New method for the quantitative assessment of axonal damage in focal cerebral ischemia

Quantification of damage in both grey and white matter is required for comprehensive assessment of neuroprotective drug efficacy. Although methods for quantification of neuronal perikaryal damage after ischemia are well established, assessment of axonal damage has been limited. This article describes a new method for quantitation of axonal injury after middle cerebral artery (MCA) occlusion in rats and its application to the study of the antioxidant ebselen. The methodology is based on immunohistochemical detection of amyloid precursor protein (APP) accumulation in deformed, swollen axons in zones of ischemia. Sixty-five axon-rich sites throughout the MCA territory are assessed for the presence (scored 1) or absence (scored 0) of accumulated APP in axonal swellings. Scores for individual sites are summated in predefined neuroanatomic regions (e.g. corpus callosum), stereotaxic levels, or for a total hemisphere APP score. Both intra-rater and inter-rater reproducibility were high (r = 0.87 and 0.80, respectively). Ebselen (1 mg kg(-1) hr(-1), intravenously) significantly reduced the volume of neuronal perikaryal damage (24%, P < 0.01) and axonal damage (total APP score reduced from 27 [23.9 to 35.1, 95% CI] to 21.5 [18.2 to 23.3], P = 0.002 with ebselen treatment). In conclusion, a robust and generally applicable method is described for assessing pathologic features in myelinated fiber tracts that is sensitive for detection of drug effects on axonal damage.