Orbicularis Oculi Morphological Alterations in Affected and Nonaffected Sides in Hemifacial Spasm

Background: Although the nonaffected side appears to be clinically normal in hemifacial spasm (HFS), it is not known whether this side can be considered normal regarding histopathological findings. The purpose of this study was to objectively evaluate and compare orbicularis oculi samples of patients with HFS (not previously treated with botulinum toxin) and control patients undergoing cosmetic upper eyelid blepharoplasty. Methods: Orbicularis oculi samples from 22 eyelids were evaluated. There were 7 samples from the affected and 7 samples from the nonaffected sides of patients with HFS who had not been previously treated with botulinum toxin, and 8 samples from normal control patients. Muscle samples were prepared using hematoxylin and eosin staining, and a digital image analysis software was used for objective analyses. Results: When compared with normal controls, endomysial and perimysial connective tissue areas were significantly increased (P = 0.015) on the affected side in HFS, suggesting that this disorder is associated with chronic alterations that lead to muscle degeneration. Cell density was significantly reduced on the affected (P = 0.028) and also on the nonaffected sides in HFS (P = 0.003) compared with normal controls. This was observed, although, clinically, there were no signs or symptoms of increased muscular contraction on the nonaffected sides in any of the patients with HFS studied. Conclusions: Significant morphological differences in the orbicularis oculi muscle in patients with HFS were observed on both the affected and nonaffected sides. Our findings suggest a potential role for muscle homeostasis disturbances on both sides for patients with HFS. Affected sides in patients with HFS did, however, demonstrate muscle degeneration that was not present on the nonaffected sides.

Original Contribution Orbicularis Oculi Morphological Alterations in Affected and Nonaffected Sides in Hemifacial Spasm Midori H. Osaki, MD, Tammy H. Osaki, MD, Teissy Osaki, MD, Gustavo R. Gameiro, Rubens Belfort Jr, MD, Suely K. N. Marie, MD Background: Although the nonaffected side appears to be clinically normal in hemifacial spasm (HFS), it is not known whether this side can be considered normal regarding histopathological findings. The purpose of this study was to objectively evaluate and compare orbicularis oculi samples of patients with HFS (not previously treated with botulinum toxin) and control patients undergoing cosmetic upper eyelid blepharoplasty. Methods: Orbicularis oculi samples from 22 eyelids were evaluated. There were 7 samples from the affected and 7 samples from the nonaffected sides of patients with HFS who had not been previously treated with botulinum toxin, and 8 samples from normal control patients. Muscle samples were prepared using hematoxylin and eosin staining, and a digital image analysis software was used for objective analyses. Results: When compared with normal controls, endomysial and perimysial connective tissue areas were significantly increased (P = 0.015) on the affected side in HFS, suggesting that this disorder is associated with chronic alterations that lead to muscle degeneration. Cell density was significantly reduced on the affected (P = 0.028) and also on the nonaffected sides in HFS (P = 0.003) compared with normal controls. This was observed, although, clinically, there were no signs or symptoms of increased muscular contraction on the nonaffected sides in any of the patients with HFS studied. Conclusions: Significant morphological differences in the orbicularis oculi muscle in patients with HFS were observed on both the affected and nonaffected sides. Our findings suggest a potential role for muscle homeostasis disturbances on both sides for patients with HFS. Affected sides in patients with HFS did, however, Department of Ophthalmology and Visual Sciences (MHO, THO, TO, RB), Paulista School of Medicine/EPM, Federal University of São Paulo/UNIFESP, São Paulo, São Paulo, Brazil; University of São Paulo Medical School (GRG), University of São Paulo/USP, São Paulo, São Paulo, Brazil; and Laboratory of Molecular and Cellular Biology (SKNM), Department of Neurology, University of São Paulo/ USP, São Paulo, São Paulo, Brazil. The authors report no conflicts of interest. Address correspondence to Midori H. Osaki, MD, Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Osaki Ophthalmology Clinics, Vergueiro St, 2045, St 1009, São Paulo, SP 04101-000, Brazil; E-mail: [e-mail] Osaki et al: J Neuro-Ophthalmol 2020; 40: 193-197 demonstrate muscle degeneration that was not present on the nonaffected sides. Journal of Neuro-Ophthalmology 2020;40:193-197 doi: 10.1097/WNO.0000000000000823 © 2019 by North American Neuro-Ophthalmology Society H emifacial spasm (HFS) is characterized by involuntary, tonic, and clonic synchronous spasms of the muscles innervated by the ipsilateral facial nerve (1,2). This condition is often associated with facial nerve root exit zone compression caused by an aberrant loop of the anterior inferior cerebellar, posterior inferior cerebellar, vertebral, or basilar artery (1,3,4). Spasms usually originate in the orbicularis oculi muscle (90%) before affecting the midface and lower third of the face over months to years (1). Initially, contractions in the orbicularis oculi muscle may mimic persistent eyelid myokymia. Although bilateral cases have been described, most cases are unilateral. Botulinum toxin-A (BTX-A) injections in the affected muscles are considered first-line treatment for this condition (1,2). HFS is a unique condition. Because of the nature of the disease, most cases present with an affected and a nonaffected side. Although the nonaffected side appears to be clinically "normal" in most cases, it is unknown whether it can be considered normal regarding histopathological findings. We are unaware of previous studies in which morphological findings in HFS patients' orbicularis oculi muscles have been evaluated. The purpose of this study was to assess and compare orbicularis oculi samples of nonprevious BTX-A-treated HFS and control patients. METHODS After obtaining Federal University of Sao Paulo Review Board approval and written informed consent, 22 orbicularis oculi samples from 11 patients were examined. All subjects were treated in accordance with the tenets of the 193 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution Declaration of Helsinki and expressed the desire to undergo upper eyelid blepharoplasty for cosmetic purposes. Seven patients with moderate and moderate-to-severe HFS, according to the Jankovic rating scale (5), who had not been previously treated with BTX-A injections, and 4 normal control patients were included in the study. All patients who underwent the surgical procedure were clinically evaluated and underwent standard preoperative examinations to identify any potential surgical risks. Exclusion criteria were as follows: (1) patients with bilateral HFS, (2) previous treatment with BTX-A injections in the affected muscles, (3) previous eyelid surgery, (4) no evident dermatochalasis, (5) lagophthalmos, (6) neuromuscular junction and peripheral neuropathic diseases, (7) local hemostasis disorders, and (8) high systemic surgical risk (9) and patients who did not wish to undergo upper eyelid blepharoplasty before receiving BTX-A applications on the affected side of the face. All patients underwent cosmetically associated upper eyelid blepharosplasty that was associated with the excision of a thin strip of preseptal orbicularis oculi muscle. All patients with HFS underwent surgery before receiving BTX-A injections. These patients were scheduled to undergo BTX-A applications in the affected muscles 1 month after upper eyelid blepharoplasty. Surgical procedures were performed in the operating room by a single surgeon, following standard aseptic conditions and local anesthesia using 1.0 mL of 2% lidocaine with epinephrine (1:100,000) on each side. Upper eyelid incisions were made using a #15 blade, and meticulous dissection of the orbicularis from the overlying skin was performed. After skin-only excision, a thin strip of preseptal orbicularis oculi muscle, obtained from all cases, was snap frozen in liquid nitrogen and stored at 280°C until further analysis. The skin was closed with 6-0 interrupted nylon sutures. Sutures were removed on the 7th postoperative day, and all patients were re-evaluated 1 month after the procedure. Specimens were serially cryosectioned and subsequently stained with hematoxylin and eosin for analysis. Morphological characterization was performed through subjective observation in optic microscopy followed by objective (digital photographs and Image J software) evaluation of the muscle fiber regarding number of cells and determination of perimysial and endomysial connective tissue area by 2 observers. Photographs of the histological slides under the ·400 power field were taken. The area containing the highest number of cells was standardized for analysis. Digital photographs were taken using the Nikon image retrieval system. Cell numbers and endomysial and perimysial connective tissue areas, obtained with the use of Image J, version 1.34s image processing software, were recorded for the affected and nonaffected sides in the HFS and control groups. This software, which was developed by the National Institutes of Health, is available at http://rsb.info.nih.gov/ij/. The connective tissue contour was delineated in its whole extension, and its area was then calculated. Three delineations followed by 3 measurements were performed by 2 investigators, blinded to specimen type and identity, for each standardized photograph, and the mean measurement was considered for statistical analysis. Independent or paired t tests were used to compare several parameters: (1) affected side of the HFS group vs control group; (2) nonaffected side vs control group; and (3) affected vs nonaffected sides in patients with HFS. Analyses were performed with SPSS Statistics V22.0 (IBM, Armonk, NY), and P values less than 0.05 were considered statistically significant. RESULTS The mean participant age was 61.0 ± 7.9 years (range: 47- 70 years) and 69.7 ± 6.4 years (range: 65-79 years) in the HFS and control groups, respectively. Three patients from the HFS group were men; all control patients were women. Four patients presented with moderate and 3 patients with moderate-to-severe HFS, according to the Jankovic rating scale (5). The mean disease duration was 7.1 ± 4.8 years. Table 1 shows demographic data from both groups. TABLE 1. Demographic data from both groups Patient Age Gender Disease Severity Disease Duration (yrs) HFS HFS HFS HFS HFS HFS HFS Control Control Control Control 58 67 65 64 56 47 70 79 65 69 66 Female Male Male Male Female Female Female Female Female Female Female Moderate Moderate to severe Moderate Moderate to severe Moderate Moderate Moderate to severe N/A N/A N/A N/A 6 13 15 4 3 5 4 N/A N/A N/A N/A HFS, hemifacial spasm; N/A, not applicable. 194 Osaki et al: J Neuro-Ophthalmol 2020; 40: 193-197 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution FIG. 1. A and B. Orbicularis oculi samples from control patients: preserved morphology. C and D. Orbicularis oculi samples from the nonaffected side of patients with hemifacial spasm: Lower cell density can be noted when compared with the normal control samples. E and F. Orbicularis oculi samples from the affected side of patients with hemifacial spasm: A reduction in the number of muscle fibers and increase in the endomysial and perimysial connective tissue areas when compared with normal control samples can be observed (hematoxylin and eosin, ·400). All patients reported satisfaction with the aesthetic results 1 month postoperatively and subjectively reported reduction in eyelid spasms after the surgical procedure. No complications, such as lagophthalmos, blepharoptosis, infection, and/or wound dehiscence, were observed during the follow-up period in either group. Morphological Analysis A significant morphological difference in muscle fibers was observed between control and patient groups (Fig. 1 and Table 2) as described below. Table 2 shows the mean values and SDs for cell numbers and connective tissue areas in normal controls and patients with HFS. Table 3 shows comparisons and P values between groups. Intraclass correlation coefficients showed high levels of agreement between the measurements performed by the 2 investigators: 0.99 (95% confidence interval: 0.98-0.99) for cell number and 0.83 (95% confidence interval: 0.64-0.93) for the connective tissue area. Affected Side vs Control Group While morphology was preserved in normal controls (Fig. 1), endomysial and perimysial connective tissue area was significantly increased (P = 0.015) and cell density was significantly reduced (P = 0.028) on the affected side of patients with HFS. Independent t tests were used for statistical analyses. Nonaffected Side vs Control Group A significantly lower cell density was observed for the nonaffected side of patients with HFS when compared with the control group (P = 0.003). Connective tissue area, however, was not different between these 2 groups (P = 0.26). Independent t tests were used for statistical analyses. Affected Side vs Nonaffected Side The number of cells was not different between affected and nonaffected sides in patients with HFS (P = 0.86). Nonetheless, a higher area of connective tissue was observed on the affected side (P = 0.031). Paired t tests were used for statistical analyses. DISCUSSION There are few reports in the literature that have evaluated orbicularis oculi muscles in patients with facial dystonias. The few published studies evaluated changes induced by BTX-A in essential blepharospasm (6-9). To the best of our knowledge, no previous study has evaluated morphological TABLE 2. Cell number and connective tissue area in normal controls and in patients with hemifacial spasm Case Controls Affected HFS side Nonaffected HFS side Cell Number, Mean ± SD Connective Tissue Area (%), Mean ± SD 67.07 ± 16.42 42.86 ± 19.82 41.71 ± 8.52 14.8 ± 7.3 28.7 ± 10.6 19.6 ± 7.5 HFS, hemifacial spasm. Osaki et al: J Neuro-Ophthalmol 2020; 40: 193-197 195 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution TABLE 3. Comparison between groups Case Controls vs affected HFS side Controls vs nonaffected HFS side Affected HFS side vs nonaffected HFS side P (Cell Number) P (Connective Tissue Area) Statistic Test 0.028* 0.003* 0.859 0.015* 0.256 0.031* t test t test Paired t test *Statistically significant. HFS, hemifacial spasm. findings in orbicularis oculi muscles in HFS. In our study, we evaluated orbicularis oculi morphology before BTX-A applications, so that we could study the morphology of the untreated muscle fibers in these patients. Owing to the low prevalence of disease in the literature, which varied between 7.4 and 14.5/100,000 individuals (1), and also to the inclusion criterion in which only nonpreviously treated patients were evaluated, our comparative study sample, including 22 specimens (7 samples for the affected side, 7 samples for the nonaffected side, and 8 control samples) can be considered relevant. Although the mean ages varied between HFS and control groups, it has been demonstrated that no age-related histopathological changes have been observed in orbicularis oculi samples (10). In the present study, all patients underwent upper blepharoplasty for aesthetic purposes, which permitted access to a thin strip of orbicularis oculi muscle for histopathological analysis under frozen biopsy. In other situations, it would be difficult to obtain orbicularis oculi samples for morphological analysis in patients with HFS, and this is probably one of the reasons that could explain the lack of orbicularis oculi histopathological studies related to this disease. Digital image analysis software has been described as a useful tool in several medical fields (11,12). The use of this technology has permitted an objective demonstration of the significant differences between normal controls and both affected and nonaffected sides of patients with HFS and between affected and nonaffected sides. Significant morphological differences were observed when comparing orbicularis oculi samples of the affected side of patients with HFS vs normal controls. Significant differences were observed for cell numbers and connective tissue areas, possibly reflecting differences in the orbicularis oculi muscle contraction between these 2 groups. The observation of connective tissue proliferation on the affected side in HFS suggests that this condition is associated with chronic alterations in the muscle, thus leading to muscle degeneration. Similar to previous histological studies, we considered perimysial and endomysial tissues as a whole unit since both perimysium and endomysium contribute to the formation and maintenance of the muscle environment (13). It is unknown whether the connective tissue increase observed in patients with HFS is progressive and part of its natural histological history as this phenomenon has been observed 196 in Duchenne muscular dystrophy (13). Further studies are needed to elucidate this observation. When comparing orbicularis oculi samples from affected and nonaffected sides in patients with HFS, the connective tissue area was significantly higher on the affected side. However, a significant difference in cell number was not observed. Finally, when comparing samples from the nonaffected side of patients with HFS and normal control patients, a significantly higher cell number was observed in the control group, although the connective tissue area was not different between these 2 groups. Thus, as expected, our findings showed significant differences in morphology between affected and nonaffected sides and between affected sides of patients with HFS and normal controls. Surprisingly, a significant difference in morphology between the nonaffected side in patients with HFS and normal controls was also observed. This was true, although, clinically, there were no signs or symptoms of increased muscular contraction on the nonaffected side in any of the patients with HFS in this study. Perhaps, the nonaffected side presents some degree of nonclinically evident muscular contraction or some type of muscle homeostatic disturbance. If present, this degree of contraction would not be substantial enough to lead to muscle degeneration. Gene expression profiles of those muscle samples with subtle alterations may provide clues with respect to signaling pathways involved in the initial muscular tissue modification. In a previous study, in which the effects of upper eyelid myectomy for benign essential blepharospasm (another focal dystonia that affects the eyelid area) were evaluated, it was shown that this surgical procedure acted as an adjuvant treatment and increased the efficacy of subsequent BTX-A injections (14). Similarly, in this study, although only a thin strip of preseptal orbicularis oculi muscle was excised, all patients subjectively reported reduction in the eyelid spasms. In addition to the aesthetic enhancement, HFS patients' quality of life seemed to have improved. The impact of facial dystonia-associated involuntary spasms on quality of life have been well established (15-17). CONCLUSIONS In conclusion, our findings demonstrated significant morphological differences in the orbicularis oculi muscle in patients with HFS, including alterations in the nonaffected Osaki et al: J Neuro-Ophthalmol 2020; 40: 193-197 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution sides when compared with normal control patients. Additional immunohistochemical studies evaluating metabolic, degenerative, and regenerative alterations, and nextgeneration sequencing technologies, could add relevant contributions to improve understanding HFS. REFERENCES 1. Ross AH, Elston JS, Marion MH, Malhotra R. Review and update of involuntary facial movement disorders presenting in the ophthalmological setting. Surv Ophthalmol. 2011;56:54- 67. 2. Czyz CN, Burns JA, Petrie TP, Watkins JR, Cahill KV, Foster JA. 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