after approval from institutional animal ethics committee , government medical college , bhavnagar , india , all the experiments were performed as per the guidelines issued by the committee for the purpose of control and supervision on experiments on animals , india . albino wistar rats ( 200 - 300 g ) of either sex were procured from the central animal house of the institute . they were housed in standard transparent polypropylene cages under controlled room temperature ( 242 ; relative humidity 60 - 70% ) in 12 - 12 h light - dark cycle . ahmedabad , india ) , dantrolene ( sigma aldrich , germany ) and magnesium chloride ( qualigen fine chemicals , mumbai , india ) were dissolved in double distilled water to get a final concentration of 20 , 1 and 10 mg / ml , respectively . vials of rocuronium ( 10 mg / ml , neon laboratories ltd . , mumbai , india ) , lignocaine ( 20 mg / ml , 2% w / v , 30 ml , hindustan laboratories ltd . , barauni , india ) and succinylcholine ( 50 mg / ml , healthcare pvt . ltd . , mumbai , india ) were used with appropriate dilutions in the physiological salt solution ( pss ) . rats were sacrificed and left hemidiaphragm with attached phrenic nerve was dissected as described by bulbring . the preparations were mounted on rat phrenic nerve diaphragm electrode in inner organ bath containing 40 ml of double dextrose tyrode solution ( containing nacl 136.9 mm , kcl 2.68 mm , cacl2 1.36 mm , magnesium chloride 0.98 mm , nah2po4 0.32 mm and dextrose 11.1 mm ) maintained at a temperature of 370.5 and bubbled with oxygen . in directly stimulated preparation , phrenic nerve was removed and only diaphragm was mounted . after initial stabilisation , the preparation was stimulated with square wave pulses of 0.5 ms duration , of 2 hz at every 10 s using the research stimulator rs 48 ( labotech , ambala , india ; amplitude : 2 v in indirect preparation and 20 v in direct preparation ) . the isometric muscle contractions were quantified with force displacement transducer and recorded on a student physiograph ( inco biodevice , ambala , india ) through strain gauge coupler . effect of zidovudine on contractions of the diaphragm induced by indirect and direct stimulation was recorded . initially , 1 mol / ml concentration of zidovudine was used ; subsequently , incremental doses of 0.5 logarithmic intervals at every 3 min were used till a final concentration of 30 mmol / ml was reached . thus , cumulative dose response curves ( drcs ) of zidovudine were obtained in indirectly ( group 1 ) and in directly stimulated preparations ( group 2 ) . rat phrenic nerve hemidiaphragm preparations were mounted to obtain drc of rocuronium in the absence of zidovudine ( group 3 ) and in the presence of 1.2 ( group 4 ) and 12 mmol / ml of zidovudine ( group 5 ) , and in animals treated with 50 mg / kg / day ( group 6 ) and 100 mg / kg / day ( group 7 ) for 15 days . drc of rocuronium was obtained using its incremental doses at 0.3 logarithmic intervals at every 3 min until the complete blockade is achieved . after obtaining stable twitch responses , rocuronium ( 10 mol / ml ) , succinylcholine ( 25 mol / ml ) , dantrolene ( 50 mol / ml ) , magnesium chloride ( 8 mmol / ml ) and lignocaine ( 600 mol / ml ) were added to produce neuromuscular blockade in the indirectly stimulated preparation . at around 50% reduction in height of t1 , zidovudine ( 30 mmol / ml ) or vehicle ( pss ) was added and their effects were compared on twitch responses . effect of zidovudine on neuromuscular transmission was studied by comparing the inhibition of twitch response ( % ) and train of four ratio ( t4/t1 ) at every incremental dose with that of baseline using the repeated measures of analysis of variance ( anova ) followed by tukey krammer multiple comparisons test in cumulative drc of zidovudine . inhibition of twitch responses by rocuronium was calculated and expressed as a percentage from the height of t1 of the train of four responses at every dose as compared to the height of t1 of basal twitch responses . drc of rocuronium in the absence and in the presence of zidovudine was compared by one - way analysis of covariance ( ancova ) using the statistical package for the social sciences ( spss ) 17 trial version software ( ibm corporation , usa ) . inhibitory concentration 5% ( ic5 ) , 50% ( ic50 ) and 95% ( ic95 ) were calculated from cumulative drc of rocuronium by employing regression analysis using masterplexreaderfit demo version software ( miraibio group of hitachi solutions america , ltd ) . comparisons of ic5 , ic50 and ic95 between the control group and interactions study groups 4 and 5 ; control group and interactions groups 6 and 7 were performed by anova followed by dunnett multiple comparisons test . effect of zidovudine 30 mmol / ml on twitch responses inhibited by dantrolene , magnesium chloride , rocuronium , succinylcholine and lignocaine was calculated in percentage and compared with that of vehicle added group ( pss ) by unpaired t - test . / a]100 ) , where a is the height of t1 in basal twitch response , b is the height of t1 , 3 min after the administration of zidovudine or vehicle . time required to produce complete block was recorded after administration of zidovudine as compared to vehicle added group , where reversal did not occur . comparison of time for complete block between vehicle added group and zidovudine added group was performed using unpaired t - test . rats were sacrificed and left hemidiaphragm with attached phrenic nerve was dissected as described by bulbring . the preparations were mounted on rat phrenic nerve diaphragm electrode in inner organ bath containing 40 ml of double dextrose tyrode solution ( containing nacl 136.9 mm , kcl 2.68 mm , cacl2 1.36 mm , magnesium chloride 0.98 mm , nah2po4 0.32 mm and dextrose 11.1 mm ) maintained at a temperature of 370.5 and bubbled with oxygen . in directly stimulated preparation , phrenic nerve was removed and only diaphragm was mounted . after initial stabilisation , the preparation was stimulated with square wave pulses of 0.5 ms duration , of 2 hz at every 10 s using the research stimulator rs 48 ( labotech , ambala , india ; amplitude : 2 v in indirect preparation and 20 v in direct preparation ) . the isometric muscle contractions were quantified with force displacement transducer and recorded on a student physiograph ( inco biodevice , ambala , india ) through strain gauge coupler . effect of zidovudine on contractions of the diaphragm induced by indirect and direct stimulation was recorded . initially , 1 mol / ml concentration of zidovudine was used ; subsequently , incremental doses of 0.5 logarithmic intervals at every 3 min were used till a final concentration of 30 mmol / ml was reached . thus , cumulative dose response curves ( drcs ) of zidovudine were obtained in indirectly ( group 1 ) and in directly stimulated preparations ( group 2 ) . rat phrenic nerve hemidiaphragm preparations were mounted to obtain drc of rocuronium in the absence of zidovudine ( group 3 ) and in the presence of 1.2 ( group 4 ) and 12 mmol / ml of zidovudine ( group 5 ) , and in animals treated with 50 mg / kg / day ( group 6 ) and 100 mg / kg / day ( group 7 ) for 15 days . drc of rocuronium was obtained using its incremental doses at 0.3 logarithmic intervals at every 3 min until the complete blockade is achieved . after obtaining stable twitch responses , rocuronium ( 10 mol / ml ) , succinylcholine ( 25 mol / ml ) , dantrolene ( 50 mol / ml ) , magnesium chloride ( 8 mmol / ml ) and lignocaine ( 600 mol / ml ) were added to produce neuromuscular blockade in the indirectly stimulated preparation . at around 50% reduction in height of t1 , zidovudine ( 30 mmol / ml ) or vehicle ( pss ) was added and their effects were compared on twitch responses . effect of zidovudine on neuromuscular transmission was studied by comparing the inhibition of twitch response ( % ) and train of four ratio ( t4/t1 ) at every incremental dose with that of baseline using the repeated measures of analysis of variance ( anova ) followed by tukey krammer multiple comparisons test in cumulative drc of zidovudine . inhibition of twitch responses by rocuronium was calculated and expressed as a percentage from the height of t1 of the train of four responses at every dose as compared to the height of t1 of basal twitch responses . drc of rocuronium in the absence and in the presence of zidovudine was compared by one - way analysis of covariance ( ancova ) using the statistical package for the social sciences ( spss ) 17 trial version software ( ibm corporation , usa ) . inhibitory concentration 5% ( ic5 ) , 50% ( ic50 ) and 95% ( ic95 ) were calculated from cumulative drc of rocuronium by employing regression analysis using masterplexreaderfit demo version software ( miraibio group of hitachi solutions america , ltd ) . comparisons of ic5 , ic50 and ic95 between the control group and interactions study groups 4 and 5 ; control group and interactions groups 6 and 7 were performed by anova followed by dunnett multiple comparisons test . effect of zidovudine 30 mmol / ml on twitch responses inhibited by dantrolene , magnesium chloride , rocuronium , succinylcholine and lignocaine was calculated in percentage and compared with that of vehicle added group ( pss ) by unpaired t - test . / a]100 ) , where a is the height of t1 in basal twitch response , b is the height of t1 , 3 min after the administration of zidovudine or vehicle . time required to produce complete block was recorded after administration of zidovudine as compared to vehicle added group , where reversal did not occur . comparison of time for complete block between vehicle added group and zidovudine added group was performed using unpaired t - test . drc of zidovudine was taken from the concentration of 1 mol / ml to 30 mmol / ml . zidovudine in the concentration range of 1 mol / ml to 1 mmol / ml reduced the twitch responses ( without affecting train of four ratio ) to the extent of 8 and 30% in indirect and direct stimulation experiments , respectively . in indirectly stimulated preparation , depression of twitch responses was nonsignificant whereas significant in directly stimulated preparation ( p<0.05 ) from the concentration of 3 - 30 mmol / ml , zidovudine significantly enhanced the twitch responses ( p<0.05 ) . up to 80% and 40% augmentation of twitch responses were noted with indirect and direct stimulation , respectively ( fig . 1 ) . however , this difference of augmentation between indirect and directly stimulated preparations was nonsignificant . interaction with rocuronium exhibited that at a concentration of zidovudine ( 1.2 mmol / ml ) , there was a trend of shift of drc towards the right whereas at concentration ( 12 mmol / ml ) , the trend of the shift was toward the left . 2 ) . differences between ic5 , ic50 and ic95 values of rocuronium in the absence and presence of zidovudine 1.2 and 12 mmol / ml were also nonsignificant ( table 1 ) . in the set of experiment where zidovudine was given in an intact animal for 15 days and phrenic nerve hemidiaphragm was obtained for in vitro experiment , there was the shift of drc of rocuronium toward right ( fig . 3 ) . however , the shift was statistically nonsignificant . as shown in table 2 , ic95 of rocuronium pattern of train of four ratios for rocuronium was not affected in the presence of zidovudine . effect of rocuronium in control and zidovudine treated groups effect of rocuronium in control and zidovudine treated groups zidovudine ( 30 mmol / ml ) has significantly augmented the twitch responses inhibited up to 50% by dantrolene ( 50 mol / ml ) , magnesium chloride ( 8 mmol / ml ) and rocuronium ( 10 mol / ml ) ( fig . 4 ) . block produced by succinylcholine ( 25 mol / ml ) was not reversed with zidovudine ( 30 mmol / ml ) , but it significantly reduced the total time for the complete blockade of succinylcholine as compared to control ( 238.757.7 vs. 192.511.3 s ; p<0.05 ) . zidovudine ( 30 mmol / ml ) neither reversed the 50% blockade nor affected the total time for the complete blockade of lignocaine ( 390100 vs. 332.547.5 s ) . effect of zidovudine on the resonses of dantrolene , magnesium chloride and rocuronium on diaphragm preparation . grey colored bars represent ( a ) dantrolene ( 50  mol / ml ) , ( b ) magnesium chloride ( 8 mmol / ml ) and ( c ) rocuronium ( 10  mol / ml ) . data expressed as mean standard error of mean and * represents significance at p > 0.05 . chronic zidovudine therapy is known to cause clinically significant myopathy and mechanisms proposed for the zidovudine - induced myopathy are depletion of mitochondrial dna , oxidative stress , direct inhibition of mitochondrial bioenergetics machinery and mitochondrial depletion of l - carnitine . in the present experiments , zidovudine has augmented the indirect stimulated preparation and exhibited a biphasic response ( initial depression followed by augmentation ) in the direct stimulated preparation . from the concentration of 1 mol / ml to 1 mmol / ml , it shown significant depression of twitch responses up to 30% in the direct stimulated preparation suggests its mild inhibitory action on the skeletal muscle . augmentation of contractions was seen with zidovudine from 3 mmol / ml concentration in both types of the preparations in a dose - dependent manner . as the augmentation was noted in both indirect as well as directly stimulated preparations , site of action could be on skeletal muscle . however , the higher augmentation of twitch responses on in the indirectly stimulated preparation suggests neuroumuscular component is also involved in its mechanism . it is worth noting that mitochondrial inhibitors are known to augment the miniature end plate potential and neurotransmitter release . interference with mitochondrial function can lead to release of already sequestered calcium in the mitochondria resulting in a higher calcium concentration in the nerve terminals , which might cause the release of higher amounts of the neurotransmitter . mefloquine augments the neurotransmitter release by increasing intracellular calcium level due to inhibition of mitochondrial adenosine triphosphate production . this may have partly contributed in the augmentation of twitch responses . observed effect of zidovudine on twitch responses inhibited by dantrolene and magnesium chloride suggests the involvement of calcium related mechanism . dantrolene , a direct muscle relaxant produces its action by inhibition of calcium release from sarcoplasmic reticulum . it also markedly depresses the frequency of spontaneous miniature potentials at neuromuscular junction due to its action at intracellular calcium stores in the presynaptic terminals . in the present study , caffeine and theophylline releases calcium from sarcoplasmic reticulum , whereas 3,4-diaminopyridine inhibits potassium conductance and releases calcium from nerve and muscle membranes . magnesium chloride inhibits entry of extracellular calcium into the nerve terminals and decreases the neurotransmitter release . it also stabilises the postjunctional membrane and reduces excitability of muscle fiber . in the present study , block of magnesium chloride was reversed with zidovudine . from the above observations it can be speculated that zidovudine increases the calcium level at neuromuscular junction and skeletal muscle . reversal of rocuronium block and potentiated succinylcholine block with zidovudine ( 30 mmol / ml ) may be explained by its role in facilitation of release of acetyl choline ( ach ) by affecting the calcium channel at the neuromuscular junction they also promote the shift of nicotinic receptors from a normal state to desensitised state . zidovudine may not be able to overcome the neuromuscular block produced by lignocaine because of sodium channel blockade or desensitised state of nicotinic receptors . trend of shift of drc was toward the right in both sets of interaction studies except for zidovudine 12 mmol / ml . in the drc , of zidovudine of indirect stimulation augmentation of twitch responses however , zidovudine 12 mmol / ml did not shift the drc of rocuronium toward the right . this dose of zidovudine may have cause desensitisation of receptors and potentiate the block of rocuronium by shifting the type of block from competitive to noncompetitive type . results of pretreated animals with zidovudine were also in line with in vitro addition of zidovudine where 50 mg / kg has shown more effect on drc of rocuronium than 100 mg / kg . dose - dependent effect observed on drc of rocuronium was not reflected on the interaction studies . zidovudine 50 mg / kg for 15 days ( equivalent human dose 10 mg / kg , a therapeutic dose ) nonsignificantly increased ic5 and ic50 and significantly increased ic95 ( p<0.05 ) . zidovudine 100 mg / kg for 15 days ( equivalent human dose 20 mg / kg , two times higher therapeutic dose ) led to nonsignificant higher values of ic5 , ic50 and ic95 , but comparatively lower than zidovudine 50 mg / kg group ( table 2 ) . some differences observed in interaction studies were quite large , statistical significant difference was not found . confirmatory studies with plasma concentration of zidovudine and level of ach at the neuromuscular junction in in vivo preparation will be helpful to study the interaction with neuromuscular blocker . augmentation of twitch responses is possibly due to its effect on the neuromuscular junction and skeletal muscle .
neuromuscular weakness is often found in patients receiving zidovudine therapy due to mitochondrial damage . 
 effect of zidovudine was evaluated in indirectly and directly stimulated isolated rat phrenic nerve hemidiaphragm preparations , by cumulative dose response curve with square wave pulses , 0.5 ms duration of 2 hz at every 10 s. to understand the observed effect of zidovudine , interaction studies were carried out with rocuronium . 
 dose response curve of rocuronium was compared in the absence and in the presence of 1.2 and 12 mmol / ml zidovudine . in another set of experiment , 
 intact animals were treated with zidovudine 50 and 100 mg / kg for 15 days and phrenic nerve hemidiaphragm was obtained for in vitro experiment . 
 effect of zidovudine ( 30 mmol / ml ) on twitch responses inhibited by dantrolene 50 mol / ml , magnesium chloride 8 mmol / ml , rocuronium 10 mol / ml , succinylcholine 25 mol / ml and lignocaine 600 mol / ml was studied . 
 zidovudine ( 3 - 30 mmol / ml ) significantly augmented the twitch responses up to 80 and 40% in indirectly and directly stimulated preparations , respectively ( p<0.05 ) . 
 zidovudine did not show significant interaction with rocuronium in any group as per dose response curve and inhibitory concentration 5% , inhibitory concentration 50% and inhibitory concentration 95% of rocuronium . 
 zidovudine ( 30 mmol / ml ) augmented the twitch responses inhibited by dantrolene , magnesium chloride and rocuronium . 
 it reduced the time for complete block of succinylcholine ( p<0.05 ) . 
 zidovudine affects the neuromuscular transmission . 
 no conclusive interaction between rocuronium and zidovudine was found .