Post-Traumatic Hypoxia Exacerbates Brain Tissue Damage: Analysis of Axonal Injury and Glial Responses

Traumatic brain injury (TBI) resulting in poor neurological outcome is predominantly associated with diffuse brain damage and secondary hypoxic insults. Post-traumatic hypoxia is known to exacerbate primary brain injury; however, the underlying pathological mechanisms require further elucidation. Using a rat model of diffuse traumatic axonal injury (TAI) followed by a post-traumatic hypoxic insult, we characterized axonal pathology, macrophage/microglia accumulation, and astrocyte responses over 14 days. Rats underwent TAI alone, TAI followed by 30 min of hypoxia (TAI+Hx), hypoxia alone, or sham-operation (n=6/group). Systemic hypoxia was induced by ventilating rats with 12% oxygen in nitrogen, resulting in a similar to 50% reduction in arterial blood oxygen saturation. Brains were assessed for axonal damage, macrophage/microglia accumulation, and astrocyte activation at 1,7, and 14 days post-treatment. Immunohistochemistry with axonal damage markers (beta-amyloid precursor protein [beta-APP] and neurofilament) showed strong positive staining in TAI+Hx rats, which was most prominent in the corpus callosum (retraction bulbs 69.8 +/- 18.67; swollen axons 14.2 +/- 5.25), and brainstem (retraction bulbs 294 +/- 118.3; swollen axons 50.3 +/- 20.45) at 1 day post-injury. Extensive microglia/macrophage accumulation detected with the CD68 antibody was maximal at 14 days post-injury in the corpus callosum (macrophages 157.5 +/- 55.48; microglia 72.71 +/- 20.75), and coincided with regions of axonal damage. Astrocytosis assessed with glial fibrillary acidic protein (GFAP) antibody was also abundant in the corpus callosum and maximal at 14 days, with a trend toward an increase in TAI+Hx animals (18.99 +/- 2.45 versus 13.56 +/- 0.81; p=0.0617). This study demonstrates for the first time that a hypoxic insult following TAI perpetuates axonal pathology and cellular inflammation, which may account for the poor neurological outcomes seen in TBI patients who experience post-traumatic hypoxia.