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The site of action of the venom of the sea snake Enhydrina schistosa
153 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 55.
No. 1.
March, 1961.
T H E S I T E O F A C T I O N O F T H E V E N O M O F T H E SEA S N A K E E N H Y D R I N A
SCHISTOSA BY
J. E. CAREY AND E. A. WRIGHT*
(From St. Mary's Hospital Medical ,School, London) T h e peripheral action of many neurotoxic snake venoms has been demonstrated but a central action has also been postulated. T h e early work on this problem has been reviewed by KELLAWAY, CHERRY and WILLIAMS (1932). These authors presented further evidence o f the peripheral action of a n u m b e r of neurotoxic snake venoms, but were also able to produce changes in the respiratory activity of animals by direct application of cotton wool soaked in venom solutions to the floor of the fourth ventricle. T h e y suggested that these results may have been indirect and due to local vasoconstriction. We have tried to elucidate this problem by direct intra-arterial injections of crude venom and also by injecting crude and fractionated venom into the medulla oblongata. MATERIALS AND METHODS
Venom.--The dried crude venom of Enhydrina schistosa was supplied by Dr. H. A. Reid, Penang General Hospital, who has given details of its collection (REID, 1956). The crude venom was made up in physiological saline before use. Two fractions of the venom were prepared by dialysis and cellulose column chromatography (CAREY and WEIGHT, 1960a). The first fraction contained 95 to 100 per cent. of the toxicity of the whole venom as found by intraperitoneal injection into mice, but no lecithinase activity. The second fraction had lecithinase activity equivalent to that of the whole venom, but less than 5 per cent. of the initial toxicity of the whole venom. The toxicity of the venom fractions was tested by injecting mice intraperitoneaUy with the solutions and comparing the survival times with mice injected with whole venom. The LDs0 for mice and rabbits had been previously determined to be between 50 and 120 btg./kg. (CAREYand WmCHT, 1960b). Animals.--Rabbits of various breeds and sexes between 2 and 3.6 kg. were used. For comparative experiments the animals were as nearly matched for weight as possible and the doses calculated on a weight basis. Anaesthetic.--Intravenous " Nembutal " (0.4 ml./kg.) fortified by open ether was used for all operative procedures. Femoral artery injections.--The standard procedure was to cannulate the femoral artery high in the thigh and inject 0.02 ml. of heparin BP (5,000 IU/ml.) made up to 0.5 ml. in physiological saline. A slow saline drip was then maintained into the peripheral circulation of the limb through the cannula. Following this a shaped perspex clamp was fitted round the lower abdomen and a rubber bag inside this clamp was inflated to 200 ram. Hg. pressure thereby cutting off the flow of arterial blood into the limb. The femoral vein was now cannulated and fluid which drained was collected, Various doses * We are very grateful to Dr. H. A. Reid for supplying us with dried venom. The Wellcome Foundation made this work possible by: a grant for the salaryofone of us (J~E.C.).
154
A C T I O N OF V E N O M O F
Enhydrina schistosa
of venom were then injected into the arterial cannula at several different rates. The artery and vein were then tied off and the same procedure carried out on the opposite side, the venom being replaced by an equal volume of saline. The clamp was removed at the end of the operation and also occasionally for a short period after completing the first side.
Vertebral artery injections.--Using the method described by BRIDGEWATER,MORGAN, ROSEN and PAYLING WRIGHT (1955) the first part of one vertebral artery was exposed and tied proximally. Venom in volumes ranging from 0.1 to 0.5 ml. was then injected into the distal part of the artery during the course of 10 to 30 seconds. Medullary injections.--The obex of the medulla oblongata was exposed and injections of venom in volumes of 0.001 to 0.005 ml. were made 2 ram. lateral to the obex and at a depth of 2 ram., using a fine glass capillary needle (WRIGHT, 1954). I n one case, 0.005 ml. was injected into one side and 0. 004 ml. was injected into the other. Pharmacological preparations.--The rat phrenic nerve diaphragm preparation as described by B/3LBRING (1946) was used and the arrangement for the close injection of acetylcholine was prepared according to BURGEN, DICKENS and ZATMAN (1949). RESULTS
Injections into the medulla oblongata of rabbits T a b l e I shows the results of i n j e c t i n g whole v e n o m a n d fractions of v e n o m i n t o t h e m e d u l l a o b l o n g a t a of rabbits. W h o l e v e n o m i n j e c t e d i n doses of 1/10th to 1/5th LDs0 caused r a p i d death. T h e dose h a d to be r e d u c e d to a p p r o x i m a t e l y 1/200th LDs0 before animals survived permanently. TABLE I. The results of injecting various doses of whole venom and venom fractions into the medulla oblongata of rabbits. The numbers in brackets are the survival times in hours of animals dying within 24 hours. (50 ~g./kg. = 1 LDs0). Doses of whole venom or fractions obtained from these initial doses. Rabbit LDs0's
Number of animals surviving more than 24 hours/Number of animals used. Whole venom
Toxic fraction
0.2
0/4
(2, 3, > 18, > 19)
0.1
0/4
(2, 3, 3, 4)
O. 08-0.07
0/1
(<21)
0.05-0.04
0/1
(4)
2/2 b
0.03-0.02
1/3 c
(2, <24)
2/2
O. 01-0.009
0/2
(6, <21)
1/1
0. 005
1/2 e
(5)
0. 002
2/2
1/1 a
Lecithinase fraction
0/1
(3)
0/2
(6, 8)
0/1
(<17)
1/1 d
a = killed after 3 days showing slight weakness of neck muscles ; b = one animal was killed at 31 hours showing slight weakness in right forelimb ; the other rabbit killed after 15 days showing extreme torticollis to the left ; c = one animal killed after 28 hours showing occasional convulsions and weakness ; d = animal survived 2 days ; e = one animal killed after 5 hours because it started rolling over and over violently ; the other survived.
155
j . E. CAREY AND E. A. W R I G H T
When the lecithinase activity of the whole venom was removed by fractionation the toxicity, as judged by intraperitoneal injection into mice, remained. When this fraction was injected into the medulla no rapid deaths occurred over a wide range of doses, although three rabbits receiving high doses were showing slight signs of medullary damage when they were killed. However when doses of the lecithinase fraction equivalent to the lecithinase activity of the whole venom doses were injected, comparable death times were recorded. It should be emphasized that this lecithinase fraction contained 95 to 100 per cent. of the lecithinase activity of the original whole venom, but when injected intraperitoneally into mice it contained less than 5 per cent. of the original toxicity. On the other hand, no lecithinase activity could be detected in the toxic fraction. Rabbits receiving either whole venom or fractions containing the lecithinase activity showed a similar clinical picture. The respiration rate was at first increased and was frequently associated with gasping or wheezing, but in the final stages before death the respiration rate became very slow and the animal became prostrated and died in coma. Most animals exhibited torticollis to the injected side and some of them made persistent circling movements in that direction. Many of them also showed convulsions in the later stages, which may have been due to anoxia owing to the failure of respiration. Rabbits injected with saline alone showed no ill effects from the operations. Most of the survivors from the venom injections were kept for a week.
Injections into the vertebral artery Two rabbits were given 0.7 LDs0 of whole venom into one vertebral artery. One showed no ill effects from this procedure and one showed some transitory generalized weakness and gasping 24 hours later, but no unilateral signs were present and the animal was fully recovered after 1 week. No abnormal signs followed the injection of 0.1 LDs0. The rabbit LDs0 was calculated on the basis of 50 ~g./kg. and in a previous experiment 0.7 I, Dso had produced a slight transitory weakness when given intravenously.
Injections into the femoral artery Rabbits were given injections of venom into the femoral artery in doses ranging from 6.25 to 50 ~g./kg. (Table II). The clamp was applied for periods ranging from 10 to 35 minutes and the venom injected over times ranging from 1 to 20 minutes. The loss of fluid from the venous cannula was usually slight, but on two occasions 1/3rd of the dose of venom was detected in this fluid. No doubt the variability in these factors accounted for at least some of the differences in the degree of paralysis at each dose level. TABLE II.
The results of localized injections of venom solution into the femoral artery of rabbits. Dose ~tg./kg.
50
Number of animals used Number of animals showing complete local paralysis immediately after recovery from anaesthesia
4
25
12.5
625
156
ACTION O F VENOMOF Enhydrina schistosa
(i) Animals receiving 50 ~tg./kg.--Rabbits 1 and 2 died of generalized paralysis within 2 days. Rabbits 3 and 4 were still completely paralysed in the injected leg when killed 63 and 97 days later. Rabbit 5 showed a transitory equivocal local paralysis. (i~) Animals receiving 25 ~tg./kg.--Rabbit 6 developed complete local paralysis, but was accidentally killed by an overdose of anaesthetic after 8 days. Rabbit 7 showed persistent paralysis but developed gross local infection and was killed after 87 days. Rabbits 8 and 9 showed only partial paralysis originally and this completely recovered after 8 to 15 days. (iii) Animals receiving 12.5 ~Lg./kg.--Rabbit 10 was completely paralysed locally and there was no recovery after 81 days. Rabbit 11 showed partial local paralysis on the 2 days following the venom injection, but was killed after 16 days owing to spastic extension of both hind limbs and an ulcerated area on the back. The spastic extension may have been due to the clamping procedure, but there were no other examples of this in any of the experimental or control animals. Rabbit 12 showed slight paralysis of the injected limb which recovered after 3 days. (iv) Animals receiving 6.25 gg./kg.--Rabbits 13 and 14 : neither animal showed any paralysis after recovery from the anaesthetic.
Electrical and histological changes in calf muscles The electromyographic response in the calf muscles following a sharp tap to the Achilles tendon was observed with a cathode ray oscilloscope. In three of the rabbits showing complete paralysis the tendon jerk trace was found to be reduced or absent immediately after recovery from the anaesthetic. Direct stimulation of the paralysed muscle with up to 9 volts (1 to 10 m.sec.) immediately on recovery from the anaesthetic caused contraction of the muscle. Direct stimulation was tried 1 day after paralysis in only one animal and in this case there was a failure of the paralysed muscle to contract. Biopsy material was taken from the calf muscles of Rabbit 6 one day and 8 days after the injection of venom. The control side showed normal muscle except for some possible infection on the 8th day. The side injected with venom showed patchy areas of fibre degeneration giving a " ghost " appearance and a heavy infiltration with polymorphonuclear leucocytes and macrophage cells. At 8 days the biopsy showed many of the fibres intact but others appeared to be replaced by basophilic granular material probably derived from degenerate inflammatory cells. The connective tissue about the fibres was infiltrated with polymorphonuclear leucocytes and granular basophilic material. Rabbit 9, which had shown a partial but transient paralysis, was biopsied 18 days after the venom injection when recovery was complete. The muscle on both sides was normal.
Pharmacological studies Rat phrenic nerve diaphragm preparations were bathed in Tyrode's solution in paraffin wax troughs so that the nerve and muscle were each in separate compartments with the distal part of the nerve running through a short Vaselined tunnel. When venom was placed in the muscle compartment, paralysis to nerve stimulation ensued, but the response to direct stimulation was unimpaired. No effect on nerve conduction was observed when the nerve alone was bathed in venom solution.
157
]'. E. CAREY AND E. A. WRIGHT
STIMULATION TO NERVE
l
MUSCLE II II
I I
l io HINS. I
FIC. 1 . - - A kymograph tracing of a rat phrenic nerve diaphragm preparation showing the development of paralysis to nerve stimulation following exposure to venom. V = v e n o m 3.5 ~tg./ml. applied, bath temperature 22°C., nerve stimulation 7/minute, pulse width 0.1 m.sec., 7 volts. Direct muscle stimulation 7/minute, pulse width 1.0 m.sec., 70 volts.
The rat phrenic nerve diaphragm preparation was exposed to various concentrations of venom for various times at 21°C. and 37°C. (Table Ill). At 37°C. the time to obtain complete paralysis increased from 12 minutes at 3.5 ~g./ml. to 88 minutes at 0.1 ~tg./ml. When preparations were exposed to the venom in the higher doses for short periods and then thoroughly washed, there was a delay in the onset of paralysis and an increase in the time for complete paralysis. With lower doses there was only very slight or no paralysis under these conditions. Using the lowest concentration of venom (0.1 ~g./ml.) preparations were washed when the trace size was reduced to approximately half. The paralysis continued to develop after washing and finally became complete, but after further washing partial recovery occurred in approximately 3 hours. With the higher doses no recovery was demonstrated even in preparations followed for over 4 hours. In all cases the muscle gave a full contraction when it was stimulated directly up to 4 hours later. Fig. 1 shows the typical picture of onset of paralysis and the unimpaired response of the muscle to direct
158
ACTION OF VENOM OF
Enhydrina schistosa
stimulation. Fig. 2 shows the effect of close injections of acetylcholine into the preparation before and during the development of paralysis. A dose of acetylcholine was chosen which caused a contraction equal to the maximal response from nerve stimulation. T h e response to acetylcholine fell off in the same manner as did the response to stimulation of the nerve. Eserine up to a dose of 50 ~g. given to this same preparation had no effect when the paralysis was complete.
Effect of anticholinesterase substances on the survival time of mice given a lethal dose of venom F o u r groups of 10 mice were given 3 ~g. of venom intraperitoneally. One group served as a control and the others received respectively 1.5 ~zg. " Prostigmin . . . . Roche," 125 ~g. D F P and 67 ~g. DFP. All drugs were given subcutaneously immediately after the venom. ( T h e D F P was in the form of eye drops 0.05 per cent. w/v Dyflos BPC in arachis oil). T h e doses of drug were the largest that mice could tolerate. No significant difference in survival times was observed. T w o to four animals survived in each group. DISCUSSION
T h e paralysing action of venom on the isolated rat phrenic nerve diaphragm preparation indisputably demonstrates a peripheral action of the venom of E. schistosa. With this ~reparation paralysis is obtained with concentrations of venom which are comparable to
30 SECS
I0 HINS
I
A
I
AAA
FIG. 2. A kymograph tracing of a rat phrenic nerve diaphragm preparation showing the effects of close retrograde injections of acetylcholine during the course of the development of paralysis following exposure to venom. Nerve stimulation and venom concentration as for Fig. 1. A = injection of acetylcholine 30 Vtg. in 0.15 ml.
j. E. CAREYAND E. A. WRIGHT TABLE III.
159
Shows the neuromuscular blocking effect of various concentrations of venom on the rat phrenic nerve diaphragm preparations, a = mean of five preparations.
Concentration of venom in the bath ~tg./ml.
Temperature of bath
Exposure time
Time for complete paralysis
°C.
rains.
rains.
3.5
21
Continuous
31
3.5
37
Continuous
12
1.75
21
Continuous
35
1.75
21
10
1.75
21
20
1.0
37
Continuous
1.0
37
23
No paralysis up to 90 rains.
0.1
37
Continuous
88
0.1
37
62
I01
0.1
37
Only very slight paralysis after 114 rains.
0.1
37
Only very slight paralysis after 154 rains.
96 Almost complete paralysis 129 Almost complete paralysis 58 a
doses which just cause death in mice receiving venom intraperitoneally. 0.1 ~tg. /ml.of venom in the bath caused complete paralysis of an isolated preparation in approximately 1.5 hours, whereas in the intact animal doses of 0.1 ~g./g. was approximately the LDs0 dose. It is also of interest to note that if the LDs0 dose killed, it usually did so in 1 to 3 hours. T h e local paralysis obtained from the injection of venom into the femoral artery provides further evidence for the peripheral action. Although this peripheral action has been demonstrated previously (KELLAWAY et al., 1932) the results of direct application of neurotoxic venoms to the medulla oblongata have suggested that the venoms also have a central action (FRAZER and ELLIOT, 1904). We have observed a striking effect of whole venom when it is injected into the medulla oblongata directly ; by this route death is caused by exceedingly small doses. However we have shown that this medullary damage is caused by a fraction of the venom which has little or no general toxicity when injected intraperitoneally. Further, doses of the toxic fraction have no effect on the medulla until very large doses are employed. F r o m these results we have concluded that the observed medullary action of this venom at least is due to the special methods of application and is unlikely to represent a mode of action following a naturally occurring snake bite. In addition the failure to produce any effect from injections of venom into the vertebral artery supports the view that this venom has no important central action when the venom enters via the blood stream. F r o m the studies of the isolated nerve muscle preparation and from the results of the femoral artery injections, the initial primary damage appears to be at the neuromuscular
160
aCTION OF VENOM OF
Enhydrina schistosa
junction. The in vitro studies indicate that the peripheral action of the venom is different from that of curare in that the paralysis is not antagonized by close injections of acetylcholine or choline esterase inhibitors, and that the preparations once paralysed show little or no recovery when the venom is removed. These two differences from the action of curare have been noted for other venoms (ARTHUS, 1910 ; CUSHNYand YAGI, 1918). Transitory generalized paralysis with apparent complete recovery can be produced in rabbits when small doses of venom are given intravenously or intraperitoneally. This phenomenon was also demonstrated locally following femoral artery injections. Subsequent histological examination showed normal muscle. It has been shown for both in vivo and in vitro preparations that even after large doses of venom causing complete paralysis the muscle can respond normally to direct stimulation for at least several hours. However in a number of cases following femoral artery injection gross muscle damage was observed later in that the response to direct stimulation was lost 1 day after paralysis, histological damage was observed and the affected muscles became grossly wasted. In a number of these animals there was no recovery after several months. It would appear from these results that there is an initial action on the neuromuscular junction which may be transitory, but higher doses can directly affect the muscle permanently. SUMMARY
When the whole venom of Enydrina schistosa is injected into the medulla oblongata of rabbits death results even with small doses. If equivalent doses of a fraction containing the lecithinase activity are used, similar effects on the medulla are observed although this fraction is non-toxic when injected intraperitoneally. On the other hand, the neurotoxic fraction, which acts similarly to the whole venom when injected intraperitoneally, has virtually no effect on the medulla. Evidence is presented that the initial effect of the venom on the muscle is at the neruromuscular junction and that this may be temporary. With high doses a permanent muscle damage develops later. REFERENCES ARTHUS,M. (1910). Arch. int. Physiol., 10, 161. BRIDGEWATER,F. A. J., MORGAN, R. S., ROSEN, K. E. K. & PAYLINGWRIGHT, G. (1955). Brit. y. exp. Path., 36, 5. Bi3LBRING,E. (1946). Brit. y. Pharmacol., 1, 38. BItumEN, A. S. V., DICKENS, F. & ZATMAN,L. J. (1949). ft. Physiol., 109, 10. CAmSY,J. E. & WRIGHT,E. A. (1960a). Nature, Lond., 185, 103. - & (1960b). Trans. R. Soc. trop. Med. Hyg., 54, 50. CUSHNY,A. R. & YaGI, S. (1918). Phil. Trans., B, 208, 1. FRAZER,J. R. & ELLIOTT, R. H. (1904). Ibld., B, 197, 249. KELLaWaY, C. H. (1937). Bull. Johns. Hopk. Univ., 60, 18. , CHERRY,R. O. & WILLIAMS,F. E. (1932). Aust. y. exp. Biol. reed. ,.qci., 10, 181. REID, H. A. (1956). Trans. R. Soc. trop. Med. Hyg., 50, 517. WRIGHT, E. A. (1954). ft. Path. Bact., 68, 131.
Vol. 55.
No. 1.
March, 1961.
T H E S I T E O F A C T I O N O F T H E V E N O M O F T H E SEA S N A K E E N H Y D R I N A
SCHISTOSA BY
J. E. CAREY AND E. A. WRIGHT*
(From St. Mary's Hospital Medical ,School, London) T h e peripheral action of many neurotoxic snake venoms has been demonstrated but a central action has also been postulated. T h e early work on this problem has been reviewed by KELLAWAY, CHERRY and WILLIAMS (1932). These authors presented further evidence o f the peripheral action of a n u m b e r of neurotoxic snake venoms, but were also able to produce changes in the respiratory activity of animals by direct application of cotton wool soaked in venom solutions to the floor of the fourth ventricle. T h e y suggested that these results may have been indirect and due to local vasoconstriction. We have tried to elucidate this problem by direct intra-arterial injections of crude venom and also by injecting crude and fractionated venom into the medulla oblongata. MATERIALS AND METHODS
Venom.--The dried crude venom of Enhydrina schistosa was supplied by Dr. H. A. Reid, Penang General Hospital, who has given details of its collection (REID, 1956). The crude venom was made up in physiological saline before use. Two fractions of the venom were prepared by dialysis and cellulose column chromatography (CAREY and WEIGHT, 1960a). The first fraction contained 95 to 100 per cent. of the toxicity of the whole venom as found by intraperitoneal injection into mice, but no lecithinase activity. The second fraction had lecithinase activity equivalent to that of the whole venom, but less than 5 per cent. of the initial toxicity of the whole venom. The toxicity of the venom fractions was tested by injecting mice intraperitoneaUy with the solutions and comparing the survival times with mice injected with whole venom. The LDs0 for mice and rabbits had been previously determined to be between 50 and 120 btg./kg. (CAREYand WmCHT, 1960b). Animals.--Rabbits of various breeds and sexes between 2 and 3.6 kg. were used. For comparative experiments the animals were as nearly matched for weight as possible and the doses calculated on a weight basis. Anaesthetic.--Intravenous " Nembutal " (0.4 ml./kg.) fortified by open ether was used for all operative procedures. Femoral artery injections.--The standard procedure was to cannulate the femoral artery high in the thigh and inject 0.02 ml. of heparin BP (5,000 IU/ml.) made up to 0.5 ml. in physiological saline. A slow saline drip was then maintained into the peripheral circulation of the limb through the cannula. Following this a shaped perspex clamp was fitted round the lower abdomen and a rubber bag inside this clamp was inflated to 200 ram. Hg. pressure thereby cutting off the flow of arterial blood into the limb. The femoral vein was now cannulated and fluid which drained was collected, Various doses * We are very grateful to Dr. H. A. Reid for supplying us with dried venom. The Wellcome Foundation made this work possible by: a grant for the salaryofone of us (J~E.C.).
154
A C T I O N OF V E N O M O F
Enhydrina schistosa
of venom were then injected into the arterial cannula at several different rates. The artery and vein were then tied off and the same procedure carried out on the opposite side, the venom being replaced by an equal volume of saline. The clamp was removed at the end of the operation and also occasionally for a short period after completing the first side.
Vertebral artery injections.--Using the method described by BRIDGEWATER,MORGAN, ROSEN and PAYLING WRIGHT (1955) the first part of one vertebral artery was exposed and tied proximally. Venom in volumes ranging from 0.1 to 0.5 ml. was then injected into the distal part of the artery during the course of 10 to 30 seconds. Medullary injections.--The obex of the medulla oblongata was exposed and injections of venom in volumes of 0.001 to 0.005 ml. were made 2 ram. lateral to the obex and at a depth of 2 ram., using a fine glass capillary needle (WRIGHT, 1954). I n one case, 0.005 ml. was injected into one side and 0. 004 ml. was injected into the other. Pharmacological preparations.--The rat phrenic nerve diaphragm preparation as described by B/3LBRING (1946) was used and the arrangement for the close injection of acetylcholine was prepared according to BURGEN, DICKENS and ZATMAN (1949). RESULTS
Injections into the medulla oblongata of rabbits T a b l e I shows the results of i n j e c t i n g whole v e n o m a n d fractions of v e n o m i n t o t h e m e d u l l a o b l o n g a t a of rabbits. W h o l e v e n o m i n j e c t e d i n doses of 1/10th to 1/5th LDs0 caused r a p i d death. T h e dose h a d to be r e d u c e d to a p p r o x i m a t e l y 1/200th LDs0 before animals survived permanently. TABLE I. The results of injecting various doses of whole venom and venom fractions into the medulla oblongata of rabbits. The numbers in brackets are the survival times in hours of animals dying within 24 hours. (50 ~g./kg. = 1 LDs0). Doses of whole venom or fractions obtained from these initial doses. Rabbit LDs0's
Number of animals surviving more than 24 hours/Number of animals used. Whole venom
Toxic fraction
0.2
0/4
(2, 3, > 18, > 19)
0.1
0/4
(2, 3, 3, 4)
O. 08-0.07
0/1
(<21)
0.05-0.04
0/1
(4)
2/2 b
0.03-0.02
1/3 c
(2, <24)
2/2
O. 01-0.009
0/2
(6, <21)
1/1
0. 005
1/2 e
(5)
0. 002
2/2
1/1 a
Lecithinase fraction
0/1
(3)
0/2
(6, 8)
0/1
(<17)
1/1 d
a = killed after 3 days showing slight weakness of neck muscles ; b = one animal was killed at 31 hours showing slight weakness in right forelimb ; the other rabbit killed after 15 days showing extreme torticollis to the left ; c = one animal killed after 28 hours showing occasional convulsions and weakness ; d = animal survived 2 days ; e = one animal killed after 5 hours because it started rolling over and over violently ; the other survived.
155
j . E. CAREY AND E. A. W R I G H T
When the lecithinase activity of the whole venom was removed by fractionation the toxicity, as judged by intraperitoneal injection into mice, remained. When this fraction was injected into the medulla no rapid deaths occurred over a wide range of doses, although three rabbits receiving high doses were showing slight signs of medullary damage when they were killed. However when doses of the lecithinase fraction equivalent to the lecithinase activity of the whole venom doses were injected, comparable death times were recorded. It should be emphasized that this lecithinase fraction contained 95 to 100 per cent. of the lecithinase activity of the original whole venom, but when injected intraperitoneally into mice it contained less than 5 per cent. of the original toxicity. On the other hand, no lecithinase activity could be detected in the toxic fraction. Rabbits receiving either whole venom or fractions containing the lecithinase activity showed a similar clinical picture. The respiration rate was at first increased and was frequently associated with gasping or wheezing, but in the final stages before death the respiration rate became very slow and the animal became prostrated and died in coma. Most animals exhibited torticollis to the injected side and some of them made persistent circling movements in that direction. Many of them also showed convulsions in the later stages, which may have been due to anoxia owing to the failure of respiration. Rabbits injected with saline alone showed no ill effects from the operations. Most of the survivors from the venom injections were kept for a week.
Injections into the vertebral artery Two rabbits were given 0.7 LDs0 of whole venom into one vertebral artery. One showed no ill effects from this procedure and one showed some transitory generalized weakness and gasping 24 hours later, but no unilateral signs were present and the animal was fully recovered after 1 week. No abnormal signs followed the injection of 0.1 LDs0. The rabbit LDs0 was calculated on the basis of 50 ~g./kg. and in a previous experiment 0.7 I, Dso had produced a slight transitory weakness when given intravenously.
Injections into the femoral artery Rabbits were given injections of venom into the femoral artery in doses ranging from 6.25 to 50 ~g./kg. (Table II). The clamp was applied for periods ranging from 10 to 35 minutes and the venom injected over times ranging from 1 to 20 minutes. The loss of fluid from the venous cannula was usually slight, but on two occasions 1/3rd of the dose of venom was detected in this fluid. No doubt the variability in these factors accounted for at least some of the differences in the degree of paralysis at each dose level. TABLE II.
The results of localized injections of venom solution into the femoral artery of rabbits. Dose ~tg./kg.
50
Number of animals used Number of animals showing complete local paralysis immediately after recovery from anaesthesia
4
25
12.5
625
156
ACTION O F VENOMOF Enhydrina schistosa
(i) Animals receiving 50 ~tg./kg.--Rabbits 1 and 2 died of generalized paralysis within 2 days. Rabbits 3 and 4 were still completely paralysed in the injected leg when killed 63 and 97 days later. Rabbit 5 showed a transitory equivocal local paralysis. (i~) Animals receiving 25 ~tg./kg.--Rabbit 6 developed complete local paralysis, but was accidentally killed by an overdose of anaesthetic after 8 days. Rabbit 7 showed persistent paralysis but developed gross local infection and was killed after 87 days. Rabbits 8 and 9 showed only partial paralysis originally and this completely recovered after 8 to 15 days. (iii) Animals receiving 12.5 ~Lg./kg.--Rabbit 10 was completely paralysed locally and there was no recovery after 81 days. Rabbit 11 showed partial local paralysis on the 2 days following the venom injection, but was killed after 16 days owing to spastic extension of both hind limbs and an ulcerated area on the back. The spastic extension may have been due to the clamping procedure, but there were no other examples of this in any of the experimental or control animals. Rabbit 12 showed slight paralysis of the injected limb which recovered after 3 days. (iv) Animals receiving 6.25 gg./kg.--Rabbits 13 and 14 : neither animal showed any paralysis after recovery from the anaesthetic.
Electrical and histological changes in calf muscles The electromyographic response in the calf muscles following a sharp tap to the Achilles tendon was observed with a cathode ray oscilloscope. In three of the rabbits showing complete paralysis the tendon jerk trace was found to be reduced or absent immediately after recovery from the anaesthetic. Direct stimulation of the paralysed muscle with up to 9 volts (1 to 10 m.sec.) immediately on recovery from the anaesthetic caused contraction of the muscle. Direct stimulation was tried 1 day after paralysis in only one animal and in this case there was a failure of the paralysed muscle to contract. Biopsy material was taken from the calf muscles of Rabbit 6 one day and 8 days after the injection of venom. The control side showed normal muscle except for some possible infection on the 8th day. The side injected with venom showed patchy areas of fibre degeneration giving a " ghost " appearance and a heavy infiltration with polymorphonuclear leucocytes and macrophage cells. At 8 days the biopsy showed many of the fibres intact but others appeared to be replaced by basophilic granular material probably derived from degenerate inflammatory cells. The connective tissue about the fibres was infiltrated with polymorphonuclear leucocytes and granular basophilic material. Rabbit 9, which had shown a partial but transient paralysis, was biopsied 18 days after the venom injection when recovery was complete. The muscle on both sides was normal.
Pharmacological studies Rat phrenic nerve diaphragm preparations were bathed in Tyrode's solution in paraffin wax troughs so that the nerve and muscle were each in separate compartments with the distal part of the nerve running through a short Vaselined tunnel. When venom was placed in the muscle compartment, paralysis to nerve stimulation ensued, but the response to direct stimulation was unimpaired. No effect on nerve conduction was observed when the nerve alone was bathed in venom solution.
157
]'. E. CAREY AND E. A. WRIGHT
STIMULATION TO NERVE
l
MUSCLE II II
I I
l io HINS. I
FIC. 1 . - - A kymograph tracing of a rat phrenic nerve diaphragm preparation showing the development of paralysis to nerve stimulation following exposure to venom. V = v e n o m 3.5 ~tg./ml. applied, bath temperature 22°C., nerve stimulation 7/minute, pulse width 0.1 m.sec., 7 volts. Direct muscle stimulation 7/minute, pulse width 1.0 m.sec., 70 volts.
The rat phrenic nerve diaphragm preparation was exposed to various concentrations of venom for various times at 21°C. and 37°C. (Table Ill). At 37°C. the time to obtain complete paralysis increased from 12 minutes at 3.5 ~g./ml. to 88 minutes at 0.1 ~tg./ml. When preparations were exposed to the venom in the higher doses for short periods and then thoroughly washed, there was a delay in the onset of paralysis and an increase in the time for complete paralysis. With lower doses there was only very slight or no paralysis under these conditions. Using the lowest concentration of venom (0.1 ~g./ml.) preparations were washed when the trace size was reduced to approximately half. The paralysis continued to develop after washing and finally became complete, but after further washing partial recovery occurred in approximately 3 hours. With the higher doses no recovery was demonstrated even in preparations followed for over 4 hours. In all cases the muscle gave a full contraction when it was stimulated directly up to 4 hours later. Fig. 1 shows the typical picture of onset of paralysis and the unimpaired response of the muscle to direct
158
ACTION OF VENOM OF
Enhydrina schistosa
stimulation. Fig. 2 shows the effect of close injections of acetylcholine into the preparation before and during the development of paralysis. A dose of acetylcholine was chosen which caused a contraction equal to the maximal response from nerve stimulation. T h e response to acetylcholine fell off in the same manner as did the response to stimulation of the nerve. Eserine up to a dose of 50 ~g. given to this same preparation had no effect when the paralysis was complete.
Effect of anticholinesterase substances on the survival time of mice given a lethal dose of venom F o u r groups of 10 mice were given 3 ~g. of venom intraperitoneally. One group served as a control and the others received respectively 1.5 ~zg. " Prostigmin . . . . Roche," 125 ~g. D F P and 67 ~g. DFP. All drugs were given subcutaneously immediately after the venom. ( T h e D F P was in the form of eye drops 0.05 per cent. w/v Dyflos BPC in arachis oil). T h e doses of drug were the largest that mice could tolerate. No significant difference in survival times was observed. T w o to four animals survived in each group. DISCUSSION
T h e paralysing action of venom on the isolated rat phrenic nerve diaphragm preparation indisputably demonstrates a peripheral action of the venom of E. schistosa. With this ~reparation paralysis is obtained with concentrations of venom which are comparable to
30 SECS
I0 HINS
I
A
I
AAA
FIG. 2. A kymograph tracing of a rat phrenic nerve diaphragm preparation showing the effects of close retrograde injections of acetylcholine during the course of the development of paralysis following exposure to venom. Nerve stimulation and venom concentration as for Fig. 1. A = injection of acetylcholine 30 Vtg. in 0.15 ml.
j. E. CAREYAND E. A. WRIGHT TABLE III.
159
Shows the neuromuscular blocking effect of various concentrations of venom on the rat phrenic nerve diaphragm preparations, a = mean of five preparations.
Concentration of venom in the bath ~tg./ml.
Temperature of bath
Exposure time
Time for complete paralysis
°C.
rains.
rains.
3.5
21
Continuous
31
3.5
37
Continuous
12
1.75
21
Continuous
35
1.75
21
10
1.75
21
20
1.0
37
Continuous
1.0
37
23
No paralysis up to 90 rains.
0.1
37
Continuous
88
0.1
37
62
I01
0.1
37
Only very slight paralysis after 114 rains.
0.1
37
Only very slight paralysis after 154 rains.
96 Almost complete paralysis 129 Almost complete paralysis 58 a
doses which just cause death in mice receiving venom intraperitoneally. 0.1 ~tg. /ml.of venom in the bath caused complete paralysis of an isolated preparation in approximately 1.5 hours, whereas in the intact animal doses of 0.1 ~g./g. was approximately the LDs0 dose. It is also of interest to note that if the LDs0 dose killed, it usually did so in 1 to 3 hours. T h e local paralysis obtained from the injection of venom into the femoral artery provides further evidence for the peripheral action. Although this peripheral action has been demonstrated previously (KELLAWAY et al., 1932) the results of direct application of neurotoxic venoms to the medulla oblongata have suggested that the venoms also have a central action (FRAZER and ELLIOT, 1904). We have observed a striking effect of whole venom when it is injected into the medulla oblongata directly ; by this route death is caused by exceedingly small doses. However we have shown that this medullary damage is caused by a fraction of the venom which has little or no general toxicity when injected intraperitoneally. Further, doses of the toxic fraction have no effect on the medulla until very large doses are employed. F r o m these results we have concluded that the observed medullary action of this venom at least is due to the special methods of application and is unlikely to represent a mode of action following a naturally occurring snake bite. In addition the failure to produce any effect from injections of venom into the vertebral artery supports the view that this venom has no important central action when the venom enters via the blood stream. F r o m the studies of the isolated nerve muscle preparation and from the results of the femoral artery injections, the initial primary damage appears to be at the neuromuscular
160
aCTION OF VENOM OF
Enhydrina schistosa
junction. The in vitro studies indicate that the peripheral action of the venom is different from that of curare in that the paralysis is not antagonized by close injections of acetylcholine or choline esterase inhibitors, and that the preparations once paralysed show little or no recovery when the venom is removed. These two differences from the action of curare have been noted for other venoms (ARTHUS, 1910 ; CUSHNYand YAGI, 1918). Transitory generalized paralysis with apparent complete recovery can be produced in rabbits when small doses of venom are given intravenously or intraperitoneally. This phenomenon was also demonstrated locally following femoral artery injections. Subsequent histological examination showed normal muscle. It has been shown for both in vivo and in vitro preparations that even after large doses of venom causing complete paralysis the muscle can respond normally to direct stimulation for at least several hours. However in a number of cases following femoral artery injection gross muscle damage was observed later in that the response to direct stimulation was lost 1 day after paralysis, histological damage was observed and the affected muscles became grossly wasted. In a number of these animals there was no recovery after several months. It would appear from these results that there is an initial action on the neuromuscular junction which may be transitory, but higher doses can directly affect the muscle permanently. SUMMARY
When the whole venom of Enydrina schistosa is injected into the medulla oblongata of rabbits death results even with small doses. If equivalent doses of a fraction containing the lecithinase activity are used, similar effects on the medulla are observed although this fraction is non-toxic when injected intraperitoneally. On the other hand, the neurotoxic fraction, which acts similarly to the whole venom when injected intraperitoneally, has virtually no effect on the medulla. Evidence is presented that the initial effect of the venom on the muscle is at the neruromuscular junction and that this may be temporary. With high doses a permanent muscle damage develops later. REFERENCES ARTHUS,M. (1910). Arch. int. Physiol., 10, 161. BRIDGEWATER,F. A. J., MORGAN, R. S., ROSEN, K. E. K. & PAYLINGWRIGHT, G. (1955). Brit. y. exp. Path., 36, 5. Bi3LBRING,E. (1946). Brit. y. Pharmacol., 1, 38. BItumEN, A. S. V., DICKENS, F. & ZATMAN,L. J. (1949). ft. Physiol., 109, 10. CAmSY,J. E. & WRIGHT,E. A. (1960a). Nature, Lond., 185, 103. - & (1960b). Trans. R. Soc. trop. Med. Hyg., 54, 50. CUSHNY,A. R. & YaGI, S. (1918). Phil. Trans., B, 208, 1. FRAZER,J. R. & ELLIOTT, R. H. (1904). Ibld., B, 197, 249. KELLaWaY, C. H. (1937). Bull. Johns. Hopk. Univ., 60, 18. , CHERRY,R. O. & WILLIAMS,F. E. (1932). Aust. y. exp. Biol. reed. ,.qci., 10, 181. REID, H. A. (1956). Trans. R. Soc. trop. Med. Hyg., 50, 517. WRIGHT, E. A. (1954). ft. Path. Bact., 68, 131.