Life Sciences Vol . 16, pp . 1731-1736 Printed in the U.S .A .

Pergamon Press

EVALUATION OF ANTIHYPERTENSIVE AGENTS IN THE STRESS-INDUCED HYPERTENSIVE RAT James L. Perhach, Jr ., Hugh C. Ferguson and Gordon R. McKinney Department of Pharmacology, mead Johnson Research Center Evansville, Indiana

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(Received in final form May 15, 1975) Summary Hypertension was induced in male, albino, Sprague-Dawley rats by exposure to environmental stimuli consisting of motion, noxious sounds and flashing lights at intervals over a period of ten weeks. Systolic blood pressure levels exceeded 150 mm Hg by the end of this period and.remained elevated for 20 weeks after cessation of eùposure to these noxious stimuli. The stress-induced hypertensive rat was found to be a useful model for the screening of antihypertensive drugs. Decreases in systolic pressure occurred following administration of guanethidine, catapres, chlorisondamine, hydralazine, hydrochlorothiazide, phentolamins, propranolol, or sotalol, s.c . to these hypertensive animals. This rat model of hypertension offers the advantages of longevity, stability and is acquired without drug administration or genetic management . The physiological effects of environmental stimuli on the living organism have been the subject of intensive study for many years. The development of hypertension as a consequence of exposure to environmental stimuli was first demonstrated by Farris et al . (1) in 1945 when hypertension was induced in emotional, gray, Norway rats after exposure to air blasts . Further evidence of the involvement of noxious stimuli in the development of hypertension in rats was provided by Buckley et al . (2) in 1953 when they showed that rats exposed to discontinuous decompression, equivalent to an altitude of 30,000 feet, for 2 hours per day for 19 weeks became progressively hypertensive. Although decompression was terminated, blood pressures remained elevated for S weeks. Hudak and Buckley (3) modified the method of Herrington and Nelbach (4) and subjected rats to a combination of audiogenic, visual, and motion stimuli. These animals became hypertensive, irritable and exhibited excessive salivation, Incrimation, urination and defecation . Further studies by Buckley et al. (5) showed that reserpine phosphate, 0.1 mg/kg i.p ., and chloroprcmazine hydrochloride, 4 mg/kg i.p ., administered 1 hr prior to subjecting rats to a 4 hr variable stress program (noise, motion, lights), not only failed to decrease the pressor effect induced by these stressors over a 27 week period, but also appeared to potentiate the lethal effect of the stressors. However, Buckley et al . (6) demonstrated, in a 20 week study, that reserpins phosphate, 0.1 mg/kg i.p ., when administered once daily starting with the seventh week of stress, not only prevented a further increase in the systolic blood pressure for the next 11 weeks, but also produced a marked decrease in blood pressure during the 19th and 20th weeks. Buckley et al . (7) also demonstrated that sodium pentobarbital, 20 mg/kg orally, daily, and acetylsalicylic acid, 100 mg/kg orally, daily, failed to prevent the development of hypertension in rats subjected to environmental stimuli . 1731

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Stress Induced Hypertensive Rat

Vol. 16, No . 11

Subsequent studies by Smookler and Buckley (8) showed that the administration of armethylp-tyrosine, 100 mg/kg p.o ., several hours prior to the start of each exposure to the stressful stimuli, prevented the development of hypertension in rats . Additional studies by Smookler et al . (9), demonstrated that treatment of rats with 6-hydroxydopamine, yielding an effective sympathectomy, was also capable of preventing the development of stress-induced hypertension . Although several pharmacologic agents have been examined for the possibility of preventing the development of hypertension in rats exposed to noxious stimuli, no comprehensive evaluation of therapeutic agents has been performed using stress-induced hypertensive rats . Since a representative animal model for essential hypertension is not yet available, it was felt that compounds known to exert an antihypertensive action in man should be evaluated to determine if they lower blood pressure in the stress-induced hypertensive rat. If such drugs are active, it might be indicative of the utility of the model for evaluating potential antihypertensive agents . Methods and Materials The stress chamber consisted of a 1.8 x 1.8 x 1 .8 m audicoetric examination roam (1200-A series, Industrial Acoustics Company, Bronx, New York) . Two Eberbach metabolic shakers were placed on a metal table (0 .9 m high) within the chamber and set to shake at 140 oscillations per min. A 150-watt indoor/outdoor Sylvania spot light was located on each wall at cage level. Each opposite pair of these lights was programmed to flash on at 1 sec intervals for 1/4 sec duration . Thus, light was present for 1/2 sec total at 1/4 sec intervals. This provided an illumination of approximately 140 footcandles at the center of the cage . Five speakers were installed, one on each wall and one on the ceiling directly over the cages. Audiogenic stimulation was produced by tape recordings of noxious sounds (compressed air blasts, bells, buzzers and tuning fork impulses) for 1/2 min periods at 5 min intervals. The intensity of this stimulation at the center of each cage was approximately 100 decibels . The three types of stimuli were automatically programmed into a control unit so that a variable sequence of stimulation could be applied for a 4 hr period . Stressed animals received 3 sessions of 4 hr duration per week during the light phase of their diurnal cycle, Monday through Saturday, on a random schedule . The animals employed for these studies were male, albino rate, Sprague Dawley descendants, weighing 60 to 80 g, and obtained from Charles River, Inc. They were housed 4 to a cage with food and water ad Zibiturt and acclimatized to the new environment for 1 week, then numbered with an oar code, and subjected to weekly blood pressure measurement for 3 weeks prior to the initiation of the stress procedure . Systolic blood pressure was determined indirectly via the tail cuff technique utilizing the Narco Biosystems apparatus . Animals were prewarmed at 33 °C for 20 min prior to recording their pressure . Evaluation of antihypertensive agents was performed by recording blood pressure initially (0 hr) and at 4 and 24 hr following each daily drug administration except on the 5th day when only the 4 hr measurement was made . Doses were calculated as the respective salts with the exception of hydrochlorothiazide which was administered as the free base . Drugs were dissolved in saline or suspended in 0 .25% methylcellulose in saline and all agents were ministered subcutaneously . Control animals received injections of saline . Statistical calculations were performed utilizing the Student's "t" test .

Vol . 16, No . 11

Stress Induced Hypertensive Rat

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Results The effect of the noxious stimuli an the biweekly mean of the indirectly measured systolic blood pressure is compared with non-treated control animas in Fig. 1. The systolic pressure of the rate exposed to the environmental

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FIG. 1 The Development of Hypertension in Rats Exposed to Noxious Stimulation and Continuation of the Hypertension after Cessation of the Stress stimuli gradually increased over the course of the 12 weeks' exposure to the stimuli. From the 6th week to the termination of the study systolic blood pressure of the stressed rats was significantly higher (p