Toxlcas, 1976, VoL 14, Dp. 133-138 . Per¢smon Pros . Printed In Gnat Hritein.

STUDIES ON THE CENTRAL VASOMOTOR REFLEXES IN CATS AFTER INTRAVENTRICULAR ADMINISTRATION OF WHOLE VENOM OF DENDROASPIS JAMESONI B. V. TE1.aNG, R. J. M. Lu~rvxYa, D. NJOROGE Department of Medicine (Pharmacology and Therapeutics), University of Nairobi, P.O. Box 30588, Nairobi, Kenya and L. GALZIGNA Department of Biochemistry, University of Padova, Via Marazola 3, Padova, Italy (Accepted forpublication 16 October 197 B. V. Telaxo, R. J. M. LmnvYn, D. NJOIt0C31i and L. G~l zlox~. Studies on the central vasomotor reflexes in cats after intravontricular administration ofwhole venom of Dendroaspis jamesoni. Toxicon 14,133-138,1976.-Administration into the lateral ventricles ofwhole venom (100Fig) of Dendroaspis jamesonioonsistently producedhypotensionand bradycardia in cats in a dose one-tenth of that required to produce the same effect by the intravenous route. No hypotension or bradycardia oaurr+ed after the perfllsate was tapped through the caudal and ofthe aqueduct of Sylvias . The cardiovascular response was again observed after injection of venom intothethird ventricle and the perfusate tapped through the cisterns magna. Injection ofvenom directly into the cisternsmagna did not produce any cardiovascular response. Tho site ofaction of the venom appeared to be on the floorof the fourth ventricle, possiblythevasomotor centre. This was confirmed when intraventricular administration of the venom depressed centrally mediated cardiovascular reflexes, viz. presser response following carotid occlusion and following electrical stimulation ofthe central ~d ofthe left vagus nerve after bilateral cervical vagotomy and carotid denervation. The venom also decreased the depressor response to electrical stimulation of the central cut end of the right vague nerve. These cardiovascular responses were central effects and from the results obtained after bilateral vagotomy removal ofboth stellate ganglion and transaction of the spinal cord (G2) it was evident that the efferent nervous pathway for the central cardiovascular effects of the venom was the sympathetic nervous system. INTRODUCTION Ir xas been reported ~MELDRUM, 196 that injection of 10 mg of the venom of Naja naja, or Lachesis alternates into the subarachnoid space in dogs produced a rapid rise in arterial blood pressure, slowing of the pulse and later diminution in and arrest of respiration . Administration of Crotalus terrificus venom in the `isolated' dog head produced transient vagal inhibition of the heart and stimulation of respiration with a latency of less than 1 min. Intraventricular injections of up to 5 mg of venom from various Crotalidae produced parasympathetic and locomotor effects in cats ~RUSSII,L and Bore, 1962) . At present very little is known about the pharmawlogical action ofthe neurotoxin of Dendroaspisjamesoni on the central nervous system or whether the venom has the ability to penetrate the bloodbrain barrier. This paper deals with the action of whole venom on the vasomotor centre and the effect it produces on the centrally mediated vasomotor reflexes. MA~.RrA~s AND METHODS Eighty-three cats of either sex weighing between 2 and 4 kg were anaesthetized with ether followed by chloralose (80 mg per kg) intravenously. The cats were kept on artificial respiration and the rectal tempera133 TOXICON 1976 Yol. 14

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B. V. TELANG, R. J. M. LUTUNYA, D. NJOROGE and L. GALZIGNA

tore maintained between 36 and 37°C throughout the experiment. The left common carotid artery was cannulated for recording the blood pressure on a smoked kymograph. Heart rata was recorded from the electrocardiogram (Lead In using a Phillips Cardiopan electrocardiograph (Model 531) . Intravenous igjections of snake venom were made through the right femoral vein . Lgjections into the right vertebral artery were made retrogradely through the right brachial artery according to the method of Gnrrormá et al. (196 . Intraventricular and intracisternal igjections were given in a volume of 0~2 ml of normal saline ; for all other routes the snake venom was administered in a volumo of Oó ml normal saline . Intraventricular and intracisteraal carmulations Lateral ventricular cannulation was carried out according to the method Of Fe~nHERG and SxeRwoon (1953) . Cannulation of the third ventricle was carried out according to the following parameters : 7 mm in front of the interauricular plane and 3 mm lateral to the sagittal suture ; the length of the cannula was 17 mm and was directed vertically downwards. To cannulate the caudal end of the aqueduct of Sylvius, the occipital plate was chipped off after cutting tho neck muscles in the midlino, the cerebellum was gently lifted up by a blunt spatula and a thin polyethylene tube (outer diameter 2 mm) was pushed gently into the aqueduct for about 3-4 mm distance. Confirmation that the Collison's cannula was in the ventricles or the polyethylene tube in the aqueduct of Sylvius was obtained after injection of methylene blue and subsequent post mortem examination of the brain. The cerebrospinal fluid was tapped out of the cisterns magna by a No . 18 hypodermic needle with a etilette, pushed through the atlanto-occipital membrane in the midliline and firmly held in position . Central vasomotor reflexes The action of snake venom on the central vasomotor areas was assessed by observing the effects of the venom on (a) the reflex presser response due to occlusion of the right common carotid artery, (b) depression of blood pressure due to electrical stimulation of the central cut end of the right vages nerve, (c) the reflex presser response to electrical stimulation of the central cut end of the left vages nerve after bilateral carotid denervation and vagotomy. The electrical pulses were delivered through bipolar shielded electrodes from a square-wave stimulator (C. F. Pahner, Mode18044) . Effective stimulus parameters for (b) varied from 8 to 10 V at a constant frequency of 10 Hz and a pulse width duration of 0~ 1 msec and for (c) they were 10 V at a constant frequency of SO Hz and a pulse width duration between 0~1 and 0~4 meet. In both (b) and (c) the stimulation was maintained for a period of 20 sec. Surgical interruption of efferent pathways To find out if the efferent nervous pathway for the cardiovascular response produced by intraventricular igjections of the venom was the vague or the sympathetic, three surgical procedures for interrupting nervous pathways were used. (a) bilateral vagoto~y in the neck, (b) transverse cervical section of the spinal wrd at G2, (c) bilateral ganglionectomy of the stellate ganglia . For this purpose the sympathetic chain up to the third thoracic ganglion was exposed through a curved incision in the axilla extending from the coatoclevicular junction to just beyond the mid-axillary line. The first, second and third ribs were cut at their angles and gently retracted, the stellate ganglion was removed on each side with its branches and a piece of sympathetic chain about 2~5 cm long including the 3rd thoracic ganglia. Statistical analysis of the data and the level of significance was determined at 95 ~ confidence limits . Values of P greater than 005 were considered not significant. Materials Atropine sulphate (Sigma, London) and propantheline bromide (G . D. Seeds Co ., England) were dissolved in normal saline . Desiccated whole venom was obtained from Baringo Snake Farm, Nakuru, Kenya and were kept in the re&igerator at 4°C prior to use. Suitable quantities of venom were weighed and dissolved in normal saline, for every experiment . RESULTS

Experiments with intravenous and intravertebral injections

Intravenous injection of whole snake venom in a dose of 100 itg did not produce any perceptible change in blood pressure or heart rate (3 cats). Administration of 1 mg of venom intravenously produced a steep fall in blood pressure within 1 min and was accompanied by bradycardia (5 cats). The normal mean blood pressure and heart rate were 109 ~ 4~5 mm of Hg (mean ~ SE) and 202 ~ 9~2 beats per min (mean ~ SE), respectively, and the decreases induced by the venom were 358 ~ 4~5 mm of Hg and 424 ~ 0~9 beats TOXICON 1976 Yol. 14

DendroaapLrJamesoni and vasomotor reRexes

135

per min, respectively, from the normal . Although the blood pressure recovered partially, it did not return to normal levels even after 60 min, whereas the heart rate returned to normal in an average period of 15 min. Intravenous administration of snake venom (1 mg) in cats 1 hr after pretreatment with propantheline bromide (6 mg per kg intravenously) produced only a small fall in blood pressure and heart rate (21"4 f 5 " 8 mm of Hg and 15"4 ~ 1 "2 beats per min, respectively ; 6 cats). Propantheline was administered intravenously slowly in three divided doses at intervals of 10 min. Propantheline caused a rapid fall in blood pressure, which returned to preinjection levels within 6 min ; there was no significant change in the heart rate (201 t 9" 7 to 209 f 5 "4 beats per min). The mean blood pressure and heart rate 1 hr after injection of propantheline and before intravenous administration of snake venom (1 mg) was 114 f 2"4 mm of Hg and 209 ~ 5 "4 beats per min, respectively . Statistical analysis showed a significant difference between the effect of venom (1 mg) in control and in propantheline pretreated cats (blood pressure, P < 005 ; heart rate, P < 0"001). Injection of snake venom (100 ltg) into the intravertebral artery produced a fall in blood pressure of 24"2 f 1"8 mm of Hg accompanied by a fall in heart rate of 23 "4 ~ 3"3 beats per min (5 cats). The normal mean blood pressure and heart rate before injection ofthe venom were 114 ~ 1 "9 mm of Hg and 212 ~ 6"9 beats per min, respectively. Experiments with intraventricular (lateral and third ventricle) and intracisternal injections

Lateral ventricular administration of 1001íg snake venom in cats produced a persistent fall in blood pressure and heart rate . The fall in blood pressure occurred within 2 min and the blood pressure and heart rate returned gradually to normal levels after an average time of 62 min after administration of venom (Fig. 1). The results of administration of snake venom into the lateral ventricle, 3rd ventricle and cisterns magna are su mmari~rrl in Table 1. TAatE 1 . INTRAVBNTRICULAR ADAQNISIRATION OF SNAKE VENOM (100 pg) Arm rrs EFFECT' ox E(AOD rxESSVRE AND HEART AATE

N 9 S S 3

Injection of snake venom into

Lateral v~tricle (wntrls) Lateral and third ventricle' Third ventriclet CisDeana magna'

Blood pressure (mm Hg f SE) Decrease Normal after venom

109 ~ 4" S 104 t 4"8 100 f S" 6 113 f 3" á

24 " 8 f 2"9 0 f 0$ 23 "4 f 2~7 0 f 0$

Heart rate (beats per min f SE) Docrcgae Normal after venom

202 f 9"2 209 f 3~4 201 f 8"9 20á f 4"7

22"4 f 3"4 0 f 0$ 234 f 2"2 0 f 0$

N donotea total number of cats used . 'The perfusate tapped out through the aqueduct of Sylvius. j~Tho pecfusate tapped out through the cisterns magna. $These values are signiScantly different (P < 0"OS) from the decreases in the controls (lateral ventricle injection) .

Central pretreatment with atropine

Snake venom (100 Ftg) injected into the lateral ventricle of 4 cats 1 hr after pretreatment with atropine (1 mg intraventricularly into the lateral ventricle) caused only a slight fall in blood pressure (7 ~ 2"3 mm Hg; mean normal blood pressure 82 ~ 1 "4 mm of Hg) and no fall in the heart rate (mean normal heart rate 202 ~ 19"0 beats per min). Statistical analysis of the decrease of blood pressure by venom in the atropinized and control cats TOXICON7976 Vol.

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B. V. TELANG, R. J. M. LUTUNYA, D. NJOROGE and L. GALZIGNA

showed a significant difference (P < 0"001). On central pretreatment with a higher dose of atropine (5 mg) there was no hypotension and bradycardia after intraventricular (lateral ventricle) administration of 100 !Ag snake venom (4 cats). The mean normal blood pressure and heart rate before administration of venom was 82"5 ~ 1 "4 mm of Hg and 169 X2"2 beats per min, respectively. Surgical interruption of nervous pathways The results are summarized in Table 2. It is evident that bilateral vagotomy did not alter the cardiovascular response to intraventricular administration of venom. After bilateral TASLB 2. EFFECT OF SURGICAL INTERRUPTION OF NERVOUS PATHWAYS ON THB BLOOD PRESSURE AND HEART RATE AFTER LATBRAL VENTRICULAR ADMINISTRATION OF SNAKH VENOM (100 /lg)

N 9 6 5 7

I~jaction of snake venom into the Lateral v~tricle (controls) Spinal cord transection Bilateralstellateganglionectomy Bilateral vagotomy

Blood pressers (mm Hg f SE) Decrease Normal after venom 109 f 4"S 24 "8 f 2"9 82 f 1 "1 0 f 0' 26"6 f 2"2 91 f 5~1 108 f 4~2 23 "6 f 3"9

Heart rate (beats per min f SE) Decrease Normal aftervenom 202 f 9"2 22~4 f 3"4 135 f 10"2 6"0 f 0"82' 162 f 14"3 4"0 f 3"1" 214 f 11 "S 24"4 f 3"3

N denotes total number of cats used for the experiment . "These values are signiScantly different (P < 0~0~ from the decreases in the controls (lateral ventricle injection) .

stellate ganglionectomy there was a fall in blood pressure but this procedure prevented the fall in heart rate . Spinal cord transaction at cervical level (C-2) prevented both the fall in blood pressure and heart rate . The tests of significance were applied (Tables 1 and 2) by comparing the decreases in blood pressure and heart rate after lateral ventricular administration of venom with the decreases in the other groups . Effect of snake venom on centrally mediated cardiovascular reflexes The effects of snake venom on the central vasomotor responses to carotid occlusion and central vagal stimulation were studied in eleven cats . The snake venom (100 ltg) was administered into the lateral ventricles in order to get direct access to the central vasomotor areas. The results of one such study are shown in Fig. 2a and 2b . When the carotid sinuses were denervated and the vagi cut, electrical stimulation of the central cut end of the left vaues nerve elicited a pressor response (5 cats). The pressor response was greatly reduced when the vaues was stimulated after intraventricular injection of snake venom (100 lIg). The results of one such study are shown in Fig. 3. DLSCUSSION

The most common experimental pharmacological effect of snake venoms, with few exceptions, is a profound cardiovascular depression which predominates over the paralytic action of the venom. Intravenously injected snake venoms generally cause a precipitous fall in blood pressure in 15-30 sec followed by a partial recovery and then a gradual or rapid descent of systemic arterial pressure terminating in death (Jn~NEZ-PORRAS, 1968). It is currently agreed that the abrupt hypotension is attributable to a peripheral action upon the capillaries (RUSSELL et al., 1962 ; BI-IANGANADA and PERRY, 1963 ; HALMAGYI et al., 1962). On the other hand, the mechanism of abrupt hypotension evoked by V. palestinae TOXICON 1976 Vol. 1~

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Cat: 3~2 kg. Anaesthesia chloralose (80 mg per kg). Records of arterial blood pressure (A) and the E.C .G . (B) before and 5 min after igjection of snake venom. The arrow indicates the point at which snake venom (100 fig) was administered into the lateral ventricle. The fall in blood pressure was from 100 to 75 mm Hg and the corresponding decrease in heart rate from 166 to 142 beats per min. The blood pressure and heart rate T+eturned gradually to normal levels in S8 min.

TOXICON1976 Vol. 1~

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lhndroaspir jameaorei and vasomotor reflexes

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venom is exceptional and consists of a peripheral vasodilatioa due to a primary action of viperatoxin on medullary vasopressor centres (BicF»at et al., 196 . In our experiments, intravenous administration of 1 mg snake venom produced hypotension and bradycardia, whereas 100 pg had no effect. On the other hand, 100 Rg snake venom administered into the lateral ventricle consistently produced hypotension and bradycardia; thus the dose of snake venom required to produce an effect was one-tenth of that required by the intravenous route. The effect of intraventricular administration of 100 pg snake venom thus appears to be central rather than peripheral in origin . Prior intravenous administration of propantheline did not completely abolish the hypotension and bradycardia due to intravenous injection of snake venom (1 mg) even though the dose ofpropantheline was as high as 6 mg per kg. Propantheline is aquaternary ammonium compound with powerful antimuscarinic actions; it does not readily penetrate the blood-brain barrier (INNE4 and Nicx~esox, 1970). Thus the attenuation of cardiovascular response to intravenous injection of snake venom (1 mg) may be due to blockade of muscarinic sites in the heart and blood vessels by propantheline. The ability of the venom to penetrate the blood-brain barrier is borne out by the fact that intravertebral administration of 100 pg snake venom produced hypotension and bradycardia. It is therefore possible that a small portion of the intravenously administered venom may penetrate the blood-brain barrier to reach the crucial sites in thecentralnervous system. Administration of snake venom into the lateral ventricles consistently produced hypotension and bradycardia. No change in blood pressure or heart rate was noticed when the snake venom had no access to the floor of the fourth ventricle (perfusate tapped through aqueduct of Sylvius) . Injection of 100 pg snake venom into the third ventricle (the perfusate tapped out through the cisterns magna) produced hypotension and bradycardia. No change in blood pressure or heart rate was noticed after injection into the cisterns magna (see Table 1). Thus the site of action of snake venom appears to be the floor of the fourth ventricle, probably the vasomotor centre. There is ample evidence that the vasomotor centre is cholinergic in nature (Sixxn et al., 1967 ; ROZBnR et al., 1968 ; $RIMAL et al., 1969). Acetylcholine when administered intraventricularly produces a pressor response and has an excitatory effect on the medullary vasomotor centre (Szxx~ et al., 1967). Crude venom contains various components such as phospholipase A, cardiotoxin, etc. it is possible therefore that the inhibitory effect of whole venom on the vasomotor centre is due to these components present in the venom and not due to an acetylcholine like component. The absence of cardiovascular response to intraventricular snake venom after central pretreatment with atropine (5 mg) may be due to prior blockade of the vasomotor centre by atropine preventing the access of one or more venom components to the postsynaptic receptors in the vasomotor centre. En>utY and GUERTZEN31'8IN (1974) has shown that central pretreatment with atropine blocks the depressor effects of Dyflos (isoflurophate) applied topically to the ventral surface of the bramstem of the cat. Intraventricular administration of snake venom reduced the depressor response to central vagal stimulation and the reflex blood pressure rise to carotid occlusion thus suggesting that depression by the snake venom is at the brain stem level probably on the vasomotor centre. This was confirmed, after bilateral carotid denervation and vagotomy, when the pressor response to stimulation of the central cut end of the left vagus nerve was reduced by the prior intraventricular administration of snake venom. The sympathetic nervous system appears to be the efferent pathway for mediation of TOXICON 19~6 Yd. 1~

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B. V. TELANG, R. J. M. LiTT[JNYA, D. NJOROGE and L. GALZIGNA

hypotension and bradycardia after intraventricular injection of snake venom (see Table 2), because the effect still persisted after bilateral vagotomy . On the other hand, hypotension without bradycardia occurred after stellate ganglionectomy, whereas neither hypotension nor bradycardia occurred after spinal cord transection at the G21eve1. Thus inhibition of the sympathetic vasomotor tone of the lower half of the body by snake venom, administered intraventricularly, appears responsible for the hypotension. According to classical concepts, the vasomotor centre consists of a vasopressor and vasodepressor area. The rise of blood pressure and heart rate is due to an increase in the tonic discharge from the vasopressor area which excites the thoracolumbar sympathetic vasoconstrictor fibres and the thoracic sympathetic fibres destined for the heart . The vasodepressor area inhibits the tonic discharge from the vasopressor centre. However, KEH,13 and N>3rI. (1972) have pointed out that there is no clear anatomical separation between the pressor and depressor areas and in the intermediate region of the upper medulla the areas overlap. Hence the authors have suggested that it is appropriate to use the term `medullary cardiovascular' centre recognizing that the area contains both neurones which excite thoracolumbar sympathetic fibres to the heart and blood vessels and neurones which inhibit these sympathetic fibres . Thus, the fall in blood pressure and heart rate may be due to a direct inhibitory effect of the venom on the so-called `medullary cardiovascular' centre reducing the tonic discharge of impulses to the sympathetic vasoconstrictor fibres and to the cardioaccelerator nerves . Acknowledgements-We thank the Dean's Committee, University of Nairobi, for the research grant (670-052) which supported this work . We thank Mr. S. K. Nvwaw and Mr . J. N. No'wNa'w for their technical assistance and Miss J. JHrrE for the photographs. REFERENCES BHwxawxwnw, K. and PExxY, J. F. (1963) Cardiovascular effects of cobra venom. J. Am. med. Ass.183, 257. Bic~x, H. L, RozH, M. and Grrr~, S. (1966) Neurotoxic activity of Vipers palestfirae venom. Depression of central vasoregulatory mechanisms . Med. Pharmac. Exp. 14, 349. EDERY, H. and GuERZZertsr~t, P. G. (1974) A central vasodepressoreffect of dyt]os . Br . J. Pluamac. S0, 481. FELDEERO, W. 8nd SHERWOOD, S. L. (1953) Permanent cannula for intraventricular igjections in cats . J. Physiol. 120, 3p. Gwrroxn~, B. B., McCwazHY, L. E. and Honisox, H. L. (1965) Central emetic action and toxic effects of digitalis in cats . J. Pharmac. exp. TRer. 147, 409. Hwt~waYt, D. F. J., Szwn~.Fre~,_ H. and Hoxxsx, G. (1965) Mechanism and pharmacology of shock due to rattlesnake venom in sheep. J. appl. Phystol. 20, 709. IrttvES, I. R. and Ntc~xgox, M. (1970) Drugs inhibiting the action of acetylcholine on structures innervated by postganglionic parasympatlretic nerves (antimuscarinic or atropinic drugs) . In : 77ee Pharmacological Bask of ?9~erapeutics, p. 536, (GoonrtwN, L. S. and Gnaiwx, A. Eds.), New York : Macmillan. JnaßxEZ-PoR.x~s, J. M. (1968) Pharmacology of peptides and proteins in snake venoms. A. Rev. Pharnwc. 8, 303. KEBt.E, C. A. and NEII., E. (1972) Neural control of the cardiovascular system. In : Samson Wright's Applied Physiology, pp. 126, 127. Oxford : Oxford University Press. MELDRUM, B. V. (1965) Tho actions of snake venoma on nerve and muscle . The pharmacology of phospholipase A and of polypeptide toxins . Pharmac. Rev. 17, 398. Rozewx, W., BuecHEx, R. P., CHAT, C. Y. and Wwxa, S. C. (1968) Effect of intracerebroventricular 1hyposcyamine, ethybenzlrapine and procaine on cardiac arrhythmias produced in dogs by pentylenetetrazol, picrotoxin and deslanoside . Int. J. Neuropharmac . 7, 1. RUSSELL, F. E. and BOHR, V. C. (1962) Intraventricular igjection of venom. Toxic. appl. Pharmac. 4, 165. RUSSELt, F. E., BUESS, F. W. and SzxwsEExa, J. (]962) Cardiovascular response to Crotalus venom. Toxkon 1, 5. Sixxw, J. N., DHwwwx, K. N., CHwxnxw, O. and Gvrrw, G. P. (1967) Role of acetylcholine in central vasomotor regulation . Can. J. Physiol. Pharmac. 45, 503. $RIMAL, R. C., Janl, B. P., S~xxw, J. N., Dnaz, K. S. and BHARGAVA, K. P. (1969) Analysis of central vasomotor effects of choline. Eur. J. Pharmac. 5, 239. TOXICON 1976 Vol. I~