The visualization of a new class of traumatically injured axons through the use of a modified method of microwave antigen retrieval

Antibodies to the amyloid precursor proteins (APP) have become routine mark ers for detecting traumatically induced axonal injury (AI) in animals and m an. Unfortunately, the techniques used to visualize these proteins are not compatible with routine electron microscopic (EM) analysis. In the current communication, we describe a method for the ultrastructural visualization o f antibodies to APP and, using this method, we identify a previously unreco gnized population of traumatically injured axons. Rats were subjected to an impact acceleration traumatic brain injury and allowed to survive 30 min t o 3h postinjury. The animals were then perfused, their brains sectioned on a vibratome and the sections prepared for immunocytochemistry using a compu ter-controlled microwave capable of temperature regulation. The use of temp erature-controlled microwave energy unmasked APP antigenic epitopes without sacrificing ultrastructural detail. The APP antibody was found in two dist inct populations of reactive axons that differed in size, morphology, locat ion, and temporal progression. Comparable to previous descriptions, one pop ulation showed traumatically related reactive changes that led to swelling and disconnection. The other population, however, revealed unanticipated ch anges reflected in nodal and paranodal swelling of small continuous fibers that showed no evidence of disconnection during the time periods assessed. These studies provide new insight into the complexity of the pathobiology o f AI, while describing a novel approach for enhancing APP immunoreactivity at the EM level.