Analysis of Connexin 32 Antigen Expression in Experimentally Degenerative Peripheral Nerve Using A Monoclonal Antibody.

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Connexin (Cx) 32 is recognized in normal peripheral nerve, and we have already reported anti-Cx32 monoclonal antibody which was named HAM8. Recently, it was reported that Cx32 was not epxressed in human degenerative peripheral nerve from the patients suffering from such as Charcot-Marie-Tooth disease. The purpose of this project was to clarify the existence or disapperance of Cx32 in degenerative peripheral nerve using a rat model and human disease.In the beginning, we examined the expression of Cx32 in normal rat peripheral nerve. We could show the existence of Cx32 in the frozen section of rat sciatic nerve using indirect immunofluorescence method. There were HAM8 positive or negative territories in the section. However, we could not show in detail the microscopic localization of Cx32 in the nerve. Then, we exmined the localization of Cx32 in the dissociated nerve fibers from normal rat with some modifications ; changes in fixative solution, duration period for fixing and concentration and duration peiriod of Triton-X.It was not being completely succeeded to visualize the Cx32 localization in normal rat peripheral nerve until now. When nerve fibers were immersed in 3 % formalin solution for 20 min or 1 hr then they were treated with 0.5,1 or 2 % Triton-X solution for 10 min or 1 hr, the signal from Cx32 was clearer than the nerve fiber preparations treated with other conditions examined. The localization was recognized along the myelin sheath besides Rvier's nodes. Furthermore, we examined the localization using con-focal LASER microscopy, but could not determine whether Cx32 was located between each myelin sheet wrapping the axons.We have established the methods to make the degenerative nerve model in rat, such as (1) nerve from irradiated rats, (2) nerve from GVH rats and (3) nerve from Waller degeneration. They and human specimen from patients were kept in deep freezer or liquid nitrogen.