dystrophic · Correspondence to: DrJosette Dangain, School ofPhysiology and Pharmacology, University of NSW, PO Box 1, Kensington, NSW2033,Australia. Received 13 January 1989andin

Journal ofNeurology, Neurosurgery and Psychiatry 1989;52:1382-1389

Long term effect of low frequency chronic electricalstimulation on the fast hind limb muscles ofdystrophic miceJ DANGAIN, G VRBOVA

From the Department ofAnatomy and Embryology, Centrefor Neuroscience, University College, Gower Street,London

SUMMARY Low frequency chronic electrical stimulation can have a beneficial effect on dystrophicmuscles. The present study was undertaken to assess the long term effect of such stimulation on thefast hind limb muscles of dystrophic mice. The relationship between the changes induced bystimulation and the initial condition of the dystrophic muscles, as well as other factors which mightcontribute to this relationship, were examined. The stimulation induced an increase in the forceoutput ofweak dystrophic muscles and a speeding of their time course ofcontraction and relaxation,as well as an increase in their fatigue resistance. In relatively strong dystrophic muscles, thestimulation induced similar changes in contractile speed and fatigue characteristics, but it led to a

slight decrease in force output. Our results suggest that the stimulation promotes the growth anddifferentiation of the small regenerating fibres known to be present in the diseased muscles and, inaddition, induces an increase in the mitochondrial content of the muscle fibres. Our results indicatethat these effects are not permanent.

It has previously been reported that low frequencyelectrical stimulation has a beneficial effect on dystro-phic muscles. Experiments carried out on dystrophicmice have shown that chronic electrical stimulation at8-10 Hz induced a functional improvement as well asan increase in the total number of fibres in the fastmuscles of the hind limbs to which it was applied.715 Inaddition, it was found that the slowing of the timecourse of contraction and relaxation caused by thedisease process in these muscles415 was reduced follow-ing stimulation and their resistance to fatigue wasincreased. Further examination of the same prepara-tions showed that both the level of glycolytic enzymes,as well as that of their different isozymes, and themembrane properties characteristic of adult fastmuscles had been restored following stimulation.312 Indystrophic chickens, a similar pattern of electricalstimulation applied chronically to the posteriorlatissimus dorsi muscle caused a reduction in the rate

Correspondence to: Dr Josette Dangain, School of Physiology andPharmacology, University of NSW, PO Box 1, Kensington,NSW 2033, Australia.

Received 13 January 1989 and in revised form 24 May 1989.Accepted 27 June 1989

of deterioration of its fibres as well as in the level ofAChE activity, usually elevated in that particularmuscle.' Finally, and perhaps most importantly,electrical stimulation at 5-8 Hz was shown to producea significant increase in the maximum voluntary forcedeveloped by the Tibialis anterior (TA) muscle inpatients with Duchenne muscular dystrophy(DMD).'4

In view of these findings, it seems important todiscover ifthe changes induced by such stimulation arepermanent. In only one of these studies were thesubjects re-examined six months after the end of thestimulation treatment.'4 This examination indicatedan overall deterioration in the physical characteristicsof the patients. However, as the assessment was basedon the overall strength of several muscle groups, thecontribution of the stimulated muscle, per se, was notclear.

In a previous study,'5 it was reported that functionalimprovement in dystrophic mice following electricalstimulation was only apparent when the muscles wereseverely affected by the disease process. It could bethat the improvement in function induced by chronicstimulation is due to an improved growth of theregenerated fibres that are known to be present indystrophic muscles, particularly during the early stage

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Long term effect oflowfrequency chronic electrical stimulation

of the disease,' and in a weaker muscle the number ofsuch fibres may be greater. However, this is notnecessarily so, and other factors may contribute to thedifferent response of "weak" and "strong" muscles toelectrical stimulation.

This study was undertaken to assess the long termeffect of low frequency chronic electrical stimulationof the fast hind limb muscles of dystrophic mice (dy2j),and to define further the relationship between theinitial condition of the diseased muscles and thechanges induced by electrical stimulation.

Methods

Dystrophic mice of the strain C57BI/6J dy2i aged 3-6 monthswere used in these experiments.

1) Experimental protocolsUsing chloral hydrate anaesthesia (IP, 1 ml/l00 g bodyweight of a 4 5% solution) and sterile precautions, Tefloncoated stainless steel wires were implanted either side of thelateral popliteal nerve in one hind limb, the other one servingas a control. The Teflon coating was removed from both endsof the wires over a length of 0 5 mm. A fine silk thread wasused to secure the wires, through small loops, to the lateralhead of the gastrocnemius muscle. The wires were led underthe skin and externalised at the neck of the animal, where theends were attached to two small hooks and sewn to the skin.7The animals were left to recover and stimulated daily via theimplanted electrodes at 10 Hz for 30 minutes in each hour for6-8 hours each day,7 or for one minute every second minutefor a similar period of time. No differences were notedbetween these regimes of stimulation. The stimulationparameters were such that not only contraction of the TAmuscle could be felt, but flexion of both the ankle and thedigits could be clearly observed.

Five dystrophic animals implanted with electrodes wereleft unstimulated for control experiments (DIC). Thirty onedystrophic mice were subjected to the stimulation treatmentfor periods oftime ranging from five to 21 days (DS). In 19 ofthese mice, the force output, contractile properties, weightand fatigue characteristics of both the operated and con-tralateral TA and EDL muscles were examined upon termin-ation of the stimulation treatment (Short term). In 12animals, a "rest" period of four weeks was allowed betweenthe last day of chronic electrical stimulation and the finalexperiment (Long term).At various intervals after the initial operations, the animals

were anaesthetised with chloral hydrate (as above). Thetendons ofthe Tibialis anterior (TA) and Extensor digitorumlongus (EDL) muscles were freed and attached to fine silkthreads. The sciatic nerve was dissected and cut centrally.Small rigid pins were put through the proximal and distalends of the tibia and the legs were then secured to a rigidtable. Contractions were elicited by stimulating the sciaticnerve with supramaximal stimuli. The tendons were attachedto stain gauges to record isometric contractions. These weredisplayed on an oscilloscope screen, from which they werephotographed, as well as on a Devices pen recorder. To testfatiguability, the muscles (TA only) were stimulated by trainsof 10 pulses at 40 Hz, repeated every second for three

minutes. The changes in tension were expressed as the ratio ofthe tension developed after three minutes to the initial tensionx 100. This is referred to as the Fatigue Index (FI). When therecordings were completed, the muscles were excised andweighed, then quickly frozen in isopentane precooled inliquid nitrogen.

2) Statistical analysisComparison between the values of the different parametersstudied obtained from the contralateral untreated muscles inthe different experimental groups was made using a one wayanalysis of variance (ANOVA). If significance was estab-lished (P < 0 05, 2 tails) the values were further comparedusing the Newmann-Keuls multiple comparison test.Comparison between the values obtained from the operatedmuscles and their respective unoperated contralateralcounterparts within each experimental group studied wasmade using a paired Student t-test (2 tails). In some instances,the raw data obtained from the operated muscles in thedifferent experimental groups were directly compared using aone way analysis of variance. If necessary, the Newmann-Keuls multiple comparison test was used.

Results

The values of the different parameters studiedobtained from the untreated contralateral TA andEDL muscles in the various groups of stimulated mice(that is, short term and long term) are summarised intable 1, where they are compared to those obtainedfrom the corresponding unoperated contralateralmuscles of implanted control (dystrophic) mice. Bothin the stimulated and implanted control animals, theforce output and weight of the untreated contralateralTA and EDL muscles were within the same range.These unoperated contralateral muscles will thereforebe used, within each animal, to assess the changesinduced by the treatment applied to their operatedcounterparts (that is, electrical stimulation orelectrode implantation).

For other parameters, such as the time course ofcontraction (TTP) and relaxation (1/2 Relax), and theFatigue Index (FI), however, some differences wereobserved. For instance, the TTP and half relaxation ofthe contralateral (untreated) EDL muscle in the "shortterm stimulated mice" were found to be significantlydifferent (P < 0-05, both cases) from those of thecontralateral untreated EDL muscle in the implantedmice. A similar trend could be observed with respect tothe TA muscle, but the differences were not sig-nificant. The FI of the contralateral TA muscle in theshort term stimulated mice, on the other hand, wassignificantly different (P < 0-05) from that of thecorresponding TA muscle in the implanted controlanimals. Interestingly, it was found that in musclescontralateral to the "long term stimulated" ones, thevalues of these parameters were not significantlydifferent from those of the "implanted control"

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Table Contractile properties,force output andfatigue characteristics ofthe untreated TA and EDL muscles in dystrophicmicefrom the different experimental groups studied

Twitch Tetanic Muscletension TTP 1/2 Relax tension weight Fl

Gp n (g) (ms) (ms) (g) (mg) (%)

DIC 5 14 30 (2 60) 35 60 (4 02) 54 60 (9-47) 46-20 (6 39) 28-44 (1-67) 24-20 (5-80)DS(St) 19 16-93 (1 26) 30 05 (1-22) 41 68 (3-60) 59-95 (4-06) 31-73 (1-15) 50-98 (4-97)trGPI 7 14-13 (1.90) 31-71 (2-33) 42-29 (4-69) 46-14 (1-79) 29-46 (2-32) 57-67 (9-17)

TA muscle p2 8 16-64 (1-03) 30-63 (1-72) 46 75 (6-59) 57 (1-56) 32-53 (1-63) 45-88 (7-99)p3 4 22-41 (3-65) 26 (1-41) 30-50 (5-56) 90 (5-40) 34-10 (0-82) 51-15 (9-01)

DS(Lt) 12 14-16 (1-23) 32-25 (1-46) 45-25 (3-34) 51-25 (1-97) 32-43 (1-23) 42-38 (6-75)-pI 7 11-44 (0-80) 32-85 (2-46) 43-14 (2-96) 46-36 (0-98) 31-73 (1-45) 46-86 (9-67)LGp2 5 17-97 (1-55) 31-40 (0-98) 48-20 (7-17) 58-10 (1-89) 33-42 (2-25) 36-10 (9-35)

** *

DIC 5 4-14 (0-21) 40 (4-34) 64 (11-80) 14-33 (2-63) 7-38 (0-43)DS(St) 19 4-39 (0-32) 28-32 (1-27)tt 40-53 (3-25)f 18-59 (1-39) 7-97 (0-34)

EDL muscle rGpl 5 3-20 (0-46) 32(2-28) 47-60(2-99) 10-82 (1-10) 6-48 (0-43)LGp2 14 4-82 (0-34) 27 (1-40) 38 (4-13) 21-37 (1-12) 8-51 (0-34)DS(Lt) 10 4-41 (0-25) 34-30 (1-73) 50-30 (2-41) 17-34 (1-40) 8-23 (0-30)rGpI 2 4-03 (0-34) 37 (7) 53 (3) 10-38 (3-30) 8-25 (1-35)LGp2 8 4-52 (0-30) 33-63 (1-65) 49-63 (2-95) 19-08 (0-78) 8-23 (0-27)

Twitch tension, Time to peak (TTP), Half relaxation (1/2 Relax), Tetanic tension, Weight and Fatigue Index (FI) of the untreatedcontralateral TA and EDL muscles, respectively, from Dystrophic implanted control (DIC) and Dystrophic Stimulated (DS) Short term (St)and Long term (Lt) mice. Values are given as mean SE (mean). Anova: (*) P < 0-05; (**) P < 0-005. Newmann-Keuls multiple comparisontest: t different from DIC, P < 0-05; (t) difference between DS(St) and DS(Lt), P = 0-05. Also summarised, but not included in thecomparison, are the values of the parameters studied in the contralateral TA and EDL muscles for each of the subgroups defined in the text.

contralateral muscles. These values were intermediatebetween the values of the two former groups.

Thus, a simple comparison between operated andcontralateral muscles might not be adequate toestablish the effect of the stimulation on theseproperties of the dystrophic muscle. The statisticalanalysis of these parameters has therefore been dealtwith accordingly.

1) Short-term effect of chronic electrical stimulationa) TA muscle The changes (%) in maximum forceoutput of the stimulated TA muscle within eachanimal studied were first examined as a function of thelength of time during which stimulation was applied(fig la). It was found that there was no correlationbetween the changes induced by chronic electricalstimulation and its duration. However, when thechanges in force output seen in the stimulated TAmuscles were examined as a function ofthe initial forcedeveloped by the muscles (taken to be that of thecontralateral control muscle within each individualanimal studied), a much clearer pattern emerged (figlb). The smaller the initial force output of thedystrophic muscle, the more likely it was to respondfavourably to electrical stimulation. On the otherhand, those muscles which initially developedrelatively large forces appeared to be adverselyaffected by the stimulation treatment. The degree ofassociation between both variables in this relationship(that is, % change and initial force output) was testedusing the Pearson's test. The correlation factorbetween the two variables was found to be significant

5050

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Fig 1 Changes (%) inforce output ofstimulated dystrophicTA muscles expressed (a) as afunction of the length of time(days) during which stimulation was applied; (b) as afunction of the maximnumforce (g) initially developed by themuscles. Each point represents an individual experiment.

1384 Dangain, Vrbova

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Long term effect oflowfrequency chronic electrical stimulation(P < 0.02). The animals were divided into threegroups according to the maximum force their TAmuscle (that is, their contralateral TA muscle) initiallydeveloped. In Gpl, animals were selected whose TAmuscles developed forces below the mean (range 35 to50 g) tetanic tension produced by all the 19 TAmuscles. Gp2 comprised animals whose TA musclesdeveloped maximum forces within this average (range51 to 75 g), and Gp3 comprised animals whose TAmuscles produced relatively large forces in comparisonwith this average (range 76 to 105 g). When thisarbitrary grouping was performed, it was found thatthe weakest muscles (Gp 1) were also the slowest, whilethe relatively "strong" dystrophic TA muscles (Gp3)were associated with a relatively faster speed ofcontraction and relaxation (table 1).We found that, indeed, the stimulation treatment

led to a significant increase (P < 0-05) in the forceoutput of the TA muscles from Gpl, t-hat is, theweakest muscles (fig 2a). On the other hand, thestimulation did not affect the force output of themuscles from Gp2, but it produced a small (17%)decrease in the force output of those dystrophicmuscles which initially developed large forces (Gp3).This difference was not significant. The implantationof electrodes, per se, had no effect on the force outputof the treated muscles, or on any of the parametersstudied. There were no differences in weight betweenoperated and contralateral muscles within any of thegroups of stimulated animals. There were no differ-ences in contractile speed and FI between operatedand contralateral muscles within each of these groups.Since stimulation might have a slight effect contra-laterally with respect to these properties, a directcomparison of the stimulated muscles from each ofthese groups with the implanted control muscles wasmade (see Methods). It could then be seen that thecontractile speed, particularly the time course ofrelaxation, became slightly, but not significantly,faster in the stimulated muscles. Similarly, there was atendency for their fatigue resistance to be enhanced(fig 2b).b) EDL muscle The changes observed in the forceoutput of dystrophic EDL muscles following chronicelectrical stimulation showed a similar pattern to thatseen in dystrophic TA muscles. There was no correla-tion between the changes in force output induced bythe stimulation and the length oftime during which thestimulation was applied (fig 3a). There was a highlysignificant correlation (P < 0 005) between thechanges in force output induced by the stimulationand the force developed by the untreated contralateralEDL muscle within each individual animal (fig 3b).The animals were divided into two groups accordingto whether the maximum force initially developed bythe EDL muscle was below (Gpl) or above (Gp2) the

0.

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U _ N ,_' N__a . a, CL Q aO CD O O CD

StimulatedShort term Long term

Fig 2 (a) Changes inforce output of TA musclefromDystrophic Implanted Control (DIC) and Stimulated, Shortterm and Long term, weak (Gpl), within average (Gp2) andrelatively strong (Gp3) dystrophic mice. The values obtainedfrom the treated muscle were expressed as % ofthoseobtainedfrom the contralateral muscle within each individualanimal. Each column therefore represents the meanSE(mean) within each group studied. The nwnbers at thebottom ofeach column indicate the number ofanimalsstudied. (*)P < 0OS (paired t-test).Note: From this "normalisation" ofthe values obtainedfromthe operated muscles, itfollows that 100% correspond tocontrol level (that is, no change). (b) TTP, 1/2 Relax andFI of the operated TA musclesfor each ofthe groupsrepresented in a. These are means (SE) ofabsolute values."n" in each group is as indicated in a, unless stated otherwise.

population's average. Again, relative differences in thecontractile speed of the muscles in each of thesesubgroups could be seen (table 1). It should be notedthat these differences were also noticeable in thecorresponding subgroups from the "long term"stimulated muscles. It was found that the stimulationtreatment led to a significant improvement in the forceoutput of the weakest muscles (P < 0 02), while itproduced a small, but significant (P < 0-01) decreasein the force output of the "strongest" dystrophicmuscles (fig 4a). As for TA muscle, these changes inforce output were not associated with any changes inweight. A direct comparison (fig 4b) of the dataobtained from the stimulated EDL muscles in thevarious groups, with that obtained from the implanted

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contractile speed between operated and contralateralmuscles within each group of stimulated animals. Theimplantation ofelectrodes, per se, had no effect on anyof the parameters studied in this muscle.These results indicated that chronic electrical

stimulation at low frequency brings about changes indystrophic muscles. The question was whether thesechanges were permanent.

2) Long term effect of electrical stimulationThis question was addressed by studyingTA and EDLmuscles which had been subjected to the same stimula-tion treatment, then left unstimulated for a period offour weeks before the final experiment.a) TA muscle It was found that four weeks after the

TTP

1/2 Relaxation(Ms)

8080 |ANOVA: p<0 02

20-

t)'- N _ N_ Q C1 Q QaCa CD CD C

2

0

ae.-I

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Fig 3 Changes (%) inforce output ofstimulated dystrophicEDL muscles expressed (a) as afunction ofthe length oftime during which stimulation was applied; (b) as afunctionofthe maximum force initially developed by the muscles.Each point represents an individual experiment.

control muscles revealed a significant decrease in thetime course of contraction (ANOVA, P < 0 05) andrelaxation (ANOVA, P < 002) in all stimulatedmuscles. While it might appear that these parameterswere more affected in the stimulated muscles from therelatively "strong" dystrophic mice, it has to beremembered that these muscles were relatively fasterto begin with (table 1). There were no differences in

StimulatedShort term Long term

Fig 4 (a) Changes inforce output ofEDL musclefromDystrophic Implanted Control (DIC) and Stimulated, Shortterm and Long term, weak (Gpl) and relatively strong(Gp2) dystrophic mice. The data are represented as infig 2a.The asterisks indicate the degree ofsignificance asfollows:(*)P < 0O05; (**)P < 0-02; (***)P < 0-01 (paired t-test). (b) TTP and 1/2 Relax (absolute values) of theoperatedEDL musclesfor each ofthe groups represented ina. The data are represented as infig 2b. The asterisksindicate differencefrom DIC: (*)P = 0-05; (**)P < 0-025;(***)P < 0 01 (Newmann-Keuls multiple comparison test).

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Long term effect oflowfrequency chronic electrical stimulationend of the stimulation treatment, the maximum forcedeveloped by the treated muscles from Gpl wassimilar to that of their contralateral control muscles,that is, that the increase in force output that hadpresumably occurred was no longer apparent (fig 2).As in the previous series ofexperiments, the maximumforce developed by the stimulated TA muscles fromGp2 was similar to that developed by their con-tralateral control muscles after this period of "rest".As we were not able to predict the total amount offorce a dystrophic muscle would develop from theclinical examination ofthe sick animal, there is no dataavailable corresponding to Gp3. We do not know,therefore, whether the slight decrease in force outputthat had been seen in the relatively strong dystrophicmuscles following stimulation would also be observedunder these particular conditions. Fig 2b shows thatthe values of both the 1/2 Relaxation time and the FIin the long term stimulated TA muscles were similar tothose obtained from the implanted control muscles.Thus, the small changes seen in the values of theseproperties in the short term stimulated dystrophic TAmuscle were no longer apparent under these particularconditions.b) EDL muscle After a similar period of "rest", thestimulated EDL muscles from the weakest group ofanimals (Gpl) developed significantly greater force(P < 005) than that of their untreated contralateralmuscles. In contrast, the stimulated muscles from therelatively "strong" dystrophic animals (Gp2)developed slightly, but significantly (P = 0-05),smaller force output than their contralateral controlmuscles (fig 4a). In both groups, the contractile speedof the long term stimulated EDL muscles differedfrom that of implanted control muscles (fig 4b). Thedifference was significant on the time course ofrelaxation (Newmann-Keuls test: P < 0-05 andP < 0-025, respectively), but not on the time course ofcontraction. These values were not significantly dif-ferent from those of the short term stimulated musclesexamined previously. However, a tendency towards areturn to "normality" could be seen.

Discussion

Our results indicate that chronic electrical stimulationat low frequency (10 Hz) brings about an increase inthe force output of the weakest TA and EDL musclesfrom the dystrophic mice studied, while it induces aslight, but significant, decrease in the force output ofrelatively strong dystrophic muscles. They alsoindicate that the percentage change in force outputobserved in the dystrophic stimulated muscles bearsno relationship to the duration of the stimulation.Finally, our results show that the stimulation has noeffect on the weight of these muscles, while it increases

their contractile speed and their resistance to fatigueindependently of whether or not their force outputimproves.That such stimulation can improve the performance

of dystrophic muscles agrees with reports in theliterature on a similar preparation715 or on childrensuffering from DMD.'4 The lack of correlationbetween the extent of these changes and the length oftime during which stimulation was applied differsfrom the finding of Scott et al'4 who reported that inyoung children suffering from DMD, the stimulationtreatment had to be applied for at least six weeksbefore any effect could be seen. Our finding, however,is similar to that of Luthert et ar who suggested thatthis lack of correlation could be because the mice usedin their study were not a homogeneous group. Thisargument may also apply to our study, since the age ofthe animals used ranged from three to six months, theseverity of their affliction was variable and the range oftension developed by the control, unstimulated TAand EDL muscles of individual animals in our studywas even greater than in their study. The lack of effectof the stimulation on the weight of dystrophic musclesis similar to that reported by Luthert et ar on a similarpreparation. They suggested that this observationcould simply be explained in view of the large amountof connective tissue and degenerating fibres present inthe diseased muscles.'6 The speeding of the time courseofcontraction and relaxation of the muscles followingstimulation agrees with that reported for the dystro-phic mouse. A similar trend was observed for the timecourse of relaxation of the stimulated TA muscle ofchildren suffering from DMD. The increase in thefatigue resistance of the dystrophic TA muscle follow-ing stimulation was similar to that previously reportedfor the EDL muscle in dystrophic mice.'5 Such anincrease in fatigue resistance was not seen in thestimulated TA muscle of children suffering fromDMD.'4 Unlike muscle from adult dystrophic mice,however, the muscles of normal children or fromchildren suffering from DMD already have a.greatability to maintain tension.'3 415While our results confirm the relationship between

the initial weakness of the dystrophic muscle and itslikeliness to respond favourably to stimulation, firstemphasised by Vrbova and Ward,'5 they also suggestthat the performance of relatively strong dystrophicmuscles might be unfavourably influenced by thestimulation treatment. This is important because,according to these authors, the TA and the EDLmuscles in dystrophic mice do not deteriorate at thesame rates. Thus, it is possible that while the stimula-tion could be beneficial to one muscle, it could bedetrimental to the other one. In our study, seven out of19 animals had a TA muscle whose force output,initially below the average tension developed by all

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1388muscles studied in that group, improved followingstimulation. Only two ofthese animals also had a weakEDL muscle whose performance improved followingstimulation, and five of these animals had relativelystrong EDL muscles whose force output decreasedslightly following the treatment. This decrease in forceoutput was, however, small and since it was notaccompanied by a proportional decrease in muscleweight, it is possible that it is due to the replacement ofcontractile proteins by mitochondria, as suggested bythe increased resistance to fatigue of these muscles.This property is known to reflect the oxidativecapacity, that is, the mitochondrial volume of themuscle fibres.8 In view of the poor performancedystrophic muscles already have, compared withnormal muscles, this small decrease in force outputshould perhaps be considered. It should be noted thatour data also indicate that in most of the dystrophicmice studied, the TA muscle was more severelyaffected than the EDL muscle. From our results, italso appears that the contractile speed of thedystrophic EDL muscle is more readily affected by thestimulation than that of the dystrophic TA muscle.Finally, our results show that there is a markedcontralateral effect of the stimulation on both thecontractile speed and the fatigue resistance of themuscles. The extent of the changes induced in theuntreated contralateral muscles are similar to thoseinduced in the stimulated muscles. Such an effect ofthe stimulation, applied to the nerve via implantedelectrodes, as carried out in the present experiments,has not been reported previously, at least in dystrophicanimals. The stimulated animals did not appear to usetheir contralateral limb preferentially during thestimulation periods and thus, it is possible that thiscontralateral effect of the stimulation is related tosome cross-reflex activity.Four weeks after the end of the stimulation treat-

ment, the changes that had been seen in the stimulateddystrophic TA muscle were no longer apparent. Thesechanges, therefore, are not permanent. Interestingly,the response of the dystrophic EDL muscle wasslightly different from that of the TA muscle as after asimilar period of "rest", the changes in force outputand contractile properties seen in the stimulated EDLmuscles of both subgroups are still present. We realisethat subgroup one contains only two samples.However, we feel that the difference in force outputbetween operated and untreated contralateral controlmuscles is large enough to be reliable. Our data suggestthat while outlasting the changes induced by thestimulation in the dystrophic TA muscle by at leastfour weeks, the changes induced in the dystrophicEDL muscle are unlikely to be permanent.The TA muscle deteriorates more severely than the

EDL muscle'6 (and present study) and the changes it

Dangain, Vrbova

undergoes following stimulation disappear morequickly than those induced in the EDL muscle. This isinteresting since both muscles have a similar functionand fibre type composition. Their anatomical posi-tion, however, is different as the TA muscle coversonly one joint (ankle) while the EDL muscle coversboth the knee and the ankle joints. Since in thesedystrophic mice the ankle joint is incapable of dorsi-flexion while the knee retains some flexibility, the TAmuscle may be subjected to more stretch than the EDLmuscle. Thus, it is possible that mechanical stretchmight contribute to the deterioration of the diseasedmuscles and might also influence the response of themuscle to stimulation.

It is known that low frequency electrical stimulationcan induce the changes normally undergone by mam-malian skeletal muscle during postnatal development,9in particular, the increase in the time course ofcontraction and relaxation that has been shown tooccur in developing fast skeletal muscles.2 The dystro-phic muscles whose force output improved followingstimulation, that is, the weakest muscles, have arelatively slower speed of contraction and relaxation.It is known that, at least during the early stages of thedisease, the sick muscles contain regenerating musclefibres. The latter, like immature muscle fibres contractand relax slowly. Thus, the slower contractile speed ofthe weakest dystrophic muscles reflects the presence ofthese fibres. It is possible that stimulation will inducenot only the growth of these fibres, but also theirdifferentiation. This would account for both theincrease in force output and for the changes incontractile properties seen in these muscles in thisstudy and for the increase in the number of fibrespreviously reported to occur in these muscles.7 Therelatively faster contractile speed of the strongerdystrophic muscles suggests that they might notcontain such fibres and it is possible that in thesemuscles, the stimulation acts on the remainingrelatively healthy fibres. This, however, seems unlikelysince electrical stimulation of a similar kind applied tonormal fast muscles from adult rabbits or rats5"0 doesnot lead to such changes in contractile speed. On thecontrary, low frequency electrical stimulation of nor-mal adult fast muscles produces a slowing of the timecourse of contraction and relaxation which resultsfrom the transformation of fast muscle fibres intofibres with morphological, biochemical and histo-chemical properties similar to those of slow musclefibres." Thus, in these muscles too it is likely that thestimulation acts on a population of regenerating orimmature muscle fibres.

This work was supported by a grant from the MedicalResearch Council.

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Long term effect oflowfrequency chronic electrical stimulation

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