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The effects of forebrain multiple lesions on the pressor response induced by bilateral carotid occlusion in conscious rats
243
Brain Research, 612 (1993) 243-246 © 1993 Elsevier Science Publishers B,V. All rights reserved 0006-8993/93/$06.00
BRES 18849
The effects of forebrain multiple lesions on the pressor response induced by bilateral carotid occlusion in conscious rats Maria Tereza Barbieri B e d r a n de Castro, Jo~o Cesar B e d r a n de Castro a n d Jos4 V a n d e r l e i M e n a n i Department of Physiology, School of Dentistry, Paulista State University,Aragatuba, SP (Brazil) (Accepted 22 December 1992)
Key words: Carotid occlusion; AV3V; Hypothalamus; Baroreceptor; Cerebral ischemia; Cardiovascular reflex
In the present study, the effects of electrolytic lesions of the anteroventral third ventricle (AV3V) region and of the medial forebrain bundle (MFB) on the pressor response induced by bilateral carotid occlusion (BCO) in conscious intact and aortic baroreceptor-denervated (AD) rats were investigated. In intact control rats, BCO during 60 s produced a pressor response that could be divided into an early response (ER = 50+3 mmHg) that reachs a peak during the first 20 s and a sustained late response (LR), smaller than ER (32 + 2 mmHg), observed during the last 30 s. In intact-innervated rats, AV3V lesion (2 days) reduced ER (22 + 3 mmHg) and LR (16 + 2 mmHg), whereas the bilateral MFB lesions (6 days) mainly reduced LR (9 + 1 mmHg). Rats with simultaneous lesion of both the AV3V region and the MFB showed additional reduction of the ER (15 5:3 mmHg), but not LR (11 + 1 mmHg) when compared to the effect of MFB lesions alone. Compared to the AV3V lesion alone, LR but not ER was reduced in rats with a double lesion. In sham-lesioned rats, AD induced a significant increase in the pressor response to BCO (ER = 75 5:4 mmHg and LR = 65 + 3 mmHg) when compared to intact controls. A similar reduction in ER and LR was observed in AD rats after AV3V (ER = 355:3 mmHg and LR=405:2 mmHg) and MFB (ER= 49_+6 mmHg and LR =41_+5 mmHg) lesions alone or combined (ER = 40 5:6 mmHg and LR = 35_+7 mmHg). The results showed that simultaneous lesions of both the AV3V region and the MFB practically abolished the pressor response to BCO. They also suggested that aortic baroreceptor activity plays a significant role in the effects of AV3V and MFB lesions on the pressor response to BCO.
INTRODUCTION
The pressor response produced by bilateral common carotid occlusion (BCO) is usually used to demonstrate reflexes involved in the cardiovascular regulation of laboratory animals. Although the pressor response to BCO has been known for a long time, the central mechanisms and pathways involved in this response are not yet well understood. In rats, the BCO during 60 s produces a pressor response that can be divided into two components: (1) an early response (ER) that reachs a peak within the first 20 s, and (2) a sustained late response (LR), smaller than ER, observed during the last 30 s. In dogs, cats and rabbits, the pressor response to BCO seems to be dependent on the reflex mechanisms of the carotid receptors. In rats, it has been suggested that the ER is more dependent on the
car o t i d r e c e p t o r s , w h e r e a s t h e L R is mainly r e l a t e d to c e r e b r a l i s c h e m i a 2'1u4. It is well a c c e p t e d the i m p o r t a n c e o f l o w er p a r t s o f t h e b r ai n in t h e i n t e g r a t i o n o f c a r d i o v a s c u l a r reflexes. F o r B C O , not only the l o w er parts o f brain, but also m o r e rostral structures, mainly at t h e h y p o t h a l a m i c level, play an i m p o r t a n t sponse 2.5,6,9,10,13,14.
role
in
the
p r essor
re-
T h e i n v o l v e m e n t o f h y p o t h a l a m i c pathways in t he c o n t r o l o f t h e p r e s s o r r e s p o n s e to B C O was first studied by L o p e s et al. 9. T h e s e
authors
o b s e r v e d that
m e d i a l h y p o t h a l a m i c knife cuts that d i s c o n n e c t e d t he a n t e r i o r h y p o t h a l a m u s f r o m the m i d d l e h y p o t h a l a m u s r e d u c e d the p r e s s o r r e s p o n s e to B C O in p e n t o b a r b i t a l a n e s t h e t i z e d rats. T h e y also d e s c r i b e d that large (2 m m radius), but n o t small (1 m m radius), h y p o t h a l a m i c d i s c o n n e c t i o n r e d u c e s t h e p r e s s o r r e s p o n s e to B C O .
Correspondence: M.T.B. Bedran de Castro, Department of Physiology, School of Dentistry, Paulista State University, Rua Jos6 Bonifficio 1193, 16015-050 Ara~atuba, SP, Brazil.
244 Since the hypothalamic disconnection reduced only the LR, Lopes et al. 9 suggested that the two components of the pressor response to BCO could be integrated at different levels. Then, one component (ER) would be integrated at the medullary level and the other (LR) at the hypothalamic level. In accordance with this idea, further studies have shown that electrolytic bilateral lesions of the medial forebrain bundle (MFB) also reduced mainly the LR during BCO in either conscious or anesthetized rats 2,t°. Recent studies from our laboratory have shown the involvement of a more rostral area, the anteroventral third ventricle (AV3V) region, on the pressor response to BCO in conscious rats 14. The AV3V region is one of the most important areas of the rat forebrain involved in cardiovascular regulation 2'4. The electrolytic AV3V lesion also reduced the pressor response to BCO, demonstrating that not only the lateral hypothalamic, but the medial hypothalamic areas play a significant role in the pressor response to BCO in rats. The results observed in AV3Vlesioned rats also suggested that not only the LR, but the E R could depend on some integration at the hypothalamic level. Considering the differences observed in the pressor response to BCO after either the lateral or medial hypothalamic lesions, we proposed to investigate the effects of simultaneous lesions of both the AV3V region and the bilateral MFB. Since the aortic baroreceptors play an important role buffering the increase in arterial pressure and vascular resistance induced by BCO2 ~J,H, and their activity seems to be important for the effects of central lesions, we studied the pressor response to BCO in intact and aortic baroreceptor-denervated (AD) rats. The study was performed in conscious freely moving rats by using special pneumatic cuffs implanted around the carotid arteries on the day before the experiment 2't2. MATERIALS AND METHODS Animals Male Wistar rats weighing 230-250 g were used in this study. All animals were housed under standard laboratory conditions and received laboratory chow diet and water ad libitum.
Arterial pressure recording In rats anesthetized with ether, a polyethylene tube (PE III connected to a PE 50) was inserted into the abdominal aorta through the femoral artery. The polyethylene tubing was tunneled subcutaneously and exposed on the back of the rat. Twenty four hours later, the mean arterial pressure (MAP) was recorded in conscious and unrestrained rats by means of a strain-gauge transducer (model P 23 Db, Statham Instruments, Hato Rey, Puerto Rico) connected to a multichannel recorder (Narcotrace 40, Narco Bio-Systems Inc, USAI.
Cerebral lesions The electrolytic AV3V lesion was performed under ether anesthesia according to Brody et al. 3. A monopolar stainless-steel wire electrode (0,4 mm in diameter) bared at the tip was positioned in a stereotaxic apparatus (David Kopf, USA) 0.0-0.3 mm caudal to the bregma in the midline to a depth of 7.0 mm from the duramater. The head was adjusted to obtain bregma and lambda at the same level. The anodal lesion was made with a direct 2-mA current for 15 s. A clip attached to the tail was used as an indifferent electrode. In the sham-lesioned rats, the electrode was placed along the same coordinates except that the depth was 5.5 mm from the duramater and no electrical current was passed. Bilateral MFB lesions were performed using an electrode (0.2 mm in diameter) positioned 5.0 mm rostral to the interaural line, 2.0 mm lateral to the midline, and 2.8 mm below the interaural plane (tooth bar 5.0 mm above 0.0), according to De Groot's atlas 7. The MFB lesions were made using a 2-mA current for 15s.
Aortic denercation Aortic denervation was performed as described elsewhere ~. Briefly, the rats were anesthetized with ether, and the carotid arteries and vagus nerves were exposed through a ventral cervical incision. The sympathetic trunk and the recurrent laryngeal nerve were identified and cut.
Histology The positions of the lesions were confirmed by histological examination of the brain tissue. At the end of the experiments, under ether anesthesia, the hearts were exposed for intracardiac perfusion with saline followed by 10% formalin. The brains were removed and stored in 10% formalin for at least 1 week. The tissue was then frozen and transverse sections (20-30/xm) were stained with hematoxylin and eosin for examination by light microscopy. Only the results observed in rats with typical MFB and AV3V lesions were used. The MFB lesions usually destroyed both the medial forebrain bundle and part of the lateral hypothalamus. The typical AV3V lesion was located below the level of the anterior commissure, with bilateral destruction of the periventricular tissue surrounding the optic recess of the 'lamina terminalis' through the preoptic and anterior hypothalamus, never extending caudally to the arcuate nucleus-median eminence region. The structures destroyed by the AV3V lesion included the preoptic and anterior hypothalamic periventricular nuclei, the median preoptic nucleus, and the anterior wall of the third ventricle with the associated organum vasculosum of the 'lamina terminalis '3.
Stat&tical analysis Bilateral carotid occlusion The bilateral common carotid arteries were occluded in conscious freely moving rats by means of pneumatic cuffs implanted 24 h before the experiment. In rats anesthetized with ether, the common carotid arteries were exposed through an incision in the ventral cervical region. A special cuff was adjusted around each carotid and a polyethylene tubing (PE 50) connected to the cuff was exteriorized through the back of the rat. For carotid occlusion the two cuffs were connected using a Y-shaped polyethylene tubing. Details about the preparation of the cuffs and the carotid occlusion were described elsewhere 2.~z.
Data are reported as mean + S.E.M. Statistical significance was calculated by analysis of variance and S t u d e n t - N e w m a n - K e u l s test. Differences were considered significant at P < 0.05.
Experimental protocol The pressor response to BCO was studied in different groups of intact-innervated and aortic baroreceptor-denervated (AD) rats submitted to either AV3V or MFB lesions alone (AV3V or MFB) or associated (AV3V + MFB lesions in the same rat). The pressor response to BCO was studied in the following groups of rats:
245 (a) Intact innervated rats: Sham lesion (MFB; 6 days + AV3V; 6 h) MFB lesion (6 days) AV3V lesion (6 h and 2 days) MFB lesion (6 days)+AV3V lesion (6 h and 2 days) (b) AD rats: Sham lesion (MFB; 6 days + AV3V; 6 h)+ AD (6 days) MFB lesion (6 days)+ AD (6 days) AV3V lesion (6 h)+ AD (6 days) MFB lesion (6 days)+ AV3V lesion (6 h)+ AD (6 days)
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RESULTS
Effect of AV3V and MFB lesions on the pressor response to B C O in intact-innervated rats
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Fig. 1. Increase in mean arterial pressure produced during 1 rain of BCO in intact-innervated sham-lesioned rats (S) and in rats submitted to MFB lesion (L-MFB, 6 days), AV3V lesion (L-AV3V, 6 h or 2 days) or L-MFB (6 days)+ L-AV3V (6 h or 2 days). The number of animals is indicated in parentheses at the top of the columns. ER, early response; LR, late response. (*), compared to sham (P < 0.05); (o), compared to L-AV3V (P<0.05); (e), compared to L-MFB
(P < 0.05).
80 70 60
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The effects of A V 3 V and bilateral MFB lesions alone or associated on the pressor response to BCO in intact-innervated rats are presented in Fig. 1. MFB lesions alone (6 days) in intact-innervated rats (basal MAP= 112+3 mmHg) abolished the LR ( 9 + 1 mmHg), but produced only a small reduction of the E R (36 + 2 m m H g ) when compared to the pressor response produced by B C O in sham rats ( E R = 50 + 3 m m H g and LR = 32 + 2 mmHg, basal M A P = 122 + 3 mmHg). A V 3 V lesion alone (6 h and 2 days, basal M A P --- 112 + 6 and 121 + 3 mmHg, respectively) reduced both components ( E R = 20 + 2 and 22 + 3 mmHg, and LR = 21 + 2 and 16 + 2 mmHg, 6 h and 2 days after lesion, respectively). In rats submitted to simultaneous lesions of the A V 3 V region and bilateral MFB, an additional reduction in the pressor response to BCO was observed when compared to rats with A V 3 V or M F B lesions alone. A reduced E R (14 + 2 and 15 + 3 mmHg, respectively) was observed in rats
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90
o
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ER
AD +
S
AD +
L-MFB
AD + L-AV3V
AD + L -MFB + L-AV3V
Fig. 2. Increase in mean arterial pressure produced during 1 min of BCO in intact-innervated sham-lesioned rats (S), aortic denervated
(AD, 6 days) sham-lesioned rats and AD rats (6 days) submitted to lesion of the MFB (L-MFB, 6 days), AV3V (L-AV3V, 6 h) or L-MFB (6 days)+L-AV3V (6 h). The number of animals is indicated in parentheses at the top of the columns. ER, early response; LR, late response. (*), compared to control (P<0.05); (o), compared to sham-lesioned AD rats (P < 0.05).
with lesions of the MFB (6 days) + A V 3 V (6 h and 2 days, basal M A P = 114 + 3 and 114 + 4 mmHg, respectively) when compared to rats with MFB lesions alone submitted to BCO. On the other hand the LR (8 _+ 1 and 11 + 1 mmHg) observed in rats with MFB + A V 3 V lesions, was reduced when compared to the rats submitted to A V 3 V lesion alone.
Effect of AV3V and MFB lesions on the pressor response to B C O in aortic-denervated rats
The effects of A V 3 V and MFB lesions alone or associated with the pressor response to BCO in aorticdenervated rats are presented in Fig 2. An increased pressor response ( E R = 7 5 + 4 and L R = 6 5 _ + 3 mmHg) was observed in sham-lesioned A D rats (basal M A P = 124 + 3 mmHg) when compared to sham-lesioned intact-innervated rats (ER = 50 + 3 and LR = 32 + 2 mmHg). Either A V 3 V (6 h, basal M A P = 124 + 5 mmHg) or bilateral MFB lesions alone (6 days, basal M A P = 123 _+ 3 mmHg) or associated (basal M A P = 124 _+ 5 mmHg) reduced the pressor response to BCO in A D rats. A similar reduction of the pressor response to BCO in A D rats was observed after both MFB (ER=49+6 m m H g and L R = 4 1 + 5 mmHg), and A V 3 V lesions (ER = 35 _+ 3 m m H g and LR = 40 _+ 2 mmHg), and MFB + A V 3 V lesions ( E R = 40_+ 6 m m H g and LR = 35 _+ 7 mmHg). DISCUSSION The present results show a profound involvement of hypothalamic pathways in both components of the
246 p r e s s o r r e s p o n s e to B C O in conscious rats. Lesions of e i t h e r the A V 3 V region or M F B a l o n e r e d u c e d the p r e s s o r r e s p o n s e to B C O in intact or aortic b a r o r e c e p t o t d e n e r v a t e d rats as previously d e s c r i b e d 2'~4. In int a c t - i n n e r v a t e d rats s u b m i t t e d to s i m u l t a n e o u s lesions of the A V 3 V region a n d M F B , the p r e s s o r r e s p o n s e to B C O was practically abolished. T h e s e results suggest that the p r e s s o r r e s p o n s e to B C O in conscious rats is almost totally d e p e n d e n t on h y p o t h a l a m i c i n t e g r a t i o n , which is d i f f e r e n t from t h a t p r o p o s e d by L o p e s et al. ~. T h e s e a u t h o r s s u g g e s t e d that the i n t e g r a t i o n of L R would be in the h y p o t h a l a m u s , w h e r e a s the E R w o u l d be i n t e g r a t e d at m e d u l l a r y level. F u r t h e r m o r e , L o p e s et al. 9 also showed that lateral, but not m e d i a l hypothalamic lesions c h a n g e d the p r e s s o r r e s p o n s e to B C O in rats. T h e p r e s e n t results clearly d e m o n s t r a t e t h a t m e dial a n d l a t e r a l h y p o t h a l a m i c a r e a s a r e involved in the control of p r e s s o r r e s p o n s e to BCO. Since it is well r e c o g n i z e d that a n e s t h e t i c s i m p a i r reflex p r e s s o r response to BCO1, this could be an e x p l a n a t i o n for the d i f f e r e n c e s o b s e r v e d w h e n c o m p a r i n g the results of L o p e s et al. ~ with the p r e s e n t findings. A n o t h e r i m p o r t a n t o b s e r v a t i o n in the p r e s e n t results is c o n c e r n e d a b o u t the c e r e b r a l a r e a s involved in t h e i n t e g r a t i o n of the E R and LR. R a t s with simultan e o u s lesions o f b o t h the A V 3 V region a n d the M F B s h o w e d a r e d u c e d E R w h e n c o m p a r e d to rats with M F B lesions alone, b u t no d i f f e r e n c e was f o u n d w h e n c o m p a r i n g rats with A V 3 V lesion alone. O n the o t h e r hand, rats with d o u b l e lesion h a d a r e d u c e d L R w h e n c o m p a r e d to rats with A V 3 V lesion alone, but no d i f f e r e n c e was o b s e r v e d w h e n c o m p a r e d to rats with M F B lesion alone. T h e s e findings allow us to c o n c l u d e that in i n t a c t - i n n e r v a t e d rats the w h o l e E R is d e p e n d e n t on the A V 3 V region, w h e r e a s the w h o l e L R d e p e n d s on M F B . T h e i n c r e a s e d p r e s s o r r e s p o n s e o b s e r v e d in A D rats was also r e d u c e d a f t e r A V 3 V or M F B lesions a l o n e or associated. H o w e v e r , the effects of lesions w e r e very d i f f e r e n t in A D rats w h e n c o m p a r e d to t h e effects of lesions in i n t a c t - i n n e r v a t e d rats. First, in A D rats, A V 3 V or M F B lesions a l o n e or a s s o c i a t e d r e d u c e d b o t h E R a n d L R to t h e s a m e d e g r e e . S e c o n d , the r e d u c t i o n in E R a n d L R p r o d u c e d by A V 3 V o r M F B lesions in A D rats w e r e s m a l l e r t h a n t h o s e o b s e r v e d in i n t a c t - i n n e r v a t e d rats (e.g. t h e r e d u c t i o n of the L R after M F B lesion in i n t a c t - i n n e r v a t e d rats was 70%,
a n d the r e d u c t i o n of this c o m p o n e n t o b s e r v e d in A D rats with the s a m e lesion was 35%). T h e s e results suggest that the inhibitory activity of the aortic b a r o r e c e p t o r s may play an i m p o r t a n t role in the effects of h y p o t h a l a m i c lesions r e d u c i n g the p r e s s o r r e s p o n s e to BCO. The authors thank Reginaldo E. Queiroz for the technical assistance and Eliane Rodrigues dos Santos for typing the manuscript.
Acknowledgements.
REFERENCES 1 Bedran de Castro, M.T.B., Farah, V.M.A. and Krieger, E.M., Influence of general anesthetics on baroreflex control of circulation, Braz. J. Med. Biol. Res., 23 (1990) 1185-1193. 2 Bedran de Castro, M.T.B., Moreira, E.D. and Krieger, E.M., Reflex and central components of carotid occlusion in conscious rats. Effect of lesion of the medial forebrain bundle, Hypertension, 8 (Suppl. I) (1986) 147-I51. 3 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, Cir. Res., 43 (Suppl. I) (1978) 1-13. 4 Brody, M.J. and Johnson, A.K., Role of the anteroventral third ventricle region in fluid and electrolyte balance, arterial pressure regulation and hypertension. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinology, Raven, New York, 1980, pp. 249-292. 5 Chai, C.Y., Share, N.N. and Wang, S.C., Central control of sympathetic cardiac augmentation in lower brain stem of the cat, Am. J. Physiol., 205 (1963) 749-753. 6 Chai, C.Y., and Wang, S.C., Integration of sympathetic cardiovascular mechanisms in medulla oblongata of the cat, Am. J. Physiol., 215 (1968) 1310-1315. 7 De Groot, J., The rat forebrain in stereotaxic coordinates, Trans. R. Neth. Acad. Sci., 52 (1959) 1-40. 8 Krieger, E.M. and Marseillan, R.F., Aortic depressor fibers in the rat: an electrophysiological study, Am. Z Physiol., 205 (1963) 771-774. 9 Lopes, O.U., Cipola-Neto, J, Rocha e Silva, M.Jr., Hypothalamic component in pressor response to carotid occlusion in the rat, Am. J. Physiol., 233 (1977) H240-H247. 10 Lopes, O.U., Timo-Iaria, C., Leit~o Filho, H.A.P., Suppression of the late component of the carotid occlusion reflex by lesion of the medial forebrain bundle in the rat, Braz. J. Med. Biol. Res., 14 (1981) 37-41. 11 Machado, B.H., Bonagamba, L.G.H, Castania, J.A. and Menani, J.V., Changes in vascular resistance during carotid occlusion in normal and baroreceptor-denervated rats, Hypertension, 19 (suppl II) (1992) 149-153. 12 Maio, A.A., Moreira, E.D., Salgado, H.C. and Krieger, E.M., Cardiovascular responses of conscious rats due to arterial occlusion, (Abstract), Braz. J. Meal. Biol. Res., 14 (1981) 115. 13 Manning, J.W., Cardiovascular reflexes following lesions in medullary reticular formation, Am. J. Physiol., 208 (1965) 283288. 14 Menani, J.V., Bedran de Castro, M.T,B. and Krieger, E.M., Influence of the anteroventral third ventricle region and sinoaortic denervation on the pressor response to carotid occlusion, Hypertension, 11 (Suppl I) (1988) I178-I181.
Brain Research, 612 (1993) 243-246 © 1993 Elsevier Science Publishers B,V. All rights reserved 0006-8993/93/$06.00
BRES 18849
The effects of forebrain multiple lesions on the pressor response induced by bilateral carotid occlusion in conscious rats Maria Tereza Barbieri B e d r a n de Castro, Jo~o Cesar B e d r a n de Castro a n d Jos4 V a n d e r l e i M e n a n i Department of Physiology, School of Dentistry, Paulista State University,Aragatuba, SP (Brazil) (Accepted 22 December 1992)
Key words: Carotid occlusion; AV3V; Hypothalamus; Baroreceptor; Cerebral ischemia; Cardiovascular reflex
In the present study, the effects of electrolytic lesions of the anteroventral third ventricle (AV3V) region and of the medial forebrain bundle (MFB) on the pressor response induced by bilateral carotid occlusion (BCO) in conscious intact and aortic baroreceptor-denervated (AD) rats were investigated. In intact control rats, BCO during 60 s produced a pressor response that could be divided into an early response (ER = 50+3 mmHg) that reachs a peak during the first 20 s and a sustained late response (LR), smaller than ER (32 + 2 mmHg), observed during the last 30 s. In intact-innervated rats, AV3V lesion (2 days) reduced ER (22 + 3 mmHg) and LR (16 + 2 mmHg), whereas the bilateral MFB lesions (6 days) mainly reduced LR (9 + 1 mmHg). Rats with simultaneous lesion of both the AV3V region and the MFB showed additional reduction of the ER (15 5:3 mmHg), but not LR (11 + 1 mmHg) when compared to the effect of MFB lesions alone. Compared to the AV3V lesion alone, LR but not ER was reduced in rats with a double lesion. In sham-lesioned rats, AD induced a significant increase in the pressor response to BCO (ER = 75 5:4 mmHg and LR = 65 + 3 mmHg) when compared to intact controls. A similar reduction in ER and LR was observed in AD rats after AV3V (ER = 355:3 mmHg and LR=405:2 mmHg) and MFB (ER= 49_+6 mmHg and LR =41_+5 mmHg) lesions alone or combined (ER = 40 5:6 mmHg and LR = 35_+7 mmHg). The results showed that simultaneous lesions of both the AV3V region and the MFB practically abolished the pressor response to BCO. They also suggested that aortic baroreceptor activity plays a significant role in the effects of AV3V and MFB lesions on the pressor response to BCO.
INTRODUCTION
The pressor response produced by bilateral common carotid occlusion (BCO) is usually used to demonstrate reflexes involved in the cardiovascular regulation of laboratory animals. Although the pressor response to BCO has been known for a long time, the central mechanisms and pathways involved in this response are not yet well understood. In rats, the BCO during 60 s produces a pressor response that can be divided into two components: (1) an early response (ER) that reachs a peak within the first 20 s, and (2) a sustained late response (LR), smaller than ER, observed during the last 30 s. In dogs, cats and rabbits, the pressor response to BCO seems to be dependent on the reflex mechanisms of the carotid receptors. In rats, it has been suggested that the ER is more dependent on the
car o t i d r e c e p t o r s , w h e r e a s t h e L R is mainly r e l a t e d to c e r e b r a l i s c h e m i a 2'1u4. It is well a c c e p t e d the i m p o r t a n c e o f l o w er p a r t s o f t h e b r ai n in t h e i n t e g r a t i o n o f c a r d i o v a s c u l a r reflexes. F o r B C O , not only the l o w er parts o f brain, but also m o r e rostral structures, mainly at t h e h y p o t h a l a m i c level, play an i m p o r t a n t sponse 2.5,6,9,10,13,14.
role
in
the
p r essor
re-
T h e i n v o l v e m e n t o f h y p o t h a l a m i c pathways in t he c o n t r o l o f t h e p r e s s o r r e s p o n s e to B C O was first studied by L o p e s et al. 9. T h e s e
authors
o b s e r v e d that
m e d i a l h y p o t h a l a m i c knife cuts that d i s c o n n e c t e d t he a n t e r i o r h y p o t h a l a m u s f r o m the m i d d l e h y p o t h a l a m u s r e d u c e d the p r e s s o r r e s p o n s e to B C O in p e n t o b a r b i t a l a n e s t h e t i z e d rats. T h e y also d e s c r i b e d that large (2 m m radius), but n o t small (1 m m radius), h y p o t h a l a m i c d i s c o n n e c t i o n r e d u c e s t h e p r e s s o r r e s p o n s e to B C O .
Correspondence: M.T.B. Bedran de Castro, Department of Physiology, School of Dentistry, Paulista State University, Rua Jos6 Bonifficio 1193, 16015-050 Ara~atuba, SP, Brazil.
244 Since the hypothalamic disconnection reduced only the LR, Lopes et al. 9 suggested that the two components of the pressor response to BCO could be integrated at different levels. Then, one component (ER) would be integrated at the medullary level and the other (LR) at the hypothalamic level. In accordance with this idea, further studies have shown that electrolytic bilateral lesions of the medial forebrain bundle (MFB) also reduced mainly the LR during BCO in either conscious or anesthetized rats 2,t°. Recent studies from our laboratory have shown the involvement of a more rostral area, the anteroventral third ventricle (AV3V) region, on the pressor response to BCO in conscious rats 14. The AV3V region is one of the most important areas of the rat forebrain involved in cardiovascular regulation 2'4. The electrolytic AV3V lesion also reduced the pressor response to BCO, demonstrating that not only the lateral hypothalamic, but the medial hypothalamic areas play a significant role in the pressor response to BCO in rats. The results observed in AV3Vlesioned rats also suggested that not only the LR, but the E R could depend on some integration at the hypothalamic level. Considering the differences observed in the pressor response to BCO after either the lateral or medial hypothalamic lesions, we proposed to investigate the effects of simultaneous lesions of both the AV3V region and the bilateral MFB. Since the aortic baroreceptors play an important role buffering the increase in arterial pressure and vascular resistance induced by BCO2 ~J,H, and their activity seems to be important for the effects of central lesions, we studied the pressor response to BCO in intact and aortic baroreceptor-denervated (AD) rats. The study was performed in conscious freely moving rats by using special pneumatic cuffs implanted around the carotid arteries on the day before the experiment 2't2. MATERIALS AND METHODS Animals Male Wistar rats weighing 230-250 g were used in this study. All animals were housed under standard laboratory conditions and received laboratory chow diet and water ad libitum.
Arterial pressure recording In rats anesthetized with ether, a polyethylene tube (PE III connected to a PE 50) was inserted into the abdominal aorta through the femoral artery. The polyethylene tubing was tunneled subcutaneously and exposed on the back of the rat. Twenty four hours later, the mean arterial pressure (MAP) was recorded in conscious and unrestrained rats by means of a strain-gauge transducer (model P 23 Db, Statham Instruments, Hato Rey, Puerto Rico) connected to a multichannel recorder (Narcotrace 40, Narco Bio-Systems Inc, USAI.
Cerebral lesions The electrolytic AV3V lesion was performed under ether anesthesia according to Brody et al. 3. A monopolar stainless-steel wire electrode (0,4 mm in diameter) bared at the tip was positioned in a stereotaxic apparatus (David Kopf, USA) 0.0-0.3 mm caudal to the bregma in the midline to a depth of 7.0 mm from the duramater. The head was adjusted to obtain bregma and lambda at the same level. The anodal lesion was made with a direct 2-mA current for 15 s. A clip attached to the tail was used as an indifferent electrode. In the sham-lesioned rats, the electrode was placed along the same coordinates except that the depth was 5.5 mm from the duramater and no electrical current was passed. Bilateral MFB lesions were performed using an electrode (0.2 mm in diameter) positioned 5.0 mm rostral to the interaural line, 2.0 mm lateral to the midline, and 2.8 mm below the interaural plane (tooth bar 5.0 mm above 0.0), according to De Groot's atlas 7. The MFB lesions were made using a 2-mA current for 15s.
Aortic denercation Aortic denervation was performed as described elsewhere ~. Briefly, the rats were anesthetized with ether, and the carotid arteries and vagus nerves were exposed through a ventral cervical incision. The sympathetic trunk and the recurrent laryngeal nerve were identified and cut.
Histology The positions of the lesions were confirmed by histological examination of the brain tissue. At the end of the experiments, under ether anesthesia, the hearts were exposed for intracardiac perfusion with saline followed by 10% formalin. The brains were removed and stored in 10% formalin for at least 1 week. The tissue was then frozen and transverse sections (20-30/xm) were stained with hematoxylin and eosin for examination by light microscopy. Only the results observed in rats with typical MFB and AV3V lesions were used. The MFB lesions usually destroyed both the medial forebrain bundle and part of the lateral hypothalamus. The typical AV3V lesion was located below the level of the anterior commissure, with bilateral destruction of the periventricular tissue surrounding the optic recess of the 'lamina terminalis' through the preoptic and anterior hypothalamus, never extending caudally to the arcuate nucleus-median eminence region. The structures destroyed by the AV3V lesion included the preoptic and anterior hypothalamic periventricular nuclei, the median preoptic nucleus, and the anterior wall of the third ventricle with the associated organum vasculosum of the 'lamina terminalis '3.
Stat&tical analysis Bilateral carotid occlusion The bilateral common carotid arteries were occluded in conscious freely moving rats by means of pneumatic cuffs implanted 24 h before the experiment. In rats anesthetized with ether, the common carotid arteries were exposed through an incision in the ventral cervical region. A special cuff was adjusted around each carotid and a polyethylene tubing (PE 50) connected to the cuff was exteriorized through the back of the rat. For carotid occlusion the two cuffs were connected using a Y-shaped polyethylene tubing. Details about the preparation of the cuffs and the carotid occlusion were described elsewhere 2.~z.
Data are reported as mean + S.E.M. Statistical significance was calculated by analysis of variance and S t u d e n t - N e w m a n - K e u l s test. Differences were considered significant at P < 0.05.
Experimental protocol The pressor response to BCO was studied in different groups of intact-innervated and aortic baroreceptor-denervated (AD) rats submitted to either AV3V or MFB lesions alone (AV3V or MFB) or associated (AV3V + MFB lesions in the same rat). The pressor response to BCO was studied in the following groups of rats:
245 (a) Intact innervated rats: Sham lesion (MFB; 6 days + AV3V; 6 h) MFB lesion (6 days) AV3V lesion (6 h and 2 days) MFB lesion (6 days)+AV3V lesion (6 h and 2 days) (b) AD rats: Sham lesion (MFB; 6 days + AV3V; 6 h)+ AD (6 days) MFB lesion (6 days)+ AD (6 days) AV3V lesion (6 h)+ AD (6 days) MFB lesion (6 days)+ AV3V lesion (6 h)+ AD (6 days)
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Fig. 1. Increase in mean arterial pressure produced during 1 rain of BCO in intact-innervated sham-lesioned rats (S) and in rats submitted to MFB lesion (L-MFB, 6 days), AV3V lesion (L-AV3V, 6 h or 2 days) or L-MFB (6 days)+ L-AV3V (6 h or 2 days). The number of animals is indicated in parentheses at the top of the columns. ER, early response; LR, late response. (*), compared to sham (P < 0.05); (o), compared to L-AV3V (P<0.05); (e), compared to L-MFB
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The effects of A V 3 V and bilateral MFB lesions alone or associated on the pressor response to BCO in intact-innervated rats are presented in Fig. 1. MFB lesions alone (6 days) in intact-innervated rats (basal MAP= 112+3 mmHg) abolished the LR ( 9 + 1 mmHg), but produced only a small reduction of the E R (36 + 2 m m H g ) when compared to the pressor response produced by B C O in sham rats ( E R = 50 + 3 m m H g and LR = 32 + 2 mmHg, basal M A P = 122 + 3 mmHg). A V 3 V lesion alone (6 h and 2 days, basal M A P --- 112 + 6 and 121 + 3 mmHg, respectively) reduced both components ( E R = 20 + 2 and 22 + 3 mmHg, and LR = 21 + 2 and 16 + 2 mmHg, 6 h and 2 days after lesion, respectively). In rats submitted to simultaneous lesions of the A V 3 V region and bilateral MFB, an additional reduction in the pressor response to BCO was observed when compared to rats with A V 3 V or M F B lesions alone. A reduced E R (14 + 2 and 15 + 3 mmHg, respectively) was observed in rats
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Fig. 2. Increase in mean arterial pressure produced during 1 min of BCO in intact-innervated sham-lesioned rats (S), aortic denervated
(AD, 6 days) sham-lesioned rats and AD rats (6 days) submitted to lesion of the MFB (L-MFB, 6 days), AV3V (L-AV3V, 6 h) or L-MFB (6 days)+L-AV3V (6 h). The number of animals is indicated in parentheses at the top of the columns. ER, early response; LR, late response. (*), compared to control (P<0.05); (o), compared to sham-lesioned AD rats (P < 0.05).
with lesions of the MFB (6 days) + A V 3 V (6 h and 2 days, basal M A P = 114 + 3 and 114 + 4 mmHg, respectively) when compared to rats with MFB lesions alone submitted to BCO. On the other hand the LR (8 _+ 1 and 11 + 1 mmHg) observed in rats with MFB + A V 3 V lesions, was reduced when compared to the rats submitted to A V 3 V lesion alone.
Effect of AV3V and MFB lesions on the pressor response to B C O in aortic-denervated rats
The effects of A V 3 V and MFB lesions alone or associated with the pressor response to BCO in aorticdenervated rats are presented in Fig 2. An increased pressor response ( E R = 7 5 + 4 and L R = 6 5 _ + 3 mmHg) was observed in sham-lesioned A D rats (basal M A P = 124 + 3 mmHg) when compared to sham-lesioned intact-innervated rats (ER = 50 + 3 and LR = 32 + 2 mmHg). Either A V 3 V (6 h, basal M A P = 124 + 5 mmHg) or bilateral MFB lesions alone (6 days, basal M A P = 123 _+ 3 mmHg) or associated (basal M A P = 124 _+ 5 mmHg) reduced the pressor response to BCO in A D rats. A similar reduction of the pressor response to BCO in A D rats was observed after both MFB (ER=49+6 m m H g and L R = 4 1 + 5 mmHg), and A V 3 V lesions (ER = 35 _+ 3 m m H g and LR = 40 _+ 2 mmHg), and MFB + A V 3 V lesions ( E R = 40_+ 6 m m H g and LR = 35 _+ 7 mmHg). DISCUSSION The present results show a profound involvement of hypothalamic pathways in both components of the
246 p r e s s o r r e s p o n s e to B C O in conscious rats. Lesions of e i t h e r the A V 3 V region or M F B a l o n e r e d u c e d the p r e s s o r r e s p o n s e to B C O in intact or aortic b a r o r e c e p t o t d e n e r v a t e d rats as previously d e s c r i b e d 2'~4. In int a c t - i n n e r v a t e d rats s u b m i t t e d to s i m u l t a n e o u s lesions of the A V 3 V region a n d M F B , the p r e s s o r r e s p o n s e to B C O was practically abolished. T h e s e results suggest that the p r e s s o r r e s p o n s e to B C O in conscious rats is almost totally d e p e n d e n t on h y p o t h a l a m i c i n t e g r a t i o n , which is d i f f e r e n t from t h a t p r o p o s e d by L o p e s et al. ~. T h e s e a u t h o r s s u g g e s t e d that the i n t e g r a t i o n of L R would be in the h y p o t h a l a m u s , w h e r e a s the E R w o u l d be i n t e g r a t e d at m e d u l l a r y level. F u r t h e r m o r e , L o p e s et al. 9 also showed that lateral, but not m e d i a l hypothalamic lesions c h a n g e d the p r e s s o r r e s p o n s e to B C O in rats. T h e p r e s e n t results clearly d e m o n s t r a t e t h a t m e dial a n d l a t e r a l h y p o t h a l a m i c a r e a s a r e involved in the control of p r e s s o r r e s p o n s e to BCO. Since it is well r e c o g n i z e d that a n e s t h e t i c s i m p a i r reflex p r e s s o r response to BCO1, this could be an e x p l a n a t i o n for the d i f f e r e n c e s o b s e r v e d w h e n c o m p a r i n g the results of L o p e s et al. ~ with the p r e s e n t findings. A n o t h e r i m p o r t a n t o b s e r v a t i o n in the p r e s e n t results is c o n c e r n e d a b o u t the c e r e b r a l a r e a s involved in t h e i n t e g r a t i o n of the E R and LR. R a t s with simultan e o u s lesions o f b o t h the A V 3 V region a n d the M F B s h o w e d a r e d u c e d E R w h e n c o m p a r e d to rats with M F B lesions alone, b u t no d i f f e r e n c e was f o u n d w h e n c o m p a r i n g rats with A V 3 V lesion alone. O n the o t h e r hand, rats with d o u b l e lesion h a d a r e d u c e d L R w h e n c o m p a r e d to rats with A V 3 V lesion alone, but no d i f f e r e n c e was o b s e r v e d w h e n c o m p a r e d to rats with M F B lesion alone. T h e s e findings allow us to c o n c l u d e that in i n t a c t - i n n e r v a t e d rats the w h o l e E R is d e p e n d e n t on the A V 3 V region, w h e r e a s the w h o l e L R d e p e n d s on M F B . T h e i n c r e a s e d p r e s s o r r e s p o n s e o b s e r v e d in A D rats was also r e d u c e d a f t e r A V 3 V or M F B lesions a l o n e or associated. H o w e v e r , the effects of lesions w e r e very d i f f e r e n t in A D rats w h e n c o m p a r e d to t h e effects of lesions in i n t a c t - i n n e r v a t e d rats. First, in A D rats, A V 3 V or M F B lesions a l o n e or a s s o c i a t e d r e d u c e d b o t h E R a n d L R to t h e s a m e d e g r e e . S e c o n d , the r e d u c t i o n in E R a n d L R p r o d u c e d by A V 3 V o r M F B lesions in A D rats w e r e s m a l l e r t h a n t h o s e o b s e r v e d in i n t a c t - i n n e r v a t e d rats (e.g. t h e r e d u c t i o n of the L R after M F B lesion in i n t a c t - i n n e r v a t e d rats was 70%,
a n d the r e d u c t i o n of this c o m p o n e n t o b s e r v e d in A D rats with the s a m e lesion was 35%). T h e s e results suggest that the inhibitory activity of the aortic b a r o r e c e p t o r s may play an i m p o r t a n t role in the effects of h y p o t h a l a m i c lesions r e d u c i n g the p r e s s o r r e s p o n s e to BCO. The authors thank Reginaldo E. Queiroz for the technical assistance and Eliane Rodrigues dos Santos for typing the manuscript.
Acknowledgements.
REFERENCES 1 Bedran de Castro, M.T.B., Farah, V.M.A. and Krieger, E.M., Influence of general anesthetics on baroreflex control of circulation, Braz. J. Med. Biol. Res., 23 (1990) 1185-1193. 2 Bedran de Castro, M.T.B., Moreira, E.D. and Krieger, E.M., Reflex and central components of carotid occlusion in conscious rats. Effect of lesion of the medial forebrain bundle, Hypertension, 8 (Suppl. I) (1986) 147-I51. 3 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, Cir. Res., 43 (Suppl. I) (1978) 1-13. 4 Brody, M.J. and Johnson, A.K., Role of the anteroventral third ventricle region in fluid and electrolyte balance, arterial pressure regulation and hypertension. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinology, Raven, New York, 1980, pp. 249-292. 5 Chai, C.Y., Share, N.N. and Wang, S.C., Central control of sympathetic cardiac augmentation in lower brain stem of the cat, Am. J. Physiol., 205 (1963) 749-753. 6 Chai, C.Y., and Wang, S.C., Integration of sympathetic cardiovascular mechanisms in medulla oblongata of the cat, Am. J. Physiol., 215 (1968) 1310-1315. 7 De Groot, J., The rat forebrain in stereotaxic coordinates, Trans. R. Neth. Acad. Sci., 52 (1959) 1-40. 8 Krieger, E.M. and Marseillan, R.F., Aortic depressor fibers in the rat: an electrophysiological study, Am. Z Physiol., 205 (1963) 771-774. 9 Lopes, O.U., Cipola-Neto, J, Rocha e Silva, M.Jr., Hypothalamic component in pressor response to carotid occlusion in the rat, Am. J. Physiol., 233 (1977) H240-H247. 10 Lopes, O.U., Timo-Iaria, C., Leit~o Filho, H.A.P., Suppression of the late component of the carotid occlusion reflex by lesion of the medial forebrain bundle in the rat, Braz. J. Med. Biol. Res., 14 (1981) 37-41. 11 Machado, B.H., Bonagamba, L.G.H, Castania, J.A. and Menani, J.V., Changes in vascular resistance during carotid occlusion in normal and baroreceptor-denervated rats, Hypertension, 19 (suppl II) (1992) 149-153. 12 Maio, A.A., Moreira, E.D., Salgado, H.C. and Krieger, E.M., Cardiovascular responses of conscious rats due to arterial occlusion, (Abstract), Braz. J. Meal. Biol. Res., 14 (1981) 115. 13 Manning, J.W., Cardiovascular reflexes following lesions in medullary reticular formation, Am. J. Physiol., 208 (1965) 283288. 14 Menani, J.V., Bedran de Castro, M.T,B. and Krieger, E.M., Influence of the anteroventral third ventricle region and sinoaortic denervation on the pressor response to carotid occlusion, Hypertension, 11 (Suppl I) (1988) I178-I181.