Friedrich Clemens, Lemm Barbara, Soukup Tomas, Asmussen Gerhard
University of Leipzig, Carl-Ludwig-Institute of Physiology, Leipzig, Germany.
Exp Eye Res. 2007 Jul;85(1):54-64. doi: 10.1016/j.exer.2007.02.017. Epub 2007 Mar 19.
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, alpha-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 alpha-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.
我们已经验证了我们的假设，该假设表明：（i）对于在光学显微镜水平可靠地确定和计数眼外肌（EOM）中的肌梭（Msp），分析梭内纤维的梭特异性肌球蛋白重链（MyHC）免疫反应性，特别是在用抗慢张力MyHC抗体染色后，是最便捷的工具；（ii）通过EOM中梭内纤维的慢张力MyHC免疫反应性确定的Msp数量远低于基于组织学检查的数量；（iii）先前报道的基于EOM组织学检查的Msp数量可能被高估。为了确定猪EOM中Msp的数量和分布，并分析梭内纤维的MyHC同工型免疫反应性，对三只9月龄猪的内侧直肌（MR）、外侧直肌（LR）、提上睑肌（LP）和眼球退缩肌（RB）的石蜡切片进行组织学染色或使用针对MyHC同工型的特异性单克隆抗体（mAb）染色。通过免疫细胞化学研究的直肌和LP肌中的Msp包含与抗慢张力、慢收缩、α-心肌和新生MyHC的mAb反应的核袋（NB）纤维，但不与抗快收缩MyHC的mAb反应，相反，后者染色核链（NC）纤维。基于对梭特异性慢张力MyHC同工型免疫反应性的测定，我们在MR肌中发现了72个Msp，在LR肌中发现了68个Msp，在LP肌中发现了12个Msp，根据组织学检查，这分别仅占Msp总数的62%、55%和32%。在RB肌中，我们甚至仅发现了15个由包囊的细肌纤维组成的梭样结构，它们仅与抗快收缩MyHC mAb有反应，但缺乏慢张力、慢收缩或α-心肌MyHCs免疫反应性。我们对猪EOM进行的分析证实了上述假设，首次在猪中证明了在组织学切片上存在模仿包囊肌纤维的“假Msp”，这些结构缺乏梭特异性MyHC免疫反应性。与其他肌肉类似，我们认为“假Msp”没有被感觉轴突支配，因此对肌肉长度变化的生理感觉没有贡献。因此，我们的结果表明，并在EOM中可靠地识别功能有效的Msp必须涉及对梭内纤维的梭特异性MyHC同工型进行免疫组织化学分析，因为在组织学染色切片上作为包囊肌纤维出现的“假”梭可能被视为“真”Msp，从而在早期研究中增加了梭数量计数。
