were given to the rats. The authors firther observed that prolonged treatment with corticosterone was needed to abolish the stressresponseoftheACTH.They proposedthat thesynthesisandthe basal level release of ACTH Is directly controlled by the concentration of corticosteroids In the blood-but the corticosteroids exert only a delayed effect In modulating the stress Induced release of the hormone. Hallback aid Folkow (I974) studied the ca‘diovascular responses to acute mental stress In spontaneously hypertensive rats. They cornpa‘edtheresponses of 7monthold5l-Rrats thathada manifest hypertension ,wIth those of control prehypertensive (IO-ll week old Sl-R) aid normotenslve rats. Blood presslre and heart rate were messindafter ligit, noise and vibration stimuli. The tachycardia (due to accentuated sympathetic and centrallY sippressed vagal discha‘ge) was wed as m Index of neural stimulation It was fomd thatthematire Sl-Rsrespondedmore stronglythaitheothertwog'oms, suggesting that they had a lower threshold for defense reactions. The authors proposed that the sympathetic hyperactivity in the SHR rats tended to trigger structlral aid vascular admtive chaiges which resulted in a manifest hypertension They firther suggested that these changes were genetically produced and were not a sequel to hypertension-~because they were also shown In the prehypertensive, but not In the renal hypertensive rats 18 Hirata gt at, (I975) measured the plasma levels of B-MSH and ACTH during acute stresses and after administration of metyrapone [2-methyl-I,2-bis-(3-pyridyI)-propanone], an adrenal steroidogenesis blocking compond, in man. Simultaneous measlrements of plasma level of FISH and ACTH were made under insulin-induced hypoglycemic condition, and lysine-vasopressin or metyraione Injection as the stressors It was observed that Insulin hypoglycemia caused a marked Increase In ACTH and a slight but sigiificant rise In plasma rISI-L Lysine-vasopressin however, caused a sigiificait rise in plasma ACTH levels without a sigiificant response from B-I'ISI-L Finally, the metyrmone caused a rise in both B-rISH and ACTH levels. The serial blood sampling showed that peak rise of the hormones occlrred together-pointing to the similar mechanism or pathway of release for both of them. Lysine-vasopressin, on the other hand, caused a sImIficant rise In the ACTH level I5 minutes after the Injection (ACTH rising from 9I.3:l3.2 to I65.7:I9.7 pg/ml). This rise was followed by the rise In plasma cortisol from 8.0: L3 - to I9.6:3.3 jig/I00 ml. Lastly, the metyralone, which Is at Inhibitor of llB-hycroxylation of adrenal steroidogenesis and causes the shift of the products from cortisol and corticosterone to deoxycorticosterone and ll-deoxycortisol, was shown in this experiment to remove the feecback inhibition on ACTH and In this way leading to a higi level of plasma ACTH with the concomitant riSH secretion Le Hevel gta], (I978) observed the dynarnlc changes In plasma alrenocorticotropin after sibjectlng 90-day old male and female Sherman rats to netrotropic stress. The stress was composed of removing the rats from their cages and putting them In a glass jar for 3 minutes. It was observed that the stress Increased the levels of ACTH, reaching 19 the maximum level of 2711 unit/ml (equivallent to 270 pg/ml), S-lo minutes post-stress, and falling siglificantly after I5 minutes (while remaining constant for the next 40 minutes). McCarty £1 a]. (I978) studied the sympatho-adrenal activity of SHR and WKY rats dlring recovery from a 2-hour forced Immobilization stress. They observed that SI-R rats responded simificantly to the stress by a higler level of norepinephrine, epinephrine and dopamine. While the plasma corticosterone response was not different In the two rat ms. This suggested to the authors that the sympathoad‘enomedullary system of the Sl-R rats may be more sensitive to Immobilization stress, and that this system remains In a heigltened state for a longer period of time following the stressful stimulation In mother experiment using forced Immobilization and haidling stress, Kvetnaisky aid colleagues (I978) attempted to measlre the plasma levels of epinephrine, norepinephrine and dopanine B-hydnxylase In Sprague-Dawley rats They observed a statistically sigiificmt and a time-dependent rise In the level of norepinephrine and epinephrine dtring and after the stress. This suggested to them that plasma catecholamines may be a good measlre for reflecting the degee of activation of the sympathoaa‘enal system dlring stress Kvetnansky gt :1, (I979) In a father study of Immobilization stress in Sl-R rats, measu‘ed the blood presswe, hea‘t rate aid plasma norepinephrine, epinephrine and corticosterone, before, dlring did after seven periods of Immobilization stress ( l50 minutes of stress per day). They found a sigiificantly higher level for the all measlred hormones In the SHR rats compa‘ed to the WKY rats and In all the stress periods However they also observed that the meal arterial presswe, being himest In the first period, g‘adually fell In 20 the successive later stages in the SHR rat. This suggested to them that the adaptive changes In the cardiovascular and sympathoad‘enomedullary systems of the repeatedly stressed rats is geater in the SHR than the WKY rats. Falkner gt 3], (I979) studied the cardiovascular response of normal adolescent mum of hypertensive wants to mental stress, which consisted of performing a‘IthinetIc calculations within a short time period The findings showed that the subjects with labile hypertension demonstrated a sustained Increase In systolic and diastolic presslre aid heart rate diring stress The analysis of the plasma level of catecholamines showed that the post-stress levels were hlgier In the labile hypertensive Individuals and subjects that had at least one hypertensive parent. This suggested to the authors that Increased attenergic activity was the mediator in the response of the hypertension prone slbjects Finally, Hausler gt a1. (I983) studied the ether-stress induced ' secretion of ACTH and corticosterone diring the development of spontaneous hypertension In rats. They observed that the ACTH response was markedly enhanced In 4-weeks-old but not In I2 or l6 weeks old Sl-R rats. This suggested to them that the Imbalance of the pituitay-acrenal axis may be the major cause of the onset aid development of hypertension In Sl-Rrats. C- STRESS, SALT INTAKE All) HYPERTEHSION In the previous two sections the relationship of stress and higl blood presslre was discussed However the connection between stress and salt Intdte, aid salt Intake aid him blood presslme remain to be explored 21 Stressors cause the release of ACTH -and this Is almost unanimously accepted by stress researchers (Selye gtgL, I936; Kendall, l97l; Riegle gt at, I973; Vates gt at, l974; Le Novel gt at, I978; Fagin gtat, I 983; Hausler gtgl, I983 and I984). Denton and his colleagues (I980) and Blaine gt 3], (I975) observed high Intakes of sodium, potassiun aid calciun chloride dlring mm and lactation (presunably to compensate for the needs of the developing fetus) In rabbits and ewes These authors were further able to demonstrate that the pregiaicy hormones and exogenous ACTH by Itself could produce similar results In control rabbits, and stress could produce results comparable to exogenous ACTH Since aldosterone did not stimulate salt mpetitre In rabbits aid since the effect of ACTH occtrred In ad'enalectomized rabbits maintained with glucocorticoids and mlneralocortlcoids, the authors suggested that ACTH, either via a central or a peripheral netral mechanism or both, may be Involved In the regulation of salt wpetite. Weisinger gta], (I980), however, confirmed therole of ACTH In salt appetite, but pointed out that ACTH had no extra-arrenal effect In rats-that Is, It produced Its effect solely througl the aa‘enal gland, thus Indicating a species difference In ACTH action mechanisms Since ACTH Is extensively used as a measlre of stress in the ailmals ( Selye, I936; Yates gt at, I974), the link between stress and higi salt aipetlte should not come as a suprise. Firthermore, the epidemiological finding that In societies with very low sodiun chloride intake hypertension is almost totally absent ( snaper, I967; Lovell, I967; Priorgtgt, I968; Truswellgtgt, I972; PagegtaL, I974and 22 TobIan, I975); or studies that have shown a higier Incidence of hypertension In the acculturated/developed countries wherein people consune a high anoint of salt in their diet ( Lowenstein, I96I ; I‘Iacrlahon gt at, I973; WIlhelmsen gt at, I973; Hatano, I975); or the salt restriction studies-wherein a subsequent crop In blood pressu‘e has been observed (Allen, I920; Perera, I947; Dahl, I972); or the dilretic therapy studies, wherein elimination of salt from the body has been accompanied by a reduction In the intensitity of hypertension (Dahl, I972); or salt loading experiments that have Indicated a concomitant rise In the blood pressure of animals (Dahl, I958, I960 and I964); or the conviction that hypertension rests on processes In the body that Increase the demand for salt ( Fallis gt at, I962; Schecter gt at, I973); or the possibility that salt is a factor In hypertension only In the presence of stress--which would be the most consistent factor In modern and developing societies ( Epstein, I963; Cobb aid Rose, I973; Pickering, I977; Eyer, I975); or the possibility that higi level of ACTH may cause hypertension-meted by the experiments In which blood pressure was elevated In rats (Freeman gt gt, I980) or sheep (Scoggins gt at, I974) or dogs (I'IcCaa, I978) or hunm (Ram gtgt, I979) all provided direct or indirect proof for the link anong stress, hypertension and salt Intate. In conclusion, a critical review of the literatire on the Interrelationship anong hypertension, stress, salt Intake and helm-endocrine physiology shows a la‘ge number of experimental studies, many of which are In disagreement, either qualitatively or ' 23 quantitatively, regarding the exact causal Interrelationship among these factors. Part of the problem Is due to the complexity of the Issues that are being studied( P399 fiat, I958; Bemard, I985) and part Is due to the methodological variations In the desigl of the studies (Reid gt at, I978). 0n the extremes of the controversy, one sees Sjoerdsma (I972) writing that " whereesnoconvincing chemical evilhrloeoul beobteined to Impliceteexoess nativity of vmistrictor systems (such as eympetlio-ed-aiel system) in pethnpnesis or mummwmmmidunmmmmmmmbymim Wistar ctivlty, especially that mediated by winpeti'ietic nervous system". Or Doyle (l978) writing that " Increased levels of circulating oeteeholaninee In hyperteneion lb Motmvuyfrinmlvimiheprimaymofraised bloodprmewhatitdnas Iiowevr,istounphuizetheoentrel roleofsympetheticnervoussyslemintllernsinmof W'. Or Oglesby (l977) who wrote: ' To do. however theepkhrniolnpic MWOWMdmeWMMMUWM MWNMMNMMUW'Cr Ddil (l972) whowrltes: 'Tlnevidmeihdselthlmeseprmufetelhyprmisdireet. dilitituiveuid mivoeel In the rat". Or Folkow 0982) who writes: " For neatly loommmmmmmlmmmprimmwmis Initiated aid maintained Proves was really stimulated by Goldilett‘s introdiction of mimic“ WWW In I930’s; unfu‘tllldelyalthusium fit the Michell miuimmuelmalqdunmlizdmwmprimmmbmwd multifmriel origin“. This seems to be the state-of-the-a‘t on the mderlylng mechanisms for the Interrelationship among hypertension, stress, and salt mtite. 24 D- STRESS, HYPERTEHSIOH All) SPONTANEOUS” HYPERTEHSIVE (Silt) RATS Spontaneously hypertensive rats (SI-R) were first developed by selective Inbreeding of normotensive Wistar rats by the Kyoto g‘oup In Jam, mder the direction of Okamoto and Aoki (Okamoto and Aoki, I963; Okamoto gtgt, I966). These rats show a mo: Incidence of hypertension with a markedly higi blood pressue and spontaneously elevated salt appetite (Catalanotto gtaL, I972; Bernard gtaL, I980). The presence of a very higi frequency of hypertensive cadiovascular disease In them suggested that they may be good models for the study of hypertension In mm (Okanoto, I972). Traditionally there have been two control ms for the SH? rats The normotensive Wistar rats (NC) aid Wista-Kyoto strain of normotensive rats (WKY). These two control guns are not quite simila‘ biochemically aid physiologically, but together can act as a safe control for the SHR rats (Frohlich and Edwa‘d, I977). At the time of matlrity, approximately 2-3 months of age, when the period of raiid development Is over, the a'terial blood presstre will become higily elevated In the SHR rats 083/ I26 mmHg), compared to WKY (I34/90 mmHg) or with NC control rats ( I40/97 mmHg) [Pfeffer and Frohlich, l9731 The SI-Rs at the age of 5-I0 weeks are said to have developed a labile phase of hypertension At the age of 3-4 months the hypertension becomes estailished , and at 4-6 months It is well estalished They show aims of Increased sympathetic discharge, reduced vagal tone, Increased heart rate and cardiac output ( Folkow and I-lallback, I977). In vitra comparisons of SI-R and WKY rats for the 25 mechanical and morphological properties of the resistance vessels show that In SI-Rs the hypertension Is associated with Increased peripheral resistance, which In tirn Is related to structural changes such as na'rowed lunen, a thickened media aid Increased number of smooth muscle cell layers In these vessels (I‘Iulvany gt at, I978). Also morphological studies on the endocrine organs of the SHR rats have demonstrated a condition of hyperactivity In both adeno hypophysIo-aa‘enocortical and the adenohypophyseo-thyroldal systems, together with a hypersecretion of vasopressin aid Sympathetic overactivity. These charges are suggested by Tabei gt 3], (I972) to possibly pu'tIcipate In the pathogenesis of the SI-R‘s hypertension ' Spontaneously hypertensive rats have a nattrally elevated salt wpetite (Catalanotto, gtgL, I972; Bernad gt at, I980; aid Hogenson gt at, I980)--but It is not quite clea‘ If the occurence of this alpetite Is seconday to hypertension or not. Catalanotto gtg], (I972) suggested that the estailishrnent of elevated salt appetite In the SIR happens after the establishment of hypertension (that Is, after maturity). However, I'Iogenson, gt :1, (I980) who observed enhaiced sodiun mpetlte In immatire (5 to 7-week old) SI-Iil rats, suggested that It starts before the we of hypertension This controversy may be due to the lack of homogeneity of the SHR (and WKY) population--that Is, the breeding history and the smpliers of the rats could somehow participate In the generation of these controversies. Studies of catecholanine tu‘nover rate In SHR rats of different age have shown that under mdlstu‘bed conditions only the young sens show sigis of Increased sympathetic activity--whereas the ttrnover rate Is mchaiged or even reduced In matire SI-Rs when they compared 26 with the control rats (Folkow, I975 and I977). Consistent with this finding, Conway's group (I975)--based on the assumption of sympathetic hyperactivity In very young SHR rats, treated them with B-aa‘energic blocker, propranolol . It was fomd that the treatment to a great extent hindered the later development of hypertension However, the same treatment had relatively no effect In the mature SHRs. This experiment not only supported Folkow's point of view, but further suggested to Its authors that the adrenergic B-blocking agents, beside their Interference with sympathetic cardiac control and renIn release, might also centrally damp the sympathetic discharge. Hausler gtg], (I984), In a search for anatomical differences in the alterior-pituitary of the 4-weeks-old SIR aid WKY rats, quantitatively aialysed the ACTH-Immutareactive cells The findings showed a lager ulterior lobe and more Malt Immtnoreactive cells In the Silks compred to the control rats. This suggested to the authors that at enhaiced availability of ACTH In the anterior pitruitary may explain the markedly enhmced stress-induced release of ACTH In these Immatlre rats. They ftrther mentioned that the Instdiility of the hypothalano-pituitay-aa‘enal axis may contribute to the development of genetically proganmed hypertension. E- CHRONIC CATHETERIZATION IN RAT The natire of the experiments on hypertension and stress calls for mdisturbed conditions of observation The sympathetic nervous response of the rats to handling and minor distlrbances is often Immediate and usually masks the effect of the factors that the experimentors are trying to measure. Therefore considerable precaution 27 Is recommended In the studies of stress hormones in general and catecholamines In particular (KvetnanSkY it al., I978). It Is easily conceivable here that one of the most difficult situations encountered by an experimentor may be the problem of measuring the basal level ( that is zero and undisturbed level of stress hormones In the rats or other experimental animals. Furthermore, the need for constantly monitoring the blood pressure of rats (or other lab animals) as an Integral part of the hypertension/stress studies has always indicated the need for finding a somd method that will Impose the least disttrbance on the animals. The tail-cuff manometeric methods, plethysmogaphy (Heymann and Salehar, I949) and other methods of direct blood presstre measirement other thai indwelling catheterizatlon, however, have had the negative effects of altering the hemodynanics of blood inder the Influence of anaesthesia and sirgical procedtres, or damaging the vessels of small animals (Still stat. I956). ' In response to these problems, chronic catheterization of rats has gained Increasing Interest among stress/hypertension students. Catheters are usually constructed from polyethylene (or tygon) tubing with a shorter aid narrower tIp (as described later In the chapter on Materials, I‘Iethods and Procedures) to be Inserted in the artery of concern--usually the cantid, abdominal aorta, femoral or tail artery of rat (Buckingian, I976), with the distal end being externallzed through the rat's skim-and stpported within a shield--usually a metal spring (Kvetnaisky et al., I978 and I979; HacCa'ty et al., I978; Fagin gt at, I983). In short, chronic cannulation (catheterizatlon) of rats allows 28 for repeated blood sampling and pressure measrrement on one hand (Fagin gtgt, I983) and more accurate meastrement of the resting circulatory level of stress hormones (Carnba gtg], , I98I) on the other. For this reason chronic cannulation Is an Integ‘al element in the experimental design of this thesis research Mfl'l'EfllfllS, METHOIIS flNll PROCEDURES 1) PROTOCG. OF THE EXPERIMENT The overall experiment was composed of two major parts; part one was desigied to measure and study the hormonal and concomitant hemodynarnic hypertensinogenic responses of rats to ether and vibration stresses (I l:00 to I200 AH); and hat 2, to exarnlne the rats' response to manual-restraint stress (I I:00 to I200 AH) and also to determine the resting level of the aforementioned responses between II:00 to I200 PM Two g‘oms (batches) of rats, as described In the next section, were used for the parts of the experiment. They were first physically examined and then housed In the laboratory. Their readiness for the stress experiments was based on two criteria one, their response to two-bottle salt preference tests, and, second, their full matirity--evidenced by the plateau of their wowth rate. Rats ( In grows of 6--half control and half experimental) were then chronically cannulated and connected to Infusion punps to facilitate blood sanpling, blood pressure, heart rate and hematocrit measurement, and Infusion processes. Upon recovery from the surgery, usually 3-6 days after the cannulation, the rats were suijected to one of the aforementioned three types of stress, and blood samples were obtained from them before and 29 30 after the stress application The blood samples were then stored for later radioimmmoassay of plasma epinephrine, norepinephrine, ACTH and corticosterone. Finally, the radiolmmmoassay results and hemodynarnic data were statistically analysed 2) EXPERII'ENTAL ANIMALS Two gems of male rats composed of a total of I9 spontaneously hypertensive (SI-R), I9 Wistar-Kyoto normotensive (WKY) and 6 Sprague-Dawley rats (50), were used for different phases of the overall experiment. Group A( to be stressed by ether and vibration) was composed of II WKY aid 9 SH? rats, all I6 weeks old before aanlsslon to the experiment; and you: 8 (to be used for manual-restraint stress and evening blood sanpling) was composed of 8 WKY, I0 SHR and 6 50 rats, all I5 weeks old before the aanission The SH? and WKY rats were all born on the sane date aid were purchased from Taconlc Farms, Germantown, NY. weight analysis of the rats at the time of their arrival showed that the m A SH? rats had an average weigit of 27417.5(meansISD), ranging from 259 to 285 game; and you) A WKY rats, had an average weight of 292:” I, ranging from 275-307 yarns. The gun 8 SHRs weighed 273s7.l (range 271-292 gains); and g‘oup 8 W3 were 286:I0.7 (range: 276-3I5 gains). The 50 rats were not age-matched with their comterparts. However, they were weiglt-matched with the WKY rats. 3- IKIISIIB All) FEEDING COMITIOII 0F TIE RATS Each two rats of the same strain were placed In a 32X35Xl6 cm plexiglass and trarispa‘ent cage with a barred -metal lid Cages were 31 placed , 4 in each shelf, on a metal rack, in a window-less room that was partially Isolated from the traffic of the main laboratory compartment. The cage bedding was made of sawdust, and distilled water and Teklado rat chow was available ad libitum. Watering, feeding, change of bedding and weighing of the rats. were performed once a week. A diunal Ilmt cycle was maintained (lights on from 0800-2000 how), and room temprattre was kept at or around 70 F’. From the moment of their arrival Into the laboratory, up to the end of the experiment, the rats were kept and cared for 2-3 months Starting from l5 to 20 days after their arrival and for a period of l to 2 weeks, a raldomlzed two-bottle preference test for NaCL was performed on the rats Due to the methodological nature of the test, one of the consequences was a transient alteration In the watering pattern of the rats dlring this period-because two bottles; one filled with distilled water and the other composed of a salt solution, were provided for each cage rather than one. After su-gical cannulation (as described later), the rats were housed singly; provided with honey and peanut butter In addition to their ordinary rat chow for the first 3 days post operation, and their externallzed cannulae were connected to Infusion pumps by Tygon tubes. The rats were kept alive between 5-I3 days after surgery, depending on their physical health status as determined, first, by their weight, blood presslre and hematocrit reading ; and second by their physical appearance and feeding behavior. The appropriate stress tests were performed on the rats during this period and not sooner than 3—5 days after the surgery. 32 4- SALT PREFERENCE TEST Two to three weeks after the arrival of each batch of rats into the laboratory, two-bottle preference tests were performed . The rationale for this test--an accessory part of the main experiment, was to make sure that the rats had developed salt appetite and that there was a difference In preference between the SHR and WKY rats before proceeding with the stress experiments. A Each rat was provided with the choice between a bottle of distilled water, aid a bottle of salt (NaCl) solution Each day the position of the flavor was changed randomly (being placed either to the left or rlg'it) so as to control for positional preference. Three different concentrations (0.03, 0.I and 0.2 I'Iolar) of the salt solution were assigned to each cage In a raidom basis and in such a way that each was smplled with all the different concentration of salt over a 6-day period (two successive days for each concentration). Each day, between lI:00 to l200 AM, the consumption rate of the rats was measlred aid results were recorded If the SI-IRs did not show a sigiiflcaltly higier preference for all the salt concentrations or at least the last two hlgler concentrations, then a second preference test was scheduled for l0 to 20 days later. 5- CIRINIIC CATI'ETERIZATIGI Catheters were chronically Implanted In the left carotid artery of the rats to Instre a direct measurement of blood presstre, serial blood sampling, and arterial dug Injection such that the animals 33 were completely undisturbed except when exposed to experimental stress. A- SURGICAL PROCEDURE The surgery for catheterization was performed under aseptic conditions. All the surgical Instruments were heat/pressure sterlized. The rats were anaesthetized with ill injection of Xylazine (Rompun’), 7.5 mg/Kg body weight, together with Ketamine hydrochloride (Vetalar’), 37.5 mg/Kg body weight For eye protection during strgery, Chloromycetin" ophthalmic ointment( with Ix chlorarnphenicol) was applied to the eye ball. The rats were then secured on a rodent surgery tale" hind-quarters higher than the fore-quarters. Two areas were shaved and cleaned with Betadlne'; the iii-st was dorsal between the ears and half-way down the neck, aid the second, the ventral skin from the pharyngeal to the thorasic area Then a horizontal Incision, I Inch In length, running cranIo-caudally on the ventral mid-line of the neck, was made. All the exposed muscle layers were pulled to the sides and secured with the help of fine hooks. After localizing the left carotid artery, It was stripped from the suromding connective tissues with exceptional care and with the help of the dissecting microscope. Then a 5 mm length of the artery was Isolated from the general circulation with micro clamps, aid the first 20 mm of a cannula (Plate la, Appendix A) as described below, was Inserted craiIo-caudally Into the artery (Plate Ic, Appendix A). The Tygon cannula was composed of a terminal piece (I Inch In length, and 0.0lx0.03 Inches In Inner and outer diameters, respectively). This piece was inserted Into a slimtly bigger tlbe, I8-20 Inches In length, 0.02 X006 Inches in Inner and outer diameters. The first inch of the latter tube was turned Into 34 a 0.5-0.7 cm loop In boiling water. Immediately before surgery, Dow Corning solution (I0 cs) was flushed through the cannula In order to reduce friction and to facilitate the flow of solutions In it. The tube was then filled up with heparin-saline solution (50 units heparin /ml 0.9% NaCl solution). After properly inserting the cannula, the two ends of the artery (the free cranial end, and the caudal end harboring the cannula) were both ligated with non-absorbable, transparent, nylon suture( Plate I d, Appendix A). A trocar was then attached to the distal(free) end of the cannula and was directed subcutaneously, from left-lateral neck direction to the cephalic aspects of the rat; peneterating through the skin somewhere close to the mid-point of the hypothetical line joining the two ears together. Panalog’ ointment (nystatIn-neornycin sulfate thiostrepton) was then generously applied to the exposed surgical sites. The muscle layers were brought in together; the swcutaneous layers were first continuosly sutlred--trapping the loop of the cannula Inorder to retard unwanted and damaging pulls over the carotid artery; finally, the skin was closed up by interrtpted sutlres. A supporting stainless steel spring (30 cm length; 3mm diameter) was then directed over the emerged catheter and Its proximal part was first patched to one or two wound clips attached to either side of the protruded catheter (Plate le, Appendix A). Then, Caulk” repair material was generously applied to the first few millimeters of the spring, the clips and the skull's skin, aid allowed to harden (Plate II, Appendix A). The distal end of the catheter was joined to a swivel--a hypodermic needle (no.23) attached to a lml plastic disposable syringe (Plate lb, Appendix A). 35 The purpose of the spring and swivel was to protect the catheter from possible damage by the rats. 8- POST- SURGICAL PROCEDURES The operated rats were weighed and housed Individually In clean plastic cages with fresh bedding, while water and food were provided for them as previously described To discotrage unnecessary post-operative hypothermia, limt bulbs were located adjacent to their cages for 2-3 hou‘s To prevent any unwanted septicemic conditions, 50 mg Ikg/day chloranphenicol was Injected Intra-arterially Into each rat for the first 3 days after Strgery. The patency of the arterial catheter was maintained by connecting the distal part of the swivel to Infusion punps, and constaitly flushing the timing systems. The Infusion fluid was composed of 58 dextrose with 5 (nits/ml Main The pimps were adjusted such that I0 ml fluid was flushed througl the catheters each day. 6- I-EART RATE, BLOW PRESSLRE AND I'EI‘IATOCRIT HEASUREI'ENT S Each day, starting with day one after catheterizatlon, between Il:00 to iz-oo AH and under a quiet and undisturbed conditions, heart rate, blood pressu-e and hematocrit of the rats were measured In this process the distal end of the catheter was attached to an Electromedic'” pressure trarisdircer which was connected to a silson‘ polygraph Calibration was performed with the aid of a mercury sphygrnomanorneter. 36 The heart rate was measured and the blood pressure was calculated from the polygraph record Mean arterial pressure was calculated from the systolic aid diastolic pressures using the formula MAN mmHg) - [2(diastoliclrisysioliclll3 The hematocrit value, an Index of the animal's health, proper blood volune and suitable cell count, was measured by aspiration of a drop of blood from the catheter onto a heparinized and clean microhematocrit cmlllay tube and centrifuging it with the International micro-tube centrifuge. (model I18). 7- BLIND SAI'PLIIB PROCEDIRES Before and during the stress tests, blood samples were taken directly from the catheter without handling the rats. The purpose of sanpling was to measue the circulating level of ACTH, corticosterone, epinephrine and norepinephrine. To Insire proper flow of the blood diring the test, 30-45 minutes before the zero sampling ( I.e. resting level), I-2 units of fibrinolysin (Plasmin') dispersed In 0.2-05 ml of distilled water was Infused Into each rat througl the catheter. A total of L5 ml of blood was rrawn into a heparinized syringe, of which I.0 ml was transferred to an Iced glass tube for ACTH and corticosterone assay, and 0.5 ml was put Into an EDTA-containing Iced tlbe for catecholarnine assay. Times were then centrifuged at 3,000 g, 4'C , for 5 minutes. The clear aliquot of plasma was then transferred to Iced plastic tiller-those, to be used for ACTH/corticosterone assay, containing I50 I“ Trasylol solution (containing 600 KIU). The samples were then frozen with dy Ice and stored at -40‘C mtil assayed for the hormones. In order to avoid anemic conditions that could jeopardize the health 37 of the rats or act as Incidental source of stress, the blood cells (Is the precipitate portion of the centrifuged blood samples) were thoroughly mixed with 0.6-l.0 ml heparin-saline solution and returned to the rats. Immediately after the cell return, 0.5 to ID ml of heparin-saline solution was Infused to each rat to clear the catheter and restore fluid volume. If a catheter remained occluded, further f Ibrinolysin treatment or catheter repair was performed. If this was successful the rat was rescheduled for the appropriate stress test and blood sampling not sooner than l-2 days. If not successful, then he was removed from the experiment Furthermore, rats showing signs of sickness evidenced by their poor hematocrit count (less than 23x cells), very low blood pressure (less than 95 mmHg In the WKY and I30 mmHg In the SHR), or severe weight loss, were given a few days rest--If not recovered, then they were also removed from the experiment 8- STRESS TESTS A- ET HER STRESS Group-A rats were used for this test. After a zero time blood sampling under non-stressful conditions each rat was placed for 2-5 minutes in a glass jar saturated with ether vapor (to achieve anaesthesia). The rats were then taken out of the glass jar, but continued to be exposed to ether by placing the muzzle into a nose cone containing ether-soaked cotton The rats were kept under close surveillance during this stage so as to avoid respiratory depression. The time period of exposure to ether 38 stress was 30 minutes. At the end of the time period a blood sample was taken. After sampling, the rats were returned to their cages and kept warm to fully recover. The subsequent stress test was not performed sooner than 3-4 days after the previous experiment. The ether and vibration stress tests were performed in a randomly alternate sequence. 8- VIBRATION STRESS After taking resting blood samples the candidate group A rats, were placed In a plexiglass cage , transferred to an EberbachO 6000 shaker, and shaken at low speed (I80 oscillations/minute). After the end of the 30-minute stress test, a blood sample was drawn and the rats were returned to their cages. ' C- MAHUAL-RESTRAIHT STRESS Subsequent to resting level blood sampling, the group-B WKY, SHR and 50 rats were held tightly In the hand and restrained from any movement for 5 minutes Post-stress blood sampling was performed I5 minutes after their release, that is, 20 minutes after the beginning of the manual-restraint stress. These rats were further used for measuring the resting evening level of their hormones, unless their physical status called for their removal from the experiment 9- RADIOII‘II'IUNOASSAY All the samples were radiolmmunoassayed with the collaboration of Dr. Raymond Nachreiner,AnImal Health Diagnostic laboratory, College of Veterinary Medicine, Michigan State University. 39 A- CATECHOLAMIHES CAT-A-leTM (The Catecholamines Radioenzymatlc Assay kitI3HI), from Upjohn Company, which Incorporates a modified Passon and Peuler methodology (I973) was employed for the RIA of epinephrine and norepinephrine. The sensitivity of the assay method for epinephrine was IO pg/ml, and for norepinephrine It was I8 pg/ml. The Intra-assay coefficient of variation was I I98 and I L48; the Inter-assay precision was I08 and I88; and the accuracy of recovery was 88-I068 and 93-ll68 for epinephrine and norepinephrine respectively. Parallelism In I:2 and I:4 dilutions resulted In I048 and I I58 recovery for epinephrine, and I098 and I l88 recovery for norepinephrine. ~ 8- CORTISOL All) CORTICOSTEROIE The Corticosterone (H3) Kit, from Radioassay Systems Laboratories was used In the RIA of plasma corticosterone. The antiserun used in this kit produces l008 cross reactivity with corticosterone and 5.38 cross reactivity with cortisol. Prallellsm of the corticosterone assay for I:2 and I:4 dilutions included II48 and I008 recovery respectively. The lnterassay coefficient of va‘iation with rat serum ls around I48. Due to the reactivity of the corticosterone assay with cortisol It was necessary to also measue the plasma level of cortisol. For this purpose, p25” Cortisol Radioimmunoassay Kit (GammaCoatm) from Travenol-Genentech Diagiostlcs, which has l008 cross reactivity of the antiserun with cortisol, was used The procedure was modified for use with bovine plasma by taking larger sample volunes (20 ill), adding an additional 0.2 mg 8-anlllno-l-naphthalene sulfonic acid per sample tube, 40 and incubating at 37C for 2 hours before decanting the tubes The Specificity tests of the antiserum Indicated 65.88, 3.88 and 2.I8 cross-reactivity for prednisolone, prednisone and corticosterone, respectively. Cross reactivity was less than l8 for cortisone, deoxycorticosterone and dexarnethasone, progestrone and betarnethasone. Precision on replicated quality control samples Indicated less than I08 Intra and Interassay coefficients of variation Sensitivity, as calculated from the standard curve at 908 of totaltrace binding was 3.8 ng/ml. C- ACTH ASSAY The ACTH kit from Immuno Nuclear Corp, which uses rabbit mti-porcine ACTH antibody, and shows a good cross-reactivity with human ACTH was employed In the ACTH assay. Prevst pilot studies of ACTH parallelism in dilutions of rat serum which had been used to deterrnlne the equivalence of rat ACTH and the hunan ACTH In Wl-IO standards, showed that a I:2 dilution of the rat sera with ACTH-free human plasma resulted In l068, l068 and I298 recoveries Dilution with borate buffer resulted in 888 and I328 recovery. In otr experiments In which pooled rat serum containing I63 pg/ml ACTH was used, recovery ranged from 74-988; pa'allellsm was 8l8 for I:2 dilution, 86 to 1098 for I:4 dilution, and l I78 for I:8 dilution The interassay coefficient of variation of the pooled plasma was I78. IO- STATISTICAL ANALYSIS Results of all the experiments were presented as meanslSD unless otherwise specified . Paired comparisons were made for the blood samples 41 taken before and after stress exposure within each group. The significance of the difference between the groups was measured by mixed design analysis of variance (P-0.05) and/or one-tail student I test (P-0.005). I I- CALIBRATING ACTHICORTICOSTERONE CONCENTRATION Addition of 0.I5 ml of Trasylol to the a tubes used for ACTH and corticosterone would have caused an unrealistic picture of their plasma concentration had no correction factor been introduced. The reported RIA concentration of these hormones would have been lower than their actual value, and this difference would have been more pronounced In the case of SHR rats that naturally possess a higher hematocrit value than their comterparts. To correct this problem, the following formula was applied to all the reported values of these two hormones: Actual Canal-b (Reported IIA “Illtl-lctSMJSI Where. Hct- Hematocrit value, expressed In percentage form RIA- Radioimmunoassay RESULTS I- silollml IIIITE Figlredeaile l showthepooled growthdataonOWKYanOZOSI-IR rats As shown, the SIR rats reached physical maturity (in terms of body weigit) at an earlier age, that is, between l35-l5l days of life. Thereafter their weiglt stayed relatively constant, close to 355 gems. However the WKY rats not only reached maximum weigit at a later age, that Is between I95-205 days of life, but get bigger and heavier than their SI-R comterparts The maximun weight attained by the WKY rats was 493: I6 grams. 2- RLIIIIII PRESSURE Figure aid Table 2 represent the findings on blood presslre of the three rat groups (age-22-34 wks.). The morning (I I:00 AM) mean arterial pressire represents the pooled blood pressure , before any stress test and 3 to 5 days after the original surgery. The evening (I l:00 PM) pressure, however, Is based on the non-pooled, 2-5 days post-stress experiment sanplings from the animals. As indicated, the morning pressu‘e of the WKY, SD and SHR rats was I07s27, I4222I and l80124mmHg respectively. Mixed analysis of 42 43 ca as 3 s 8 so so... .ass on _ a 8:8 s s. 32 a; 2n Em s :8 532° 2: ._ 952d 44 - 550: 323:2. ca 8m .8 n2 8. at E 2.. we. no. a... 8. .m. a! a: a! an. mm. 8. mm. 3. 8. 8 Tn+n1runnn+n+nwnn.»+1o .. om , , 4. oo— :2m .0. 3.: .e. s om. .. 8a emu ilelolsiroiie/o\olelioirx\e ill: I). all... Ila llQIrcrlo \e It... lllQi‘iO 0. all \OII Ilorlrtilolloll \./.\. «he: can :2: :83 .3 E.— E3930 IIOIIOVM“W 8n own Gov t omv com .2323 :a 45 TABLE I SHIRT" RRTE OF IIIKV RNII SIIR RRTS m mm W III”! M? Still IIIKV SIIII 86 286:3 273:2 8 8 l08 304:8 289:8 8 8 I I4 33 I :4 304:2 8 8 I23 345:3 3II:I l2 l2 I28 366:8 3 I 7:8 20 20 I 35 373:8 330: I I2 I 2 I 36 385:4 323:6 8 6 I42 390:7 330:3 I 2 I 2 I43 394:5 3 I 8:5 I 2 7 l 49 4I 0:5 339:2 l2 I 2 ISI 400:8 325:4 8 8 I 56 403:7 330:2 l 2 l 2 I57 4I4:7 330:3 8 8 I 63 425:8 336:2 I 2 I 2 I65 436:7 342:4 6 6 l 70 44l :5 350:3 I 8 I 8 I 77 444: I I 348:2 9 7 l 78 449:8 350:7 4 6 l85 457:5 352:4 9 8 I93 475:5 358:2 9 6 20 I 493: l 2 362:3 7 6 209 479: I 6 358:3 7 7 2I6 492:25 360:6 4 3 46 .30.. am new 5.3 Min 05 .0 9593.3 323.3 :88 9:30.. Es 8”. c 8.55 as .5 8; c 8.52.. a 8.5: BLOOD PRESSURE I60 ‘- l60 .. to z 2 tr 2 8 S fl EVENING reassures \\ \ mo - I20 - \\\\\ 47 V N\\ E \\\ .\\ \\ . fig \\ \ §‘\ \ . \\\\ \\\\\\‘ a 1m .- m 4r 60 ‘I- FIOIIIE 2 48 no.9.- ID meeeé X— as n a. m a a a. .. a. as s. .N a R .33 set *8. 0.4.. am... so. 8. 23: 2.. r... y... . r... 2.. 1.. lual rial. Idal Sign“! 05-0-0 h.- 8. Minnow-b .00.: dl-IEI ICU! N méh 49 variance and t-test of the results showed a significant difference between the rat g‘oups. The SI-IRs had a significantly higher blood pressure compared to both 50 and WKY g‘oups ( p<0.0005). The 50's blood pressure was also found to be sigiIfIcantly higher than the WKY‘s (p<0.0005). The evening blood pressure of all the groups showed a moderate decline compared to morning observation. Being l04:6, I2I: I9 and I74: I 9 mmHg for WKY, SD and SHR rats. Mixed analysis of variance and t-test of the observation Indicated a sigiIfIcant difference between the findings. The SI-R g‘om possessed a significantly higher evening blood pressure than the other two g‘oups (p<0.0005). The 50 rats, on the other hand, had a sigiiflca'itly higher pressure compared to WKY rats (p<0.05). 3- ETIIER STRESS R- RC'I'II RNII CIIITICISTEIME Figure 3 shows the ACTH response of WKY and SHR rats to 30 minutes of ether stress . The resting level of ACTH was 66 :I9 pg/ml In WKY and 73:9 pg/ml in SHR rats. Exposue to ether raised the level of ACTH sigilficantly In both rat ms. The plasma level reached 522:I66 pg/ml In WKY and 5l0:l44 pg/ml in the SHR rats. Paired, t-test analysis of the results showed no significant difference between the two rat groups before and after the ether stress (table 3). When the results are expressed in terms of percent change , the WKY rats showed a 7l8 8 Increase and SHR rats showed a lower level of response, 6I68 However the difference was not statistically sigilficant Figure and Table 3 also show the plasma level of corticosterone before and after ether stress. As indicated , both rat groups 50 .32 arm 0cm 2 5 mmogum Locuo mouse—E on go»; Dew 0.3509 88882.30 can zB< .o .96. 2.8:. .m 8.6.“. 51 n ”50...- _._E.. size-.5858 :8. ===== =22 22Wflhzuuzeu THE. E— 52. 3; Has 9... unless.— z. 33.592599 52¢ Eu 52 .s .5 an .8. .is 5.3 8mm 3. 2. we saw om. as. a 52.6 mm: 2». can 3 a... F... o.» 2. 25.... 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After 30 minutes of ether stress the SHR level rose to 7433:6757 pg/ml, an Increase of 22908l8, aid the WKY rose to 362: 273 pg/ml, al Increase of 94858 The difference was statistically sigiificant (p<0.0l ). The basal levels of norepinephrine In the WKY (l70:72 pg/ml) and the SI-R rats (I76:40 pg/ml) were not sigiificantly different. Stress caused 3l38 and l9388 increase in the basal level of the hormone in the SHR and WKY rats (not statistically significant). The final (30-minute) plasma level of this hormone, siglificaltly different In both rat grows-was 590:245 pg/ml In the WKY and 2298:2375 pg/ml in the SHR rats (p<0.05). 4- IIIBIIIITIIIN STRESS 8- limit llhlll communion: The ACTH response of the rat groups to vibration stress is shown in Table and Figure 5. There was no siglificant difference in the basal ACTH 54 .38 arm new 6.3 c. 32... 35¢ «32...... on dots can 882. 2.52.8382. new oct..ao...8 .o .26. «88:. .v 2.6.“. 55 fu¢=Oru ‘3 2 3' >8) .0. \ \ mvumJ. .« x \ \ m.m.. \ \ \\\ ........... ”3mnwvum \ . \xb .2 M \ uz.§..uz_..u..mm. m w \\m fifiMV \\\ . ; \\\\ H mmm «wrx , \ .o. geamww \ \\ ... 3 8— \\§§\‘\ \\;\:\\\. =52. I s u a E S \ \\\‘ §§V\§\ \ 55$ L.88. nous—.0 Bus 9... mule-flu.— uZ-G—r—uz:ucez az- uzaasuzzu 56 Guns arm x5 :5 5.3 mo. 9 S. 9 on... .. «on. «.3 £8 8 . 32.8 8% $5. mm 52..“ mm: mm.“ 88 a: 888 8? SK 3 :3... a: 9% 8m 3 E 29.. Km n8 8 .8...“ 2m 8.. 8m 0: 83 En Sn am :8! 3.! wozczo mozcxo mozczo mazczo a u.=.omo¢ ¢u=.u hum. u u.=.omnc aux.” ham: Janna—3% Ill—253.35% anus: Bus: 2. maze-.3.— uziasuzzucez .28 3.83.3223 9 39¢.— 57 m £59...— Em . o , .. 8. H E an .E\ .5 Hrs-£599.23“- : x a E: A. 8“ Zea-pluuzau . 8» L. 8.. 22.2.32: how.— _ a. E .7 s... anal—u zap—8.3.3 2. unless.— z— 293500.599 :2. 35¢ 59 3.15 mzm "sac. 5.3 noo— om. 0N. om . mum om. om. K .3096 .00 men 09» o _ _ Ohm m. n oov mm zen! Ila mom 3. mo. an NVN NW. N. . N0 509.9 .mo. NVM . .v Go we... own omv .o. Zen! ’8. 325.0 3255 a mb340mm< zo.h hmmm maul-h 22:...- l._. Hal-haul 8- HID-5.93.00 .2. El .553 moz hmmm n 3.2 3.3% 60 level between 5m ( 101282 pg/ml) and WKY ( 85:71 pg/ml). Both rat groups reacted similarly to vibration stress in terms of percent change and the post-stress plasma value of the hormone. The post-stress level was 4501112 WM (4628 increase) in WKY, and 4002120 pg/ml (570% increase) in the SHR Figure and Table 5 also show the corticosterone response of the rats. As it is seen them was no significant difference in the resting level between the SHR (116196 ng/ml) and WKY (69:78 ng/ml). Vibration stress also produced a simlla change in the corticosterone level. In both groups the level rose between 900-10503. The post-stress hormone level in thellln8... 325.0 325.0 825...”. 3235 R mSAOmm< zo...<¢m.> hmua x 3.3.89 20.... kmma gamuuguzfluazl .ggaaulll 64 level--which brought the final concentration of the hormone to 4131135 pg/ml in the W3 and 6801156 in the suns (p<0.025). S-MONOOI. OESTOOINT SWISS Il- IIC'III IINO COOIICOSTEIONE Table and Figure 7 present the findings on ACTH and corticosterone before and 15 minutes after a 5-minute manual-restraint stress. The SHR rats had a siglificantly higher basal level of ACTH (1821115 pg/ml) compared to 80 rats (85127 pg/ml) [p<0.05]. Stress caused the highest increase in the WKY (2661 103 pg/ml), and the least in 511123 (1691155 pg/ml). On a percentage basis the WKY and 50 rats responded almost identically (28812398 vs. 29911658 ) to the stress The SHR rats, however, responded less strongly (17912308). The final, post-stress level of ACTH was himest in the 111110! m (400190 pg/ml), lowest in the 50s (310179 pg/ml) and moderate (351179 pg/ml) in the SHRs. Statistical malysis of the post-stress observation showed that only the difference between the WKY 31d 50 rats was significant (p<0.05). The corticosterone results show that the pre-stress level of the hormone in the WKY rats (146172 ng/ml) was significantly higher than the 50 rats (80137 ng/ml) [p<0.05]. However the SHR response to stress was much stronger than the other two groups(1202113548 increase). 50 and WKY rats responded by 48614188 and 18511468 change, respectively. Mixed analysis of wine and t-test of the results of percent-change showed that both the difference between WKY and SD rats, and SHR and WKY rats were siglificant (p<0.05). 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The SHR rats had a simificantly higier resting level of epinephrine (65238 pg/ml) compared to WKY rats (241: l 2 pg/ml), and not only reached to the highest post-stress level (524:453 pg/ml) but also their reaction in terms of absolute change (4551455 pg/ml, p8:— moz ¢aflfif 99 J995 9999 $§$§99999999 99999999999.9 A 9.9 VVVVVVVVV'V'VVVVVVVVV‘VVVVVV 6%Vflflflfifivfififi§fifl?fifi?:fifigfib9§ 5". 9&5 ’ éflfififififififig 77 O 999 9 9 9 999999999999 fihfifl“§§g§§gfihfi édddddéflv % ncm (pg/mu E conncomnonmg/mn fl EPINEPIIIIINElpg/Inl) a nonsrmnunmflng/mu WY n- zoo- CONCENTIIIITIIN 150 - FIGURE IO 78 mood v 8.0 v 03.0: .— o. { t o. t .. R. .5 .o. 8 .33 SN 2 8 8. 25.2 s.“ R .m 8 mm .. mm o. R 8 5...... av. mm 8 8 2.32 3.... 2.5.... 2.52. 2.59.. . 2.52. uziasuziucez uzaasuzzu uzecflueehceu 3.5.. auto-‘80. maul—h a. dulud 02—hou- n!. 0.". 3 02.2.0! lad-.5 o. 39:. 79 7- Sill? PREFERENCE TESTS Table ii, 12 and 13 are in reference to NaCl preference tests performed on the Sl-R and WKY rats prior to the surgery and stress tests. The first table pertains to the group A rats ( used for ether and vibration stress). Twenty four ( i2 SHR and 12 WKY) rats, 24 weeks old, were used for this experiment The results show that this group of 5m rats had a higher preference and appetite for salt compared to WKY rats. This behavioral difference is statistically shown to be significant for 0.1 and 0.2 mola‘ NaCl solutions. Table .12, shows the results of the preference test on eighteen, 16-week old, g‘oup 8 rats As it is indicated, the SHR rats only showed a significmtly higier preference for and intake of salt solution at the higiest concentration, that is, 0.2 molar NaCl. However, two weeks later the results of the next preference test on the same rats showed that the Sl-Rs had a much higher salt appetite than the WKY rats for all the salt concentrations (Table 13). Furthermore, examination of the WKY preference for salt indicates that there was a decrease in their preference from i6 to l8 weeks. Thus the wpearance of a significant difference between the two you: was mainly due to a decrease in the WKY rather than an increase in the preference of SHR rats. 80 ANT-5 mzm uAN. a5 5.3 88... 88... mz m8... m8... mz 88... moood mz 2...... . m o v m w . n o. dull m n m. 8 an mm .o .o E 25.... a... N n a m o N v m N dull R 8 mm m. R mm on Q. mm 2...... 3.... 5:2... 3 33% magma...— Aanugaqzl Jud—flaufluulll ah.- . 5.0-0 .0... Man. ta 5. Hutu-Pals hula — _ man8:— ~E§ fang g «he: a .523 .39 8mm:— 0— .3 32233:.— :3 N. mam