Immunohistochemical study on the development of extraocular muscles. I. Rat

Development of extraocular muscles of rat was studied immunohistochemically using antibodies against three molecular markers of muscle differentiation: brain type (CK-B) and muscle type (CK-M) creatine kinase isoenzymes and muscle type enolase isoenzyme (β-enolase). The time of innervation of extraocular muscles was also studied immunohistochemically using antibody against neuron specific enolase (NSE). Periodic acid-Schiff (PAS) and PAS after diastase digestion stainings was used for the demonstration of glycogen. At embryonic day (E) 15, when muscle primordium of each extraocular muscle appears, β-enolase and glycogen were observed in all muscle primordia, while CK-B was immunoreactive only in lateral rectus (LR), superior rectus (SR), inferior rectus (IR) and inferior oblique (IO) muscle primordia. CK-B immunoreactivity appeared in superior oblique (SO) and medial rectus (MR) muscles at E17 and E18, respectively. By E18-19, CK-M immunoreactivity became positive in all muscles. NSE immunoreactive nerve fibers were first observed in LR, SR, IR and IO at E15, in SO at E16, and in MR at E17. Consequently, β-enolase immunoreactivity and glycogen appeared in all extraocular muscles at E15, while CK-M at E18. LR, SR, IR and IO muscles became immunoreactive to CK-B antibody at E15; however, SO and MR muscles became immunoreactive at E17 and E18, respectively. The sequence and time of appearance of CK-B in extraocular muscles were similar to those of NSE-immunoreactive nerve fibers. These findings suggest that in rats the expression of CK-B in each extraocular muscle coincides with the innervation of that muscle, while CK-M is expressed only after innervation.