Protective Effect Of Low Expression Aquaporin-4by RNA Interference On Blood-brain Barrier In Traumatic Brain Edema Rats

Trauma brain edema is the most important secondary pathophysiologicalresponse after traumatic brain injury (TBI). The pathological response of it is theaccumulation of too much water in the intracellular space, resulting in increasingbrain volume. This has become one of the important factors that wereassociated with injury and prognosis. In clinic, the damage of traumatic brainedema cause increased intracranial pressure, constriction of brain and evenbrain hernia. This has become one of the main causes of the death and thedisability. The mechanism of traumatic brain edema is still not clear. Integratedwith variety of views, the theories of the mechanism existed as follows:(1) theblood-brain barrier damage theory;(2) cellular calcium overload theory;(3) thefree radical damage theory;(4)the brain blood rheology change and the brainmicrocirculatory disturbance theory;(5) the cell energy metabolism disordertheory;(6) traumatic inflammation reaction theory. The damage of BBB involvein the pathological basis of vasogenic cerebral edema. Meanwhile, the structuraland functional changes of BBB caused by ischemia and hypoxia may be theearliest and most important reason resulted in brain edema after TBI. However,the molecular mechanism of BBB change in the early stage of TBI is not clearso far, and there is still a lack of effective drugs to prevent the formation anddevelopment of traumatic brain edema in clinic. The clinical treatment of brainedema nowadays is mainly confined to osmotic diuresis, hypertonic saline andsurgical decompression. The drug to inhibit the formation and development of brain edema has not been reported however. The discovery of aquaporinsmakes it possible to find a kind of fewer side-effect drugs that are suitable for thetreatment of various types of brain edema.The AQPs belong to transmembrane channel protein family with highselectivity and low activated energy to transport water molecules. AQP4, themost important member of AQPs, distributes widely in the central nervoussystem. It expresses mainly in the tissues contacted directly with cerebrospinalfluid and around the blood vessels of the brain parenchyma, such as pia mater,ependymal ventricles of the catheter system and choroid plexus, especially inthe foot process of astrocytes that are connected with the pia mater and thecontact surface of capillaries.AQP4has extracellular baroreceptor so as toadjust the balance of water, which is also a main factor to mediate the transportand balance of water and electrolyte. Researchers abroad transfected culturedastrocytes in vitro by siRNA interference to reduce the express of AQP4andmRNA.With the application of plasmid, similar experiments were also done indomestic research and satisfacting results came out in the end. In the AQP4gene knockout mice, the trauma and water intoxication of brain edema reducedsignificantly. To date, the literature search shows that the study of this field hasnot been found in our province. In this study, we intend to inhibit AQP4expression in astrocytes by RNA interference, in order to determine its inhibitoryeffect to traumatic brain edema and protective function to BBB. ObjectiveInvestigate the protective effect of low expression aquaporin-4(AQP4) byRNA interference(RNAi) on blood-brain barrier(BBB) in traumatic brain edemarats.MethodsA total of264healthy adult male wistar rats were involved, weighting250-300g. The traumatic brain injury (TBI) model was established byMarmarou’s method. After TBI model establishment, we injected AQP4RNAiplasmid, blank plasmid, or plasmid solvent into the rats ventricle understereotaxic machine, respectively. And to investigate the effect of AQP4RNAion the treatment of brain edema and protection of BBB.Experiment methods were designed as follows:1Brain water content detected by dry-wet weight methodAfter the brain wet weight was measured, the brains were desiccated at100°C for24h until the weight was constant, than dry weight was measured,and brain water content was calculated as follows: brain water content=(wetweight–dried weight)/wet weight×100%.2BBB permeability detected by Evans blueEvans blue solution was injected via the caudal vein, and we detected theEvans blue content in the injury brain tissue as the indicator of BBB permeability,and then analysis the changes of BBB permeability after TBI.3AQP4mRNA expression detected by in situ hybridizationAQP4mRNA expression in the center of injjury brain tissue in eachexperimental group was measured using in situ hybridization method. AndImage-proplus6.0image analysis software was used to calculate the averagepositive intensity of AQP4mRNA. 4AQP4and zonula occludens-1protein expression detectedby immunofluorescence histochemical stainingImmunofluorescence histochemical staining was used to detect theexpression of AQP4protein in various experimental conditions, and thecorelation of the AQP4protein expression and brain edema development afterTBI were analized, to identify the effect of RNAi. We also detected theexpression of zonula occludens-1protein, analized and confirmed the tendencyof BBB permeability.5Cell morphology and ultrastructure changes observed underoptical microscopy and transmission electron microscopyParaffin section and ultrathin section was prepared, and the cellmorphology and ultrastructure changes of BBB in each group at different timepoints were observed using optical microscopy and transmission electronmicroscopy.6Statistical analysisData were statistically processed using SPSS17.0software and statisticalanalyses were performed by one-way analysis of variance. The correlation ofAQP4expression and brain water content was analized using Pearson method.P value <0.05was considered to indicate a significant difference.ResultsRNAi plasmid reduced the expression of AQP4in injury brain tissue,effectively. The BBB permeability and brain water content in RNAi group at eachtime points were decreased than TBI group and control plasmid group(P<0.05).The level of zonula occludens-1protein expression in the RNAi group wassignificantly higher than TBI group and control plasmid group at each time pointafter TBI (P <0.05). The scope and degree of astrocytes and neurons edema in RNAi group were lower than TBI group and control plasmid group, no obviousglial cells and neurons edema were seen in sham-operated group. The degreeof ultrastructure change and tight junction openment were attenuated comparedwith the TBI group and control plasmid group, no obvious change was seen insham-operated group.ConclusionsAPQ4protein expression positively correlated to brain water content afterTBI. Therefore, using the RNAi method to inhibit APQ4expression could reducethe brain water content, and have the protective effect on BBB in traumatic brainedema rats.