Myogenic precursor cells in craniofacial muscles.

Craniofacial skeletal muscles (CskM), including the masticatory (MM), extraocular (EOM) and laryngeal muscles (LM), have a number of properties that set them apart from the majority of skeletal muscles (SkM). They have embryological origins that are distinct from musculature elsewhere in the body, they express a number of immature myosin heavy chain isoforms and maintain increased and distinct expression of a number of myogenic growth factors and their receptors from other adult SkMs. Furthermore, it has recently been demonstrated that unlike limb SkM, normal adult EOM and LM retain a population of activated satellite cells, the regenerative cell in adult SkM. In order to maintain this proliferative pool throughout life, CSkM may contain more satellite cells and/or more multipotent precursor cells that may be more resistant to apoptosis than those found in limb muscle. A further exciting question is whether this potentially more active muscle precursor cell population could be utilized not only for SkM repair, but be harnessed for repair or reconstruction of other tissues, such as nervous tissue or bone. This is a highly attractive speculation as the innate regenerative capacity of craniofacial muscles would ensure the donor tissue would not have compromised future function.

Reduction in vesicant-induced cellular inflammation and hyperalgesia by local injection of corticotropin releasing factor in rabbit eyelid.

OBJECTIVE AND DESIGN: This study tested the ability of corticotropin releasing factor (CRF) to mitigate inflammatory cell infiltration and hyperalgesia after a direct subcutaneous doxorubicin injection, a vesicant drug that causes localized inflammation at the site of subcutaneous exposure. SUBJECTS: This study used 62 adult New Zealand white rabbits. METHODS: Doxorubicin (1-2 mg), followed by either CRF (300, 150, 75, 35, 20 or 10 microg in 1 ml saline) or saline only, was injected into pairs of rabbit eyelids. One set of doxorubicin and CRF treated eyelid pairs also received injection of a CRF antagonist into one eyelid. The eyelid tissue was assayed from 1-7 days later for morphometric changes in CD11b-positive inflammatory cell infiltrate. Hyperalgesia was assessed using the blink response to von Frey hair stimulation. Epidermal and dermal nerve fibers were visualized immunochemically using an antibody to PGP 9.5. The cellular localization of corticotropin releasing factor was determined immunohistochemically in eyelid tissue. RESULTS: Doses of CRF from 35 to 150 microg significantly reduced inflammatory cell infiltration at all the post-injury time intervals examined, from 2 h post-injection up until 7 days. Doses of 10 and 20 microg CRF had no effect on local tissue inflammation caused by subcutaneous doxorubicin exposure. Doses of 35-150 microg CRF treatment significantly reduced the development of doxorubicin-induced hyperalgesia; this was maintained up to 4 days. Local CRF injections of 35-150 microg markedly reduced the doxorubicin induction of PGP-positive axons within the epidermis and subepidermis. CRF receptors were localized to the basal layer of the epithelium and to the subcutaneous nerve afferents. CONCLUSIONS: Local injection of CRF, after a subcutaneous exposure to the vesicant drug doxorubicin, significantly reduced the infiltration of CD11b-positive cells and reduced doxorubicin-induced hyperalgesia in the treated eyelids. This reduction in hyperalgesia appears to be a local peripheral effect, reducing nerve fibers within the subepidermal treatment area. This demonstrates the potential clinical usefulness of CRF because it could be used post-injury as a locally injected treatment; this potent local anti-inflammatory agent may have wide applications following drug, chemical, physical or surgical injury.

Physiological assessment of muscle strength in vitro after direct injection of doxorubicin into rabbit sternocleidomastoid muscle.

Doxorubicin chemomyectomy is a potent method for the permanent removal of a muscle or group of muscles after direct local injection, and has been used successfully to treat blepharospasm and hemifacial spasm patients. The efficacy of doxorubicin chemomyectomy on reducing muscle strength after direct injection of doxorubicin into rabbit sternocleidomastoid muscle was tested. One- and 6-month postinjection force assessment was performed in vitro to measure alterations in peak twitch and tetanic force generation, as well as fatigue responses for the treated muscles compared to control. There were significant reductions of both twitch and tetanic peak amplitudes in the doxorubicin-treated muscles. One month after treatment, the decreases in force were greater after 2 mg doxorubicin injections than after 1 mg doxorubicin. While there was a significant reduction in force generation after doxorubicin treatment, fatigue resistances for the doxorubicin-treated muscles were increased compared to the controls. There were significant reductions in muscle mass after doxorubicin treatment, and by 6 months, the myosin heavy chain isoform distribution was similar to normal sternocleidomastoid, except for an increase in slow myosin-positive fibers. Doxorubicin chemomyectomy resulted in a significant reduction in functional force generation in the treated sternocleidomastoid muscles. These findings suggest a potential clinical use of doxorubicin chemomyectomy to treat cervical dystonia patients.

Doxil-induced chemomyectomy: effectiveness for permanent removal of orbicularis oculi muscle in monkey eyelid.

PURPOSE: To test the safety and effectiveness of Doxil chemomyectomy in monkey eyelids using treatment schedules and doses similar to those proposed for the human blepharospasm patients in Phase I and II trials. METHODS: Bupivacaine/hyaluronidase and Doxil were injected sequentially into the eyelids of five Cynomolgus monkeys. Eyelids received 1, 2, or 3 sets of injections. The monkeys were euthanatized at a minimum of 2 months and a maximum of 12 months after the final treatment. The eyelids were prepared for histologic examination, and muscle fiber loss was quantified. RESULTS: All Doxil injections resulted in a significant loss of myofibers. No bruising, ulceration, or other skin injuries occurred, even after a third injection regimen within a single treated eyelid. Two-day preinjury with a bupivacaine/hyaluronidase mixture had a significant adjuvant effect. CONCLUSIONS: Doxil chemomyectomy is an effective protocol to permanently remove muscle from injected eyelids in nonhuman primates. Serial injections over the course of several months using the preinjury protocol combined with Doxil treatment significantly increased Doxil's myotoxic effects. Additionally, the injection of the liposome-encapsulated form of doxorubicin did not result in skin injury or ulceration. Species differences demonstrated the importance of testing these drugs in nonhuman primates. Thus, repeated doses of Doxil may prove to be as clinically effective as free doxorubicin injections in reducing muscle spasms in blepharospasm patients but with increased safety to the skin and tissue around the injection site.

Acute effects of the skeletal muscle-specific immunotoxin ricin-mAb 35 on extraocular muscles of rabbits.

PURPOSE: To determine the acute histologic and ultrastructural effects of a recently developed muscle-specific immunotoxin, ricin-mAb 35. METHODS: Graduated doses of ricin-mAb 35, composed of ricin conjugated to a monoclonal antibody against the nicotinic acetylcholine receptor of skeletal muscle, were injected into one superior rectus muscle in rabbits. After 3, 7, and 14 days, both superior rectus muscles were removed and prepared for electron microscopy and histologic examination, by using a number of immunohistochemical markers to identify inflammatory cell infiltration, muscle fiber loss, and muscle regeneration. RESULTS: Myotoxicity of the ricin-mAb 35 was focal and dose related. At the highest dose tested, there was substantial inflammatory cell infiltrate by 3 days, which largely disappeared by 7 days. Significant muscle loss was apparent by 7 days after ricin-mAb 35 treatment. Both the inflammatory reaction and muscle fiber loss were confined to the immediate injection site. Surrounding muscle appeared to be normal. At 14 days after treatment, early signs of muscle regeneration were evident within the tissue sections. No evidence of orbital or systemic toxicity was seen in any animal. CONCLUSIONS: Direct injection of ricin-mAb 35 into the extraocular muscles of rabbits results in a dose-related focal injury to the muscles, with a self-limited inflammatory component and significant muscle fiber loss. This novel immunotoxin may be useful in the treatment of strabismus if chronic studies show a sustained histologic and electrophysiologic effect.

Mucocutaneous junction as the major source of replacement palpebral conjunctival epithelial cells.

PURPOSE: The conjunctival epithelium performs an important role in the homeostasis and integrity of the eye. To protect the integrity of the ocular surface, these cells must be replaced from locally concentrated or randomly distributed foci of stem cells. These slow-cycling stem cells produce transient amplifying cells that undergo further divisions before becoming mature conjunctival epithelial cells. In the current study, the source of palpebral conjunctival cells was determined. METHODS: Adult rabbits were injected intraperitoneally with bromodeoxyuridine (BrdU) at a dose of 50 mg/kg body weight and killed after 1, 3, 5, and 7 days and 2 months. The orbital contents and eyelids were exenterated en bloc, frozen to maintain the orientation between the eyelids and globe, and sectioned in a parasagittal plane. Random midglobe sections were stained for the presence of proliferating cell nuclear antigen (PCNA). Additional sections were immunostained to detect BrdU-labeled conjunctival epithelial cells. BrdU-positive cells were counted in a series of 0.4-mm zones from the mucocutaneous junction of the eyelid, through the fornix and bulbar conjunctiva. A second set of rabbits received daily injections of BrdU for 2 or 4 weeks followed by a 2-month BrdU-free period before death and processing. RESULTS: In all eyelid sections examined, there was a focus of PCNA-positive cells in the mucocutaneous junction and a few scattered PCNA-positive cells along the length of the palpebral conjunctiva toward the fornix. In both the upper and lower eyelids, the peak concentration of BrdU-labeled cells/0.4-mm zone was located at progressively greater distances from the mucocutaneous junction in the animals killed at 1, 3, and 5 days respectively and was unidentifiable by 7 days. A focus of BrdU-labeled conjunctival cells remained within 1 to 2 mm of the mucocutaneous junction at all postinjection intervals. These were always found within one cell height of the basement membrane in the basal layer of the epithelium. In the long-term studies, BrdU-labeled nuclei were retained at the mucocutaneous junction. CONCLUSIONS: The mucocutaneous junction of the conjunctival epithelium is a source of actively dividing transient amplifying cells that migrate toward the fornix at a rate of approximately 1.7 mm/d with a transit time of approximately 6 days. Long-term retention of label at the mucocutaneous junction indicates that slow-cycling stem cells are present at this location. It appears that most palpebral conjunctival epithelial stem cells are located near the mucocutaneous junction. These results are not necessarily at variance with previous studies, but they diminish the relative importance of the forniceal region in palpebral conjunctival homeostasis. The mucocutaneous junction may provide a therapeutically significant source of replacement conjunctival cells.

Direct injection of liposome-encapsulated doxorubicin optimizes chemomyectomy in rabbit eyelid.

PURPOSE: Doxorubicin chemomyectomy presently represents the only permanent, nonsurgical treatment for blepharospasm and hemifacial spasm. The major deterrent to an otherwise extremely effective treatment protocol is the development in patients of localized inflammation, discomfort, and skin injury over the injection site. As a potential alternative therapy, Doxil (Sequus, Menlo Park, CA), a liposome-encapsulated form of doxorubicin that displays tissue-selective therapeutic effects compared with free doxorubicin, was examined. These effects have been related to its increased retention in tissues and its sustained release over time. For the skin, Doxil is classified as an irritant rather than a vesicant. METHODS: Rabbits received direct injections of 1, 2, or 3 mg Doxil alone or in sequence with other agents directly into the lower eyelids. The treated eyelids were examined daily for signs of skin injury. One month after the last injection, the rabbits were euthanatized, and their eyelids were examined histologically for the effect of Doxil on the orbicularis oculi muscle and the skin. RESULTS: At equivalent milligram doses of free doxorubicin, Doxil spared the skin from injury. Doxil was only approximately 60% as effective in killing muscles as the same milligram dose of free doxorubicin. However, either two injections of Doxil spaced 2 months apart or preinjury of the lid with bupivacaine before a single dose of Doxil treatment resulted in increased muscle loss compared with a single dose of Doxil alone and was as effective as free doxorubicin. Higher doses of Doxil did not increase the desired myotoxic effect; apparently, the dose effect levels off at a maximum. Signs of skin injury were minimal; there were small or no adverse skin changes at the maximum effective myotoxic doses. CONCLUSIONS: Injection of Doxil resulted in significant reduction of skin injury compared with doxorubicin alone. Although single injections of Doxil were myotoxic, multiple exposure of the eyelid to the liposome-encapsulated form substantially improved myotoxicity while sparing the skin. Repeated doses of the liposome-encapsulated form of doxorubicin may be as clinically effective as free doxorubicin injections and may produce fewer unwanted side effects.

Doxorubicin does not spread systemically following a local injection into the eyelids of rabbits.

PURPOSE: An experimental treatment for benign essential blepharospasm and hemifacial spasm involves the direct injection of doxorubicin into the eyelids to permanently kill muscle. This study examined the extent of local and systemic spread of doxorubicin after localized injections of low doses into the eyelid and determined the length of time doxorubicin was retained in the eyelid after injection. METHODS: Two mg doxorubicin was injected subcutaneously into the lower eyelids of rabbits. After various time periods, the eyelids were removed and dissected into three separate specimens consisting of skin, subcutaneous connective tissue including orbicularis oculi muscle, or palpebral conjunctiva. Nearby tissues were also collected, including facial muscles and extraocular muscles. Urine, blood, kidney, spleen, heart and liver samples were collected. All tissues were prepared for HPLC determination of doxorubicin concentration. RESULTS: Doxorubicin was detected in all three eyelid specimens for the first 4 days after injection, although by the fourth day the level of doxorubicin was greatly reduced. On and after the seventh day, there was no detectable doxorubicin in the treated eyelid tissues. There were no detectable levels of doxorubicin in the urine or any other body tissue at any of the post-injection intervals examined. There was no long term retention in any of the eyelid tissues examined. CONCLUSIONS: The well described array of serious systemic side effects caused by the use of high systemic doses of doxorubicin as a chemotherapeutic agent made it critical to ascertain how long doxorubicin remained within the injected eyelids, and to determine to what extent and with what time course local injections of chemically intact doxorubicin might spread systemically. The short retention of the active or unmetabolized drug at the injection site is important, since more than one set of injections has been required for satisfactory amelioration of muscle spasms in blepharospasm and hemifacial spasm patients. The lack of detectable systemic spread of the drug distant from the local site of injection as well as the lack of long term retention of the locally injected doxorubicin lends support for the safety of doxorubicin administered in this manner to blepharospasm and hemifacial spasm patients.

Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles.

Because complex structural differences in adult extraocular muscles may have physiological and pathophysiological significance, the three-dimensional pattern of myosin heavy chain (MHC) isoform expression within the orbital and global layers of the muscle bellies compared with the distal tendon ends was quantitatively assessed. Three of the six extraocular muscles of adult rabbits were examined for immunohistologic expression of all fast, fast IIA/X, slow, neonatal and developmental MHC isoforms. The percentages of myofibers positive for each of these 5 myosin isoforms were determined in the orbital and global layers. There were relatively similar patterns of fast and slow MHC expression in the orbital and global layers of each of the three muscles examined. There were high levels of developmental MHC in the orbital layers, but significantly fewer developmental MHC positive myofibers in the global layer. The most variable expression was found with the neonatal MHC. There were significant differences between the longitudinal expression of the various isoforms in the middle of each muscle compared with the tendon end. In the orbital layer of all three muscles examined, the large numbers of fibers positive for fast MHC in the middle of the muscle dramatically decreased at the tendon end, with a concomitant increase in expression of slow myosin. There was a greater number of developmental MHC-positive myofibers at the tendon end than in the middle of the muscle in all three muscles examined. In the global layer, the IIA/X-positive myofibers comprised only half of the total number of fast-positive myofibers whereas in the orbital layer they comprised all or almost all of the fast positive myofibers. The configuration of the extraocular muscles is more complex than might be indicated by previous studies. The lateral rectus muscle had the most individual pattern of MHC expression when compared with the inferior rectus and inferior oblique muscles. Together with dramatic cross-sectional MHC fiber type differences between the orbital and global layers of the muscles, there are pronounced longitudinal differences in the proportions of myofibers expressing these five MHC isoforms in the middle region of the muscles and those in the distal tendon ends. This longitudinal progression appears to occur both within single myofibers, as well as within the series of myofibers that comprise the length of the muscle. We also confirm that the number of myofibers is reduced at the tendonous end while the cross-sectional area of each of the remaining myofibers is proportionally increased with regard to those in the muscle belly. Future studies may yet require two additional schemes for anatomic classification of the named extraocular muscles. One will be based on immunohistochemical features of their constituent myofibers as a supplement to classifications based on their electron microscopic appearance, innervation patterns or relative position with regard to the globe and orbit. Another will be based on the proportional length and longitudinal position of individual myofibers within an individual extraocular muscle.

Time course of the regenerative response in bupivacaine injured orbicularis oculi muscle.

Local anesthetics, particularly bupivacaine, are known to be myotoxic to skeletal muscle. Injury is followed by satellite cell mediated regeneration. The eyelid is a common site for the injection of local anesthetics. Due to the complex anatomy of this region and the unique properties of facial musculature compared to limb skeletal muscle, the response of the orbicularis oculi to local injection of bupivacaine was examined to determine the time course of maximum satellite cell activation and division. The lower eyelids of rabbits were injected with two doses of a combination of bupivacaine and hyaluronidase, spaced 18 h apart. To assess the time course of satellite cell division, bromodeoxyuridine (BrdU) was injected immediately or, 1, 2, 3, 6 or 13 days after the second bupivacaine injury. The rabbits were sacrificed 24 h later. The eyelids were prepared for immunohistological examination and morphometric analysis of the presence of CD11-positive monocytes, neutrophils and macrophages, MyoD expression in satellite cells and/or myoblasts, and co-expression of BrdU and the developmental myosin heavy chain isoform. One day after bupivacaine injury of the orbicularis oculi, there was a large influx of CD11-positive cells which gradually decreased over time. Maximum activation of satellite cells, as defined by MyoD expression, occurred 2 and 3 days after the injury. Using double labeling techniques, the peak of BrdU incorporation occurred on day 3 and was identified in developmental myosin co-labeled cells 4 days after injury. The peak of satellite cell activation and division occurred 3 days after bupivacaine induced injury, as demonstrated by both MyoD expression and after pulse labeling with BrdU as identified in double labeled cells positive for BrdU and the developmental myosin heavy chain isoform. The process of regeneration in this muscle extended beyond the duration of this study. Muscle fibers remained small in cross-sectional area and positive for developmental myosin 2 weeks after injury, at a time when the fiber number had reached control, uninjured levels.

Doxorubicin chemomyectomy as a treatment for cervical dystonia: histological assessment after direct injection into the sternocleidomastoid muscle.

The sternocleidomastoid muscle (SCM) is one of the major muscles involved in producing abnormal head position in cervical dystonia patients. This study tested whether doxorubicin chemomyectomy, direct injection of doxorubicin into the SCM to permanently remove muscle fibers, has the potential to be a nonsurgical, permanent treatment for cervical dystonia. The right SCM of rabbits was injected with either 1 or 2 mg doxorubicin. Animals were sacrificed 1-2 months postinjection. The SCM was prepared for histological examination of muscle fiber loss and fiber type composition. In all cases, direct injection of doxorubicin resulted in significant decreases in total muscle cross-sectional areas ranging from 75% up to 98%. Individual myofiber cross-sectional areas were smaller than normal after 2 mg doxorubicin treatment, but similar to normal fiber size after 1 mg doxorubicin. There were increased numbers of myofibers that expressed slow and neonatal myosin heavy chain isoforms in these remaining muscle fibers compared to the untreated SCM on the contralateral side. Developmental myosin heavy chain (MHC) was also present in 53% of the remaining myofibers of the treated muscles. The fiber type composition of muscles contralateral to the doxorubicin injections was compared to the fiber type composition of SCM from normal, untreated controls; no difference was seen in the proportions of fast, slow, and neonatal MHC fiber types in these SCM muscles. In summary, the direct injection of doxorubicin into the SCM resulted in significant muscle loss. This supports the use of doxorubicin chemomyectomy as a potential permanent, nonsurgical treatment for cervical dystonia.

Muscle fiber type compartmentalization and expression of an immature myosin isoform in the sternocleidomastoid muscle of rabbits and primates.

The sternocleidomastoid muscle is located in the neck and is both a neck rotator and flexor. Cervical dystonia, a focal dystonia disorder, is characterized by forceful involuntary contraction of a group of neck muscles, usually including the sternocleidomastoid. Little is known about the fiber type composition, fiber type compartmentalization and innervation patterns in this muscle in rabbit and primates. Sternocleidomastoid muscles from rabbit and monkey were analyzed for muscle fiber type composition and number, muscle fiber cross-sectional area and patterns of innervation. The sternocleidomastoid muscle was composed of two distinct regions, or compartments, with different fiber type compositions: an outer or superficial region composed of mostly type 2 myofibers and an inner deep region composed of both type 2 and type 1 myofibers. Neonatal myosin heavy chain isoform was detected in approximately 25% of the myofibers in both regions of the muscle. Neuromuscular junctions were located in seven endplate bands approximately 1-3 cm apart throughout the length of the muscle. There is clear evidence of anatomical subdivisions within this muscle. Not only is there variation in fiber type composition between superficial and deep regions of the muscle, but unlike most other mature skeletal muscles, it continues to express neonatal myosin heavy chain isoform in the adult. The motor program for neck movements is extremely complex, and the histological complexity plays a role in allowing for a continuum of movements of the head and neck, from maintenance of posture to rapid head movements.

Long-term efficacy of local doxorubicin chemomyectomy in patients with blepharospasm and hemifacial spasm.

OBJECTIVE: This study examines the long-term follow-up of all patients treated with doxorubicin injections in the eyelids. DESIGN: Nonrandomized clinical trial. PARTICIPANTS: Eighteen patients with blepharospasm (12 female; 6 male) and nine patients with hemifacial spasm (4 female; 5 male). INTERVENTION: Eyelids were repeatedly injected at intervals of 10 or more weeks until the spasms were ameliorated or the patient requested discontinuation. MAIN OUTCOME MEASURE: Clinical "cure" defined as sufficient symptomatic relief to defer further paralytic treatment. All patients have been followed for more than 1 year since the last injection. RESULTS: Nine of 18 patients with blepharospasm completed the full course of treatment and are considered "cured" for more than 1 year (median, 3 years; maximum, 6 years). Six of nine patients with hemifacial spasm completed treatment. Five of these six patients are considered "cures," lasting for more than 4.5 to 6 years. Two additional patients, one with blepharospasm and one with hemifacial spasm, had significant amelioration and were untreated for more than 3 years after the last doxorubicin injection, but occasionally request botulinum toxin supplementation. The minimum effective dose per treated eyelid ranged from 1.0 to 4.2 mg (median, 2.25 mg). The treatment-related discontinuations and complications were related to skin inflammation. Four of the 14 "cured" patients required some surgical "touch-up" on 1 eyelid. However, all the patients who completed treatment are either cured or have had significant amelioration of symptoms. CONCLUSIONS: Doxorubicin chemomyectomy is an evolving technique and an effective treatment for essential blepharospasm and hemifacial spasms symptomatically localized to the eyelids. Sixteen (59%) of the initial series of 27 patients completed the treatment. Of these, all are apparently cured or their symptoms significantly ameliorated. In the future, an even higher proportion would be expected to complete the treatment due to improvements in the selection criteria and treatment protocols developed during this 8-year trial. While the treatment appears to be reasonably safe compared with surgical myectomy in its present form, the authors are continuing to explore and introduce additional cotreatments to minimize the acute skin changes and maximize the long-term effectiveness of the myectomy.

Doxorubicin chemomyectomy is enhanced when performed two days following bupivacaine injections: the effect coincides with the peak of muscle satellite cell division.

PURPOSE: Doxorubicin is effective in permanently removing muscle after direct injection into the eyelid for treatment of blepharospasm and hemifacial spasm. However, patients often require two or more injection series before full abatement of their spasms is achieved. Local anesthetics cause muscle necrosis, followed by regeneration, a process that requires activation and division of muscle satellite cells. This study examined whether the muscle toxicity of doxorubicin could be amplified by injection of doxorubicin into the eyelid of rabbits 2 days after a local anesthetic injury, perhaps exploiting the toxic effects of doxoribicin on satellite cells at the peak time of their division after injury. METHODS: Rabbit eyelids received two series of injections of bupivacaine and hyaluronidase spaced 18 hours apart. Two days later, the eyelids were injected with either 0.5 or 1 mg doxorubicin. Animals were monitored daily for onset and duration of skin injury. After 1 month, the eyelids were assessed for muscle loss using histologic and morphometric techniques. RESULTS: Injection of doxorubicin during the peak of satellite cell activation and division 2 days after injury significantly increased muscle loss over doxorubicin alone. This treatment did not result in increased skin injury compared with doxorubicin alone. CONCLUSIONS: Permanent muscle loss was increased when doxorubicin was injected at the peak of satellite cell division 2 days after injury of the muscle with bupivacaine in rabbit eyelid, taking advantage of the antimitotic effects of doxorubicin on satellite cell division during the period of active regeneration. When local anesthetic injection immediately preceded the doxorubicin injection, increased myotoxicity was not seen. The injection of doxorubicin into muscle 2 days after a previous injury maximizes muscle loss. The increased muscle loss provided by this double treatment may decrease the number of injection visits required by blepharospasm and hemifacial spasm patients during their course of treatment, thus reducing the number of patients with side effects, which increases with repeated exposures of the eyelid to doxorubicin.

Local injections of corticotropin releasing factor reduce doxorubicin-induced acute inflammation in the eyelid.

PURPOSE: Doxorubicin chemomyectomy is an effective alternative treatment option for patients with blepharospasm and hemifacial spasm. One side effect of the use of doxorubicin in localized injections is the development of acute inflammation and skin injury at the injection site. Corticotropin releasing factor (CRF) was reported to reduce inflammation after acute inflammatory injuries due to other causes and at other sites. This study was performed to assess the potential of CRF to prevent the development of skin injury and eyelid soreness after local doxorubicin injection. METHODS: Rabbits received lower eyelid injections of either 75 or 150 micrograms CRF followed by injection of either 0.5, 1, or 2 mg doxorubicin or doxorubicin alone. Eyelids were assessed for changes in acute inflammation by immunohistochemical localization of macrophages and monocytes using anti-CD11, an antibody specific for these cell types. Short-term alterations in vascular permeability were assessed using an Evans blue assay. Additional eyelids were followed daily for changes in the skin over the injection site to determine day of onset of skin injury and the total duration of skin injury. After 1 month, the eyelids were processed histologically for morphometric analysis of muscle fiber loss. Monkey eyelids also were examined for the effect of CRF and doxorubicin injections. RESULTS: Doxorubicin alone produced an acute inflammatory reaction in the treated eyelids, with a large influx of macrophages and monocytes throughout the connective tissue at 1 and 2 days. Corticotropin releasing factor pretreatment significantly reduced this influx of inflammatory cells into the connective tissue. Doxorubicin produced a large increase in vascular permeability in the treated eyelids, with resultant edema. Corticotropin releasing factor did not alter this change in vascular permeability, indicating that CRF appears to have a specific effect on migration of inflammatory cells rather than just a generalized effect on vascular permeability. Corticotropin releasing factor and doxorubicin cotreatments delayed the onset of skin injury and decreased the total duration of injury to the skin compared to doxorubicin alone. The effectiveness of doxorubicin chemomyectomy was maintained; muscle loss was significant at all doses of CRF combined with doxorubicin. CONCLUSIONS: Corticotropin releasing factor dramatically decreased the acute inflammatory reaction that results in the eyelid from local doxorubicin injections. Not only did CRF reduce the acute influx of monocytes and macrophages, but it protected the skin overlying the injection site, substantially reducing the extent of skin injury. The efficacy of doxorubicin-induced muscle toxicity was maintained. A treatment protocol that combines myotoxicity with antiinflammatory activity in the treated eyelids may lead to a more effective patient treatment by increasing patient acceptance. The potential should be explored that CRF may be of clinical use in limiting tissue injury when administered immediately after extravasation during cancer chemotherapy.

Palpebral conjunctival transient amplifying cells originate at the mucocutaneous junction and their progeny migrate toward the fornix.

PURPOSE: The conjunctival epithelium performs an important role in the homeostasis and integrity of the eye. These cells need to be replaced in order to protect the integrity of the ocular surface. Epithelial cells are replaced from slow cycling stem cells which in turn produce transient amplifying cells that undergo further divisions before becoming mature conjunctival epithelial cells. The natural history of the bulbar palpebral conjunctival cells has not been previously described. METHODS: A single injection of bromodeoxyuridine (brdU), a thymidine analogue, was administered intraperitoneally to adult rabbits at a concentration of 50 mg/kg body weight. The rabbits were sacrificed at 1, 3, 5, and 7 days following the injections. The orbital contents including the eyelids were exenerated en bloc, frozen in a manner that maintained the orientation and continuity between the eyelids and globe and sectioned in a parasagittal plane. The tissue was stained immunohistochemically to detect brdU labeled conjunctival epithelial cells. The brdU-positive epithelial cells were counted in a series of 0.4 mm zones starting at the mucocutaneous junction of the eyelid and progressing through the fornix and bulbar conjunctiva. Rabbit eyelids and human eyelid surgical specimens were stained for cyclin D1, a marker for cells that are in the G1 phase of the cell cycle. RESULTS: In both the upper and lower eyelids, the peak number of brdU labeled cells/0.4 mm zone was located at progressively greater distances from the mucocutaneous junction in the animals sacrificed at 1, 3 and 5 days respectively, and gone by 7 days. A focus of brdU-labeled conjunctival cells remained within 1-2 mm of the mucocutaneous junction at all post-injection intervals. Foci of cyclin 1-positive cells were found almost exclusively near the mucocutaneous junction, but not in the fornix. CONCLUSIONS: The mucocutaneous junction of the conjunctival epithelium is a source of actively dividing transient amplifying cells that migrate toward the fornix at a rate of about 1.7 mm/day as replacement conjunctiva so that at least some conjunctival epithelial stem cells must be located near the mucocutaneous junction. The presence of cyclin D1 staining cells at the mucocutaneous junction supports this view. These results are not necessarily at variance with previous studies, but they do diminish the relative importance assigned the forniceal region in palpabral conjunctival homeostasis. Moreover, the mucocutaneous junction might provide a therapeutically significant source of replacement conjunctival cells. The transit time of conjunctival epithelial cells is about 6 days.

Doxorubicin chemomyectomy in orbicularis oculi: increasing drug infiltration at the injection site.

PURPOSE: Doxorubicin is an effective agent for chemomyectomy of the orbicularis oculi muscle when injected directly into the eyelid. However, the amount of muscle loss often differs throughout the medial to lateral extent of the eyelid. In order to increase uniform muscle loss in rabbit eyelids, doxorubicin was injected together with agents known to increase the spread of drugs at their injection site in an attempt to increase infiltration of the doxorubicin in the plane of the orbicularis oculi muscle. METHODS: Three agents were injected, singly or in combination, 20 min prior to doxorubicin administration. Drugs included: collagenase, hyaluronidase, bupivacaine, and hyaluronidase and bupivacaine together. Doxorubicin was injected at a dose of either 0.5 mg, 1 mg or 2 mg. Muscle loss from the co-treatments was compared with that seen with doxorubicin alone as assessed by light microscopic morphometric analysis of the treated eyelids one month after treatment. RESULTS: Injection of doxorubicin alone resulted in a dose-related muscle loss. Injections of either collagenase or hyaluronidase prior to doxorubicin treatment did not alter muscle loss compared with doxorubicin alone. The combination of hyaluronidase and bupivacaine slightly increased muscle loss over doxorubicin alone at the lower doses of doxorubicin. Muscle loss was increased further after doubling the dose of hyaluronidase. Collagenase pretreatment exacerbated eyelid skin injury. Hyaluronidase did not influence the skin toxicity of doxorubicin at doses of 1 or 2 mg, but did result in a delayed onset and a shorter duration of skin injury at the 0.5 mg dose of doxorubicin. CONCLUSIONS: Only hyaluronidase combined with bupivacaine increased doxorubicin induced myotoxicity in the orbicularis oculi muscle, presumably by increasing doxorubicin infiltration at the intramuscular injection site throughout the length of the muscle fibers. Thus, the combined injection of hyaluronidase and bupivacaine prior to doxorubicin treatment improves doxorubicin chemomyectomy by increasing drug infiltration throughout the full extent of the orbicularis oculi muscle. Future studies will be directed at achieving better penetration of the drug into individual muscle cells.

Orbicularis oculi muscle fibers are relatively short and heterogeneous in length.

PURPOSE: The anatomy of individual myofibers within the orbicularis oculi muscle was examined to determine individual myofiber lengths in the different regions of the muscle. A wide variety of eyelid conditions require eyelid surgery or drug injections directly into the eyelid. Knowledge of regional myofiber anatomy and physiology is important for accurate treatment of these conditions. METHODS: Eyelid specimens from rabbits were treated with collagenase, fixed, and stained for neuromuscular junction location. Individual myofibers were dissected from these muscle specimens and were measured to determine individual myofiber length and neuromuscular junction position. Additional eyelid specimens of rabbits and humans were stained en bloc to visualize neuromuscular junction location in the pretarsal and preseptal regions of the orbicularis oculi muscle. RESULTS: The myofibers showed variable lengths, shorter in the pretarsal region of the muscle and longer in the preseptal region. The average individual myofiber length in the pretarsal region was 36% as long as the entire length of the pretarsal muscle region. In the preseptal region, the myofibers were slightly longer, covering 54% of the entire length of this region of the muscle. In both the pretarsal and preseptal regions of the rabbit and human orbicularis oculi muscle, there were many clusters of neuromuscular junctions throughout the medial to lateral length of the muscle, with the majority of the neuromuscular junctions in the medial and lateral canthal regions of the preseptal portion of the lid. This indicates that the muscle is composed of relatively short, overlapping myofibers, and that the shortest myofibers reside in the medial and lateral canthal regions of the eyelid. Multiple innervation of one rabbit myofiber was observed as a rare occurrence. CONCLUSIONS: Individual myofibers of the orbicularis oculi muscle are relatively short, end intrafascicularly, and are of heterogeneous lengths varying regionally within the muscle. Thus, for drug injections into the eyelid, optimal drug effectiveness may require treatment of the entire lid from medial to lateral canthus to overcome the tissue barriers to diffusion. The existence of muscle fibers of heterogeneous lengths suggests that the complex organization of muscle fibers may play previously unappreciated but important roles in normal function, pathophysiology, and age-related changes in the eyelid.

N-CAM is expressed in mature extraocular muscles in a pattern conserved among three species.

PURPOSE: Extraocular muscles express a number of characteristics not normally seen in mature skeletal muscle. The expression of neural cell adhesion molecule, N-CAM, was assessed immunohistochemically in the extraocular muscles of rabbits, monkeys, and humans to examine the hypothesis that there is greater complexity to extraocular muscle fiber types than the traditional description. The presence of N-CAM in normal mature extraocular muscles may play a role in the etiology and pathobiology of Graves' ophthalmopathy and/or other ocular myopathies. METHODS: Extraocular muscles from rabbits, monkeys, and humans were obtained and processed for immunohistochemical localization of the more mature, less sialylated form of N-CAM in histologic sections and in individual, intact myofibers prepared by in vitro digestion techniques. N-CAM localization was compared to the immunohistochemical localization of fast, neonatal, and developmental myosin heavy chain isoforms on serially sectioned myofibers. Additional sections were examined for the presence of the early appearing polysialylated form of N-CAM (polyNCAM). RESULTS: N-CAM-positive myofibers were found in all six extraocular muscles of the three species examined. The rectus muscles showed an increased proportion of N-CAM-positive fibers compared to the oblique muscles. Individual myofibers in the rectus muscles were N-CAM positive extracellularly and intracellularly; in the oblique muscles, only extracellular staining was evident. Intact dissociated rectus muscle fibers were found that were N-CAM positive on their entire fiber surface. N-CAM was not regionally localized on these isolated fibers. Although polyNCAM was present in the extraocular muscles, only a few small diameter cells were positive for this form of N-CAM. CONCLUSIONS: Populations of mature myofibers from all six extraocular muscles express N-CAM homogeneously on their cell surfaces. The orbital layer was almost completely N-CAM positive. When the staining pattern of the global layer of the rectus and oblique muscles is compared, not only did the rectus muscles have greater numbers of N-CAM-positive fibers, all rectus muscles showed both extracellular and intracellular N-CAM expression. This was in contrast to the oblique muscles, in which the number of N-CAM-positive fibers was reduced but N-CAM was only present extracellularly. Because of the exclusive involvement of the extraocular muscles in Graves' ophthalmopathy, the absence of N-CAM expression on other mature skeletal muscles, and the increased expression of N-CAM in those muscles (rectus) more affected in this disease, the authors postulate that N-CAM expression may at least be a marker for susceptibility to, if not play a clinically significant role in, the etiology and pathobiology of this disease. Complex patterns of association were demonstrated between N-CAM expression and myosin types, indicating that N-CAM-positive myofibers do not conform to a specific fiber type as traditionally defined.

Cyclosporin protects the eyelid skin from injury after injection of doxorubicin.

PURPOSE: The myotoxic drug doxorubicin can treat blepharospasm and hemifacial spasm permanently when injected directly into the eyelid of patients. One side effect of this treatment is the dose-related occurrence of injury to the skin overlying the injection site. The purpose of this study was to determine if injection of the immunosuppressive agent cyclosporin into rabbit eyelids before doxorubicin treatment could reduce the occurrence of injury to the overlying skin and to determine the effect of cyclosporin pretreatment on doxorubicin-induced muscle fiber loss. METHODS: Anesthetized rabbits received injections of varying doses of cyclosporin 20 minutes before injection of either 0.5, 1, or 2 mg doxorubicin. The rabbits were examined daily, and epithelial changes were recorded as to duration, time of onset, and healing. When the skin was completely healed, the animals were killed and eyelid tissue was prepared for morphometric determination of muscle fiber number. Acute inflammation was quantitatively assessed using an Evans blue assay. RESULTS: At specified doses, cyclosporin improved the doxorubicin chemomyectomy protocol in three ways. It delayed the onset of skin injury at the higher doses of doxorubicin, and it markedly decreased the duration of skin injury. At some doses, cyclosporin completely prevented the formation of epithelial defects. The combination, however, did not increase muscle loss compared to doxorubicin alone; in fact, it had a slightly myoprotective effect. A dose range for cyclosporin administration was determined that resulted in a quantitative and dose-dependent reduction in inflammation at the injection site. CONCLUSIONS: The injection of cyclosporin into the eyelids before doxorubicin treatment delayed the onset, reduced the duration, and limited the extent of development of eyelid skin injury. Perhaps by limiting cytokine release, cyclosporin decreased the inflammatory reaction compared to that seen with doxorubicin alone. This combination has the potential to improve patient acceptance of doxorubicin chemomyectomy for the treatment of blepharospasm and hemifacial spasm.