In the penetrating ballistic-like brain injury (PBBI) model, a temporary cavity and resultant injury was formed by the rapid inflation/deflation (i.e. < 40 ms) of an elastic balloon attached to the end of the custom probe, injuring 10% of total rat brain volume. Tissue from the ipsilateral core and perilesional injury zones was collected. Total RNA was isolated at 4, 12, and 24 h, 3 and 7 days post-injury (sham and PBBI, n = 6 per group). cDNA was synthesized using oligodT primers. Quantitative real time PCR was performed using Taqman expression assays for aqp4 (recognizing all isoforms), aqp9, and hif1伪. Using separate animals, tissue lysate was collected at 4 and 24 h, 3 and 7 days post-injury and analyzed by immunoblot for protein expression of multiple isoforms of AQP4, the single known isoform of AQP9 and for expression of transcription factor HIF1伪 (sham, probe only control, and PBBI, n = 8–10 per group).
Global aqp4 mRNA was decreased at 24 h (p < 0.01) with PBBI. Three of the four known protein isoforms of AQP4 were detected, M1 (34 kDa), M23 (32 kDa) and isoform 3 (30 kDa). AQP4 M1 decreased at 3 and 7 days post-injury (p < 0.001; p < 0.01). AQP4 M23 levels were highly variable with no significant changes. AQP4 isoform 3 levels were decreased 3 days post-PBBI (p < 0.05). From 4, 12, and 24 h aqp9 mRNA levels were decreased with injury (p < 0.01, p < 0.05, p < 0.01) while AQP9 levels were decreased at 3 and 7 days after PBBI (p < 0.001, p < 0.01). At 12 and 24 h post-PBBI hif1伪 mRNA levels increased (p < 0.05, p < 0.01) but at 3 and 7 days mRNA levels decreased (p < 0.05, p < 0.01). From 24 h and 3 and 7 days HIF1伪 protein levels were decreased (p < 0.0001, p < 0.0001, p < 0.0001). In comparison to probe control, PBBI led to greater decreases in protein for AQP4 M1 (trend), AQP4 isoform 3 (trend), AQP9 (p < 0.05) and HIF1伪 (p < 0.05).
PBBI is characterized by a loss of AQP4 M1, AQP4 isoform 3 and AQP9 at delayed time-points. The severity of the injury (PBBI versus probe control) increased these effects. Therefore, AQP9 and the AQP4 M1 isoform may be regulated by HIF1伪, but not AQP4 isoform 3. This delayed loss of aquaporins may markedly reduce the ability of the brain to efflux water, contributing to the protracted edema that is a characteristic following severe penetrating TBI. Factors contributing to edema differ with different types and severities of TBI. For example, cellular based edema is more prominent in diffuse non-penetrating TBI whereas vasogenic edema is more prevalent with TBI involving hemorrhage. Molecular regulation leading to edema will likely also differ, such that treatments which have been suggested for non-hemorrhagic moderate TBI, such as the suppression of aquaporins, may be detrimental in more severe forms of TBI.