Lack of fever suppression or central AVP release in 1K1C hypertensive rats

BRAIN RESEARCH ELSEVIER

Brain Research 658 (1994) 15- 20

Research report

Lack of fever suppression or central AVP release in lKlC hypertensive rats Mary L. Earle a

a,*,

Thomas Horn

b,

Norman Kasting

c,

Rainer Landgraf

d,

Quentin J. Pittman

a

Department of Neuroscience. Faculty of Medicine, The University of Calgary, Calgary, Alta., T2N 4Nl, Canada b Department of Biosciences, University of Leipzig, Talstrasse 33, Leipzig D-04103, Germany C Department of Physiology, The University of British Columbia, Vancouver, BC, V6T lW5, Canada d Max Planck Institute of Psychiatry, Clinical Institute. Kraepelinstr. 2, D-80804, Munich, Germany

Accepted 7 June 1994

Abstract Previous studies from our laboratory showed a transient suppression of the febrile response to intracerebroventricular (i.c.v.) PGE t in the one-kidney, one-clip (IKIC) model of hypertension. This may have been due to an enhanced vasopressinergic transmission since arginine vasopressin (AVP), acting within the central nervous system (CNS), is thought to mediate endogenous antipyresis. These initial experiments utilized a protocol for the induction of lKlC hypertension which produced an initial rapid rise in blood pressure, evident by day 4 following surgery. with a corresponding inhibition of the febrile response. The present experiments utilized a more slowly developing lKlC hypertension (evident by day 12 following surgery) to firstly attempt to determine if inhibition of the febrile response is due to the actual change in blood pressure or to neural signals arising from the clipped kidney, and secondly to determine if the concentration of AVP in push-pull perfusates of the ventral septal area (VSA) of pyrogen-treated sham-operated and lKlC rats were altered. In urethane-anaesthetized rats, i.c.v. PGE z evoked brisk monophasic fevers in both lKlC and sham-operated animals, with no significant difference between fever heights. Consistent with this, we found no increase in immunoreactive AVP from perfusates of the VSA of lKlC rats. These results suggest that there is no inhibition of the febrile response to PGE z when a slower developing hypertension is induced, nor is there an elevated release of AVP into the VSA under our conditions. We conclude that a rapid increase in blood pressure, and not high blood pressure per se, is required to produce an inhibition of the febrile response.

Keywords: Arginine vasopressin; Blood pressure; Antipyresis; PUSh-pull perfusion; Ventral septal area

1. Introduction

Several drugs, such as acetylsalicylic acid (aspirin), indomethacin, and acetaminophen, act to reduce the body temperature during a febrile episode, but do not alter the normal body temperature and, as such, are defined as antipyretics. In addition to these exogenous antipyretics, some neuropeptides are thought to act as naturally occurring (endogenous) antipyretics, returning the febrile body temperature to its normal level, and / or ensuring that the febrile response per se is not

* Corresponding author. Department of Pharmacology & Therapeutics, The University of Calgary, Health Science Centre, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada. Fax: (l ) (403) 270-2211. 0006-8993/94/$07.00 © 1994 Elsevier Science B.Y. All rights reserved SSDI 0006-8993(94)00730-Z

deleterious to the organism. Arginine vasopressin (AVP) is thought to be one of these endogenous antipyretics. There is now a large body of evidence to support the original suggestion of Kasting et a1. [7] that AVP acts as an antipyretic within the ventral septal area (VSA) of the brain and thereby contributes to the homeostatic control of body temperature [5,14]. The bed nucleus of the stria terminalis (BST) has been shown to provide the major vasopressinergic innervation to the VSA [2]. Electrical stimulation of the BST suppressed fever, an effect which was blocked by the application of a Vt antagonist into the VSA [13], indicating that the vasopressinergic axonal projections from the BST to the VSA may mediate antipyresis. A recent study from our laboratory has demonstrated that in the conscious one-kidney, one-clip OK1C) hypertensive rat, there is a transient inhibition

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M. L. Earle et al. / Brain Research 658 (1994) 15-20

of fever in response to the i.c.v. administration of prostaglandin E 1 (PGE 1) compared to the non-hypertensive controls [3]. In these studies, the removal of one kidney was done 4 days prior to the clipping of the remaining kidney, so as to produce a rapidly developing hypertension [3], with systolic blood pressures rising from approximately 115 mmHg (presurgery) to 170 mmHg 4 days following renal artery stenosis. Based on experiments utilizing AVP receptor blockade, the cause of this reduction in the febrile response was attributed to an enhanced activity of AVP neurotransmission within the VSA of hypertensive rats. The present study will address two points. Firstly, we asked if this rapid development of hypertension is required for the inhibition of fever observed in the previous studies, or whether it is simply the activation of neural signals from the compromised kidney. By clipping the renal artery and removing the remaining kidney at the same time, a slower developing 1K1C hypertension can be achieved, while presumably activating similar neural signals. Secondly, in this more slowly developing 1K1C hypertension, we asked if the release of AVP into the VSA is enhanced. We therefore carried out push-pull perfusion and measured the febrile response in urethane-anaesthetized 1K1C and control rats. Cardiovascular reflexes are well maintained under urethane, compared with other anaesthetics [10], and this preparation develops a well-characterized febrile response to PGE 2 [11].

2. Materials and methods Male Sprague-Dawley rats (Charles River Inc., Oue.) weighing 100-140 g (at the time of surgery) were housed 3-4 per cage, and maintained at 20°C with access to laboratory rat chow and water ad libitum. 2.1. Blood pressure measurements Systolic blood pressure was measured, using the tail-cuff plethysmography technique, prior to and at 4, 8, 12 and 18 or 21 days following surgery. A system was chosen for a non-invasive method of blood pressure measurement that did not require significant external preheating [I), and in which the temperature of the chamber was maintained at 27°C. The rats were restrained in plexiglass cylinders, to which they were adapted for two 30 min-I h periods over 2 consecutive days prior to experimentation. Following this training period, the baseline blood pressure of the rats was measured prior to any surgery. The rats were allowed to sit restrained for 15 min before collection of data, in order to facilitate slight vasodilation. The systolic pressures throughout all experiments were always taken at the same time of day (i.e. between 08.00 and 11.00 h) in order to reduce the possible effects of diurnal variation on blood pressure. Pulse signals from the photocell were fed into an amplifier (Model 29 Pulse Amplifier, IITC Inc.) for the regulation of the gain, offset and intensity of the signal. A Model 20-NW cuff pump (lITC Inc.) maintained inflation and deflation rates constant, releasing the cuff pressure over a 30 s period to allow for the return of blood flow, and registered pressure changes with a transducer. Signals from the

photocell amplifier and cuff pressure transducer were recorded on a Gould chart recorder (Gould recorder 2000) and used to determine the point at which a pulsatile pen deflection first reappeared, thereby defining systolic blood pressure. A mean of at least five clear recordings was calculated for each animal.

2.2. One kidney-one clip (IK1C) surgery Following initial baseline blood pressure recordings rats were anaesthetized using sodium pentobarbital (50 mg z'kg i.p.) and, under aseptic conditions, an incision was made and the left renal artery, vein and nerve were isolated and ligated. The left kidney was then removed, taking care not to damage the adrenal gland, and the incision closed. A second incision was then made, the right kidney was exteriorized and the renal artery isolated by removing the overlying fascia. A small aluminum clip (slit size 0.2 mm) was then placed over the renal artery such that blood flow was reduced but not completely occluded. The kidney was then replaced into the abdomen, and the wound closed. In controls for these experiments, termed sham-operated rats, the left kidney removed, under identical conditions as described above, however, the right kidney was left unclipped. After the IKIC or sham surgery was completed, all rats were placed in a stereotaxic frame and a stainless-steel guide cannula was implanted above a lateral cerebral ventricle (23 gauge thin wall, 12 mm length, coordinates: posterior 0.2 mm and lateral 1.2 mm (from bregma), ventral 2.0 mm from dura) and secured to the skull via screws and dental acrylic. A second guide cannula (20 gauge thin wall stainless-steel tubing, 15 mm length) was then implanted above the right VSA, (coordinates posterior 2 mm from bregma, lateral - 0.8 mrn, ventral 2.0 mm from dura) and again secured using dental acrylic. Topical bacitracin (Baciguent) was applied to the wounds and the animals were allowed to recover. Acetaminophen was added to the drinking water (final concentration approximately 0.2 mgyrnl) for 24 h following surgery, for pain relief. At either day 4 or day 18 after surgery (consistent with the previous experiments), systolic blood pressures were recorded. Rats were then anaesthetized with urethane (1.6 g/kg) and placed on a heating pad, with a rectal probe to monitor body temperature. A stable baseline temperature was obtained over a period of 30-60 min by use of a heating pad attached to a variable transformer. The heat supply was maintained constant at this level for the duration of the experiment, during which time push-pull perfusion fluid was collected from the VSA as described below. During the experiment the animals rectal temperatures were monitored at 10 min intervals. Push- pull perfusion. All perfusions were carried out unilaterally on the animal's right side. The push-pull perfusion cannula consisted of an inner 30 gauge stainless-steel 'push' cannula and an outer 23 gauge, thin walled 'pull' cannula [I8). The cannulae were attached by means of polyethylene tubing to a Harvard infusion/withdrawal pump (Harvard apparatus, MA, USA) calibrated at 20 JLl/min. The system was flushed with hydrochloric acid (0.1 M) prior to commencement of the experiment, in order to ensure there was no contamination of the tubing, and then washed out with sterile, pyrogen-free saline. The perfusion fluid was artificial cerebrospinal fluid (aCSF), which was freshly prepared every day from stock solutions and consisted of the following (in mM) (9): NaCI 139, KCl 3.30, NaHC0 3 21.0, Na zHP0 4·2H zO 0.49, Urea 2.16, CaCl z 1.26, MgCl z' 6H zO 1.15 in distilled water, and was adjusted to pH 7.28 (osmolarity: 295 mosmolykg), Bacitracin (0.025 g/mn, a peptidase inhibitor, was added and the solution was filtered (Millipore sterile Millex-GV 0.22 JLm filter unit). The push-pull perfusion cannula was lowered through the guide cannula so that the tip rested in the VSA. The perfusate collected in the initial 15 min was discarded. Following this initial equilibration period, the samples for analysis were collected over 30 min periods, resulting in a total volume of 600

M.L. Earle et al. / Brain Research 658 (1994) /5-20

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I.d in each sample. The perfusates were stored immediately at - 20°C for later analysis by radioimmunoassay (RIA) for AVP. All assays were done in duplicate in the same assay, using a previously validated RIA [6]. Three sequential 30 min samples were taken. At the onset of the second collection period POE z (5 fLI injection volume, containing 100 ng POE z), was injected by gravity flow into the lateral ventricle via a stainless-steel injector needle (27 gauge) attached to PE-20 tubing. At the conclusion of the study, the rats were perfused transcardially with 0.9% saline followed by 10% formalin in saline solution. Frozen coronal brain sections of 40 fLm thickness were mounted on gelatin-coated slides, stained with Neutral red and push-pull perfusion sites identified using a light microscope. All perfusion sites were subsequently verified histologically to be within the areas of the VSA. Two experimental and two control groups were used: the IKIC group at days 4 and 18, and the sham-operated. normotensive controls for days 4 and 18. The average baseline temperature before treatment was calculated for each animal, and the results were

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Fig. I. A: systolic blood pressure before (presurgery) and 4 days after surgery ±S.E.M. in sham-operated (open bars n = 4) and IKIC (closed bars n = 10) conscious rats, prior to push-pull perfusion studies. The systolic blood pressures do not differ significantly. B: systolic blood pressure before (presurgery) and 18 days after surgery ± S.E.M. in sham-operated (open bars n = 3) and I KIC (closed bars n = 8) conscious rats, prior to push-pull perfusion studies. The systolic blood pressures of both groups of rats on day 18 were significantly different from the corresponding baseline values (* P:::; 0.05). In addition, the systolic blood pressure of l KlC rats was significantly different from that of the sham-operated animals on day 18 (# P :::; 0.05).

expressed as the deviation from this pre-treatment value. Data from individual animals were combined, and a group mean and standard error of the mean (S.E.M.l were calculated. Both the maximum change in temperature (febrile response) and blood pressure data recorded on days 4 and 18 of each group were compared statistically using a 2-way analysis of variance (ANOVA) for repeated measures. The AVP release data were analyzed using ANOVA for repeated measurements, followed by Scheffe's post-hoc analysis where appropriate. Results were considered to be significant if P:::; 0.05.

3. Results At day 4 (Fig. l A), the systolic blood pressures of both the 1KIC rats (n = 10) and the sham-operated animals tn = 4) remained unchanged compared to their pressures prior to surgery as shown in Fig. I. In addition, both pre surgery and day 4 pressures were identical between the two groups. The initial baseline ternperatures in the urethane-anaesthetized rats used in

M.L. Earle et

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at. / Brain Research 658 (I994)

the present study was maintained between 36.5 and 37°C; and did not differ significantly between groups. Subsequent PGE 2-induced fevers were identical in the 1K1C rats, when compared with their sham-operated controls (maximum fever height in 1K1C rats was 0.74 ± O.l3°C, compared with 0.87 ± 0.16°C in the sham-operated controls), as shown in Fig. 2A. AVP content of perfusates from the VSA of the day 4 rats was not significantly different in either baseline levels or following PGE 2 injection, between sham-operated rats and 1K1C rats (Fig. 2B). As expected, at day 18, the blood pressures of the 1K1C rats (n = 8) were significantly elevated compared with both their baseline levels (P < 0.05) and compared with the sham-operated rats in = 3; P < 0.05; Fig. IB). The baseline temperatures for the two groups A .......

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did not significantly differ. No inhibition of PGE 2-induced fever was seen in the 1K1C rats compared with their sham-operated controls (Fig. 3: maximum fever height 1.19 ± 0.23 1K1C; 0.91 ± 0.34 sham-operated rats). The release of AVP into the VSA in the shamoperated animals was, however, significantly increased (P::=; 0.05) compared with 1K1C hypertensive rats in sample 2, and compared with sham-operated baseline levels (prior to PGE 2 ) , as shown in Fig. 3. Push-pull perfusion sites were located within the VSA using histological verification techniques; a typical example of a push-pull perfusion site within the VSA is shown in Fig. 4. In addition to the experiments reported here, an initial study was performed in which no bacitracin was added to the perfusion fluid, resulting in undetectable levels of AVP within the VSA (this is consistent with studies by Ramirez et al. [15]). However, fever studies were performed using these animals, and the data can be combined with the present results. Even with a substantially greater sample size, no inhibition of the febrile response to PGE 2 was observed either in the day 4 animals (maximum fever height 1K1C = 1.5 ± 0.17, n = 25; sham-operated rats = 1.27 ± 0.17, n = 14) or the day 18 animals (maximum fever height 1K1C = 1.27 ± 0.19, n = 18; sham-operated rats = 1.26 ± 0.11, n = 12).

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Fig. 3. A: change in rectal temperature ± S.E.M. in response to i.c.v. injection of 100 ng PGE 2 in day 18 rats. Sham-operated rats (open circles n = 3) and IK1C-operated rats (closed circles n = 8) are shown. Arrow indicates time of PGE 2 injection; temperature responses were measured for 60 min following this. Febrile responses were not significantly different between these groups. B: AVP release ± S.E.M. (measured using push-pull perfusion) within the VSA in day 18 rats. the temperatures of which are plotted in A, prior to (sample I), during (sample 2) and after (sample 3) the i.c.v. injection of 100 ng PGE 2 • Open bars, sham-operated rats (n = 3); closed bars, lK1C rats (n = 8). AVP release during PGE 2 injection (sample 2) was significantly greater in sham-operated animals compared with that in IKIC rats during the same time period (* P $ 0.05).

The aim of the present study was firstly to determine if the transient suppression of the febrile response in 1K1C rats [3] could also be observed following the induction of a slower-developing 1K1C hypertension. Secondly, since this transient inhibition of fever had been attributed to a possible overactivity of the vasopressinergic input to the VSA, we wished to compare the release of AVP within the VSA in these hypertensive rats with their normotensive controls. The present data demonstrate that the transient inhibition of the febrile response observed following the rapid induction of lKIC hypertension [3] is not observed following the induction of a more slowly developing 1K1C hypertension. The push-pull perfusion studies described in this report also show no increase in AVP release into the VSA in response to PGE 2 , except in day 18 sham-operated rats. This latter observation is statistically significant; however, the actual level of peptide detected was within the normal baseline range [19]. In the present study, the systolic blood pressure of the rats rose less than 6 mmHg after 4 days. Fyda et al. [3] used a protocol for the induction of lKIC hypertension where one kidney was removed 4 days prior to occlusion of blood flow to the remaining kidney. Under

M.L. Earle et al. / Brain Research 658 (1994) 15-20

19

Fig. 4. Photomicrograph of a coronal section of the brain showing the VSA perfusion site . ac, anterior commissure; VSA, ventral se pta l area; dbb, diagonal band of Broca. Bar = 200 !Lm.

these conditions a more rapidly developing hypertension occurs, where the mean systolic blood pressure rose 55 mmHg over the initial time period. Since this rise in blood pressure is many times greater than that observed in the present experiment, this is the most likely explanation of the different fever responses observed in these two experimental paradigms. Indeed, this would argue for a blood pressure-related explanation for the inhibition of fever observed by Fyda et al. [3]. Furthermore, it would appear that it is the rapidity of the rise in blood pressure that is important as opposed to the actual level, since even in the previous study the febrile response returned to control levels by day 18, when blood pressure was at its highest. It should be possible to test this possibility by administering peripheral vasoconstrictor substances to mimic th e rapid rise in blood pressure seen by Fyda et al. [3]. Since there was no inhibition of the febrile response to PGE z, the observation that there was no increase in AVP release into the VSA in the lKlC rats is not surprising. The lack of an increase in AVP release into the VSA in lKIC hypertensive rats in response to a pyrogenic challenge, such as seen previously [8], is puzzling. It is possible that the more modest fevers in the present study did not activate th e central A VP pathways, thought to be important in antipyresis, to a degree sufficient for detection of increased A VP in the perfusates, however, the actual fever heights shown in the present study were identical to those shown by

Fyda et al. [3]. In addition, the actual levels of peptide detected under both baseline and febrile conditions were consistent with some other studies [17,19]. In one other study [8], an increase in AVP content of VSA perfusates was observed following the induction of fever, however, th e level of fever produced during this study was considerably more than that observed in the present study, probably the result of differing baseline temperatures. In summary, we can conclude that lKIC hypertension itself does not cause transient inhibition of the febrile response to PGE z , nor increase AVP release into the VSA. One may furthermore speculate that a much more rapid increase in blood pressure may elicit transient neural ch anges not encountered following a more slowly developing hypertension . While it is clear from previous studies that there are interactions between cardiovascular sensory signals and th e thermoregulatory system [4,12,16], it is apparent that the time-course, degree and type of hypertension may be critical variables in determining the nature of the inte raction.

Acknowledgements Supported by the Heart & Stroke Foundation, MRC, VW and NATO. M .L.E. is an AHFMR Student, TH. is a DAAD Student, N.K is an MRC Scientist, and

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M.L. Earle et a!./ Brain Research 658 (1994) 15-20

Q.J.P. is an AHFMR Scientist. We thank Dr. M. Wilkinson for preparation of the push-pull perfusion cannulae, and for his comments on this manuscript.

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[9] Landgraf, R., Neumann, 1. and Schwarzberg, H., Central and peripheral release of vasopressin and oxytocin in the conscious rat after osmotic stimulation, Brain Res., 457 (1988) 219-225. [10] Maggi, C.A and Meli, A, Suitability of urethane anaesthesia for physiopharrnacological investigations in various systems. Part 2. Cardiovascular systems, Experientia, 42 (1986) 292-297. [11] Malkinson, T.J., Cooper, KE. and Veale, W.L., Physiological changes during thermoregulation and fever in urethan-anaesthetized rats, Am. 1. Physiol., 255 (1988) R73-R81. [12] Nagasaka, T.H., Shibata, H., Nunomura, T. and Ohmae, 0., Baroreflex suppression of non-shivering thermogenesis in restrained and non-restrained rats, 1. Theor. Bioi., 9 (1984) 93-96. [13] Naylor, AM., Pittman, Q.J. and Veale, W.L., Stimulation of vasopressin release in the ventral septum of the rat brain suppresses prostaglandin El fever, J. Physiol., 399 (1988) 177189. [14] Pittman, QJ. and Wilkinson, M.F., Central arginine vasopressin and endogenous antipyresis, Can. J. Physiol. Pharmacol., 70 (1992) 786-790. [15] Ramirez, V.D., Ramirez, A.D., Rodriguez, F., Poncet, C. and Vincent, J.D., Release of vasopressin from the septum and hippocampus of freely moving rats: effects of bacitracin and synthetic vasopressin, 1. Neuroendocrinol., 2 (1990) 453-460. [16] Wasserstrum, N. and Herd, J.A, Baroreflex depression of oxygen consumption in the squirrel monkey at 10°C, Am. 1. Physiol., 232 (19m H451-H458. [17] Wilkinson, M.F., A uasopressinergic pathway within the brain and it's role in drug-induced antipyresis and pyrogenic tolerance, PhD Thesis, The University of British Columbia, 1990. [18] Wilkinson, M.F. and Kasting, N.W., Centrally acting vasopressin contributes to endotoxin tolerance, Am. J. Physiol., 258 (1990) R443-R449. [19] Wilkinson, M.F. and Kasting, N.W., Vasopressin release within the ventral septal area of the rat brain during drug-induced antipyresis, Am. 1. Physiol., 264 (1993) R1l33-R1138.