Determination of slow-tonic MyHC immunoreactivity is an important step in the evaluation of muscle spindles in porcine extraocular muscles

Abstract

We have tested our hypothesis suggesting (i) that for the reliable determination and counting of muscle spindles (Msp) at the light microscopy level in extraocular muscles (EOM), analysis of the spindle specific myosin heavy chain (MyHC) immunoreactivity of intrafusal fibers, especially after staining with anti-slow-tonic MyHC antibodies, is the most convenient tool, (ii) that the number of Msp determined by the slow-tonic MyHC immunoreactivity of intrafusal fibers in EOM is much lower than that based on histological examination and (iii) that the previously reported numbers of Msp based on histological examination of EOM could be overestimated. In order to determine the number and distribution of Msp and to analyze the MyHC isoform immunoreactivity of intrafusal fibers in porcine EOM, paraffin sections of three 9-month-old pig medial (MR) and lateral rectus (LR), levator palpebrae (LP) and retractor bulbi (RB) muscles were stained histologically or using specific monoclonal antibodies (mAbs) against MyHC isoforms. Msp in recti and LP muscles studied by immunocytochemistry contained nuclear bag (NB) fiber(s) reacting with mAbs against slow-tonic, slow-twitch, α-cardiac and neonatal MyHCs, but not with the mAb against fast-twitch MyHC, which, on the contrary, stained nuclear chain (NC) fibers. Based on determination of spindle specific slow-tonic MyHC isoform immunoreactivity we have found 72 Msp in the MR and 68 Msp in the LR and 12 Msp in LP muscles, which was only 62, 55 and 32% of the Msp total counts according to histological examination, respectively. In the RB muscle, we have even found only 15 spindle-like-structures composed of encapsulated thin muscle fibers, which possessed only a reaction with anti-fast-twitch MyHC mAb, but lacked slow-tonic, slow-twitch or α-cardiac MyHCs immunoreactivity. Our analysis of porcine EOM confirmed the above suggestions, demonstrating, for the first time in the pig, the presence of “false Msp” mimicking encapsulated muscle fibers on histological sections that lack spindle specific MyHC immunoreactivity. In analogy with other muscles we suggest that “false Msp” are not innervated by sensory axons and therefore do not contribute to the physiological sensation of the muscle length changes. Our results thus show that the reliable identification of functionally effective Msp in EOM must involve immunohistochemical analysis of spindle specific MyHC isoforms of intrafusal fibers, as “false” spindles appearing on histologically stained sections as encapsulated muscle fibers could be regarded as “true” Msp and thus increase the spindle number counts in earlier studies.