Effect Of Neostigmine On Diaphragm Weakness Of Rats With Bungatotoxin

Background and ObjectiveIn our country,there were about300000snakebite cases every year, fatality rateis3%-5%. The percentage of bungarus snakebite is8.12%[1，2].Especially in the southof China,snakebites are one of our most common emergencies. Neurotoxin is primaryin bungatotoxin[3,4,5].It contains a variety of ingredients, such as:α-Bungatotoxin,β-Bungatotoxin, κ-Bungatotoxin, P-Bungatotoxin[6,7].Patients who were bitten by abungarus snake could appear to have muscle weakness symptoms which are causedby neuromuscular block.The order of neuromuscular block was studied: Eyelid ptosisappeared early,then dysphasia appears and limb muscle strength weakening arefollowed.Death is the most serious result,which is caused by respiratory muscleparalysis.The order of arising and recovering of muscle weakness symptom isopposite.At present,the accepted methods of treatment of snake-bite are local woundtreatment and early application of antivenoms.It is necessary to take effective meas-ures,such as assisted ventilation、hemoperfusion and drugs of recovering muscleforce,to treat serious patients. Given the snake bites often happen in the mounains andsuburb,in which hospitals are not euipped with antivenom routinely,antienom can notbe used widely in certain areas.So it is necessary to seek more treatments that can beused safely and widely and save up more time for patients.In past reports, it has been controversial that the effect of neostigmine on skeletal muscle weakness which causedby different ingredients of bungatotoxin and other neurotoxins[8].It has been showedthat there was exact efficacy that neostigmine can antagonize other skeletal muscleweakness disease which caused by neuromuscular block,such as muscle relaxants[9,10，11].And the meachanisms of causing diseases are similar according to comparingneurotoxins witn muscle relaxants[8].However,there are only a few studies of the effectof neostigmine on skeletal muscle weakness that produced by bungatotoxin.Therehave been reported that it can be reduced the application of assisted ventilation whenneostigmine was used in snake bite[12].The objective of this research is to clear whether neostigmine can improveskeletal muscle force via detecting the tensions of diaphragm strips which were addedwith neostigmine of different concentrations or normal saline after being added withbungatotoxin.MethodsTwenty SPF level of healthy female rates were randomly divided into fourgroups（n=5）:normal control group (group A)、1μM neostigmine group（B组）、3μM neostigmine group（C组）、10μM neostigmine group（D组）. The drugof group A is normal saline.Female Sprague-Dawley rats were anaesthetized byintraperitoneal injections of nembutal,then took out the diaphragm and clearedresidue blood and adipose tissue.After that, longitudinal strip(~1cmX1.5cm),cutparallel to the anatomical direction of the muscle fibres,was prepared from theisolated diaphragm.One side of strip connected to a force-displacement transdu-cer,the tension was recorded by physiological recorder via a force-displacementtransducer,the other side of strip was fastened to fixed link.The whole stripwas placed into tank and constantly perfused with kreb’s salt solution bubbledwith95%O2and5%CO2.The temperature of the organ bath was maintainedat37℃.The strips were mounted in the organ bath under a resting tension of1g.The stris were allowed to equilibrate for about45-60min with changes ofKreb’s solution at15min intervals before beginning the experiments[8]. After that,10-4M acetylcholine were added to the strips and choose the strips withthe tension of less than （1±10%）g[13].Then bungatotoxin were added to thestrips and washed it after5min.In the end,the tensions of strips which wereadded with neostigmine of different concentrations or normal saline were recor-ded in various time (5min、10min、15min、30min、60min).ResultsThe tensions of strips in different period in group A, group B and group C,group D as follows:5min:（0.846±0.019）g、（0.853±0.027）g、（0.853±0.024）g、（0.858±0.013）g；10min：（0.844±0.023）g、（0.860±0.033）g、（0.863±0.030）g、（0.912±0.026）g；15min：（0.846±0.033）g、（0.870±0.033）g、（0.873±0.024）g、（0.920±0.025）g；30min：（0.848±0.019）g、（0.870±0.029）g、（0.888±0.036）g、（0.932±0.013）g；60min：（0.860±0.024）g、（0.883±0.031）g、（0.908±0.032）g、（0.954±0.015）g。(1)Compared with the group A:There was no significant difference betweengroup B and group A（P>0.05）; There was a significant differences between group Cand group A at60min（P <0.05）; There were significant differences between group Dand group A at10min、15min、30min、60min（P≤0.01）。(2) Compared with the group B:There was no significant difference betweengroup C and group B（P>0.05）; There were significant differences between group Dand group B at10min、15min、30min、60min;（At10min、15min:P <0.05； At30min、60min:P≤0.01）。(3) Compared with the group C: There was a significant difference betweengroup D and group C at60min（P <0.05）;(4) The tensions of strips in different groups were recovered differently,butthey were not recovered to initial levels.ConclusionIt is attested that the effect of neostigmine on diaphragm weakness of rats withbungatotoxin, the efficacy and dosage are relevant.