Lateral electrical surface stimulation is one of methods used in the therapy of the progressive form of idiopathic
scoliosis (IS) in children and youth. However, there are data suggesting that this method may lead to serious adverse side
effects, when used for a too long period of time per day. To clarify this issue, the present study was aimed at disclosing possible
changes in the ultrastructural appearance of rabbit supraspinal muscles undergoing long-term stimulation (9 h per day, 3
months), an animal model successfully used to mimic the situation in humans. In comparison to the control animals, muscles
of "overstimulated" rabbits exhibited clear signs of microscopical lesions, including depletion and disintegration of myofilaments,
proliferation, dilatation and, sometimes, swelling of sarcoplasmic reticulum and/or mitochondria, as well as signs of
destruction of the Z line. The above-mentioned abnormalities, especially the signs of degenerative processes associated with
the Z line and the observed microlesions strongly suggest that the failure of the long-term LESS therapy of the IS may be
attributable to these ultrastructural lesions

Andrzejewska, Anna

Babińska, Izabella

Kowalski, Ireneusz M.

Lipińska, Joanna

Majewski, Mariusz

Szarek, Józef

Wojtkiewicz, Joanna

English

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 43, No. 4, 2005pp. 243-247 Ultrastructural features of supraspinal muscles in rabbitsafter long-term transcutaneous lateral electrical surfacestimulation (LESS)Ireneusz M. Kowalski1, Józef Szarek2, Izabella Babin´ska2, Joanna Wojtkiewicz3, Anna Andrzejewska4, Joanna Lipin´ska2 and Mariusz Majewski31Provincial Childrens’ Hospital and Rehabilitation Centre, Ameryka,2Department of Forensic and Administrative Veterinary Medicine and 3Division of Clinical Physiology, Warmia and Mazury University, Olsztyn, 4Department of Clinical Pathomorphology, Medical Academy, Białystok, PolandAbstract: Lateral electrical surface stimulation is one of methods used in the therapy of the progressive form of idiopathicscoliosis (IS) in children and youth. However, there are data suggesting that this method may lead to serious adverse sideeffects, when used for a too long period of time per day. To clarify this issue, the present study was aimed at disclosing possiblechanges in the ultrastructural appearance of rabbit supraspinal muscles undergoing long-term stimulation (9 h per day, 3months), an animal model successfully used to mimic the situation in humans. In comparison to the control animals, musclesof "overstimulated" rabbits exhibited clear signs of microscopical lesions, including depletion and disintegration of myofila-ments, proliferation, dilatation and, sometimes, swelling of sarcoplasmic reticulum and/or mitochondria, as well as signs ofdestruction of the Z line. The above-mentioned abnormalities, especially the signs of degenerative processes associated withthe Z line and the observed microlesions strongly suggest that the failure of the long-term LESS therapy of the IS may beattributable to these ultrastructural lesions. Key words: Idiopathic scoliosis - Lateral electrical surface stimulation (LESS) - Muscle -  Ultrastructure - Rabbit IntroductionLateral electrical surface stimulation (LESS) has beenthought to be one of the very promising techniques inthe therapy of progressive idiopathic scoliosis (IS) inchildren [3, 4, 11, 27, 28]. However, there is a growingbody of evidence that in its "classical" form, (i.e., 9 hoursa day, rectangular impulse; duration of single impulse:0.1-0.2 ms, duration of impulse series: 4.0 s; pausebetween series: 8 s; impulsation frequency: 40 Hz; basedon [1, 4, 28]), LESS is able to cause adverse side-effectsthat drastically diminish its tolerance by the patient.Thus, dissomnia, different forms of contact dermatitis aswell as changes in muscles, are the most often observedside-effects of this therapy [1, 2, 4, 11, 12, 22, 26]. Inour previous paper, concerning the light-microscopicalevaluation of LESS-induced changes in the rabbit supra-spinal muscles, we found that this procedure led to anincrease in the muscle congestion, granular atrophy and,in approximately 20% of the affected muscle fibers, theirwave-like course [13]. Thus, the above-mentioned ef-fects raised a debate as to whether this approach shouldbe used as a therapeutic tool in its present form [11, 14,23, 24]. Furthermore, the method appears also too ex-pensive to be widely used in the clinic. As the mostpronounced adverse side-effect appear to be changes inthe stimulated muscles, the present study was aimed atdisclosing possible changes in the ultrastructural appear-ance of rabbit supraspinal muscles undergoing long-term (i.e., "classical") electrostimulation (9 h per day, 3months), an animal model successfully used to mimicthe situation in humans. Materials and methodsTen male, pure-breed (New Zealand White), age-matched (bornwithin 5 days) and clinically healthy rabbits were used in the presentinvestigation. The animals were adapted for 30 days to the newenvironment. They were kept indoors in a room with controlledCorrespondence: M. Majewski, Div. Clinical Physiology, Warmiaand Mazury University, Oczapowskiego 13, 10-717 Olsztyn, Po-land; e-mail: Mariusz.Majewski@uwm.edu.pltemperature (18˚C) and humidity (70%). Each animal was placed ina separate metal cage (50 × 50 × 50 cm) and received dry chow andwater ad libitum. At the beginning of the investigation, the rabbitswere aged approximately 3.5 months and weighed 2000-2200 g. Allexperimental and surgical procedures were approved by the LocalEthics Commission of the Warmia and Mazury University in Olsztyn(32/N, 2002).After the period of acclimatization, the animals were randomlydivided into two groups: control (C; n=5), comprising animals notundergoing electrostimulation but wearing the backpack with thedevice and the experimental one (E; n=5) where the animals weresubjected to LESS for 9 h/day, during 3 months. Localization of thestimulatory electrodes, preparation of the skin surface and all tech-nical parameters of the electrostimulator (SCOL-2, Elmech, Warsaw,PL) have previously been described in detail [11]. Briefly, skinrectangle (15 × 6 cm) located on the right side of the animal abovethe musculus longissimus thoracis (MLT; a part of musculus lon-gissimus dorsi, the most important muscle entity stabilizing thespine) has been carefully shaved and then, in order to remove hairrests, treated with a dermatologically neutral depilatory cream. Twoflat, circular electrodes have been mounted on skin surface bydouble-sided adhesive tape, approximately 2-3 cm laterally from themedian line. The distance between both electrodes was set to ap-proximately 10 cm. Electrodes were then connected with the SCOL-2device, running the "classical" LESS protocol: 9 hours per day,rectangular impulses, duration of single impulse: 0.1-0.2 ms, dura-tion of impulse series: 4.0 s; pause between series: 8 s; impulsefrequency: 40 Hz. The ultrastructural examination was done on samples collectedboth from places subjected to LESS (on the right side) and fromnon-electrostimulated ones (on the left side of the animal). Blocks(1.3 × 0.4 × 0.3 cm) of the muscle were gently stretched and pinnedto balsa wood plates, fixed by immersion in 1.5% glutaraldehyde inphosphate buffer (pH 7.2), postfixed in 2% osmium tetroxide (2 h,room temperature) and then embedded in Epon 812. Semi-thinsections were stained according to the method of Levis and Knight[15]; the appropriate place for preparing ultrathin sections wasestablished under a light microscope. The ultrathin sections werethen contrasted with uranyl acetate [25], followed by lead citrate [21].Ultrastructural examination was conducted using an Opton 900 PCtransmission electron microscope (Opton, Germany). ResultsData concerning changes evoked by LESS in stimulatedMLTd, found at the light-microscopic level, have pre-viously been published elsewhere [13]. Electron-microscopic evaluation of muscle sectionstaken from bilateral MLT of animals belonging to thecontrol group did not reveal any signs of abnormalities(Fig. 1). Electron micrographs obtained from sections pre-pared from the left MLT (i.e., contralateral to the sideof stimulation) showed no pathology in the vast ma-jority of striated myocytes (Fig. 2). Mitochondriawere usually rod-shaped (Figs. 2, 4). Sporadically,intracellular spaces were observed, which were eitherempty (Fig. 5) or contained lamellar structures (Fig.6). Clusters of glycogen particles of various sizes werevisible in the sarcoplasm (Figs. 2, 6). The spacesbetween myofibrils usually had correct dimensions(Figs. 2, 6). Only sporadically the sarcoplasm occu-pied a larger area (Fig. 4). The Z line frequently showedan irregular course and sometimes it was invisible (Figs.4, 6).On the other hand, electron microscopic examinationrevealed lesions in the ultrastructure of approx. 50% ofmuscle fibers in the right MLT of rabbits subjected tolong-term LESS (Fig. 3). Thus, from several to severaldozen of muscle fibers with depletion of myofilamentsand dilatation of intermyofibrillar space were observedwithin each analyzed section (Fig. 7). The myofibrilsystem was loose, and sometimes resembled combedhair. In about 30% of such fibers, the continuity ofmyofibrils was disrupted in various parts of the sar-comere (Fig. 7). Sporadically, entire sarcomeres fellapart (Fig. 8). Vesicular dilatation of the sarcoplasmicreticulum and its proliferation were also observed (Fig.8), however, the intensity of such changes varied signi-ficantly. In some cases, granular and fibrous remnantsof muscle fibers were observed, with macrophages lo-cated nearby (not shown). Clusters of normal or enlarged/swollen mitochondriawere observed relatively frequently (Fig. 7). Further-more, various-sized empty spaces, often filled with dis-integrated lamellar structures, were often observed(Figs. 9, 10). Relatively frequently, a thickening of theZ line was seen, often close to the lamellae-filled spaces(Fig. 9). Sporadically, the Z line showed an irregular,zig-zagging course (Fig. 3).DiscussionThe present study revealed that the failure of the "classi-cal" LESS (with the curative time-frame set for 9 hoursa day) in patients suffering from IS may be attributableto pronounced ultrastructural lesions in muscles under-going a kind of "overstimulation". Thus, the experimen-tal design allowed us to provide a clear-cut evidence thattoo long electrostimulation of skeletal muscle is respon-sible for both the atrophic (depletion of myofilaments,disorganization of the Z line) and degenerative changes(various-sized and -shaped intracellular empty spaces,dilatation and swelling of sarcoplasmic reticulum and/ormitochondria, together with the destruction of the Z line)that, taken together, may be the reason for the lowcompliance of patients, what, in turn, results in therapyfailure. Interestingly, similar but definitely less pronouncedchanges observed in a subset of myocytes of the contra-lateral (i.e., unstimulated musculus longissimus tho-racis), may be explained by the assumption that themuscle entity became injured due to the unproportionalload of work as it tried to counteract the LESS-forcedscoliosis [29]. However, this hypothesis need to beverified in detail.It should be stressed, however, that changes observedin both the muscular and connective tissues are, mostprobably, secondary to lesions of the afferent limb of the244 I.M. Kowalski et al.Fig. 1. Control group, musculus longissimus thoracis dexter (MLTd). Correct ultrastructure of muscle fibers. × 15 500. Fig. 2. Electrostimu-lated (E) group, left MLT (MLTs). The structure of the muscle is devoid of any visible lesions that may be attributed to the electrostimulationof the contralateral muscle. Note the presence of rod-shaped mitochondria (M) and clusters of glycogen. × 15 500. Fig. 3. E group, rightMLT (MLTd). Low-power electron-microscopical picture of an "overtrained" muscle, showing both the LESS-unaffected (to the left) andLESS-affected (to the right) muscle fibers, the latter with a clearly visible "zig-zag"-shaped Z lines and some dilated cisternae of thesarcoplasmic reticulum. × 15 500. Fig. 4. E group, MLTs. Dilatation of intermyofibrillar space (large arrows), proliferation and dilatation ofsarcoplasmic reticulum (SR), irregular course of the Z line (small arrows), depletion of the myofilaments (within the circle). × 15 500. Fig. 5.E group, MLTs. Empty space in the left upper corner. Disintegration of the Z line (asterisks) and its irregular course (arrowheads), depletionof the myofilaments (within the circle). T-tubules and terminal cisternae are also visible (arrows). × 19 500. Fig. 6. E group, MLTs. Spacefilled with lamellar structures in the left upper corner, disorganization of the Z line. Visible clusters of glycogen. × 27 000. Long-term LESS and skeletal muscle ultrastructure 245spinal circuit contributing to the IS etiology [5-7, 10, 13,16, 18, 20, 24]. This appears to be in line with secondarymorphological lesions described in muscles by Grimbyand co-workers [4], who also suggested their interrela-tionship with destructive action of long-term LESS onthis afferent pathway.This view may further be substantiated by observa-tion that the atrophy of the muscle fiber was paralleledby the widening of the sarcoplasmic reticulum, a phe-nomenon that is thought to be characteristic for simulta-neous lesion of muscle fiber and nerve terminal [8, 9,17-19, 23, 24]. Since the results of the present studyclosely mirrored the above-mentioned situation, it istempting to assume that at least part of the adverseinfluence(s) of long-term LESS may be explained bysuch a mechanism(s). From the etiological point of view,the chronic injury of the lower motor neuron must alsobe taken into consideration as a possible cause of theobserved changes [9]. It should also be pointed out, thatthe factor responsible for the observed degenerativechanges was most probably the functional overloadingof the supraspinal muscle due to the LESS "overstimu-lation". This may be judged not only from the increasein the number of mitochondria present in the musclefibres, changes in their shape and structure (organizationof the mitochondrial cristae) but also from the destruc-tion of the Z line, what is regarded as a sign of severedamage of the integrity of the skeletal muscle fiber (fordetails see [11]).Thus, it could be assumed that the lesions observedare, at least partly, responsible for the failure of "classi-cal" LESS therapy mentioned in the literature, especiallydue to the "overloading" of the neuro-muscular controlmechanisms by too long electrostimulation. The periodof stimulation was far too long for any of compensatorymechanisms of the muscle, thus it evoked a "myasthe-nia-like" status rather, than inducing the strengtheningof the muscle entity and, as a result, it was not onlyFig. 7. E group, MLTd. Depletion (asterisks) and degradation of myofilaments in the myofibrils (arrows), dilatation of sarcoplasmic reticulum(SR) and swollen mitochondria (M). × 27 000. Fig. 8. E group, MLTd. Depletion of some sarcomeres, proliferation and dilatation of thesarcoplasmic reticulum (SR), myelin-like structures (asterisks). × 27 000. Fig. 9. E group, MLTd. Spaces filled with disintegrated lamellarstructures (asterisks). Glycogen granules are visible in the sarcoplasm. Moreover, a thickened Z line can also be observed. × 15 500. Fig. 10.E group, MLTd. A large intracellular space filled with a myelin-like, lamellar structures (arrow) and a smaller one, devoid of any lamellae(asterisk). Visible glycogen granules. × 15 500.246 I.M. 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ChirNarz Ruchu Ortop Pol 56: 9-12[29] Z˙ uk T (1961) Electromyographic studies in conservatively andsurgically treated scoliosis (in Polish). Chir Narz Ruchu OrtopPol 26: 27-32Received: February 16, 2005Accepted after revision: June 2, 2005Long-term LESS and skeletal muscle ultrastructure 247

Ultrastructural features of supraspinal muscles in rabbits after long-term transcutaneous lateral electrical surface stimulation (LESS)

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Ultrastructural features of supraspinal muscles in rabbits after long-term transcutaneous lateral electrical surface stimulation (LESS)

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