Multiple Immunostaining Methods to Detect Traumatic Axonal Injury in the Rat Fluid-Percussion Brain Injury Model

Abstract

Immunohistochemistry using beta-amyloid precursor protein (APP) N-terminus antibodies is routinely used to detect traumatic axonal injury (TAI). The temporal and regional distributions of APP C- and N-terminus immunoreactivity were investigated in rats with experimental brain injury and compared to distribution of neurofilament (NF) immunoreactivity. Male Sprague-Dawley rats underwent right lateral fluid-percussion (FP) brain injury or sham injury. Six FP injury rats and two control rats were transcardially fixed with 10% formalin at 1, 6, 24, and 48 hours, and 1 and 2 weeks after injury and serial 6 microm-thick paraffin sections were prepared. At 6 hours after injury, APP C-terminus immunostaining labeled small neurons and axonal deposits in the injured parasagittal cortex, striatum, thalami, and dorsal medulla, whereas APP N-terminus and NF immunostaining showed few axonal deposits in the parasagittal cortex. At 24-48 hours post-injury, marked axonal damage, including axonal swelling and bulbs, was observed in the injured cerebral hemisphere, cerebellar white matter, and medulla. NF immunostaining was most sensitive for axonal damage in the injured deep cortical layers, cerebellum, and medulla. At 1-2 weeks after injury, APP N-terminus immunostaining mainly showed dot-like axonal profiles in the injured thalamus. APP C-terminus immunoreactivity may serve as an early marker of TAI, and the C-terminal fragments of APP may be involved in the evolution of TAI because C-terminal fragments of APP are neurotoxic and pro-apoptotic. Multiple immunostaining methods may be required to fully recognize the extent of TAI.