A Study On The Effects Of Botulinum Toxin A On Muscle And Nerve

Botulinum toxin A(BTXA) is a neurotoxin produced by Clostridium botulinum.At present,there are three types of BTXA on sale:Botox(made by Allergan CO.,LTD,USA.), Dysport(made by Speywood CO.,LTD,UK),and 'hengli'(made by Lanzhou Institute of Biological Products,China).It was used by Carruthers for treating hyperkinetic muscles of facial expression that result in lines and furrowsin the 1920s.Since that time,the application of botulinum toxin have grown to treat face wrinkles which has gained popularity in the field of aesthetic surgery.Botulinum toxin A binds to presynaptic terminals of the motor neurons and inhibits the release of acetylcholine from the nerve terminals.Usually,application of BTXA for target muscles will cause chemodenervation in 12-72h and it will last for 3-6m before the muscle disfunction process return to nomal.In China,botulinum toxin A,commercially named'hengli',was manufactured in 1997.The manufacturers of botulinum toxin A(Lanzhou Institute of Biological Products)recommend storing the exotoxin at -20～-5℃,avoiding light,and diluting with physiologic saline solution before application to obtain the most effective result.They also point that it must be used in approximately 4 hours after reconstitution,or the potency of the toxin will decrease.One bottle of BTXA is very expensive and the amount of BTXA used in aesthetic surgery is only about 25u and no more than 100u.Hence,the manufacturers of 'hengli' reduce 100u of BTXA to 50u of BTXA.However,a significant amount of the drug is still discarded which may cause pollution because,on average,only a small amount of it is used in a patient.Recently,many experimental studies have been performed to investigate the stored BTXA.Some studies have shown that there is no decrease in potency of the toxin when stored after reconstitution,which means there is no difference between the reconstituted and the stored drug.But some studies have shown that refrigerated toxin is not as potent as the fresh form.Whether BTXA which is stored longer than 4 hours after reconstitution will be sill effective as it is fresh,the purpose of the study was to compare fresh versus stored reconstituted botulinum toxin type A for initial potency,duration of action,and relations with the acting time in the purpose of providing experimental evidence for the clinical use of butulinum toxin A.Hence,more importance clinical significance and application prospects have been shown by the effects of fresh and stored BTXA on muscle and nerve. ObjectiveThe study was divided into two parts:visual evaluation of the maximum erect static position of the auricle;Alterations in muscle and nerve ultrastructure were evaluated with electron microscopy.The purpose of the study was to compare the efficacy of reconstituted and stored botulinum toxin type A for initial potency,duration of action,and relations with the acting time,providing experimental evidence for the use of BTXA in clinic in future.Methods1.The study was carried out on 27 New Zealand white rabbits.The animals were divided into 9 groups(n=3),Group 1 was used as the control group,and the rest 8 groups were used as the experimental groups.The right anterior auricular muscle models were used for all injections in each group,and the left ones were used as the compare.Animals in Group 1 received an injection of 0.05ml sterile saline.The amount of saline injected in Group 1 was equal to the BTXA 0.05ml(2.5u) injected in other groups.Half of the prepared agent was used for fresh injections and the remainder was stored in the refrigerator for 1,2 and 3 weeks.2.The visual inspection was used to identify the grades of the drooping ears after the injections of fresh and stored BTXA.The position of auricle was graded fromⅠtoⅢ.Ⅰindicated a normal preinjection position,Ⅱindicated a minimal/moderate paretic auricle (rabbit can raise ear more than 60degrees from skull),andⅢindicated a moderate/severe paretic auricle(rabbit cannot raise ear more than 60 degrees from the skull).3.The muscle and nerve sections obtained and alterations in muscle and nerve tissues were compared after the injection of fresh and stored BTXA on the fifth day and the 12th week..Results1.The visual inspections were:At postinjection 5 days and 12 weeks,all ears that received botulinum toxin were drooping and the drooping grade is 3.And the results showed that there was an overall quicker trend to return to baseline the longer the botulinum toxin was stored.2.Electron microscopic alterations in muscle and nerve ultrastructure:(1) Degeneration findings in muscle after botulinum toxin injection revealed no significant difference between freshly reconstituted and stored toxin in the early period(the fifth day). When stored(for 1 and 2 weeks) toxin was used,atrophic changes in the muscle were the same as the fresh toxin at 12 weeks.Muscles of the group which was injected BTXA stored for 3 weeds had less severe degenerative findings than group which was injected with BTXA stored for 1 and 2 weeks muscles.(2) On nerve evaluation,fresh and stored toxin both display significant acute changes on nerve ultrastructure;When stored(for 1 and 2 weeks) toxin was used,atrophic changes in the nerve were the same as the fresh toxin at 12 weeks.Nerves of the group which was injected with BTXA stored for 3 weeds had less severe degenerative findings than group which was injected with BTXA stored for 1 and 2 weeds nerves.ConclusionIt seems that stored BTXA for 1 weeks and stored BTXA for 2 weeks has the same initial potency,but the duration of action significantly decreased when BTXA was stored for 3 weeks or more.Stored(for 1 and 2 weeks) and fresh BTXA are more durable than the stored BTXA for 3 weeks.Hence,we command that the time for the storage of BTXA should better be shorter than 2 weeks.