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Prevention of renal hypertension and of the central pressor effect of angiotensin by ventromedial hypothalamic ablation
Brain Research, 205 (1981) 255-264 © Elsevier/North-Holland Biomedical Press
255
P R E V E N T I O N OF R E N A L H Y P E R T E N S I O N A N D OF T H E C E N T R A L P R E S S O R E F F E C T OF A N G I O T E N S I N BY V E N T R O M E D I A L H Y P O T H A L A MIC ABLATION
ALAN KIM JOHNSON, JAMES BUGGY*, GREGORY D. FINK** and MICHAEL J. BRODY Departments of Psychology and Pharmacology and The Cardiovascular Center, University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Accepted July 10th, 1980) Key words: renal hypertension - - angiotensin - - hypothalamus - - cardiovascular control
SUMMARY Various lines of research have implicated the central nervous system in the development of renal hypertension. The ablation of a periventricular region surrounding the anteroventral third ventricle (AV3V) has been shown to block the development of renal hypertension. Because the hemodynamic effects produced by AV3V electrical stimulation can be abolished by a midline lesion of the ventromedial hypothalamic-median eminence region (VMH-ME), the effect of V M H - M E ablation on the development of renal hypertension was studied. Following recovery from surgery that destroyed the V M H - M E region the lesioned rats and controls were subjected to unilateral nephrectomy and figure-of-eight wrapping of the remaining kidney. Control animals developed renal hypertension but those with V M H - M E lesions did not. When the pressor response produced by intracerebroventricular injections of angiotensin II was studied, it was found that rats with V M H - M E lesions, as compared to neurologically intact animals, showed significantly attenuated responses. The data suggest that a neural system related to cardiovascular control descends through the V M H - M E region and that the integrity of this pathway is necessary for the development of renal hypertension.
* Present address: Department of Physiology, University of South Carolina, School of Medicine, Columbia, S.C. 29208, U.S.A. ** Present address: Department of Pharmacology, Michigan State University, East Lansing, Mich. 48824, U.S.A.
256 INTRODUCFION
Alteration of the intergrity of central nervous system function by ablation of a specific neuroanatomical locus, the preoptic-hypothalamic tissue surrounding the anteroventral third ventricle (AV3V), has been shown to prevent the development and interrupt the maintenance of 1- and 2-kidney models of renal hypertension 4. The lesion also attenuates pressor and thirst responses to angiotensin and osmotic stimuli and interferes with the ability of these interventions to release antidiuretic hormone2,S, 14. The centrally mediated pressor activity of angiotensin results from a combination of increased sympathetic activity and increased release of vasopressin by the hypotha!amo-neurohypophyseal system 12,26. Neurally mediated changes in blood flow to renal hindquarter and mesenteric vascular beds produced by electrical stimulation of the AV3V are abolished after lesion of more posterior sites in the ventromedial hypothalamic-median eminence region (VMH-ME) but not lateral hypothalamus H. These studies suggest that neurogenic and neuroendocrine influences on the cardiovascular system evoked by activation of the AV3V region depend on posterior hypothalamic connections which may be interrupted by a midline ablation encompassing VMH-M E. The purpose of the present study was to determine if a VMH-ME lesion alters the development of renal hypertension. MATERIALS A N D METHODS
Studies were conducted on adult, male Sprague-Dawley rats initially weighing 270-340 g. The animals had free access to food and water and were individually housed in a room with controlled temperature and lighting cycles. Brain lesions were produced in animals (50 mg/kg sodium pentobarbital plus 0.2 mg/kg atropine sulfate i.p.) mounted in a stereotaxic instrument with the skull leveled between bregma and lambda. The rats were anesthetized for this procedure and for all subsequent surgeries with an intraperitoneal injection of 50 mg/kg sodium pentobarbital and 0.2 mg/kg of atropine sulfate. A bone flap about 1.5 mm in diameter and centered around the midline was removed above the brain site to be lesioned. The lesioning electrode, a 24-gauge nichrome wire insulated except for an exposed, beveled tip of 0.5 mm, was lowered into the brain on the midline after retraction of the midsagittal sinus. Stereotaxic coordinates were: ventromedial hypothalamus-median eminence 3.0 mm posterior to bregma, 8.4 mm below dura. Lesions were produced by passing direct current (2-3 mA for 15-20 sec) between the brain electrode and a large uninsulated cathode placed in the rectum. Daily water and weekly blood pressure measures were taken for two weeks after the lesion. Rats were then anesthetized as above, one kidney removed, and a figure-of8 ligature was placed on the other kidney to produce renal hypertension by the Grollman method as previously described 6. A neurologically intact control group was subjected to the same nephrectomy and renal wrapping procedure.~Weekly measures of systolic blood pressure were the mean determinations made on two or more days by tail-cuff plesthysmography in conscious rats prewarmed for 5 min in a chamber at 35 °C4"
257 After completing the experiment on the development of renal hypertension several rats with VMH-ME lesions were randomly selected for stereotaxic guided cannulation (23-gauge stainless-steel guide cannulae) of a lateral cerebral ventricle. This chronic implantation allowed intraventricular injection of freely moving rats 14. Identical cannulations were made in a group of intact control normotensive rats. After allowing at least 5 days to elapse for recovery, heparin-filled polyethylene catheters (PE 50) were placed in the femoral artery and subcutaneously tunnelled to exit from the back of the neck. The day after catheterization, the arterial line was connected via polyethylene tubing to an Ailtech MS-10 pressure transducer through a fluid swivel (BRS/LVE) mounted on top of a testing cage which permitted measurement of blood pressure in conscious unrestrained rats. Catheter patency during measurement sessions was maintained by a continuous flush of the arterial line (Intraflow, Sorenson Research). A 30gauge injector cannula was placed inside the cerebroventricular guide cannula and was connected via polyethylene tubing to a microliter syringe containing angiotensin I1, (Hypertension, CIBA) in artificial cerebrospinal fluid. Blood pressure was continuously monitored during cerebroventricular injections of 50, 100 and 200 ng of angiotensin II (50 ng//~l concentration). Doses were administered in random order separated by at least 20 min or until baseline blood pressure was restored. At the completion of testing, lesioned animals were anesthetized and perfused through the heart with saline followed by buffered I0 ~ formalin. Brains were removed and stored in formalin, 40 #m frozen sections were later taken through the extent of the lesion and stained with cresyl violet-luxol fast blue. Lesion location was then verified using light microscopy. The development of renal hypertension was compared in median eminence-ventromedial hypothalamic ablated rats and intact rats by analysis of variance with appropriate multiple comparisons of significant differences. Other data were analyzed with Student's t-tests. Group differences with a probability of 0.05 or less were considered significant. RESULTS As shown in Fig. 1, rats with VMH-ME lesions exhibited markedly elevated water intakes (diabetes insipidus) before wrapping. Of the 13 animals with VMH-ME lesions 10 rats had daily water intakes that exceeded 3 standard deviations of the mean of the control group. Fig. 1 also illustrates the changes in blood pressure and water intake for the lesioned animals and control group from the week preceding until 3 weeks after the renal wrap procedure. Prior to wrapping, blood pressures did not differ between the groups, but by the third and fourth weeks of the study, blood pressure in the intact rats was significantly elevated. On the other hand, animals with a lesion of the VMH-ME region showed no change in their systolic blood pressure throughout the study. Water intake before wrapping was significantly greater in lesioned rats but in the first week after wrapping (week 2), did not differ between groups. For weeks 3 and 4 of the study, water intake was again significantly greater in lesioned rats but below prewrapping values.
258 190
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180
Systolic Blood Pressure
., " 170
(mmHg)
160
~
150
d"l
/
.'""
T
SD
o"
1
s
i
125 Mean Daily Water Intake (ml)
100
75 50
I
i
i
i _ _
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i~ i ~.,,./~ i ~ ;
~o--__
/
jn~-......~ _ T ~'m S.D .I. -o
0 i
1K W r a p
Time
(weeks)
Fig. I. Blood pressure and water intake measures for neurologically intact control rats (n ~ 7) or rats with median eminence-ventromedial hypothalamic ablation (n = 13) before and after renal wrapping. Overall standard deviation from the analysis of variance is indicated in the right hand margin of each graph.
The changes in mean arterial pressure following intraventricular injections of angiotensin for VMH-ME lesioned and intact rats (both groups normotensive) are illustrated in Fig. 2. Lesioned rats showed significantly reduced pressor responses at each dose of angiotensin tested. Histological examinations revealed damage to arcuate nucleus, ventral portions of the hypothalamic periventricular nucleus, median eminence, portions of pars centralis and medialis of ventromedial hypothalamus, and ventral portions of the dorsomedial nucleus of the hypothalamus. No animals were excluded from statistical analysis on the basis of histological criteria. Typically lesions were bilaterally symmetrical with respect to the midline as illustrated by the representative histological brain sections shown in Figs. 3 and 4.
30 2O &MAP (mrnHg)
. ~ 10
"~
"~k
5'0
100 ' 200 ' IVT Angiotensin II (ng)
Fig. 2. Changes in mean arterial pressure in neurologically intact control (n -- 6) and median eminence-ventromedial hypothalamic lesioned (n = 7) rats after lateral ventricular injection of 50, 100 or 200 ng angiotensin II. Asterisks indicate statistically significant group differences.
259
Fig. 3. Coronal sections taken from the middle of 3 representative brains with ventromedial hypothalamic-median eminence lesions. Abbreviations: 3V, third ventricle; F, fornix; L, lesion; MFB, medial forebrain bundle; ML, lateral mammillary nucleus; MT, mammillothalamic tract.
260
Fig. 4. Full coronal section (upper) from a representative ventromedial hypothalamic-median eminence lesion. Higher power enlargement (lower) of the lesion and surround. Abbreviations: F, fornix; L, lesion; LT, lesion electrode tract; MT, mammillothalamic tract. DISCUSSION In previous studies we have established that a midline ablation of the periventricular tissue of the AV3V region reversed or prevented the development of Grollman renal hypertensionS, 6 as well as several other forms of experimental hypertension 4. The findings reported here extend this earlier work on central mechanisms and renal hypertension (l-kidney wrap) by showing that a lesion placed caudal to the AV3V region will also prevent this form of elevated blood pressure. Both the AV3V lesion and the V M H - M E lesion are locus specific because in other experiments it has been demonstrated that asymmetrical AV3V lesions 6, area postrema ablation 7, and midthalamic
261 destruction (Buggy, Johnson, Fink and Brody, unpublished observations) do not disrupt the development of Grollman renal hypertension. The results of the present studies are in agreement with those characterizing the neuroanatomical and functional relationships of the AV3V with other brain regions. On the basis of tritiated amino acid track tracing techniques, two primary pathways from the AV3V to lower brain areas have been describedg, ~s. A medial periventricular pathway has been defined that passes through the region of the VMH-ME lesion and eventually reaches the periventricular central gray. A second descending pathway takes a more lateral course to pass through the lateral hypothalamus and terminates in the ventral lateral tegmental areag, 28. The medial pathway appears to be involved in cardiovascular regulation. Examination of the hemodynamic effects produced by electrical st!mulation of the AV3V region indicated that stimulation of the ventromedial hypothalamus in the region of the median eminence produced patterns of regional blood flow responses that closely resemble those seen during AV3V activation11. These changes in renal and hindlimb resistance produced by AV3V electrical stimulation were significantly attenuated by lesions in the region of the ventromedial hypothalamus-median eminence region 11. Lesions of the lateral hypothalamus (i.e. the lateral pathway) had no effect on vascular responses produced by AV3V ablation. The present study also demonstrated that like AV3V destruction 14, VMH-ME lesions also produced a significant reduction in the pressor response to intracranial injections of angiotensin II. Recently, the functional studies have been extended to examine the relationship of the periventricular central gray to the AV3V 15. Central gray stimulation in the terminal region of the descending medial pathway produced regional blood flow responses that are qualitatively similar to those produced by AV3V activationlL Also, central gray ablation attenuated the hemodynamic effects of AV3V stimulation15. The lateral descending pathway from the AV3V has been suggested by Mogenson and colleagues to mediate drinking16,17. AV3V ablation produces acute adipsia 13. In contrast, as indicated by the present study, VMH-ME lesions increased postsurgery water intake. Destruction of the midlateral and the far lateral hypothalamus abolishes, respectively, angiotensin II and hyperosmotic drinking induced by intracranial injections of these stimulP6, ~7. Drinking responses to intracranial injections of angiotensin II are not abolished by ventromedial hypothalamus lesions ~6. In preliminary work, we have studied water intake in rats with VMH-ME lesions. Unlike rats with AV3V destruction, animals with VMH-ME ablations, increase drinking to subcutaneous angiotensin II and hyperosmotic thirst challenges. Thus, it appears that the medial pathway is not critical for the control of water intake whereas the lateral projection is essential. Taken together the results of lesion, stimulation, and anatomical studies suggest that cardiovascular and drinking systems which are coextensive within the AV3V are anatomically distinct at lower levels of the neuroaxis. Neither the present experiments nor those employing AV3V lesions indicate whether the lesion-related prevention of renal hypertension is due primarily to an alteration of a neurogenic or of a neuroendocrine influence on the circulation. Both AV3V lesions and VMH-ME lesions involve the hypophysiotropic zone and are therefore likely to influence anterior and posterior pituitary function.
262 Vasopressin, the antidiuretic hormone which is synthesized in the hypothalamus and released from the posterior lobe of the pituitary, has been postulated as a hypertensive factor in various forms of chronically elevated blood pressure10,1s,19, 27, including renal hypertension z0. Centrally applied angiotensin releases vasopressin and this release is attenuated by AV3V 14 and median eminence lesions 2e. Thus, it would be reasonable to hypothesize that the antihypertensive effect of AV3V and VMH-ME lesions may be due to altered vasopressin function. However, previous studies21, 24 suggest that vasopressin release from the posterior pituitary is not necessary for the development of renal hypertension. Ogden et al. 2~ reported that there was no consistent effect from removal of the posterior lobe in reducing renal hypertension and that prior posterior hypophysectomy did not prevent the development of hypertension. Sattler and Ingram 24 attempted interruption of the supraoptic hypophyseal tract in the median eminence of renal hypertensive dogs. The elevated blood pressures of 5 of 8 dogs significantly decreased following the ablation, but not to normal levels; the 3 remaining animals evidenced no significant decrease. Other experiments which assessed not only the contribution of posterior pituitary factors but also adenohypophyseal hormones have employed either total hypophysectomy or stalk section1,3,22, 2,~. Such studies show that removal of the pituitary will not prevent a rise in blood pressure with hypertension-inducing renal manipulations. In established renal hypertension, hypophysectomy will reduce but not normalize blood pressure. Taken together the data suggest that a hypothesis of altered humoral mechanisms will not, in and of itself, account for the prevention of renal hypertension following either AV3V or VMH-ME lesions. It appears that disruption of a neurogenic component is a necessary aspect of AV3V and VMH-ME ablations. In conclusion, the results presented here lend further support to the role for the central nervous system in the mediation of renal hypertension. Furthermore, the ventromedial hypothalamic-median eminence ablation studies define a descending pathway from the AV3V forebrain region through the basal hypothalamus which may exert both neuroendocrine and neurogenic influences on the cardiovascular system. The integrity of this hypothalamic pathway is necessary for centrally mediated pressor responses to angiotensin and for the expression of renal hypertension. ACKNOWLEDGEMENTS The authors thank Sandra Boutelle, Regina Knake and Bill Packwood for excellent technical assistance in various aspects of these studies. We are grateful to Gertrude Nath for her aid in preparation of this manuscript. These studies were supported in part by USPHS Grants HLP-14388 and GM00141, a Research Scientist Development Award 1 KO2 MH00064, and the Iowa Medical Research Council.
263 REFERENCES 1 Anderson, E., Page, E. W., Li, C. H. and Ogden, E., Restoration of renal hypertension in hypophysectomized rats by the administration of adrenocorticotropic hormone, Amer. J. Physiol., 141 (1944) 393-396. 2 Bealer, S. L., Phillips, M. I., Johnson, A. K. and Schmid, P. G., Effect of anteroventral third ventricular lesions on antidiuretic responses to central angiotensin II, Amer. J. Physiol., (1979) in press. 3 Braun-Menendez, E., Hypophysis and blood pressure, Cardiologia, 21 (1952) 272-283. 4 Brody, M. J., Fink, G. D., Buggy, J., Haywood, J. R., Gordon, F. J. and Johnson, A. K., The role of the anteroventral third ventricle (AV3V) region in experimental hypertension, Circulat. Res., 43 (1978) I2-I13. 5 Buggy, J., Fink, G. D., Haywood, J. R., Johnson, A. K. and Brody, M.J., Interruption of the maintenance phase of established hypertension by ablation of the anteroventral third ventricle (AV3V) in rats, Clin. exp. Hypertension, 1 (1978) 337-353. 6 Buggy, J., Fink, G. D., Johnson, A. K. and Brody, M. J., Prevention of the development of renal hypertension by anteroventral third ventricular tissue lesions, Circulat. Res., 40 (1977) I110-I117. 7 Buggy, J., Haywood, J. R., Fink, G. D., Phillips, M. I. and Brody, M. J., Central responses to angiotensin: no role for area postrema in rat, Fed. Proc., 37 (1978) 3085. 8 Buggy, J. and Johnson, A. K., Preoptic-hypothalamic periventricular lesions: thirst,deficits and hypernatremia, Amer. J. Physiol., 233 (1977) R44-R52. 9 Conrad, L. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat, J. comp. Neurol., 169 (1976) 185-220. 10 Crofton, J. T., Share, L., Shade, R. E., Lee-Kwon, W. J., Manning, M. and Sawyer, W. H., The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the rat, Hypertension, 1 (1979) 31-38. 11 Fink, G. D., Buggy, J., Haywood, J. R., Johnson, A. K. and Brody, M. J., Hemodynamic responses to electrical stimulation of areas of rat forebrain containing angiotensin on osmosensitive sites, Amer. J. Physiol., 235 (1978) H445-H451. 12 Hoffman, W. E., Phillips, M. I., Schmid, P. G., Falcon, J. and Weet, J. F., Antidiuretic hormone release and the pressor response to central angiotensin II and cholinergic stimulation, Neuropharmacology, 16 (1977) 453-471. 13 Johnson, A. K. and Buggy, J., Periventricular preoptic-hypothalamus is vital for thirst and normal water economy, Amer. J. Physiol., 234 (1978) R122-R129. 14 Johnson, A. K., Hoffman, W. E. and Buggy, J., Attenuated pressor responses to intracranially injected stimuli and altered antidiuretic activity following preoptic-hypothalamic periventricular ablation, Brain Research, 157 (1978) 161-166. 15 Kneupfer, M. M., Gordon, F. J., Johnson, A. K. and Brody, M. J., Identification of descending cardiovascular pathways from the anteroventral third ventricle (AV3V) region, Fed. Proc., 38 (1979) 1446. 16 Kucharczyk, J. and Mogenson, G. J., Separate lateral hypothalamic pathways for extracellular and intracellular thirst, Amer. J. Physiol., 228 (1975) 295-301. 17 Mogenson, G. J. and Kucharczyk, J., Central neural pathways for angiotensin-induced thirst, Fed. Proc., 37 (1978) 2683-2688. 18 M6hring, J., Kintz, J. and Schoun, J., Role of vasopressin in blood pressure control of spontaneously hypertensive rats, Clin. Sci. Molec. Med., 55 (1978) 2475-2505. 19 M6hring, J., M6hring, B., Petri, M. and Haack, D., Vasopressor role of A D H in the pathogenesis of malignant DOC hypertension, Amer. J. Physiol., 232 (1977) F260-F269. 20 M6hring, J., M6hring, B., Petri, M. and Haack, D., Plasma vasopressin concentrations and effects of vasopressin antiserum on blood pressure in rats with malignant hypertension (twokidney Goldblatt hypertension), Circulat. Res., 42 (1978) 17-22. 21 Ogden, E., Page, E. W. and Anderson, E., The effect of posterior hypophysectomy on renal hypertension, Amer. J. Physiol., 141 (1944) 389-392. 22 Page, I. H. and Sweet, J. E., The effect of hypophysectomy on arterial blood pressure o( dogs with experimental hypertension, Amer. J. Physiol., 120 (1937) 238. 23 Peck, J. W., Discussion: thirst(s) resulting from bodily water imbalances. In A. N. Epstein, H. R. Kissileff and E. Stellar (Eds.), The Neuropsychology of Thirst: New Findings and Advances in Concepts, V. H. Winston, 1973.
264 24 Sattler, D. G. and Ingrain, W. R., Experimental hypertension and the neurohypophysis, Endocrinology, 29 (1941) 952-957. 25 Schimert, P., Kezdi, P. and Nishimura, T., The effect of pituitary stalk-section on neurogenic and renal hypertension in the dog, Arch. int. Pharmacodyn., 147 (1964) 236-254. 26 Severs, W. L., Surnmy-Long, J., Taylor, J. S. and Connor, J. D., A central effect of angiotensin : release of pituitary pressor material, J. Pharmacol. exp. Ther., 174 (1970) 27-34. 27 Share, L., Crofton, J. T. and Shade, R. E., Vasopressin in the spontaneously hypertensive rat, Physiologist, 20 (1977) 86. 28 Swanson, L. W., Kucharczyk, J. and Mogenson, G. J., Autoradiographic evidence for pathways from the medial preoptic area to the midbrain involved in the drinking response to angiotensin II, J. comp. NeuroL, 178 (1978) 645-660.
255
P R E V E N T I O N OF R E N A L H Y P E R T E N S I O N A N D OF T H E C E N T R A L P R E S S O R E F F E C T OF A N G I O T E N S I N BY V E N T R O M E D I A L H Y P O T H A L A MIC ABLATION
ALAN KIM JOHNSON, JAMES BUGGY*, GREGORY D. FINK** and MICHAEL J. BRODY Departments of Psychology and Pharmacology and The Cardiovascular Center, University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Accepted July 10th, 1980) Key words: renal hypertension - - angiotensin - - hypothalamus - - cardiovascular control
SUMMARY Various lines of research have implicated the central nervous system in the development of renal hypertension. The ablation of a periventricular region surrounding the anteroventral third ventricle (AV3V) has been shown to block the development of renal hypertension. Because the hemodynamic effects produced by AV3V electrical stimulation can be abolished by a midline lesion of the ventromedial hypothalamic-median eminence region (VMH-ME), the effect of V M H - M E ablation on the development of renal hypertension was studied. Following recovery from surgery that destroyed the V M H - M E region the lesioned rats and controls were subjected to unilateral nephrectomy and figure-of-eight wrapping of the remaining kidney. Control animals developed renal hypertension but those with V M H - M E lesions did not. When the pressor response produced by intracerebroventricular injections of angiotensin II was studied, it was found that rats with V M H - M E lesions, as compared to neurologically intact animals, showed significantly attenuated responses. The data suggest that a neural system related to cardiovascular control descends through the V M H - M E region and that the integrity of this pathway is necessary for the development of renal hypertension.
* Present address: Department of Physiology, University of South Carolina, School of Medicine, Columbia, S.C. 29208, U.S.A. ** Present address: Department of Pharmacology, Michigan State University, East Lansing, Mich. 48824, U.S.A.
256 INTRODUCFION
Alteration of the intergrity of central nervous system function by ablation of a specific neuroanatomical locus, the preoptic-hypothalamic tissue surrounding the anteroventral third ventricle (AV3V), has been shown to prevent the development and interrupt the maintenance of 1- and 2-kidney models of renal hypertension 4. The lesion also attenuates pressor and thirst responses to angiotensin and osmotic stimuli and interferes with the ability of these interventions to release antidiuretic hormone2,S, 14. The centrally mediated pressor activity of angiotensin results from a combination of increased sympathetic activity and increased release of vasopressin by the hypotha!amo-neurohypophyseal system 12,26. Neurally mediated changes in blood flow to renal hindquarter and mesenteric vascular beds produced by electrical stimulation of the AV3V are abolished after lesion of more posterior sites in the ventromedial hypothalamic-median eminence region (VMH-ME) but not lateral hypothalamus H. These studies suggest that neurogenic and neuroendocrine influences on the cardiovascular system evoked by activation of the AV3V region depend on posterior hypothalamic connections which may be interrupted by a midline ablation encompassing VMH-M E. The purpose of the present study was to determine if a VMH-ME lesion alters the development of renal hypertension. MATERIALS A N D METHODS
Studies were conducted on adult, male Sprague-Dawley rats initially weighing 270-340 g. The animals had free access to food and water and were individually housed in a room with controlled temperature and lighting cycles. Brain lesions were produced in animals (50 mg/kg sodium pentobarbital plus 0.2 mg/kg atropine sulfate i.p.) mounted in a stereotaxic instrument with the skull leveled between bregma and lambda. The rats were anesthetized for this procedure and for all subsequent surgeries with an intraperitoneal injection of 50 mg/kg sodium pentobarbital and 0.2 mg/kg of atropine sulfate. A bone flap about 1.5 mm in diameter and centered around the midline was removed above the brain site to be lesioned. The lesioning electrode, a 24-gauge nichrome wire insulated except for an exposed, beveled tip of 0.5 mm, was lowered into the brain on the midline after retraction of the midsagittal sinus. Stereotaxic coordinates were: ventromedial hypothalamus-median eminence 3.0 mm posterior to bregma, 8.4 mm below dura. Lesions were produced by passing direct current (2-3 mA for 15-20 sec) between the brain electrode and a large uninsulated cathode placed in the rectum. Daily water and weekly blood pressure measures were taken for two weeks after the lesion. Rats were then anesthetized as above, one kidney removed, and a figure-of8 ligature was placed on the other kidney to produce renal hypertension by the Grollman method as previously described 6. A neurologically intact control group was subjected to the same nephrectomy and renal wrapping procedure.~Weekly measures of systolic blood pressure were the mean determinations made on two or more days by tail-cuff plesthysmography in conscious rats prewarmed for 5 min in a chamber at 35 °C4"
257 After completing the experiment on the development of renal hypertension several rats with VMH-ME lesions were randomly selected for stereotaxic guided cannulation (23-gauge stainless-steel guide cannulae) of a lateral cerebral ventricle. This chronic implantation allowed intraventricular injection of freely moving rats 14. Identical cannulations were made in a group of intact control normotensive rats. After allowing at least 5 days to elapse for recovery, heparin-filled polyethylene catheters (PE 50) were placed in the femoral artery and subcutaneously tunnelled to exit from the back of the neck. The day after catheterization, the arterial line was connected via polyethylene tubing to an Ailtech MS-10 pressure transducer through a fluid swivel (BRS/LVE) mounted on top of a testing cage which permitted measurement of blood pressure in conscious unrestrained rats. Catheter patency during measurement sessions was maintained by a continuous flush of the arterial line (Intraflow, Sorenson Research). A 30gauge injector cannula was placed inside the cerebroventricular guide cannula and was connected via polyethylene tubing to a microliter syringe containing angiotensin I1, (Hypertension, CIBA) in artificial cerebrospinal fluid. Blood pressure was continuously monitored during cerebroventricular injections of 50, 100 and 200 ng of angiotensin II (50 ng//~l concentration). Doses were administered in random order separated by at least 20 min or until baseline blood pressure was restored. At the completion of testing, lesioned animals were anesthetized and perfused through the heart with saline followed by buffered I0 ~ formalin. Brains were removed and stored in formalin, 40 #m frozen sections were later taken through the extent of the lesion and stained with cresyl violet-luxol fast blue. Lesion location was then verified using light microscopy. The development of renal hypertension was compared in median eminence-ventromedial hypothalamic ablated rats and intact rats by analysis of variance with appropriate multiple comparisons of significant differences. Other data were analyzed with Student's t-tests. Group differences with a probability of 0.05 or less were considered significant. RESULTS As shown in Fig. 1, rats with VMH-ME lesions exhibited markedly elevated water intakes (diabetes insipidus) before wrapping. Of the 13 animals with VMH-ME lesions 10 rats had daily water intakes that exceeded 3 standard deviations of the mean of the control group. Fig. 1 also illustrates the changes in blood pressure and water intake for the lesioned animals and control group from the week preceding until 3 weeks after the renal wrap procedure. Prior to wrapping, blood pressures did not differ between the groups, but by the third and fourth weeks of the study, blood pressure in the intact rats was significantly elevated. On the other hand, animals with a lesion of the VMH-ME region showed no change in their systolic blood pressure throughout the study. Water intake before wrapping was significantly greater in lesioned rats but in the first week after wrapping (week 2), did not differ between groups. For weeks 3 and 4 of the study, water intake was again significantly greater in lesioned rats but below prewrapping values.
258 190
~j > a D~
180
Systolic Blood Pressure
., " 170
(mmHg)
160
~
150
d"l
/
.'""
T
SD
o"
1
s
i
125 Mean Daily Water Intake (ml)
100
75 50
I
i
i
i _ _
% ',
i~ i ~.,,./~ i ~ ;
~o--__
/
jn~-......~ _ T ~'m S.D .I. -o
0 i
1K W r a p
Time
(weeks)
Fig. I. Blood pressure and water intake measures for neurologically intact control rats (n ~ 7) or rats with median eminence-ventromedial hypothalamic ablation (n = 13) before and after renal wrapping. Overall standard deviation from the analysis of variance is indicated in the right hand margin of each graph.
The changes in mean arterial pressure following intraventricular injections of angiotensin for VMH-ME lesioned and intact rats (both groups normotensive) are illustrated in Fig. 2. Lesioned rats showed significantly reduced pressor responses at each dose of angiotensin tested. Histological examinations revealed damage to arcuate nucleus, ventral portions of the hypothalamic periventricular nucleus, median eminence, portions of pars centralis and medialis of ventromedial hypothalamus, and ventral portions of the dorsomedial nucleus of the hypothalamus. No animals were excluded from statistical analysis on the basis of histological criteria. Typically lesions were bilaterally symmetrical with respect to the midline as illustrated by the representative histological brain sections shown in Figs. 3 and 4.
30 2O &MAP (mrnHg)
. ~ 10
"~
"~k
5'0
100 ' 200 ' IVT Angiotensin II (ng)
Fig. 2. Changes in mean arterial pressure in neurologically intact control (n -- 6) and median eminence-ventromedial hypothalamic lesioned (n = 7) rats after lateral ventricular injection of 50, 100 or 200 ng angiotensin II. Asterisks indicate statistically significant group differences.
259
Fig. 3. Coronal sections taken from the middle of 3 representative brains with ventromedial hypothalamic-median eminence lesions. Abbreviations: 3V, third ventricle; F, fornix; L, lesion; MFB, medial forebrain bundle; ML, lateral mammillary nucleus; MT, mammillothalamic tract.
260
Fig. 4. Full coronal section (upper) from a representative ventromedial hypothalamic-median eminence lesion. Higher power enlargement (lower) of the lesion and surround. Abbreviations: F, fornix; L, lesion; LT, lesion electrode tract; MT, mammillothalamic tract. DISCUSSION In previous studies we have established that a midline ablation of the periventricular tissue of the AV3V region reversed or prevented the development of Grollman renal hypertensionS, 6 as well as several other forms of experimental hypertension 4. The findings reported here extend this earlier work on central mechanisms and renal hypertension (l-kidney wrap) by showing that a lesion placed caudal to the AV3V region will also prevent this form of elevated blood pressure. Both the AV3V lesion and the V M H - M E lesion are locus specific because in other experiments it has been demonstrated that asymmetrical AV3V lesions 6, area postrema ablation 7, and midthalamic
261 destruction (Buggy, Johnson, Fink and Brody, unpublished observations) do not disrupt the development of Grollman renal hypertension. The results of the present studies are in agreement with those characterizing the neuroanatomical and functional relationships of the AV3V with other brain regions. On the basis of tritiated amino acid track tracing techniques, two primary pathways from the AV3V to lower brain areas have been describedg, ~s. A medial periventricular pathway has been defined that passes through the region of the VMH-ME lesion and eventually reaches the periventricular central gray. A second descending pathway takes a more lateral course to pass through the lateral hypothalamus and terminates in the ventral lateral tegmental areag, 28. The medial pathway appears to be involved in cardiovascular regulation. Examination of the hemodynamic effects produced by electrical st!mulation of the AV3V region indicated that stimulation of the ventromedial hypothalamus in the region of the median eminence produced patterns of regional blood flow responses that closely resemble those seen during AV3V activation11. These changes in renal and hindlimb resistance produced by AV3V electrical stimulation were significantly attenuated by lesions in the region of the ventromedial hypothalamus-median eminence region 11. Lesions of the lateral hypothalamus (i.e. the lateral pathway) had no effect on vascular responses produced by AV3V ablation. The present study also demonstrated that like AV3V destruction 14, VMH-ME lesions also produced a significant reduction in the pressor response to intracranial injections of angiotensin II. Recently, the functional studies have been extended to examine the relationship of the periventricular central gray to the AV3V 15. Central gray stimulation in the terminal region of the descending medial pathway produced regional blood flow responses that are qualitatively similar to those produced by AV3V activationlL Also, central gray ablation attenuated the hemodynamic effects of AV3V stimulation15. The lateral descending pathway from the AV3V has been suggested by Mogenson and colleagues to mediate drinking16,17. AV3V ablation produces acute adipsia 13. In contrast, as indicated by the present study, VMH-ME lesions increased postsurgery water intake. Destruction of the midlateral and the far lateral hypothalamus abolishes, respectively, angiotensin II and hyperosmotic drinking induced by intracranial injections of these stimulP6, ~7. Drinking responses to intracranial injections of angiotensin II are not abolished by ventromedial hypothalamus lesions ~6. In preliminary work, we have studied water intake in rats with VMH-ME lesions. Unlike rats with AV3V destruction, animals with VMH-ME ablations, increase drinking to subcutaneous angiotensin II and hyperosmotic thirst challenges. Thus, it appears that the medial pathway is not critical for the control of water intake whereas the lateral projection is essential. Taken together the results of lesion, stimulation, and anatomical studies suggest that cardiovascular and drinking systems which are coextensive within the AV3V are anatomically distinct at lower levels of the neuroaxis. Neither the present experiments nor those employing AV3V lesions indicate whether the lesion-related prevention of renal hypertension is due primarily to an alteration of a neurogenic or of a neuroendocrine influence on the circulation. Both AV3V lesions and VMH-ME lesions involve the hypophysiotropic zone and are therefore likely to influence anterior and posterior pituitary function.
262 Vasopressin, the antidiuretic hormone which is synthesized in the hypothalamus and released from the posterior lobe of the pituitary, has been postulated as a hypertensive factor in various forms of chronically elevated blood pressure10,1s,19, 27, including renal hypertension z0. Centrally applied angiotensin releases vasopressin and this release is attenuated by AV3V 14 and median eminence lesions 2e. Thus, it would be reasonable to hypothesize that the antihypertensive effect of AV3V and VMH-ME lesions may be due to altered vasopressin function. However, previous studies21, 24 suggest that vasopressin release from the posterior pituitary is not necessary for the development of renal hypertension. Ogden et al. 2~ reported that there was no consistent effect from removal of the posterior lobe in reducing renal hypertension and that prior posterior hypophysectomy did not prevent the development of hypertension. Sattler and Ingram 24 attempted interruption of the supraoptic hypophyseal tract in the median eminence of renal hypertensive dogs. The elevated blood pressures of 5 of 8 dogs significantly decreased following the ablation, but not to normal levels; the 3 remaining animals evidenced no significant decrease. Other experiments which assessed not only the contribution of posterior pituitary factors but also adenohypophyseal hormones have employed either total hypophysectomy or stalk section1,3,22, 2,~. Such studies show that removal of the pituitary will not prevent a rise in blood pressure with hypertension-inducing renal manipulations. In established renal hypertension, hypophysectomy will reduce but not normalize blood pressure. Taken together the data suggest that a hypothesis of altered humoral mechanisms will not, in and of itself, account for the prevention of renal hypertension following either AV3V or VMH-ME lesions. It appears that disruption of a neurogenic component is a necessary aspect of AV3V and VMH-ME ablations. In conclusion, the results presented here lend further support to the role for the central nervous system in the mediation of renal hypertension. Furthermore, the ventromedial hypothalamic-median eminence ablation studies define a descending pathway from the AV3V forebrain region through the basal hypothalamus which may exert both neuroendocrine and neurogenic influences on the cardiovascular system. The integrity of this hypothalamic pathway is necessary for centrally mediated pressor responses to angiotensin and for the expression of renal hypertension. ACKNOWLEDGEMENTS The authors thank Sandra Boutelle, Regina Knake and Bill Packwood for excellent technical assistance in various aspects of these studies. We are grateful to Gertrude Nath for her aid in preparation of this manuscript. These studies were supported in part by USPHS Grants HLP-14388 and GM00141, a Research Scientist Development Award 1 KO2 MH00064, and the Iowa Medical Research Council.
263 REFERENCES 1 Anderson, E., Page, E. W., Li, C. H. and Ogden, E., Restoration of renal hypertension in hypophysectomized rats by the administration of adrenocorticotropic hormone, Amer. J. Physiol., 141 (1944) 393-396. 2 Bealer, S. L., Phillips, M. I., Johnson, A. K. and Schmid, P. G., Effect of anteroventral third ventricular lesions on antidiuretic responses to central angiotensin II, Amer. J. Physiol., (1979) in press. 3 Braun-Menendez, E., Hypophysis and blood pressure, Cardiologia, 21 (1952) 272-283. 4 Brody, M. J., Fink, G. D., Buggy, J., Haywood, J. R., Gordon, F. J. and Johnson, A. K., The role of the anteroventral third ventricle (AV3V) region in experimental hypertension, Circulat. Res., 43 (1978) I2-I13. 5 Buggy, J., Fink, G. D., Haywood, J. R., Johnson, A. K. and Brody, M.J., Interruption of the maintenance phase of established hypertension by ablation of the anteroventral third ventricle (AV3V) in rats, Clin. exp. Hypertension, 1 (1978) 337-353. 6 Buggy, J., Fink, G. D., Johnson, A. K. and Brody, M. J., Prevention of the development of renal hypertension by anteroventral third ventricular tissue lesions, Circulat. Res., 40 (1977) I110-I117. 7 Buggy, J., Haywood, J. R., Fink, G. D., Phillips, M. I. and Brody, M. J., Central responses to angiotensin: no role for area postrema in rat, Fed. Proc., 37 (1978) 3085. 8 Buggy, J. and Johnson, A. K., Preoptic-hypothalamic periventricular lesions: thirst,deficits and hypernatremia, Amer. J. Physiol., 233 (1977) R44-R52. 9 Conrad, L. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat, J. comp. Neurol., 169 (1976) 185-220. 10 Crofton, J. T., Share, L., Shade, R. E., Lee-Kwon, W. J., Manning, M. and Sawyer, W. H., The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the rat, Hypertension, 1 (1979) 31-38. 11 Fink, G. D., Buggy, J., Haywood, J. R., Johnson, A. K. and Brody, M. J., Hemodynamic responses to electrical stimulation of areas of rat forebrain containing angiotensin on osmosensitive sites, Amer. J. Physiol., 235 (1978) H445-H451. 12 Hoffman, W. E., Phillips, M. I., Schmid, P. G., Falcon, J. and Weet, J. F., Antidiuretic hormone release and the pressor response to central angiotensin II and cholinergic stimulation, Neuropharmacology, 16 (1977) 453-471. 13 Johnson, A. K. and Buggy, J., Periventricular preoptic-hypothalamus is vital for thirst and normal water economy, Amer. J. Physiol., 234 (1978) R122-R129. 14 Johnson, A. K., Hoffman, W. E. and Buggy, J., Attenuated pressor responses to intracranially injected stimuli and altered antidiuretic activity following preoptic-hypothalamic periventricular ablation, Brain Research, 157 (1978) 161-166. 15 Kneupfer, M. M., Gordon, F. J., Johnson, A. K. and Brody, M. J., Identification of descending cardiovascular pathways from the anteroventral third ventricle (AV3V) region, Fed. Proc., 38 (1979) 1446. 16 Kucharczyk, J. and Mogenson, G. J., Separate lateral hypothalamic pathways for extracellular and intracellular thirst, Amer. J. Physiol., 228 (1975) 295-301. 17 Mogenson, G. J. and Kucharczyk, J., Central neural pathways for angiotensin-induced thirst, Fed. Proc., 37 (1978) 2683-2688. 18 M6hring, J., Kintz, J. and Schoun, J., Role of vasopressin in blood pressure control of spontaneously hypertensive rats, Clin. Sci. Molec. Med., 55 (1978) 2475-2505. 19 M6hring, J., M6hring, B., Petri, M. and Haack, D., Vasopressor role of A D H in the pathogenesis of malignant DOC hypertension, Amer. J. Physiol., 232 (1977) F260-F269. 20 M6hring, J., M6hring, B., Petri, M. and Haack, D., Plasma vasopressin concentrations and effects of vasopressin antiserum on blood pressure in rats with malignant hypertension (twokidney Goldblatt hypertension), Circulat. Res., 42 (1978) 17-22. 21 Ogden, E., Page, E. W. and Anderson, E., The effect of posterior hypophysectomy on renal hypertension, Amer. J. Physiol., 141 (1944) 389-392. 22 Page, I. H. and Sweet, J. E., The effect of hypophysectomy on arterial blood pressure o( dogs with experimental hypertension, Amer. J. Physiol., 120 (1937) 238. 23 Peck, J. W., Discussion: thirst(s) resulting from bodily water imbalances. In A. N. Epstein, H. R. Kissileff and E. Stellar (Eds.), The Neuropsychology of Thirst: New Findings and Advances in Concepts, V. H. Winston, 1973.
264 24 Sattler, D. G. and Ingrain, W. R., Experimental hypertension and the neurohypophysis, Endocrinology, 29 (1941) 952-957. 25 Schimert, P., Kezdi, P. and Nishimura, T., The effect of pituitary stalk-section on neurogenic and renal hypertension in the dog, Arch. int. Pharmacodyn., 147 (1964) 236-254. 26 Severs, W. L., Surnmy-Long, J., Taylor, J. S. and Connor, J. D., A central effect of angiotensin : release of pituitary pressor material, J. Pharmacol. exp. Ther., 174 (1970) 27-34. 27 Share, L., Crofton, J. T. and Shade, R. E., Vasopressin in the spontaneously hypertensive rat, Physiologist, 20 (1977) 86. 28 Swanson, L. W., Kucharczyk, J. and Mogenson, G. J., Autoradiographic evidence for pathways from the medial preoptic area to the midbrain involved in the drinking response to angiotensin II, J. comp. NeuroL, 178 (1978) 645-660.