Experimental Study The Role Of Hypoxia-inducible Factor-1α, Aquaporin-4, And Matrix Metalloproteinase-9in Early Brain Injury After Subarachnoid Hemorrhage In Rats

Backgroud and Objective:Subarachnoid hemorrhage (SAH)is a common encountered cerebro-vascular diseasewith high morbidity and mortality. It is a big threaten to human health,and predoiminantlyattacks young and middle-aged group. Early brain injury (EBI) is the most common causeof disability and death in patients suffering from aneurysmal subarachnoid hemorrhage(SAH). Treatment of EBI is considered as a major goal in the management of patientssurviving SAH. However, the exact molecular mechanism of EBI still remains obscure,which has hindered the development of effective and specific treatment paradigms for EBI.The possible mechanisms about EBI may include: elevation of intracranial pressure (ICP),reduction of cerebral blood flow (CBF), suppression of cerebral perfusion pressure (CPP),fall in brain oxygenation, and neuronal cell death, among which blood-brain barrier (BBB)breakdown and brain edema played an important role in the pathogenesis of EBI.Recently, hypoxia-inducible factor-1α (HIF-1α), aquaporin-4(AQP-4), and matrixmetalloproteinase-9(MMP-9) have been demonstrated to be integral components of thepathophysiology of brain edema and BBB dysfucntion and have become viable candidatesfor potential pharmacological targets in an experimental model of brain trauma. At thetime, previous studies have researched these proteins in SAH models, for example, Tait etal.(2010) investigated the role of AQP-4in brain edema in a mouse model of SAH andthat AQP-4null mice manifested increased brain edema following SAH as a consequencereduced elimination of excess brain water. Another previous study concerning MMP-9andSAH reported that MMP-9contributed to the development of EBI after SAH by promotingcerebral edema formation. However till now, none of them focused on the relationgshipamong HIF-1α, AQP-4, and MMP-9after experimental SAH and also the underlying signaling pathway related to brain edema and BBB disruption remains unclear.The purpose of this study was:1.To observe the expression of HIF-1α, AQP-4,MMP-9in early brain injury after SAH and analyzing the association between theexpression of HIF-1α, AQP-4, MMP-9and permeability of BBB and brain edema.2. To investigate the role of HIF-1α,AQP-4and MMP-9in early brain injury after SAHin rats.3. To determine minocycline and2-methoxyestradiol (2ME2) whether haveneuroprotective effect in early brain injury after SAH in rats.Methods:1. Animals and Prechiasmatic cistern SAH Model:Ninety health maleSprague-dawley(SD) rats were assigned randomly into following groups: Control group（n=18）, SAH grou（pn=18）, SAH+Anti–AQP-4antibody group（n=18）, SAH+2ME2group（n=18），SAH+minocycline group（n=18）.The SAH model was induced by injectionof0.3ml fresh arterial, non-heparinized blood into the prechiasmatic cistern in20seconds.2. Inhibition of AQP-4, MMP-9, and HIF-1α and Brain samples:Anti–AQP-4antibody（1μg/kg）, minocycline (an inhibitor of MMP-9，1mg/kg), or2ME2(an inhibitorof HIF-1α，2.5mg/kg), was administered intravenously at2h and24h after SAH. Brainsamples were extracted at48h after SAH.3. Brain water content and Blood-brain barrier permeability:BBB permeability wasdetermined by measuring brain EB contents, and the brain edema was also examined bymeasuring brain water content.4.Western blot and immunohistochemistry:We measured the expressions of HIF-1α,AQP-4, MMP-9, laminin, zona occludens-1（ZO-1）and occludin by western blot andimmune--histochemistry.Results:1. Significant increase (P <0.05) in water content was detected in the brain samples at48h after SAH when compared with rats in control group. Rats in SAH groupdemonstrated a significant increase (P <0.01) in BBB permeability to Evans blue relativeto rats of control group. 2.Compared with control group, the SAH animals have significantly up-regula--tedexpressions of HIF-1（α P<0.01）,in addition to decreased amounts of lamini（nP<0.05）,ZO-1（P<0.01）and Occludin（P<0.05）. Brain edema was repressed after inhibition of HIF-1α （P<0.01）. BBB permeability was also ameliorated after inhibition of either HIF-1α（P<0.01）. Inhibition of HIF-1α significantly suppressed the level of AQP-4（P<0.01）and MMP-9（P<0.01）, which could induce the expression of laminin （P<0.05）,ZO-1（P<0.01） and Occludin（P<0.01）.3. Compared with control group, the SAH animals have significantly up-regulatedexpressions of AQP-4（P<0.01）. Brain edema was repressed after inhibition of AQP-4,（P<0.01）. BBB permeability was not modulated after inhibition of AQP-4.4. Compared with control group, the SAH animals have significantly up-regulatedexpressions of MMP-9（P<0.01）, in addition to decreased amounts of laminin（P<0.05）,ZO-1（P<0.01）and Occludin（P<0.05）. Brain edema was repressed afterinhibition of MMP-9（P<0.01）. BBB permeability was also ameliorated after inhibition ofeither MMP-9（P<0.01）. Inhibition of MMP-9reversed the loss of laminin（P<0.05）,significantly suppressed the level of AQP-4（P<0.01）,and suppressed the level of HIF-1α（P>0.05）.ZO-1（P>0.05） and Occludin（P>0.05）was not modulated after inhibitionof MMP-9.Conclusions:1. HIF-1α and MMP-9could aggravate EBI by promoting Brain edema and BBBpermeability after SAH in rats. But AQP-4could only aggravate EBI by promoting Brainedema, and BBB permeability was not modulated.2.Our results suggest that HIF-1α plays a role in brain edema formation and BBBdisruption via a molecular signaling pathway involving AQP-4,MMP-9, laminin,ZO-1and Occludin in EBI after SAH in rats.3.2ME2injected after SAH can inhibit expression of active and total HIF-1αprotein,which could significantly suppressed the level of AQP-4and MMP-9, and inducethe expression of laminin,ZO-1and Occludin. It may be a potential direction for brain protection after SAH by reducing Brain edema and BBB permeability.4.Minocycline could have dual neuroprotective effects whereby it can ameliorateBBB disruption and reduce vasogenic brain edema by regulating MMP-9in addition toreducing edema through decreased expression of AQP-4and HIF-1α followingexperimental SAH.