Temporal alterations in aquaporin and transcription factor HIF1α expression following penetrating ballistic-like brain injury (PBBI).
OBJECTIVE
Brain edema is a primary factor in the morbidity and mortality of traumatic brain injury (TBI). The various isoforms of aquaporin 4 (AQP4) and aquaporin 9 (AQP9) are important factors influencing edema following TBI. Others have reported that these AQPs are regulated by the transcription factor hypoxia inducible factor (HIF) 1α. Therefore, we examined the temporal alterations in the multiple isoforms of AQP4 and AQP9, and its possible upstream regulation by HIF1α, and evaluated whether different severities of penetrating injury influence these mechanisms.


METHODS
In the penetrating ballistic-like brain injury (PBBI) model, a temporary cavity and resultant injury was formed by the rapid inflation/deflation (i.e. <40ms) 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 24h, 3 and 7days 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 hif1α. Using separate animals, tissue lysate was collected at 4 and 24h, 3 and 7days 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 HIF1α (sham, probe only control, and PBBI, n=8-10 per group).


RESULTS
Global aqp4 mRNA was decreased at 24h (p<0.01) with PBBI. Three of the four known protein isoforms of AQP4 were detected, M1 (34kDa), M23 (32kDa) and isoform 3 (30kDa). AQP4 M1 decreased at 3 and 7days post-injury (p<0.001; p<0.01). AQP4 M23 levels were highly variable with no significant changes. AQP4 isoform 3 levels were decreased 3days post-PBBI (p<0.05). From 4, 12, and 24h aqp9 mRNA levels were decreased with injury (p<0.01, p<0.05, p<0.01) while AQP9 levels were decreased at 3 and 7days after PBBI (p<0.001, p<0.01). At 12 and 24h post-PBBI hif1α mRNA levels increased (p<0.05, p<0.01) but at 3 and 7days mRNA levels decreased (p<0.05, p<0.01). From 24h and 3 and 7days HIF1α 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 HIF1α (p<0.05).


CONCLUSIONS
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 HIF1α, 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.
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. Electronic address: casandra.m.cartagena.civ@mail.mil.
Casandra M
CM
Cartagena
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. Electronic address: katie.phillips1@us.army.mil.
Katie L
KL
Phillips
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. Electronic address: frank.c.tortella.civ@mail.mil.
Frank C
FC
Tortella
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. Electronic address: jitendra.r.dave.civ@mail.mil.
Jitendra R
JR
Dave
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. Electronic address: kara.e.schmid.mil@mail.mil.
Kara E
KE
Schmid
D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics / Q000378:metabolism*; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D001930:Brain Injuries / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008297:Male; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014948:Wounds, Gunshot / Q000378:metabolism*
C496032:Aqp4 protein, rat; C491637:Aqp9 protein, rat; D051401:Aquaporin 4; D020346:Aquaporins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020033:Protein Isoforms; D012333:RNA, Messenger
Aquaporins; Edema; PBBI; Penetrating brain injury