Long-term effects of ricin-mAb 35 on extraocular muscles of rabbits: potential treatment for strabismus

Abstract

Purpose: The immunotoxin, ricin-mAb 35, composed of ricin conjugated to a monoclonal antibody against the nicotinic acetylcholine receptor of skeletal muscle, has been proposed as a potential new agent for treatment of focal muscle dystonias. It has been demonstrated that direct injection of ricin-mAb 35 into rabbit extraocular muscle (EOM) results in significant muscle loss within 1 week. In this study, the long-term myopathic effects of ricin-mAb 35 on extraocular muscle were investigated.

Methods: Rabbit superior rectus muscles were injected with ricin-mAb 35 at a dose of 0.2 microg/kg, with the contralateral superior rectus muscle serving as the control. After 56 days, 105 days, and 1 year, the superior rectus muscles were removed and prepared for light or electron microscopy. Postinjection changes in muscle fiber morphometry and ultrastructure were examined. Immunohistochemical markers were used to identify inflammatory cellular infiltrate and myosin heavy chain (MHC) isoform expression.

Results: Despite evidence of ongoing regeneration, treated muscles continued to show a decrease in both myofiber number and in total cross-sectional area 56 and 105 days after injection. Individual myofiber cross-sectional areas were markedly heterogeneous at 56 days. Myofiber number and muscle cross-sectional area returned to normal 1 year after injection, but pronounced heterogeneity of myofiber size remained. The most significant changes in myosin heavy chain (MHC) isoform expression occurred in the orbital layer, where, at 56 and 105 days, there were increased numbers of fast and neonatal myofibers and decreased numbers of slow myofibers. In the global layer, after both 105 days and 1 year, there was a decrease in myofibers that were positive for slow, neonatal, and developmental MHC expression.

Conclusions: EOM injection with ricin-mAb 35 results in a sustained decrease in muscle mass at 105 days after injection, with subtler morphometric changes persisting even to 1 year. Changes in muscle force development as a result of ricin-mAb 35 injection are currently under investigation. This novel immunotoxin may be useful in the treatment of strabismus if these studies show sustained weakness in treated muscles.