Journal of Neuro-Ophthalmology, June 1992, Volume 12, Issue 2

Effects of repeated botulinum toxin injections on orbicularis oculi muscle.

Histologic evaluation was conducted on 12 orbicularis oculi specimens from 11 patients with essential blepharospasm and Meige's disease who had received an average of 11.3 injections of botulinum A toxin over 3.5 years. Denervation was demonstrated by the spread of acetylcholinesterase staining on muscle fibers when specimens were evaluated within 11 weeks of the last injection. When specimens were taken after 12 weeks, spread of acetylcholinesterase was confined to the neuromuscular junctions, with little fiber size variability resembling normal muscle. Fibrosis seen in three specimens could be correlated to prior surgery. Repeated injections of botulinum toxin into human muscle do not appear to cause irreversible muscle atrophy or other degenerative changes. Denervation changes (fiber size variability, acetylcholinesterase spread) appear to correlate to the time interval since the last injection.

Journal of Clillical Neuro- ophthalmology 12( 2): 121- 127, 1992. ' 91992 Raven Press, Ltd., New York Effects of Repeated Botulinum Toxin Injections on Orbicularis Oculi Muscle Gary E. Borodic, M. D. and Robert Ferrante, M. S. Histologic evaluation was conducted on 12 orbicularis oculi specimens from 11 patients with essential blepha­rospasm and Meige's disease who had received an av­erage of 11.3 injections of botulinum A toxin over 3.5 years. Denervation was demonstrated by the spread of acetylcholinesterase staining on muscle fibers when specimens were evaluated within 11 weeks of the last injection. When specimens were taken after 12 weeks, spread of acetylcholinesterase was confined to the neu­romuscular junctions, with little fiber size variability re­sembling normal muscle. Fibrosis seen in three speci­mens could be correlated to prior surgery. Repeated in­jections of botulinum toxin into human muscle do not appear to cause irreversible muscle atrophy or other de­generative changes. Denervation changes ( fiber size variability, acetylcholinesterase spread) appear to corre­late to the time interval since the last injection. Key Words: Blepharospasm- Meige's syndrome, Botu­linum A toxin- Dystonia- Acetylcholinesterase- Hemi­facial spasm From the Massachusetts Eye and Ear Infirmary ( G. E. B.) and Neuropathology Unit ( R. F.), Massachusetts General Hospital, Boston, Massachusetts, U. S. A. Address correspondence and reprint requests to Dr. Gary E. Borodic, 100 Charles River Plaza, Boston, MA 02114, U. S. A. 121 Botulinum A toxin has been used to treat a num­ber of movement disorders, including benign es­sential blepharospasm, facial dyskinesia associated with Meige's disease, spasmodic torticollis, adult­onset spasmodic torticollis, occupational hand dystonias, and spasmodic dysphonia ( 1- 9). Many of these conditions are chronic movement disor­ders that require repeated injections of the toxin to maintain a beneficial effect. The toxin effect gener­ally lasts 2-- 4 months ( 1- 2). The U. s. Food and Drug Administration has re­cently approved botulinum A toxin for use in blepharospasm after 8 years of clinical trials. Short­term side effects of botulinum A toxin for many of these conditions result from local toxin spread to contiguous muscles ( 7,8). Dysphagia results from toxin spread to the peripharyngeal muscles after higher dose injections over the sternocleiodomas­toid muscle used for the treatment of spasmodic torticollis ( 8). Diplopia may be a consequence of lower eyelid injections when toxin used to treat involuntary blepharospasm spreads to the inferior oblique muscle ( 7). Long- term or latent clinical complications of bot­ulinum toxic injections have not been reported. However, because therapy for many of these movement disorders requires repeated injections, there is a potential for a long- term effect of toxin administration on human muscle, In this study, we report the chronic histopathologic effects in pa­tients who have received repeated botulinum A toxin injections into the orbicularis oculi muscle for benign essential blepharospasm or Meige's disease for at least several years. MATERIALS AND METHODS Patients involved in this study required surgical intervention in two clinical situations. The first was involutional ptosis caused by disinsertion of 122 G. E. BORODIC AND R. FERRANTE RESULTS " Botulinum toxin failures underwent orbicularis myec­tomy procedures of the upper lid and brow. Human Specimens Treated with Repeated Botulinum Toxin Injections The histologic response appeared to be depen­dent on the time interval between the last injection and the surgical procedure. The degree of acetyl­cholinesterase activity on muscle fibers was most prominent within a period of 7- 15 weeks following a therapeutic injection of botulinum toxin. Nor­mally, the cholinesterase staining pattern is con- Indication for intervention Botox failure" Botox failure Ptosis Botox failure Botox failure Partial botox failure Ptosis Botox failure Ptosis Ptosis Botox failure Ptosis Prior surgery None Myectomy None None Blepharoplasty Myectomy None Blepharoplasty None None Blepharoplasty None 71 59 65 91 71 76 61 78 71 56 62 Age TABLE 1. Case history summary 1 1a 2 3. 4. 5. 6. 7. 8. 9. 10 11 late the histochemical findings using various stains. Morphometric measurements were made with the bioquant II system. Muscle fiber size was eval­uated by measuring cross- sectional diameters in 200 fibers in each specimen. Statistical compari­sons were made using an F test, and the results were expressed as the mean +/ - standard devia­tion with variance. The age of patients and indications for surgical therapy are shown in Table 1. The number of bot­ulinum toxin injections and the duration of ther­apy are shown in Table 2, which also presents the intervals between the last injection and the surgi­cal procedure. The control values were taken from specimens removed during routine ptosis proce­dures from patients who never received botulinum toxin injections. Control data were derived from four separate muscle specimens from three differ­ent patients. Each control patient demonstrated ptosis associated with elevated lid crease, and re­tracted preaponeurotic fat pads with good levator function indicating the diagnosis of involutional ptosis. None of the control patients had blepharo­spasm. Patient number the attachment of the levator aponeurosis to the tarsal plate. To correct this condition, which often involves reinsertion of the levator aponeurosis to the tarsal plate, a small strip of orbicularis muscle is removed to debulk the lid. The second clinical situation was unsatisfactory or incomplete symp­tomatic response to botulinum toxin, which re­sulted in the selection of an orbicularis myectomy procedure as an alternative method of therapy. Each patient who became a surgical candidate for the correction of involutional disinsertion pto­sis or a myectomy procedure was advised that a histologic analysis of resected muscle for botuli­num toxin activity would be conducted. Comori trichrome stain and hematoxylin and eosin were used to evaluate fiber size variability, and acetyl­cholinesterase enzyme histochemistry was done to assess evidence of denervation. The acetylcholin­esterase stain has been previously reported as be­ing a valuable method of assessing denervation in animal ( 9,10) and human striated muscle after the injection of botulinum toxin ( 11,12). Specimens taken from orbicularis oculi muscle were immediately placed in cold ( 4° C) formol­calcium ( Baker's solution) and fixed for 6- 12 hours at 4° C. Muscle specimens were cryoprotected in gum sucrose solution for 3 hours or until the tissue sank in solution. The muscle was oriented in a longitudinal plane and frozen in a cryostat chuck using OTC compound ( Tissue Tek). When the tis­sue sample size permitted, the muscle was subdi­vided and cut in longitudinal and cross- section. Cut tissue sections ( 10 f. lm) were adhered to gel­coated slides, allowed to air dry for 2 minutes, and subsequently stained for acetylcholinesterase ac­tivity, using the method of Ceneser- Jensen and Blackstad ( 13). Sections were incubated in a solu­tion containing 13 ml maleic buffer ( 1.96 g maleic acid, 0.8 g NaOH, 10.8 ml IN NaOH, 200 ml dis­tilled water), 10 mg acetylthiocholine iodide, 2 ml 0.03 M cupric sulfate, 1 ml 0.1 sodium citrate, and 0.5 mM potassium ferricyanide for 1 hour at 37° C. Contiguous cryostat sections were stained either with hematoxylin and eosin or with Comori tri­chrome stain to assess tissue morphology. To correlate the acetylcholinesterase staining p~ ttern with other histochemical enzyme stains, histochemistry for myofiber ATPase activity was conducted according to the method of Brooke and Kaiser ( 14), and NADH activity was assessed ac­cording to the method of Scarpelli, Hess, and Pearse ( 15). Acetylcholinesterase, ATPase, and NADH staining were conducted on five albino rab­bit longissimus dorsi muscle specimens at 5 weeks after 10 lU botulinum toxin was injected to corre- ! Clin NeurfHJPhthalmol, Vol. 12, No. 2, 1992 STRIATED MUSCLE AFTER BOTULINUM TOXIN INJECTIONS TABLE 2. Effects of repeated botulinum toxic injections Histologic Findings 123 Patient number Control 1: H01 2: H02 3: AK 4: RG 5: MS 6: RN 7: EP 8: TC 9: GC 10: TP 11 : CP Time since last injection 1 wk 8wk 7wk 22wk 52wk 52wk 18 wk 7wk 16 wk 22wk 7wk Number of injections/ du ration of therapy 7 injections, 1.25 yr 16 injections, 3.3 yr 14 injections, 3.4 yr 19 injections, 5.5 yr 11 injections, 5.0 yr 4 injections, 2.7 yr 12 injections, 3.0 yr 11 injections, 4.0 yr 16 injections, 4.5 yr 6 injections, 2.0 yr 2 injections, 1.0 yr Fiber size voriability ( median diameter in microns) 28.43 s = 5.29, v = 28 31.8 s = 8.2, v = 66 30 s = 14, v = 213 27.0 s = 15.5, v = 237 25.7 s = 6.8, v = 46 34.4 s = 7, v = 48 28.1 s = 8, v = 64 32.0 s = 9, v = 81 28.5 s = 17.0, v = 289 26.0 s = 13.5, v = 172 28.1 s = 8, v = 63 23.5 s = 11, v = 121 Cholinesterase fibrosis staining ( median size in microns) Focal, 12.5, s = 5.4 Focal, 21, s = 8 Diffuse, 35.2, s = 18 Diffuse, ND Focal, 20, s = 8 Focal, 25 Focal, 22.5, s = 6 Focal, 19.3, s = 6.7 Diffuse, 161.3, s = 124 Diffuse, 34, s = 13 ND Diffuse, 147, s = 70 ++ + S, standard deviation; v, variance; ND, not determined. fined to a focal area on the muscle fiber within the neuromuscular junction ( see Fig. 2). Within 15 weeks after botulinum toxin injection however, there appeared to be extensive spread of enzyme activity throughout large surface areas of muscle fibers, indicating denervation ( Fig. 1). This obser­vation was especially evident in patient 7 ( TC) and patient 11 ( CP). When the time interval between these procedures and the last botulinum toxin in­jection was longer than 3 months, there was no substantial spread of acetylcholinesterase activity from the neuromuscular junction ( Fig. 2) as com­pared with normal control patterns ( see Table 2). The degree of acetylcholinesterase spread on mus­cle fibers was inversely related to the time interval from injection to biopsy ( Fig. 3). Muscle fiber variability was directly related to the time from the last injection. Muscle fiber diam­eter variability in a pattern consistent with neuro­genic atrophy was noted in specimens within 3 months of botulinum toxin injection ( Fig. 4). Sta­tistical quantification demonstrated a significant difference in fiber diameter variability relative to controls ( p < .05, F test) for patients injected within 15 weeks of biopsy ( see patients 1 ( H02), 3, 8, 11). The degree of muscle fiber size variability FIG. 1. Spread of acetylcholinesterase staining activity on human orbicularis oculi muscle tissue 7 weeks after therapeutic botulinum toxin injections. ] Clin Neuro- ophthalmol. Vol. 12, No. 2, 1992 124 G. E. BORODIC AND R. FERRANTE FIG. 2. Acetylcholinesterase activity is confined to the neuromuscular junction ( ar­row) after 52 weeks since the last botulinum toxin injection. relative to intervals since injections is shown in Fig. 5. In patients not receiving botulinum toxin for 6 months, the fiber size variability was not signif­icantly different from controls ( F test, specimens in cases 4, 5, 6, 10) after an average of 10.0 injec­tions over an average of 3.8 years. Acetylcholines­terase staining pattern was also not significantly different from control specimens in this group. These observations indicate no evidence of perma­nent denervation after multiple botulinum injec­tions over several years if botulinum toxin had not been injected within 6 months before the evalua­tion. Fibrosis was apparent, however, in specimens 1, 3, 4, each of which were from patients who under­went previous surgical procedures ( see Table 1). of type l: type 2 fibers increased significantly as compared to control specimens, with type 1 fibers representing 22% of the total population. The NADH activity also demonstrated alter­ations in fiber size and fiber typing. In addition, this method identified changes in the intermyo­fibrillary network that were consistent with dener­vation. Acetylcholinesterase staining of these animal specimens after 5 weeks demonstrated diffuse staining characteristic on fiber surfaces that was associated with increased fiber diameter size vari­ation ( Fig. 6B). ATPase staining and NADH reactivity showed findings typically associated with denervation in animal studies, which correlated and substantiated 100 ' 50 50 ~--+---.----+ --+-- Choll nes t Spread 8 8 16 18 16 23 24 52 52 Time Since last Injection o FIG. 3. Denervation reflected by cholinesterase spread. ATPase and NADH Staining on Animal Tissues Showing Intense Spread of Acetylcholinesterase Activity After Botulinum Toxin Injections In control specimens, myofibrillary ATPase ac­tivity at pH = 9.4 demonstrated both type 1 and type 2 fibers. The number or percent ratio of type 1- 2 fibers was 3.5% of the total. Type 1 fibers were distributed throughout the muscle specimens in saline injected controls. At the injection site there was marked variation of muscle fiber size of both fiber types. The pattern of fiber typing was altered in that more grouped fiber typing and the presence of grouped atrophy strongly suggested denevation ( Fig. 6A). The ratio I elin NeuTo- vplll/ w! nwL VoL 12, No. 2, 1992 STRIATED MUSCLE AFTER BOTULINUM TOXIN INJECTIONS 125 300 Microns 350r------------------- 16 18 22 22 52 52 Time from Injection /' I o ~---'-~~---'-----'-------'----'-------'-_ _'______'_ __'__ L_ Contro 1 wI- 50 100 150 250 200 temporally with the clinical response seen after ad­ministering botulinum A toxin for the treatment of movement disorders in humans ( 16). Generally, the duration of effect of botulinum toxin for the treatment of benign essential blepharospasm and facial dyskinesia associated with Meige's disease is 2- 4 months. The temporal effect of botulinum toxin in the treatment of this disease correlates very well with the histologic phenomena of func­tional denervation as reflected by the histologic specimens demonstrated in this study over a sev­eral month period. Two muscle specimens from patient 1 were an­alyzed: one taken 8 weeks after injection and one taken 1 week after injection. After 8 weeks, spread of acetylcholinesterase was noted with increased FIG. 5. Orbicularis muscle fiber size, variations after botulinum toxin. DISCUSSION the result found with acetylcholinesterase staining pattern after 5 weeks. After injection of nonlethal doses of botulinum toxin, Duchen ( 9,10) had demonstrated denerva­tion followed by reinnervation with collateral ax­onal sprouting. The initial effect of botulinum toxin is to block the release of acetylcholine from the presynaptic membrane at the neuromuscular junction ( 9). Shortly after this block, within several weeks, collateral axonal sprouting has been dem­onstrated in both animal ( 10) and human orbicu­laris oculi tissue ( 11,12). This collateral sprouting is followed by the regeneration of neuromuscular junctions on muscle fibers. With the denervation, spread of acetylcholinesterase enzyme activity on muscle fibers is demonstrated after 3-- 4 weeks and appears in normal levels after 12- 14 weeks in ro­dent studies with botulinum A injections ( 9,10). Earlier reports have indicated that sprouting from the preterminal axon can be noted 6 weeks to 3 years since the last botulinum toxin injection ( 12). Although reversible muscle fiber atrophy and acetylcholinesterase activity spread were demon­strated in this study, permanent dysmorphic alter­ations in the preterminal axon or ultrastructural changes of muscle fiber are still possible. The histologic cycle, consisting of denervation­reinnervation and increased acetylcholinesterase staining activity on muscle fibers, correlates well JClin Neuro- ophthalmol, Vol. 12, No. 2, 1992 126 A B G. E. BORODIC AND R. FERRANTE FIG. 6. A: ATPase staining showing an increased proportion of type I fibers compared to controls ( longissimus dorsi in albino rabbit). Also. notice type I fiber grouping and fiber atrophy suggesting denervation. B: Acetylcholinesterase staining after 5 weeks showing diffuse staining characteristic on fiber surfaces away from the neuromuscu­lar junctions. The diffuse staining pattern was associated with increased fiber size and fiber variation. fiber size variability, which indicated active dener­vation. After 1 week, no appreciable spread in ace­tylcholinesterase was seen, and there was minimal fiber size variability. It appears that a latency pe­riod of longer than 2 weeks is needed for acetyl­cholinesterase spread to occur after botulinum toxin injections. A similar latency was noted in the ] Clm N£ u, o'Dphtlralmo! Vol. 12, No. 2, 1992 longissimus dorsi muscles in albino rabbits ( 8) and the soleus muscle in white mice ( 9,10). Orbicularis oculi muscle fiber size variability has been found to be greater than in other muscles ( 16,17). Hence, fiber size variability alone must be viewed with caution unless the variation is so great as to represent grouped muscular atrophy occur- STRIATED MUSCLE AFTER BOTULINUM TOXIN INJECTIONS 127 ring with denervation. Histochemical analysis with the acetylcholinesterase stain is therefore par­ticularly useful in confirming the denervation ef­fect created by botulinum toxin. The potential long- term effects of repeated injec­tions of botulinum toxin on the structure and in­tegrity of human muscle are of particular concern. When muscle is subjected to repeated botulinum toxin injections, long- term chronic irreversible changes may occur; for example, permanent neu­rogenic muscle atrophy with resulting fibrosis and permanent muscular scarring. These phenomena, however, were not demonstrated in the group of patients who had received multiple botulinum toxin injections over a number of years. One ex­planation could be that during reinnervation there is enough neurotrophic influence on muscle fibers to prevent long- term contractures from denerva­tion. This may explain why botulinum toxin rarely causes complete paralysis of the muscles injected for therapeutic purposes. Antibody formation may be instrumental in the reduced effect of repeated botulinum toxin in some patients ( 16). In such sit­uations, repeated injections of botulinum toxin may be less effective and may result in lesser de­grees of denervation over a period of time. Some evidence of fibrosis was noted in several of the muscle specimens. However, it could not be clearly attributed to the botulinum toxin injections. The presence of fibrosis in the muscles studied ap­peared to correlate most closely to the prior lid surgery, and it could have represented a portion of the surgical scar. In summary, repeated injections of botulinum A toxin over several years do not seem to be associ­ated with irreversible denervation or changes in the morphologic character of human orbicularis oc­uli muscle fiber size and acetylcholinesterase stain­ing pattern. The diffuse atrophy seen in muscle fibers in the short term appears to be a reversible phenomenon. Reduced acetylcholinesterase activ­ity increases to normal levels as the effects of the toxin diminish. These histologic studies generally parallel the clinical duration of action of botulinum toxin when used to treat blepharospasm, as well as other forms of segmental dystonias. REFERENCES 1. Scott AB, Kennedy RA, Stubbs AA. Botulinum A toxin for the treatment of blepharospasm. 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