Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin

• Reactive protoplasmic and fibrous astrocytes show high levels of calpain-cleaved alpha 2 spectrin by double-immunohistochemistry and immunofluorescent study
• Protoplasmic astrocytes, although known not to be detected with antibodies for astrocytes commonly used for clinical specimens in pathology practice, are demonstrated by immunohistochemistry with antibody against alpha spectrin breakdown product in formalin fixed and paraffin embedded tissue.
• The study also showed evidence for transformative emergence of reactive fibrous astrocytes from protoplasmic astrocytes.

ABSTRACTCalpain, a family of calcium-dependent neutral proteases, plays important roles in neurophysiology and pathology through the proteolytic modification of cytoskeletal proteins, receptors and kinases. Alpha 2 spectrin (αII spectrin) is a major substrate for this protease family, and the presence of the αII spectrin breakdown product (αΙΙ spectrin BDP) in a cell is evidence of calpain activity triggered by enhanced intracytoplasmic Ca2 + concentrations. Astrocytes, the most dynamic CNS cells, respond to micro-environmental changes or noxious stimuli by elevating intracytoplasmic Ca2 + concentration to become activated. As one measure of whether calpains are involved with reactive glial transformation, we examined paraffin sections of the human cerebral cortex and white matter by immunohistochemistry with an antibody specific for the calpain-mediated αΙΙ spectrin BDP. We also performed conventional double immunohistochemistry as well as immunofluorescent studies utilizing antibodies against αΙΙ spectrin BDP as well as glial fibrillary acidic protein (GFAP). We found strong immunopositivity in selected protoplasmic and fibrous astrocytes, and in transitional forms that raise the possibility of some of fibrous astrocytes emerging from protoplasmic astrocytes. Immunoreactive astrocytes were numerous in brain sections from cases with severe cardiac and/or respiratory diseases in the current study as opposed to our previous study of cases without significant clinical conditions that failed to reveal such remarkable immunohistochemical alterations. Our study suggests that astrocytes become αΙΙ spectrin BDP immunopositive in various stages of activation, and that spectrin cleavage product persists even in fully reactive astrocytes. Immunohistochemistry for αΙΙ spectrin BDP thus marks reactive astrocytes, and highlights the likelihood that calpains and their proteolytic processing of spectrin participate in the morphologic and physiologic transition from resting protoplasmic astrocytes to reactive fibrous astrocytes.