Movement Disorders Vol. 7, No. 2, 1992, pp. 171-177. 0 1992 Movement Disorder Society
Severe But Temporary Injury to Rabbit Orbicularis Oculi Muscle Using Dihematoporphyrin Ether and Laser Photochemomyectomy Jonathan D. Wirtschafter, David Paul Slovut, Leif Stordal, *Joseph Valentino, and Linda Kirschen McLoon Department of Ophthalmology and *Department of Otolargyngology, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Summary: The use of local dihematoporphyrin ether (DHE) injections, followed by laser light activation, was investigated as a potential permanent myectomy treatment for muscle spasms, in particular blepharospasm and hemifacial spasm. DHE was injected into the eyelids of rabbits, followed by laser activation, as used in photochemotherapy . Four days after treatment, histological examination indicated that doses of 20.5 mg of DHE and laser treatment with an energy density of at least 100 J/cm2 resulted in an almost total destruction of the orbicularis oculi muscle in the treated eyelid. The amount of muscle injury was dependent on both dose of DHE and energy density levels. Histologically, the tarsal glands and conjunctiva were damaged. Glandular tissue was markedly reduced, and the conjunctival epithelium showed hyperplasia and a loss of mucous cells. Six months after DHE and laser treatment, the majority of the muscle tissue had regenerated, although there was evidence of previous injury. While DHE injections combined with laser light activation were lethal to muscle at the site of treatment, this treatment was not permanent. The orbicularis oculi muscle retained its ability to regenerate. However, photochemomyectomy may be studied further as an adjuvant treatment to temporarily injure and debulk large muscles when botulinum toxin is contraindicated due to the large doses involved or as a permanent treatment when used together with an antimitotic agent such as doxorubicin. Key Words: Facial muscles-Blepharospasm-Photodynamic therapy.
Blepharospasm can be a debilitating form of dystonia characterized by intense, involuntary spasms of the orbicularis oculi muscle and functional blindness. The etiology of the disorder is usually unknown, although blepharospasm has been associated with some brainstem lesions (1,2). Multiple surgical and medical treatments are available, but none provide a fully satisfactory, permanent result. Blepharospasm can be treated with botulinum toxin
injections into the eyelids (3,4), but this treatment is temporary. Spasms return, usually between 9 and 12 weeks ( 4 8 ) . Botulinum toxin (BT) injections are also only effective in 70430% of patients treated (5,6). When BT injections fail, or when patients seek a more permanent result, surgical myectomy is an option (5). In addition to surgical complications, reoperation or supplementary BT injections are often required to relieve spasms. Lack of permanence of BT injections, coupled with potential complications of surgical intervention, suggested a need for an alternative treatment for blepharospasm. One new experimental therapy, the local injection of
Address correspondence and reprint requests to Dr. L. McLoon, Department of Ophthalmology, University of Minnesota, Box 493, UMHC, Minneapolis, MN 55455, U.S.A.
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doxorubicin into the eyelid, results in substantial muscle loss in animals (9,lO) and spasm relief in selected human patients (1 1). Another possible approach would involve the use of photoradiation therapy, the light-activated chemicals that are used in cancer treatment. We tested whether this approach could be used to locally remove muscle to treat this type of disease. Photoradiation therapy is a treatment for neoplasms that uses light-activated fluorescent compounds known as photosensitizers. The most welldescribed antineoplastic photosensitizers are hematoporphyrin derivative (HPD) and extracts of HPD, such as dihematoporphyrin ether (DHE). These are relatively inert compounds that preferentially concentrate in most neoplastic tissue 24-72 h after intravenous administration (12,13). Neoplasms are selectively destroyed because they concentrate and retain HPD longer than the normal, surrounding tissue, probably due to their more active metabolism and less efficient lymph drainage (12). The dye is activated with 630 nm coherent light. This wavelength allows dye activation with maximal tissue penetration, and the light can easily be channeled by fiber optics to specific locations. The light results in activation of the HPD, causing the release of free oxygen radicals, which in turn initiates a process leading to cell death (14). The destruction as a result of this treatment also stems from damage to the tumor vasculature ( 1 9 , which causes localized ischemia and tissue necrosis. To decrease the risk of skin phototoxicity, HPD has been administered locally (16-18). After intratumor HPD administration, there are substantially greater amounts of HPD concentrated in the tumor than after intravenous administration (17,18). Muscle has been shown to take up HPD (19), and muscle injury and necrosis after phototherapy have been described (20). Although previous investigations have used photodynamic therapy to treat tumors, there is a potential for its use in selective muscle destruction at a specified and controlled location. We examined the effect of a local injection of DHE in the eyelids of rabbits, followed by laser light application in an attempt to produce selective muscle loss, or photochemomyectomy . Local injection into the eyelid should produce a high extracellular concentration of the DHE. This should increase the uptake of the drug by the muscle cells at the injection site, as well as potentially damage the vasculature that supports this muscle. Local injection would decrease the po-
Movement Disorders, Vol. 7, No. 2 , 1992
tential for skin phototoxicity. We examined the eyelids histologically either 4 days or 6 months after DHE and laser treatment to assess muscle loss. MATERIALS AND METHODS Sixteen adult New Zealand white rabbits were obtained from Birchwood Laboratory and housed with the University of Minnesota Research Animal Resources. Animals were handled in accordance with the guidelines set up by the National Institutes of Health on the use of animals in research. Animals were deeply anesthetized with an equal mixture of ketamine HCl(lO0 mg/ml) at a dose of 10 mg/kg and xylazine (Rompun, 20 mg/ml) at a dose of 2 mgfkg. After shaving the areas to be injected, DHE (Photofrin 11, QLT Phototherapeutics, Lederle Labs., Vancouver, B.C., Canada) was injected along the entire length of the pretarsal portion of the right upper and left lower eyelids (21). Doses were graded in a series of rabbits from 0.1 to 1 .O mg in 0.1 cc of a 5% dextrose solution. The eyelids of the untreated, opposite eyes were used as controls. Prior to laser treatment, a 4-0 silk traction stitch was placed through the medial or lateral portion of the lid to flatten the eyelid and maximize exposure of the lid to the laser light. Artificial tears were applied to maintain corneal lubrication during laser treatment. A local anesthetic, proparacaine HCI, was applied to the cornea. The laser target was marked using carbol fuchsin solution. None of the marker dye was irradiated directly during laser application. To serve as controls, one rabbit eyelid only received an injection of 0.5 mg DHE and a second eyelid was only subjected to laser treatment. Eyelids were illuminated with a defocused 630 nm wavelength light from a Coherent medical 920 laser (Palo Alto, CA). The output of the laser at the fiber optic was calibrated using a Diamond Ophir Optics Inc. model DG power meter (Wilmington, MA). Adjusting for a power drop of 15%, across the system, the power densities delivered to the tissue ranged from 53-213 J/cm2. For the first six rabbits (10 lids), a 2.0 cm2 area was exposed to laser light. Using this protocol, the amount of muscle damage in the pretarsal and presepta1 regions of the eyelid was inconsistent, varying with the distance from the center of the laser treatment area. With the 10 subsequent animals (20 lids), three 0.2 cm2 spots were subjected to laser treatment. These spots touched each other but did
PHOTOCHEMOM YECTOMY IN EYELID MUSCLE
not overlap. This laser pattern provided more consistent muscle damage. Initially, a 2-3-h interval between injection and laser illumination of the eyelids was used (22) to determine the optimal range of energy densities and DHE doses. Once the optimal ranges for dose and energy density were established, different intervals between injection and laser application were examined in two rabbits (four lids): 20 min, 2,8, and 24 h. After two upper eyelids became infected, all rabbits were treated prophylactically with Maxitrol (dexamethasone 0.1%, neomycin sulfate, polymyxin B sulfate; Alcon, Fort Worth, TX, U.S.A.) applied topically to the DHE-treated eyelid. Eleven rabbits were killed 4 days after treatment. One rabbit treated using the large laser spot protocol and four rabbits subjected to the three laser spot protocol were killed 6 months after treatment. The rabbits were deeply anesthetized with intramuscular ketamine and xylazine, as previoudy described. Eyelid tissue was harvested. Samples of the eyelids from lateral, central, and medial parts of the lid were frozen immediately in 2-methyl-butane chilled on liquid nitrogen, sectioned at 12 pm on a cryostat, and processed for ATPase histochemistry under alkaline conditions (23). Additional eyelid specimens were fixed in Omnifix, embedded in paraffin, sectioned at 8 pm, and stained with hematoxylideosin or Masson trichrome stains. The eyelids were examined using light microscopy for changes in the orbicularis oculi muscle, skin, conjunctiva, and tarsal glands. The globes were also removed for histo-
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logical examination. They were fixed in Omnifix, embedded in paraffin, and stained with hematoxylin/eosin or Masson trichrome. RESULTS Four Days After DHE and Laser Treatment Four days after DHE and laser treatment, all eyelids were edematous, with erythema found in all cases. In the first experimental rabbit, a combination of 0.5 mg DHE and an energy density of 200 J/cm2 focused on a 2.0 cm2 spot resulted in considerable muscle fiber injury and death. The muscle injury was limited to the site of laser light application. In one rabbit, where the laser application was on the lateral aspect of the eyelid, there was significant muscle loss in the lateral portion of the eyelid, but the amount of muscle that remained increased toward the medial angle of the eyelid (Fig. 1). To damage a greater amount of muscle, the protocol was changed to include three 0.2 cm2 spots across the mediolateral dimension of the eyelid in an effort to more evenly distribute the laser application across the entire muscle and increase the amount of muscle exposed to treatment. The use of this experimental protocol resulted in greater muscle injury when examined at 4 days posttreatment, which was both DHE dose-dependent and laser energy-dependent. At a dose of 0.25 mg DHE and laser illumination of 63.5 J/cm2, there was muscle injury in the preseptal portion of the orbicularis oculi, but little muscle injury in the pretarsal region of
FIG. 1. Photomicrograph of the orbicularis oculi of an eyelid 4 days after treatment with DHE and a 2.0 cmz spot at a laser energy density of 200 Jicm’. A Significant muscle disruption at the site of laser application. B: Large amount of muscle left uninjured at a site not included in the laser application. Arrows indicate muscle fibers in cross-section. Bar is 100 km.
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the muscle (Fig. 2A). When the laser power was increased to 127 J/cm2 with a DHE dose of 0.25 mg, preseptal portions of the orbicularis oculi suffered slight injury to the pretarsal region (see Fig. 2A). When the dose of DHE was increased to 0.5 mg, there was increased muscle loss at both laser power levels, although there was greater muscle injury at the higher laser illumination level (Fig. 2B,C). At this dose of DHE, laser activation resulted in significant edema in the treated eyelid. One interesting phenomenon of the laser-induced muscle injury was that the muscle injury was most severe in the superficial eyelid. This probably related to decreasing penetration of the laser light
through the tissue. If muscle remained after treatment, it was always in the deepest portion of the orbicularis oculi close to the Meibomian glands (Fig. 2C). At a dose of 0.75 mg DHE and a laser energy level of 127 J/cm2, almost 100% of the muscle was destroyed (Fig. 2D). There was, however, severe edema in these eyelids. A series of rabbits was injected with 0.5 mg DHE and subjected to laser activation after 20 min, 2 h, 8 h, or 24 h. Maximal muscle injury was seen with the 2- and 8-h intervals (Fig. 2B,C). By 24 h, it appeared that sufficient DHE had been cleared from the eyelid tissue to minimize the amount of muscle loss. Unfortunately, the injury to the eyelid was not
FIG. 2. Photomicrograph of the orbicularis oculi of eyelids 4 days after treatment with three 0.2 cm2 spots of laser illumination. A: A dose of 0.25 mg DHE and a laser treatment of 63.5 J/cm2(shown) or 127 J/cm2 resulted in little injury to the pretarsal region of the muscle. B: At a dose of 0.5 mg DHE and an energy density of 63.5 J/cm2 there was greater muscle damage in the pretarsal area. C: At a dose of 0.5 mg DHE and an energy density of 127 Jicm’ there was significant muscle disruption in the pretarsal area. D At a dose of 0.75 mg DHE and an energy density of 127 J/cm2 almost all of the muscle was injured. Conjunctival surface is toward the bottom of the photomicrographs. Arrows indicate muscle fibers in cross-section. Bar is 100 pm.
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confined to the orbicularis oculi muscle. There was damage to the Meibomian glands that was also dose and light dependent. The most serious changes involved the conjunctiva (Fig. 3A). There was acantholysis, with an apparent loss in definition of the basement membrane of the epithelium. There was concomitant hyperplasia, elongation of the rete pegs, and loss of mucous cells. The conjunctival changes were seen in rabbits that just received laser application but no DHE, although they were always more severe with both laser and DHE treatment. They were not present when DHE was injected without laser activation. Few changes were seen in the skin, however, except for slight skin ulceration at the center of the laser application site. Laser application alone resulted in no obvious muscle injury. Injection of DHE alone, however, resulted in some muscle injury, presumably because of activation by ambient light. The amount of muscle loss was clearly less than that seen with DHE followed by laser treatment. The globes were examined histologically. No retinal damage was observed. In one case, mild corneal edema was seen in one eye (not shown). Six Months After DHE and Laser Treatment Six months after treatment with 0.5 mg DHE and an energy density of 127 J/cm2, there was no apparent loss of muscle fibers in the orbicularis oculi muscle compared to controls. Thus, even after the nearly total disruption of the muscle that was seen 4 days after treatment, the muscle appeared fully regenerated. There was some evidence of previous muscle injury, however, in the form of fibers of
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irregular shape and size, as well as split muscle fibers (Fig. 4A,B). The skin and Meibomian glands appeared normal, but abnormalities of the conjunctival epithelium persisted (Fig. 3B). Abnormalities included hyperplasia with the presence of some atypical cells, although polarity seemed to be maintained. DISCUSSION
Local injection of DHE, followed by laser light activation of the DHE, can significantly damage the orbicularis oculi muscle in situ. We determined optimal treatment parameters, including dosage, time interval between injection and laser treatment, laser spot size, and aiming pattern. Four days after treatment, at a DHE dose of 0.5 mg and a laser energy level of 127 J/cm2, there was almost total destruction of the muscle within the eyelid. The destruction was temporary, however, and 6 months after the treatment the majority of the orbicularis oculi muscle had regenerated. Injury to the eyelid muscle was dependent on both DHE dose and the energy density of the laser light. The first laser delivery method was suboptimal because of the nonuniform distribution of laser light across the eyelid. When the laser target was changed to three spots of smaller size, injury to the eyelid muscle was more consistent. To injure an even larger area of muscle, a lens-tipped fiber optic would make possible more uniform energy distribution to a larger area of the eyelid. Since laser illumination alone did not produce muscle injury, it is unlikely that thermal damage is responsible.
FIG. 3. A: Photomicrograph of the conjunctival changes seen after DHE and laser treatment 4 days after treatment. There was a loss of mucous cells and hyperplasia of the epithelium (asterisk indicates the conjunctival epithelium). B: Abnormalities of the conjunctiva are still present 6 months after treatment (arrowhead), although mucous cells have regenerated (arrow). Bar is 50 pm.
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FIG. 4. Photomicrograph of the orbicularis oculi of an eyelid treated with 0.5 mg DHE and 127 J/cm2 6 months after treatment stained with the alkaline ATPase protocol. A Low power photomicrograph shows that most of the muscle has regenerated. Evidence of previous injury in the orbicularis oculi is seen in the form of fibers of irregular shape and size (arrow). Bar is 100 km. B: Higher power photomicrograph shows split muscle fibers (arrowhead). Bar is 50 pm.
The effects of photodynamic therapy in the eyelid were not specific to the muscle. Adjacent structures, including the skin, conjunctiva, and Meibomian glands were also damaged. Most of the damage was temporary, although long-term changes were seen in the conjunctival epithelium. It may be possible to alter the dose, energy level, or time interval between injection and laser application to improve the selectivity of this photochemomyectomy . Part of the reason that this technique works so well for cancer treatment is that the cancer cells preferentially take up the DHE and are thus made more susceptible to damage by laser light (12,24). There is evidence from tissue culture studies that myocardial cells take up HPD rapidly over a 2-h period (22). There is no evidence, however, that skeletal muscle would have preferential uptake of DHE over other cells in the eyelid. Thus, all cells that take up the compound in the eyelid would be injured with subsequent activation of the DHE by the laser light. Local injection of HPD allows the use of doses five times lower than the typical tumoricidal doses used in cancer therapy (17,18). In this way, the risk of systemic toxicity and cutaneous phototoxicity is decreased. One possible danger of using photodynamic therapy in the orbital region is inadvertant irreversible injury to the cornea or retina. The injection of DHE directly into the eyelid, as opposed to systemic administration, decreases the possibility of spread
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into surrounding orbital structures and the globe itself. However, great care must be used since it has been shown that if HPD is in the retina, photoradiation therapy will result in retinal damage at the site of light activation (25). N o damage to the lens or cornea were reported in this study. The one case of corneal edema seen in our experimental animals may have been due to spillage of the DHE onto the cornea at the time of injection, which then may have been activated during the laser application. An opaque scleral shield may prevent this type of injury, as would irrigation of the cornea before laser treatment. The major drawback of using DHE and laser activation for destroying eyelid muscle is that the muscle regenerated fully within 6 months. A more diffuse illumination of the entire eyelid width, rather than the spot illumination done in this study, may increase the muscle injury and potentially make it permanent by injuring more of the muscle. It may be possible to combine photodynamic therapy with the use of an antimitotic drug to remove muscle permanently. Edell and Cortese (26) have demonstrated synergy between doxorubicin and photodynamic therapy. Doxorubicin has been shown to be an effective myotoxin after local injection into the eyelid (9,27). A single 2 mg injection of doxorubicin can remove as much as 70% of the muscle in the treated eyelid (10). The effective dose of doxorubicin for muscle injury is also close to the
PHOTOCHEMOMYECTOMY IN EYELID MUSCLE toxic level for the overlying eyelid skin, and was a source of ulceration in both animals and patients (9,ll). It may be possible to combine doxorubicin injection with photodynamic therapy to increase muscle loss while decreasing the possibility of injury to the eyelid skin. We believe that the results of this study suggest limited applicability of photochemomyectomy in the treatment of eyelid spasms. However, it points toward two other areas of investigation. First, as a primary or adjuvant treatment, photomyectomy could be used to temporarily reduce the strength and spasms of large appendicular muscles such as the leg adductors in patients with multiple sclerosis, another condition where Botulinum A toxin has proven to be of temporary benefit (28). This would be particularly useful in large muscles, where the dose of botulinum toxin required could cause weakness in distant muscles, including those involved in respiration, and thus be potentially fatal to the patient. Second, photochemomyectomy in appendicular muscles could be combined with treatment with an antimitotic agent, such as doxorubicin, to prevent regeneration and produce permanent myectomy. Acknowledgment: This w o r k w a s supported b y NEI grant EY07935 to L.K.M. and J.D.W., t h e Donald a n d Louise Gabbert Neuro-ophthalmology Research Fund, Minnesota Lions and Lionnesses, a n d a n unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.
REFERENCES 1. Spector GJ, Smith PG, Burde RM. Selective facial neurectomy for essential blepharospasm. Laryngoscope 1981;91: 1896-1903. 2. Jankovic P, Pate1 SC. Blepharospasm associated with brainstem lesions. Neurology 1983;33:1237-1240. 3. Scott AB, Kennedy RA, Hubbs HA. Botulinum toxin injection as a treatment for blepharospasm. Arch Ophthalmol 1985 ;103~347-350. 4. Perman KI, Baylis HI, Rosenbaum AL, Kirschen DG. The use of botulinum toxin in the medical management of benign essential blepharospasm. Ophthalmology 1986;93:1-3. 5 . Elston JS. Long-term results of treatment of idiopathic blepharospasm with botulinum toxin injections. Br J Ophthalmol 1987;71 :664-668. 6. Grandas F, Elston J, Zuinn N , Marsden CD. Blepharospasm: a review of 264 patients. J Neurol Neurosurg Psychiatry 1988;51:767-772. 7 . Geller BD, Hallett M, Ravits J. Botulinum toxin therapy in hemifacial spasm: clinical and electrophysiologic studies. Muscle Nerve 1989;12:716-722. 8 . Seiff SR. Freeman LN, Bluestone DL, Berg BO. Use of botulinum toxin to treat blepharospasm in a 16 year old with dystonic syndrome. Pediatr Neurol 1989;5:121-123.
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9. McLoon LK, Wirtschafter J. Doxorubicin chemomyectomy: injection of monkey orbicularis oculi in selective muscle injury. Invest Ophthalmol Vis Sci 1988;29:185k1859. 10. McLoon LK, Bauer G, Wirtschafter J. Quantification of muscle loss in the doxorubicin treated orbicularis oculi of the monkey. Invest Opthalmol Vis Sci 1991;32:1667-1673. 11. Wirtschafter JD. Clinical doxorubicin chemomyectomy. An experimental treatment for benign essential blepharospasm and hemifacial spasm. Ophthalmology 1991 ;98:357-366. 12. Bugelski PJ, Porter CW, Dougherty TJ. Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse. Cancer Res 1981;41:4606-4612. 13. Gomer CJ, Doiron DR, Rucker N, Razum NJ, Fountain SW. Action spectrum (620-640 nm) for hematoporphyrin derivative induced cell killing. Phofochem Photobiol 1984;39:365368. 14. Dougherty TJ. Photodynamic therapy (PDT) of malignant tumors. CRC Crit Rev Oncol Hematol 1984;2:83-116. 15. Star WM, Marijnissen HPA, Van Den Berg-Blok AE, Versteeg JAC, Franken KAP, Reinhold HS. Destruction of rat mammary tumor and normal tissue microcirculation by hematoporphyrin derivative photoradiation observed in invivo sandwich observation chambers. Cancer Res 1986;46: 2532-2540. 16. Kostron H, Bellnier DA, Lin CW, Swartz MR, Martuza RL. Distribution, retention and phototoxicity of hematoporphyrin derivative in a rat glioma: intraneoplastic versus intraperitoneal injection. J Neurosurg 1986;64:768-774. 17. Amano T, Prout GR, Lin CW. Intratumor injection as a more effective means of porphyrin administration for photodynamic therapy. J Urol 1988;139:392-395. 18. Lin CW, Amano T, Rutledge AR, Shulok JR, Prout GR. Photodynamic effect in an experimental bladder tumor treated with intratumor injection of hematoporphyrin derivative. Cancer Res 1988;48:6115-6120. 19. Gomer CJ, Dougherty TJ. Determination of (3H) and (14C) hematoporphyrin derivative distribution in malignant and normal tissue. Cancer Res 1979;39:146-151. 20. Zhou C, Yang W, Ding Z, et al. The biological effects of photodynamic therapy on normal skin in mice. I. A light microscopic study. Adv Exp Med Biol 1985;193:105-109. 21. McLoon LK, Wirtschafter J. Regional differences in the orbicularis oculi muscle: conservation between species. J Neurol Sci 1991;104:197-202. 22. Berns MW, Dahlman A, Johnson FM, et al. In vitro cellular effects of hematoporphyrin derivative. Cancer Res 1982;42: 2325-2329. 23. Brooke MH, Kaiser KK. Muscle fiber types. How many and what kind? Arch Neurol 1970;23:369-379. 24. Dougherty TJ, Grindey GB, Fie1 R, Weishaupt KR, Boyle DG. Photoradiation therapy. 11. Cure of animal tumors with hematoporphyrin and light. J Natl Cancer Inst 1975;55:115121. 25. Gomer CJ, Doiron DR, Jester JV, Szirth BC, Murphree AL. Hematoporphyrin derivative photoradiation therapy for the treatment of intraocular tumors: examination of acute normal ocular tissue toxicity. Cancer Res 1983;43:721-727. 26. Edell ES, Cortese DA. Combined effects of hematoporphyrin derivative phototherapy and Adriamycin in a murine tumor model. Lasers Surg Med 1988;8:413417. 27. Baker L, Wirtschafter JD. Experimental doxorubicin myopathy: a permanent treatment for eyelid spasms? Arch Ophthal 1987;105:1265-1268. 28. Scott AB, Tsui JKC, Bhatt MM, et al. Treatment of spasticity with botulinum toxin: a double blind study. Ann Neurol 1990;28:512-5 15.
Movement Disorders, Vol. 7, N o . 2, 1992
Severe But Temporary Injury to Rabbit Orbicularis Oculi Muscle Using Dihematoporphyrin Ether and Laser Photochemomyectomy Jonathan D. Wirtschafter, David Paul Slovut, Leif Stordal, *Joseph Valentino, and Linda Kirschen McLoon Department of Ophthalmology and *Department of Otolargyngology, University of Minnesota, Minneapolis, Minnesota, U.S.A.
Summary: The use of local dihematoporphyrin ether (DHE) injections, followed by laser light activation, was investigated as a potential permanent myectomy treatment for muscle spasms, in particular blepharospasm and hemifacial spasm. DHE was injected into the eyelids of rabbits, followed by laser activation, as used in photochemotherapy . Four days after treatment, histological examination indicated that doses of 20.5 mg of DHE and laser treatment with an energy density of at least 100 J/cm2 resulted in an almost total destruction of the orbicularis oculi muscle in the treated eyelid. The amount of muscle injury was dependent on both dose of DHE and energy density levels. Histologically, the tarsal glands and conjunctiva were damaged. Glandular tissue was markedly reduced, and the conjunctival epithelium showed hyperplasia and a loss of mucous cells. Six months after DHE and laser treatment, the majority of the muscle tissue had regenerated, although there was evidence of previous injury. While DHE injections combined with laser light activation were lethal to muscle at the site of treatment, this treatment was not permanent. The orbicularis oculi muscle retained its ability to regenerate. However, photochemomyectomy may be studied further as an adjuvant treatment to temporarily injure and debulk large muscles when botulinum toxin is contraindicated due to the large doses involved or as a permanent treatment when used together with an antimitotic agent such as doxorubicin. Key Words: Facial muscles-Blepharospasm-Photodynamic therapy.
Blepharospasm can be a debilitating form of dystonia characterized by intense, involuntary spasms of the orbicularis oculi muscle and functional blindness. The etiology of the disorder is usually unknown, although blepharospasm has been associated with some brainstem lesions (1,2). Multiple surgical and medical treatments are available, but none provide a fully satisfactory, permanent result. Blepharospasm can be treated with botulinum toxin
injections into the eyelids (3,4), but this treatment is temporary. Spasms return, usually between 9 and 12 weeks ( 4 8 ) . Botulinum toxin (BT) injections are also only effective in 70430% of patients treated (5,6). When BT injections fail, or when patients seek a more permanent result, surgical myectomy is an option (5). In addition to surgical complications, reoperation or supplementary BT injections are often required to relieve spasms. Lack of permanence of BT injections, coupled with potential complications of surgical intervention, suggested a need for an alternative treatment for blepharospasm. One new experimental therapy, the local injection of
Address correspondence and reprint requests to Dr. L. McLoon, Department of Ophthalmology, University of Minnesota, Box 493, UMHC, Minneapolis, MN 55455, U.S.A.
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doxorubicin into the eyelid, results in substantial muscle loss in animals (9,lO) and spasm relief in selected human patients (1 1). Another possible approach would involve the use of photoradiation therapy, the light-activated chemicals that are used in cancer treatment. We tested whether this approach could be used to locally remove muscle to treat this type of disease. Photoradiation therapy is a treatment for neoplasms that uses light-activated fluorescent compounds known as photosensitizers. The most welldescribed antineoplastic photosensitizers are hematoporphyrin derivative (HPD) and extracts of HPD, such as dihematoporphyrin ether (DHE). These are relatively inert compounds that preferentially concentrate in most neoplastic tissue 24-72 h after intravenous administration (12,13). Neoplasms are selectively destroyed because they concentrate and retain HPD longer than the normal, surrounding tissue, probably due to their more active metabolism and less efficient lymph drainage (12). The dye is activated with 630 nm coherent light. This wavelength allows dye activation with maximal tissue penetration, and the light can easily be channeled by fiber optics to specific locations. The light results in activation of the HPD, causing the release of free oxygen radicals, which in turn initiates a process leading to cell death (14). The destruction as a result of this treatment also stems from damage to the tumor vasculature ( 1 9 , which causes localized ischemia and tissue necrosis. To decrease the risk of skin phototoxicity, HPD has been administered locally (16-18). After intratumor HPD administration, there are substantially greater amounts of HPD concentrated in the tumor than after intravenous administration (17,18). Muscle has been shown to take up HPD (19), and muscle injury and necrosis after phototherapy have been described (20). Although previous investigations have used photodynamic therapy to treat tumors, there is a potential for its use in selective muscle destruction at a specified and controlled location. We examined the effect of a local injection of DHE in the eyelids of rabbits, followed by laser light application in an attempt to produce selective muscle loss, or photochemomyectomy . Local injection into the eyelid should produce a high extracellular concentration of the DHE. This should increase the uptake of the drug by the muscle cells at the injection site, as well as potentially damage the vasculature that supports this muscle. Local injection would decrease the po-
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tential for skin phototoxicity. We examined the eyelids histologically either 4 days or 6 months after DHE and laser treatment to assess muscle loss. MATERIALS AND METHODS Sixteen adult New Zealand white rabbits were obtained from Birchwood Laboratory and housed with the University of Minnesota Research Animal Resources. Animals were handled in accordance with the guidelines set up by the National Institutes of Health on the use of animals in research. Animals were deeply anesthetized with an equal mixture of ketamine HCl(lO0 mg/ml) at a dose of 10 mg/kg and xylazine (Rompun, 20 mg/ml) at a dose of 2 mgfkg. After shaving the areas to be injected, DHE (Photofrin 11, QLT Phototherapeutics, Lederle Labs., Vancouver, B.C., Canada) was injected along the entire length of the pretarsal portion of the right upper and left lower eyelids (21). Doses were graded in a series of rabbits from 0.1 to 1 .O mg in 0.1 cc of a 5% dextrose solution. The eyelids of the untreated, opposite eyes were used as controls. Prior to laser treatment, a 4-0 silk traction stitch was placed through the medial or lateral portion of the lid to flatten the eyelid and maximize exposure of the lid to the laser light. Artificial tears were applied to maintain corneal lubrication during laser treatment. A local anesthetic, proparacaine HCI, was applied to the cornea. The laser target was marked using carbol fuchsin solution. None of the marker dye was irradiated directly during laser application. To serve as controls, one rabbit eyelid only received an injection of 0.5 mg DHE and a second eyelid was only subjected to laser treatment. Eyelids were illuminated with a defocused 630 nm wavelength light from a Coherent medical 920 laser (Palo Alto, CA). The output of the laser at the fiber optic was calibrated using a Diamond Ophir Optics Inc. model DG power meter (Wilmington, MA). Adjusting for a power drop of 15%, across the system, the power densities delivered to the tissue ranged from 53-213 J/cm2. For the first six rabbits (10 lids), a 2.0 cm2 area was exposed to laser light. Using this protocol, the amount of muscle damage in the pretarsal and presepta1 regions of the eyelid was inconsistent, varying with the distance from the center of the laser treatment area. With the 10 subsequent animals (20 lids), three 0.2 cm2 spots were subjected to laser treatment. These spots touched each other but did
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not overlap. This laser pattern provided more consistent muscle damage. Initially, a 2-3-h interval between injection and laser illumination of the eyelids was used (22) to determine the optimal range of energy densities and DHE doses. Once the optimal ranges for dose and energy density were established, different intervals between injection and laser application were examined in two rabbits (four lids): 20 min, 2,8, and 24 h. After two upper eyelids became infected, all rabbits were treated prophylactically with Maxitrol (dexamethasone 0.1%, neomycin sulfate, polymyxin B sulfate; Alcon, Fort Worth, TX, U.S.A.) applied topically to the DHE-treated eyelid. Eleven rabbits were killed 4 days after treatment. One rabbit treated using the large laser spot protocol and four rabbits subjected to the three laser spot protocol were killed 6 months after treatment. The rabbits were deeply anesthetized with intramuscular ketamine and xylazine, as previoudy described. Eyelid tissue was harvested. Samples of the eyelids from lateral, central, and medial parts of the lid were frozen immediately in 2-methyl-butane chilled on liquid nitrogen, sectioned at 12 pm on a cryostat, and processed for ATPase histochemistry under alkaline conditions (23). Additional eyelid specimens were fixed in Omnifix, embedded in paraffin, sectioned at 8 pm, and stained with hematoxylideosin or Masson trichrome stains. The eyelids were examined using light microscopy for changes in the orbicularis oculi muscle, skin, conjunctiva, and tarsal glands. The globes were also removed for histo-
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logical examination. They were fixed in Omnifix, embedded in paraffin, and stained with hematoxylin/eosin or Masson trichrome. RESULTS Four Days After DHE and Laser Treatment Four days after DHE and laser treatment, all eyelids were edematous, with erythema found in all cases. In the first experimental rabbit, a combination of 0.5 mg DHE and an energy density of 200 J/cm2 focused on a 2.0 cm2 spot resulted in considerable muscle fiber injury and death. The muscle injury was limited to the site of laser light application. In one rabbit, where the laser application was on the lateral aspect of the eyelid, there was significant muscle loss in the lateral portion of the eyelid, but the amount of muscle that remained increased toward the medial angle of the eyelid (Fig. 1). To damage a greater amount of muscle, the protocol was changed to include three 0.2 cm2 spots across the mediolateral dimension of the eyelid in an effort to more evenly distribute the laser application across the entire muscle and increase the amount of muscle exposed to treatment. The use of this experimental protocol resulted in greater muscle injury when examined at 4 days posttreatment, which was both DHE dose-dependent and laser energy-dependent. At a dose of 0.25 mg DHE and laser illumination of 63.5 J/cm2, there was muscle injury in the preseptal portion of the orbicularis oculi, but little muscle injury in the pretarsal region of
FIG. 1. Photomicrograph of the orbicularis oculi of an eyelid 4 days after treatment with DHE and a 2.0 cmz spot at a laser energy density of 200 Jicm’. A Significant muscle disruption at the site of laser application. B: Large amount of muscle left uninjured at a site not included in the laser application. Arrows indicate muscle fibers in cross-section. Bar is 100 km.
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the muscle (Fig. 2A). When the laser power was increased to 127 J/cm2 with a DHE dose of 0.25 mg, preseptal portions of the orbicularis oculi suffered slight injury to the pretarsal region (see Fig. 2A). When the dose of DHE was increased to 0.5 mg, there was increased muscle loss at both laser power levels, although there was greater muscle injury at the higher laser illumination level (Fig. 2B,C). At this dose of DHE, laser activation resulted in significant edema in the treated eyelid. One interesting phenomenon of the laser-induced muscle injury was that the muscle injury was most severe in the superficial eyelid. This probably related to decreasing penetration of the laser light
through the tissue. If muscle remained after treatment, it was always in the deepest portion of the orbicularis oculi close to the Meibomian glands (Fig. 2C). At a dose of 0.75 mg DHE and a laser energy level of 127 J/cm2, almost 100% of the muscle was destroyed (Fig. 2D). There was, however, severe edema in these eyelids. A series of rabbits was injected with 0.5 mg DHE and subjected to laser activation after 20 min, 2 h, 8 h, or 24 h. Maximal muscle injury was seen with the 2- and 8-h intervals (Fig. 2B,C). By 24 h, it appeared that sufficient DHE had been cleared from the eyelid tissue to minimize the amount of muscle loss. Unfortunately, the injury to the eyelid was not
FIG. 2. Photomicrograph of the orbicularis oculi of eyelids 4 days after treatment with three 0.2 cm2 spots of laser illumination. A: A dose of 0.25 mg DHE and a laser treatment of 63.5 J/cm2(shown) or 127 J/cm2 resulted in little injury to the pretarsal region of the muscle. B: At a dose of 0.5 mg DHE and an energy density of 63.5 J/cm2 there was greater muscle damage in the pretarsal area. C: At a dose of 0.5 mg DHE and an energy density of 127 Jicm’ there was significant muscle disruption in the pretarsal area. D At a dose of 0.75 mg DHE and an energy density of 127 J/cm2 almost all of the muscle was injured. Conjunctival surface is toward the bottom of the photomicrographs. Arrows indicate muscle fibers in cross-section. Bar is 100 pm.
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confined to the orbicularis oculi muscle. There was damage to the Meibomian glands that was also dose and light dependent. The most serious changes involved the conjunctiva (Fig. 3A). There was acantholysis, with an apparent loss in definition of the basement membrane of the epithelium. There was concomitant hyperplasia, elongation of the rete pegs, and loss of mucous cells. The conjunctival changes were seen in rabbits that just received laser application but no DHE, although they were always more severe with both laser and DHE treatment. They were not present when DHE was injected without laser activation. Few changes were seen in the skin, however, except for slight skin ulceration at the center of the laser application site. Laser application alone resulted in no obvious muscle injury. Injection of DHE alone, however, resulted in some muscle injury, presumably because of activation by ambient light. The amount of muscle loss was clearly less than that seen with DHE followed by laser treatment. The globes were examined histologically. No retinal damage was observed. In one case, mild corneal edema was seen in one eye (not shown). Six Months After DHE and Laser Treatment Six months after treatment with 0.5 mg DHE and an energy density of 127 J/cm2, there was no apparent loss of muscle fibers in the orbicularis oculi muscle compared to controls. Thus, even after the nearly total disruption of the muscle that was seen 4 days after treatment, the muscle appeared fully regenerated. There was some evidence of previous muscle injury, however, in the form of fibers of
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irregular shape and size, as well as split muscle fibers (Fig. 4A,B). The skin and Meibomian glands appeared normal, but abnormalities of the conjunctival epithelium persisted (Fig. 3B). Abnormalities included hyperplasia with the presence of some atypical cells, although polarity seemed to be maintained. DISCUSSION
Local injection of DHE, followed by laser light activation of the DHE, can significantly damage the orbicularis oculi muscle in situ. We determined optimal treatment parameters, including dosage, time interval between injection and laser treatment, laser spot size, and aiming pattern. Four days after treatment, at a DHE dose of 0.5 mg and a laser energy level of 127 J/cm2, there was almost total destruction of the muscle within the eyelid. The destruction was temporary, however, and 6 months after the treatment the majority of the orbicularis oculi muscle had regenerated. Injury to the eyelid muscle was dependent on both DHE dose and the energy density of the laser light. The first laser delivery method was suboptimal because of the nonuniform distribution of laser light across the eyelid. When the laser target was changed to three spots of smaller size, injury to the eyelid muscle was more consistent. To injure an even larger area of muscle, a lens-tipped fiber optic would make possible more uniform energy distribution to a larger area of the eyelid. Since laser illumination alone did not produce muscle injury, it is unlikely that thermal damage is responsible.
FIG. 3. A: Photomicrograph of the conjunctival changes seen after DHE and laser treatment 4 days after treatment. There was a loss of mucous cells and hyperplasia of the epithelium (asterisk indicates the conjunctival epithelium). B: Abnormalities of the conjunctiva are still present 6 months after treatment (arrowhead), although mucous cells have regenerated (arrow). Bar is 50 pm.
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FIG. 4. Photomicrograph of the orbicularis oculi of an eyelid treated with 0.5 mg DHE and 127 J/cm2 6 months after treatment stained with the alkaline ATPase protocol. A Low power photomicrograph shows that most of the muscle has regenerated. Evidence of previous injury in the orbicularis oculi is seen in the form of fibers of irregular shape and size (arrow). Bar is 100 km. B: Higher power photomicrograph shows split muscle fibers (arrowhead). Bar is 50 pm.
The effects of photodynamic therapy in the eyelid were not specific to the muscle. Adjacent structures, including the skin, conjunctiva, and Meibomian glands were also damaged. Most of the damage was temporary, although long-term changes were seen in the conjunctival epithelium. It may be possible to alter the dose, energy level, or time interval between injection and laser application to improve the selectivity of this photochemomyectomy . Part of the reason that this technique works so well for cancer treatment is that the cancer cells preferentially take up the DHE and are thus made more susceptible to damage by laser light (12,24). There is evidence from tissue culture studies that myocardial cells take up HPD rapidly over a 2-h period (22). There is no evidence, however, that skeletal muscle would have preferential uptake of DHE over other cells in the eyelid. Thus, all cells that take up the compound in the eyelid would be injured with subsequent activation of the DHE by the laser light. Local injection of HPD allows the use of doses five times lower than the typical tumoricidal doses used in cancer therapy (17,18). In this way, the risk of systemic toxicity and cutaneous phototoxicity is decreased. One possible danger of using photodynamic therapy in the orbital region is inadvertant irreversible injury to the cornea or retina. The injection of DHE directly into the eyelid, as opposed to systemic administration, decreases the possibility of spread
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into surrounding orbital structures and the globe itself. However, great care must be used since it has been shown that if HPD is in the retina, photoradiation therapy will result in retinal damage at the site of light activation (25). N o damage to the lens or cornea were reported in this study. The one case of corneal edema seen in our experimental animals may have been due to spillage of the DHE onto the cornea at the time of injection, which then may have been activated during the laser application. An opaque scleral shield may prevent this type of injury, as would irrigation of the cornea before laser treatment. The major drawback of using DHE and laser activation for destroying eyelid muscle is that the muscle regenerated fully within 6 months. A more diffuse illumination of the entire eyelid width, rather than the spot illumination done in this study, may increase the muscle injury and potentially make it permanent by injuring more of the muscle. It may be possible to combine photodynamic therapy with the use of an antimitotic drug to remove muscle permanently. Edell and Cortese (26) have demonstrated synergy between doxorubicin and photodynamic therapy. Doxorubicin has been shown to be an effective myotoxin after local injection into the eyelid (9,27). A single 2 mg injection of doxorubicin can remove as much as 70% of the muscle in the treated eyelid (10). The effective dose of doxorubicin for muscle injury is also close to the
PHOTOCHEMOMYECTOMY IN EYELID MUSCLE toxic level for the overlying eyelid skin, and was a source of ulceration in both animals and patients (9,ll). It may be possible to combine doxorubicin injection with photodynamic therapy to increase muscle loss while decreasing the possibility of injury to the eyelid skin. We believe that the results of this study suggest limited applicability of photochemomyectomy in the treatment of eyelid spasms. However, it points toward two other areas of investigation. First, as a primary or adjuvant treatment, photomyectomy could be used to temporarily reduce the strength and spasms of large appendicular muscles such as the leg adductors in patients with multiple sclerosis, another condition where Botulinum A toxin has proven to be of temporary benefit (28). This would be particularly useful in large muscles, where the dose of botulinum toxin required could cause weakness in distant muscles, including those involved in respiration, and thus be potentially fatal to the patient. Second, photochemomyectomy in appendicular muscles could be combined with treatment with an antimitotic agent, such as doxorubicin, to prevent regeneration and produce permanent myectomy. Acknowledgment: This w o r k w a s supported b y NEI grant EY07935 to L.K.M. and J.D.W., t h e Donald a n d Louise Gabbert Neuro-ophthalmology Research Fund, Minnesota Lions and Lionnesses, a n d a n unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.
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