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 Botulinum A toxin has been used to treat a num­ oculi specimens from 11 patients with essential blepha­ ber of movement disorders, including benign es­ rospasm and Meige's disease who had received an av­ sential blepharospasm, facial dyskinesia associated erage of 11.3 injections of botulinum A toxin over 3.5 years. Denervation was demonstrated by the spread of with Meige's disease, spasmodic torticollis, adult­ acetylcholinesterase staining on muscle fibers when onset spasmodic torticollis, occupational hand specimens were evaluated within 11 weeks of the last dystonias, and spasmodic dysphonia (1-9). Many injection. When specimens were taken after 12 weeks, of these conditions are chronic movement disor­ spread of acetylcholinesterase was confined to the neu­ ders that require repeated injections of the toxin to romuscular junctions, with little fiber size variability re­ sembling normal muscle. Fibrosis seen in three speci­ maintain a beneficial effect. The toxin effect gener­ mens could be correlated to prior surgery. Repeated in­ ally lasts 2--4 months (1-2). jections of botulinum toxin into human muscle do not The U.s. Food and Drug Administration has re­ appear to cause irreversible muscle atrophy or other de­ cently approved botulinum A toxin for use in generative changes. Denervation changes (fiber size blepharospasm after 8 years of clinical trials. Short­ variability, acetylcholinesterase spread) appear to corre­ late to the time interval since the last injection. term side effects of botulinum A toxin for many of Key Words: Blepharospasm-Meige's syndrome, Botu­ these conditions result from local toxin spread to linum A toxin-Dystonia-Acetylcholinesterase-Hemi­ contiguous muscles (7,8). Dysphagia results from facial spasm toxin spread to the peripharyngeal muscles after higher dose injections over the sternocleiodomas­ toid 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 bot­ ulinum 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 pa­ tients 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 From the Massachusetts Eye and Ear Infirmary (G.E.B.) and Neuropathology Unit (R.F.), Massachusetts General Hospital, Patients involved in this study required surgical Boston, Massachusetts, U.S.A. Address correspondence and reprint requests to Dr. Gary E. intervention in two clinical situations. The first Borodic, 100 Charles River Plaza, Boston, MA 02114, U.S.A. was involutional ptosis caused by disinsertion of 121 122 G. E. BORODIC AND R. FERRANTE the attachment of the levator aponeurosis to the late the histochemical findings using various tarsal plate. To correct this condition, which often stains. involves reinsertion of the levator aponeurosis to Morphometric measurements were made with the tarsal plate, a small strip of orbicularis muscle the bioquant II system. Muscle fiber size was eval­ is removed to debulk the lid. The second clinical uated by measuring cross-sectional diameters in situation was unsatisfactory or incomplete symp­ 200 fibers in each specimen. Statistical compari­ tomatic response to botulinum toxin, which re­ sons were made using an F test, and the results sulted in the selection of an orbicularis myectomy were expressed as the mean +/- standard devia­ procedure as an alternative method of therapy. tion with variance. Each patient who became a surgical candidate The age of patients and indications for surgical for the correction of involutional disinsertion pto­ therapy are shown in Table 1. The number of bot­ sis or a myectomy procedure was advised that a ulinum toxin injections and the duration of ther­ histologic analysis of resected muscle for botuli­ apy are shown in Table 2, which also presents the num toxin activity would be conducted. Comori intervals between the last injection and the surgi­ trichrome stain and hematoxylin and eosin were cal procedure. The control values were taken from used to evaluate fiber size variability, and acetyl­ specimens removed during routine ptosis proce­ cholinesterase enzyme histochemistry was done to dures from patients who never received botulinum assess evidence of denervation. The acetylcholin­ toxin injections. Control data were derived from esterase stain has been previously reported as be­ four separate muscle specimens from three differ­ ing a valuable method of assessing denervation in ent patients. Each control patient demonstrated animal (9,10) and human striated muscle after the ptosis associated with elevated lid crease, and re­ injection of botulinum toxin (11,12). tracted preaponeurotic fat pads with good levator Specimens taken from orbicularis oculi muscle function indicating the diagnosis of involutional were immediately placed in cold (4°C) formol­ ptosis. None of the control patients had blepharo­ calcium (Baker's solution) and fixed for 6-12 hours spasm. 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 RESULTS longitudinal plane and frozen in a cryostat chuck using OTC compound (Tissue Tek). When the tis­ Human Specimens Treated with Repeated sue sample size permitted, the muscle was subdi­ Botulinum Toxin Injections vided and cut in longitudinal and cross-section. Cut tissue sections (10 f.lm) were adhered to gel­ The histologic response appeared to be depen­ coated slides, allowed to air dry for 2 minutes, and dent on the time interval between the last injection subsequently stained for acetylcholinesterase ac­ and the surgical procedure. The degree of acetyl­ tivity, using the method of Ceneser-Jensen and cholinesterase activity on muscle fibers was most Blackstad (13). Sections were incubated in a solu­ prominent within a period of 7-15 weeks following tion containing 13 ml maleic buffer (1.96 g maleic a therapeutic injection of botulinum toxin. Nor­ acid, 0.8 g NaOH, 10.8 ml IN NaOH, 200 ml dis­ mally, the cholinesterase staining pattern is con- tilled water), 10 mg acetylthiocholine iodide, 2 ml 0.03 M cupric sulfate, 1 ml 0.1 sodium citrate, and TABLE 1. Case history summary 0.5 mM potassium ferricyanide for 1 hour at 37°C. Contiguous cryostat sections were stained either Patient Indication for with hematoxylin and eosin or with Comori tri­ number Age Prior surgery intervention chrome stain to assess tissue morphology. 1 71 None Botox failure" To correlate the acetylcholinesterase staining 1a Myectomy Botox failure 2 59 None p~ttern Ptosis with other histochemical enzyme stains, 3. 65 None Botox failure histochemistry for myofiber ATPase activity was 4. 91 Blepharoplasty Botox failure conducted according to the method of Brooke and 5. 71 Myectomy Partial botox failure 6. 76 None Ptosis Kaiser (14), and NADH activity was assessed ac­ 7. 61 Blepharoplasty Botox failure cording to the method of Scarpelli, Hess, and 8. 78 None Ptosis Pearse (15). Acetylcholinesterase, ATPase, and 9. 71 None Ptosis 10 56 Blepharoplasty Botox failure NADH staining were conducted on five albino rab­ 11 62 None Ptosis bit longissimus dorsi muscle specimens at 5 weeks " Botulinum toxin failures underwent orbicularis myec­ after 10 lU botulinum toxin was injected to corre- tomy procedures of the upper lid and brow. ! Clin NeurfHJPhthalmol, Vol. 12, No.2, 1992 STRIATED MUSCLE AFTER BOTULINUM TOXIN INJECTIONS 123 TABLE 2. Effects of repeated botulinum toxic injections Histologic Findings Number of Cholinesterase Patient Time since injections/duration Fiber size voriability fibrosis staining number last injection of therapy (median diameter in microns) (median size in microns) Control 28.43 s = 5.29, v = 28 Focal, 12.5, s = 5.4 1:H01 1 wk 7 injections, 1.25 yr 31.8 s = 8.2, v = 66 Focal, 21, s = 8 2:H02 8wk 16 injections, 3.3 yr 30 s = 14, v = 213 Diffuse, 35.2, s = 18 + 3:AK 7wk 14 injections, 3.4 yr 27.0 s = 15.5, v = 237 Diffuse, ND + 4:RG 22wk 19 injections, 5.5 yr 25.7 s = 6.8, v = 46 Focal, 20, s = 8 5:MS 52wk 11 injections, 5.0 yr 34.4 s = 7, v = 48 Focal,25 + 6:RN 52wk 4 injections, 2.7 yr 28.1 s = 8, v = 64 Focal, 22.5, s = 6 7:EP 18 wk 12 injections, 3.0 yr 32.0 s = 9, v = 81 Focal, 19.3, s = 6.7 8:TC 7wk 11 injections, 4.0 yr 28.5 s = 17.0, v = 289 Diffuse, 161.3, s =124 9:GC 16 wk 16 injections, 4.5 yr 26.0 s = 13.5, v = 172 Diffuse, 34, s = 13 10:TP 22wk 6 injections, 2.0 yr 28.1 s = 8, v = 63 ND 11 :CP 7wk 2 injections, 1.0 yr 23.5 s = 11, v = 121 Diffuse, 147, s = 70 S, standard deviation; v, variance; ND, not determined. fined to a focal area on the muscle fiber within the The degree of acetylcholinesterase spread on mus­ neuromuscular junction (see Fig. 2). Within 15 cle fibers was inversely related to the time interval weeks after botulinum toxin injection however, from injection to biopsy (Fig. 3). there appeared to be extensive spread of enzyme Muscle fiber variability was directly related to activity throughout large surface areas of muscle the time from the last injection.