Increased calpain expression in experimental demyelinating optic neuritis: an immunocytochemical study

Abstract

Since calcium activated neutral proteinase (calpain) is present in the central nervous system (CNS) and degrades myelin proteins, this endopeptidase has been suggested to play a role in myelin destruction in demyelinating diseases such as multiple sclerosis (MS). In the present study, calpain immunocytochemical expression was examined in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS and optic neuritis. To identify cells expressing calpain, we labeled rat optic nerve sections for calpain with a polyclonal myelin calpain antibody and with monoclonal antibodies for glial (GFAP, OX42) and inflammatory (CD2, ED2, ED1, IFN-γ) cell-specific markers. The results showed increased calpain expression in microglia (OX42) and infiltrating macrophages (ED1,2) in EAE compared to normal controls. Astrocytes constitutively expressed calpain in controls and acute EAE. Reactive astrocytes in EAE located in or near inflammatory foci, exhibited markedly increased calpain expression. Most T cells in acute EAE showed low level calpain expression while activated IFN-γ-producing lymphocytes in inflammatory foci exhibited elevated levels of calpain expression. Thus, our results demonstrate increased calpain expression (at transcriptional and/or translational levels) in a rat model of optic neuritis. A role for calpain in myelin destruction during optic neuritis may be relevant to the pathogenesis of this disorder.

Introduction

Optic neuritis is a demyelinating condition of the optic nerve that often appears as a presenting feature of multiple sclerosis (MS). Symptoms of optic neuritis include blurred vision, pain on eye movement, and blindness, which may develop over periods of hours to days and improve during the following months. Visual abnormalities are accompanied by axonal conduction block and increased blood brain barrier permeability which are often associated with demyelination [9]. Experimental allergic encephalomyelitis (EAE) has been used as an animal model for uveitis and optic neuritis 7, 22, 23, 24. Although the mechanism of demyelination is unknown, the demyelinating features present in the optic nerve in this disease are similar to those found in the spinal cord. Matsumoto et al. [19], showed microglial proliferation during the initial and peak stages of EAE with astrocyte activation occurring during the onset of the recovery stage. Using flow cytometry and histochemical techniques, Banati et al. [1], observed secretory proteinases including cathepsins B and L in microglia which may participate in myelin destruction if released during initial and peak stages of EAE. In addition to microgliosis, infiltrating T cells are observed in EAE lesions. Activated T cells have been shown to release proteinases including calpain and may also participate in demyelination [11].

Calpain is present in all cell types so far studied, and has been shown to degrade axonal and myelin proteins including myelin basic protein (MBP), neurofilament proteins, and myelin associated glycoprotein 3, 14. The degradation of these proteins at physiological pH, suggests calpain may play a role in demyelinating diseases (e.g., MS) and tissue destruction in spinal cord injury 4, 15. Until activated by increased calcium levels calpain remains a proenzyme, but calpain expression and secretion may be upregulated in stimulated glial and inflammatory cells such as those occurring in optic neuritis lesions. Optic nerve is composed of myelinated axons and glial cells, the latter undergoing activation and proliferation during the peak stage of optic neuritis. In order to examine the role of calpain in optic neuritis, we employed immunoperoxidase and fluorescent double-labeling techniques to explore changes in calpain expression in glial and infiltrating inflammatory cells. We found markedly increased calpain immunoreactivity in microglia, macrophages, and astrocytes in the optic nerves of rats with experimental optic neuritis compared to controls. A preliminary report of this work has been previously presented [26].