Cholinesterase activity after DFP application in botulinum poisoned, surgically denervated or normally innervated rat skeletal muscles

Lüe Sciences Yol . 7, Part I, pp . 411-417, 1968 . Printed in Great Britain.

Perga.mon Press

ChDLIf~ESTERASE ACTIVITY AFTER OFP APPLICATI~I IN BOTULINUM POISONm, SURGICALLY OENERIIATED ~ NORM4LLY IN~ERVATED RAT SKELETAL M15CLES B. Saneason and S. Thesleff Oepartrnents of Anetony and Pharmacology, University of Lund, Lund, Sweden

(Received 6 November 1967 ; in final form 29 January 1968) Cholinesterase activity at the motor end plate has been found to decrease after denervation and to increase following reinnervation, indicating a neural influence on the production of the enzyme (1, 2, 3, 4) . With denervation the entire muscle msrrtirene becomes sensitive to applied acetylcholine (5), an effect which hoe been found also when the muscle is poisoned with Clostridium botulinum toxin. This toxin is known to prevent the release of acetylchbliné from the nerve terminals (6, 7) . In the present series of experiments the rate of reappearance of cholinesterase activity in muscles following diisopropyl phosphocofluoridate (OFP) treatment has been studied histochanically in botulinum poisoned, surgically denervated and normally innecvated muscles. Methods Adult white tuts (Wistar) weighing between 150 and 200 g were used . These were placed into three groups . Group I - In 22 rets the sciatic nerve was divided on one aide and tha two stumps were ligated. After 1 week all the animals were given 1 .5 mg of OFP in 0.1 ml physiological NaCl solution locally to the surface of the anterior tibialis muscle of the denervated leQ . This annum of OFP is tolerated by only about 65~ of the rats despite the administr,etion of atropine before and after DFP application . The OFP used was synthetized by the Researçh Institute of National Oefer;;e, Deot . 1 . Sundbybetp and a fresh solution was prepared for

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each experiment . The animals were then divided into 5 aubgroupe, being killed after 1, 2, 7, 14 and 21 days respectively . The anterior ti.bialis marls on both sides was c~enwed, weighed and fixed in 10~ neutral formol for 2 hours, For the histochemical demonatret'ion of cholinester~e the method of Koelle and Friedenwald (B) was used. The length of the incubation time needed for the motor end plates to became visible was used as a rough estimate of the relative ertnunt of cholinesterase at the end plates. Sections from the rtascles were incubated for 5, 10, 15, 30, 45, 60 and 120 min . In addition sections were incubated for 20 hours in those cases in which 120 min . of incubation gave negative results . The contralaterol muscle was taken as control for canparison of the incubation time. Group II - 23 rats were locally poisoned with Clostridiun botulinum toxin type A dissolved in sterile phosphate buffer as described by Arrbache (9) . The toxin solution (0 .25 ml) was applied locally to the anterior tibialis muscle in one leg . The dosage given was such as to oroduce paralysis of the anterior tibial muscle within 24 hours without causing marked generalized effects . Five days later the animals were given OFP locally in the same way as in group I . The animals were killed after 1, 7, 14 and 21 days respectively. The poisoned and the normal contrelateral muscles were then treated in the same way as in Rroup T: Group III - In this group 23 rate which had normally innervated rtuscles served as control animals . They were only given DFP locally as in the two other groups and the rats were killed after 1, 7, 14 and 21 days respectively . Results In table 1 the mean wet merle weight of treated muscles is given in per cent of contrelater,al normal muscles . As can be seen from tha table, there was a gradual decrease in the

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wet weight of the denetvated as well as of the botulinum poisoned muscles . The weight reduction was moat pronounced in the denetvaticm group. The deoreese was seen 1 week after dividing of the sciatic nerve, and after 26 days the muscle weight was approximately one third of the normal weight . The decrease in weight of the botulinun poisoned muscle was lass pronounced, being reduced to about 2/3 of the nornel weight 26 days after the poisoning. The muscles treated only with DFP showed no change in weight .

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In figure 1 e diegnam is given of the incubation times necessary in the different groups to visualize cholinesterase activity et the motor end plates . In each incubation procedure a muscle from a nor+nel rat was also incubated together with the experirtent muscles serving as a reference for the incubation time. In norRel muscle an incubation time of 5 min, was sufficient to clearly visualize cholinesterase activity at motor end plates . This is indicated in the figure by the horizontal "Control level" . One day after the application of the DFP the incubation time woes prolonged in all the groups, being most pronounced in the denerveted muscle group ( -'_ 120 min .) . In the botulinun treated group the incubation time varied between 45 and 120 min ., mean BO min . In the control group, the incubation time varied between 30 and 60 min. with a mean of 45 min . Innib. time. (min) Control lehl s _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~»=cx) =ai >s xi ~c»

FIG . 1 The figure shows the incubation times required for histochemical visualization of cholinesterase in nuacles 1, 2, 7, 14 and 21 days after local application of e DFP . " Muscle with normal innervationj o surgically denervated muscles botulinun poisoned rtuscle . The "Control level" represents the incubation time which visualized motor end plates in cantrelaterol muscles without application of DFP . The mean tirtss are given and the bars r~epr~esent the range of measurements . The fipur~es in parenthesis ere the number of muscles studied .

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It can be seen that after 1 week the incubation time wee shortened to between 15 end 30 min, in all the groups, indicating an increase in the cholineeter~ese activity when capered with that observed one day after the DFP application . Fourteen days after the OFP application the normally innervated rtuscles ahawed normal cholinesterase activity, whereas in the botulinun poisoned and the denervated grnups there woes a slight lengthening of the incubation time es capered with that in normal muscle . This prolongation rerteined unchanged 21 days after the oFP application . The artount of DFP used for inhibition of the cholinesterase activity et the motor end plates was accanpanied by a mortality rate of about 35; in the primary materiel reducing the nurtber of nets in the subsequent subgrnups to that given in the figure .

Discussion The present observation of the restitution of cholinesterase activity after deneNation end application of DFP is not in full agrearent with the findings made by Filogamo end Gabella (10, 11) . They reported that in chicken nusclea, treated in a similar way, no cholinesterase activity could be denrnstroted even after 90 days . The diverging results may be due to the fact that Filogamo et al . and we have used different species with possible different reaction patterns . The restitution of cholinesterase activity in ell the three groups 7 days after the application of OFP is quite evident . Flowever, there is a tendency for the cholinesterase to be restituted more rapidly in the normally innervated muscle then in botulinun poisoned and in denerveted muscle, as seen in the figure . After 14 days the normally innerveted muscle had a normal incubation time, whereas in the botulinum poisoned muscle end particularly in the denervated muscle the restitution of cholinesterase seamed to be elawer . On the other heed, the histochemical method used in the present study is not quantitative and therefore the different lengthen of incubation times registered

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here can serve only ae rough estimates of the relative cholinesterase activity . The restitution of cholinesterase activity found 7 days after OFP application could be explained by reeynthesis of the enzyme and/or by the possibility that DFP, was reversibly bound to the cholinesterase and that therefore the observed recovery is the result of cholinesterase regaining fts activity . The lastmentioned possibility is, however, made unlikely by the elegant studies of Blaber and Creasey (12,

13) who showed that DFP apparently

irreversibly inhibits the cholinesterase activity of rat erythrocytes and brain tissue in vivo . If resynthesis of the enzyrrg accounts for the recovery of cholinesterase activity the reduced rates of recovery that were observed in botulirnm poisoned and in chronically denervated muscles could be explained by the absence of a 'trophic' factor narnelly released by the motor nerve. The tepidity, however, with which the recovery of cholinesterase activity returned in the denervated and the botulinum poisoned groups and approached that of the untreated, normally innervated group suggests that rtuscular canponents in addition to neurotrophic factors govern the rate of resynthesis of cholineaterose at the muscle end plate.

Acknowledganents We are indepted to Dr . J . Zélena fran the Institute of Physiology of the Czechoslovak Acadeny of Sciences,_Pregue, who initiated this study and also took part in the preliminary experinents. The study was supported by a grant from the Maggie 5tephen's Foundation .

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