Effects Of P38Pathway On The Expression Of Aquaporin4in Rats Of Fluid Percussion Brain Injury

Section one The expression tendency of p38MAPK and its relationshipwith AQP4expression of fluid percussion brain injury in ratsObjective: To observe the expression tendency of p38MAPK and itsrelationship with AQP4expression of fluid percussion brain injury in rats.Methods:56adult male SD rats, weighting250g-300g, were randomlydivided into2groups: Normal control group(NC group) and Traumatic braininjury group (TBI group). Traumatic brain injury (TBI group) were dividedinto6subgroups according to the time points of sacrifice such as1h,6h,12h,24h,3d and7d after fluid percussion. Each group included8rats. TBI groupanimal were given general anesthesia and craniotomy, and then establishedinto the models of fluid percussion brain injury. NC group were given generalanesthesia and craniotomy without fluid percussion.After animals of NC group after shock, and thoses of TBI groupwere made neurological score at corresponding points, animals of two groups were over-anesthesed with10%chloralhydrate and decapitated. Animal brains were taken out and3mm thick coronal slices were harvested in the brain injury area. The examples were stained with HE for theobservation of morphology and the expression AQP4,p38MAPK and p-p38MAPK were detected by immunohistochemistry staining. About100mg brain tissues were taken from the front of the injury area to measurethe brain water content by dry-wet weight method. The brain tissue behind the injury area was divided into two parts, one of which was usedtodetecte the expression of AQP4,p38MAPK and p-p38MAPK protein byWestern Blot, and the other of which was to detect AQP4mRNA expression by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).Results: 1Neurological score: Compared with the NC group, the neurological score ofTBI group decreased at the6th hour after shock (2.875±1.126,P <0.05),showed inflection point at the12th hour (4.375±0.916,P <0.05)after shock,which was lower than that of the NC group. The neurological score becamelower again at the24th hour after shock,(2.125±0.835,P <0.05), then gradelyinceased. Until the3rd day the score is still lower than the NC group(5.75±0.886,P <0.05).2Histological observations: Cellular edema was observed at6h after shock.Brain edema became much severe at12th hour after shock than before,andcellular edema was the most obvious visible at24th after shock, whensignificantly increased cell volume, cytoplasmic dye, and vacuolardegeneration were observed, accompanied by the proliferation of glial cells.Edema began to reduce at the3rd day and significantly decreased at the7thday, but the proliferation of glial cells gradually inceased significantly.3Brain water contentThere was not significant difference at1st hour (P>0.05)between TBIgroup and NC group. At6th hour, brain water content ascended slightly(79.54±0.715, P<0.05), and rose significantly higher to12th hour(81.89±0.581, P<0.05), then came up to its peak at24th hour (83.31±1.115,P<0.05). Brain water content began to decease at the3rd day(82.13±0.593,P<0.05), becamed lower significantly at7th day(81.24±1.010,P<0.05).4ImmunohistochemistryThe results showed that AQP4mainly expressed in the membrane ofastrocytes and ependymal cell, especially abundantly expressed in astrocytefootprocess which was directly contact with the capillary and soft brain. InTBI group, AQP4expression began to increase at6th hou(r0.424±0.021), andreached its peak at24th hour (0.909±0.052), then began to decease.p38MAPK mainly expressed in neurons and glial cells, especially on bothsides of the ventricle and choroid plexus. After fluid percussion brain injury,P38MAPK expression gradually increased, especially at3d(0.746±0.040),7d(0.917±0.040)after shock. P-p38MAPK mainly expressed in the nucleus and almost didn't express in normal brain tissue. After fluid percussion braininjury, P-p38MAPK expression increased and its trendency was similar tothat of AQP4. At6-12h (0.292±0.022,0.499±0.019)after shock, P-p38MAPKexpression gradually increased, reached its peak at24h (0.911±0.049), beganto decline at3days(0.666±0.037,P<0.05).5Western BlotCompared with that of NC group, AQP4expression in those of TBIgroup increased at6th hour(1.639±0.161), continued to rise at12th hour(2.303±0.211, P<0.05), reached its peak at24th hour (2.822±0.150, P<0.05),decreased(2.112±0.195, P<0.05) at the3rd day, significantly deceased(1.554±0.199) at the7th day,but was still higher than that of NC group(P<0.05).Compared with that of NC group, the p-p38MAPK expression in animalsof TBI group increased too. It gradually began to increase from the6th hourafter injury (1.581±0.146), reached a peak at the24th hour after injury (2.178±0.124, P <0.05), decreased(1.913±0.161, P<0.05) at the3rd day,andsignificantly deceased (1.673±0.100) at the7th day,but still higher than thatof NC group (P<0.05).Compared with NC group, p38MAPK expression gradually increasedwith the time extension. The main data were showed as followed:1.417±0.123at6h,(P <0.05),1.755±0.079(P <0.05) at12h,2.082±0.106(P <0.05)at24h,2.211±0.107(P <0.05) at3d and2.373±0.064,(P <0.05) at7d.6RT-PCRAQP4mRNA expression in those of TBI group began to increase at1sthour (0.186±0.016), expressd obviously at6th hour(0.422±0.039), continuedto increase at12th hour (0.620±0.068, P<0.05),reached its peak at the24thhour (1.060±0.140,P<0.05)and lasted to the3rd day (0.739±0.109, P<0.05). Atthe7th day AQP4mRNA expression decreased to0.429±0.088(P<0.05),butwas still higher than NC group (P<0.05). There was significantly difference (P<0.05) among subgroups of TBI group.7the correlation analysis between brain water content and AQP4and p-p38MAPKBrain water content in those of TBI group increased with the increasingexpression of AQP4and its mRNA. The water content has a positivecorrelationship with expression of AQP4protein and its mRNA (rwestern blot=0.904, rprotein=0.935; r mRNA=0.924, P <0.01).Brain water content in those of TBI group increased with the increasingexpression of p-p38MAPK. The water content has a positive correlationship withexpression of p-p38MAPK (rprotein=0.949; rwestern blot=0.969,P<0.01)。AQP4expression increased with the increasing expression ofp-p38MAPK, P-p38MAPK has a positive correlationship with the expressionof AQP4and its mRNA (rprotein=0.978; r mRNA=0.943,P<0.01)。Conclusions:1AQP4and its mRNA expression tend to increase in edemous brain after fluidpercussion brain injury in rats.2Fluid percussion injury activate the p38pathway, and the p-p38MAPKshows the up-regulating tendency in rats.3P-p38MAPK has a positive correlationship with the expression of AQP4expression of AQP4and its mRNA.4Regulation of p38pathway may be a new way to treat cerebral edema afterfluid percussion injury. Section two Effects of SB203580on aquaporin4expression in rats withfluid percussion brain injuryObjective:To observe the effects of SB203580, a specific inhibitor ofp38MAPK, on AQP4expression after fluid percussion brain injury in rats.Methods:152adult male SD rats, weighting250g-300g, were randomlydivided into4groups: Normal control group(NC group),Traumatic braininjury group(TBI group),SBI group and SBII group。The TBI group, SBI group and SBII group were divided into six subgroups according to1hours,6hours,12hours,24hours,3days and7days after shok. Each subgroup wasmade up with8rats. At thirty minutes before establishing models, a total of10μl SB203580was injected stereotaxically into the right lateralcerebroventricle with the concentration of100μmol/L and10mmol/Lrespectively in SBI group and SBII group.The animals in NC group were performed craniotomy without fluidpercussion and those of other groups were respectively accepted fluidpercussion. According to the corresponding time points after shock,neurological score were valued. All animals were over-anesthesed by10%chloralhydrate and decapitated. Animal brains were quickly removed and3mm-thick coronal slices were harvested in injury area. The examples werestained with HE for the observation of morphology. AQP4,p38MAPK andP-p38MAPK were detected by immunohistochemistry staining. About100mgbrain tissues were taken from the front of the injury area to measure the brainwater content by dry-wet weight method. The brain tissue behind the injuryarea was divided into two parts, one of which was used to detect AQP4,p38MAPK and P-p38MAPK protein by Western Blot, and the other of whichwas employed to detect AQP4mRNA expression by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).Results:1Neurological scoreCompared with that of TBI group, the neurological function scores ofSBI group and SBII group at all points increased and was statisticallysignificant between them (P <0.05). Between animals of SBI group and SBIIgroup, only at12h (4.875±0.835,5.125±0.835),24h (2.500±0.926,2.75±0.707),3d (6.125±0.996.38±0.744), the difference of neurological functionscores was statisticsly significance (P <0.05). Compared with those of NCgroup, the difference of neurological function scores in animals of TBI group,SBI group and SBII group was not statistically significant (P>0.05) at7th day(9.000±0.00,8.375±0.744,8.500±0.535,8.625±0.518). 2Histological observationCompared with TBI group, the degree of brain edema in SBI group andSBII group reduced at each time point, and the range of brain edema reducedtoo. Inflammatory cell exudation, proliferation of glial cells and the gaparound neurons in SBI group and SBII group arrived at the lighter state thanthat in TBI group. The nerve cells of the SBII group at each time point becamemilder edema than those of the SBI group.3Brain water contentCompared with the TBI group, brain water content of SBI group andSBII group were slightly lower at the1st hour after shock(P>0.05), wassignificantly lower than at the6th hour, and the difference of brain watercontent between them was statistically significant (P <0.05) but had notdifference between the SBI group and SBII group (P>0.05). At the12th hour,brain water content between three groups (81.89±0.58,80.77±0.79,79.48±0.76) was statistically different (P <0.05). At the24^th hour, the brain watercontent in three groups reached the highest (83.31±1.11,82.12±0.90,80.92±0.94), and was statistically different (P <0.05). At the3rd day, they began todecline and the same statistical differences existed among three groups (82.13±0.59,81.12±0.93,79.94±0.99, P <0.05). At the7th day, brain watercontent became significantly lower (81.24±1.01,80.35±0.58,78.94±0.67)in TBI group, SBI group and SBII group, but which of later two group wasstill slightly higher than that of group with the statistical difference (P<0.05).The difference between the SBI group and the the SBII group groupwas not statistically significant (P>0.05).4ImmunohistochemistryCompared with the TBI group, AQP4protein expression in SBI groupand the SBII group was not statistically significant (P>0.05) at the1th hour,became lower at12h (0.535±0.027,0.489±0.333,0.583±0.016),24h(0.856±0.023,0.800±0.038,0.909±0.052),3d(0.776±0.025,0.708±0.018,0.804±0.016)(P <0.05). There were statistically significance differencebetween SBI group and SBII group at12^th hour,24^th hour,3rd day (P <0.05). The expression of P-p38MAPK in each group was similar to AQP4withtrendency of increasing at the6th hour after shock, arriving at peak at24^thhour, then decreasing gradually. Compared with the TBI group, P-p38MAPKexpression in SBI group and SBII group were lower at6th hour (0.265±0.024,0.261±0.020,0.292±0.022),12th hour(0.426±0.028,0.350±0.026,0.499±0.019),24th hour (0.747±0.044,0.665±0.026,0.911±0.049),3rdday(0.580±0.042,0.483±0.039,0.666±0.037) and7th hour (0.342±0.021,0.256±0.018,0.378±0.026) and the difference between them was statisticallysignificant (P <0.05).Only at2th hour,24th hour,3rd day, the difference ofP-p38MAPK expression was statistically significant between SBI group andthe the SBII group (P <0.05).Compared with the TBI group, the p38MAPK expression in SBI groupand SBII group were lower than the TBI group at other time points except at1th hour (P <0.05). Compared with SBI group, p38MAPK expression of SBIIgroup showed lower at at other time points except at1th hour (P <0.05) withstatistical significance (P <0.05).5Western BlotCompared with TBI group, the expression of AQP4protein in the SBIgroup and SBII group decreased and the differences were idenfied to bestatistically significant at the other time points except at1st hour (P <0.05).The difference was statistically significant between the SBI group and theSBII group at12^th hour,24^th hour and at3rd day (P <0.05). The expression ofp-p38MAPK was consistent with that of AQP4.At1st hour, the expression of p38MAPK between three groups had notsignificant difference (P>0.05). At6th hour, the expression of p38MAPKbeween the TBI group and SBI group had also no significant difference (P>0.05), but the difference between TBI group and SBI Group, the SBII Groupand SBI group became statistically significant (P <0.05).At the other timepoints the differences among three groups were statistically significant (P<0.05).6RT-PCR Compared with TBI group, the expression of AQP4in the SBI group andthe SBII group was statistically significant different at1th hour (P>0.05). At7thday, the expression of AQP4in SBII group and TBI group was no differenteither (P>0.05). The differences among three groups were statisticallysignificant at other time points (P <0.05).Conclusion:1SB203580, the specifical inhibitor of p38MAPK, can knock down theexpression of AQP4mRNA in rats with fluid percussion brain injury.2There is certain amount-effect relationship in preventive effect of SB203580on p38MAPK pathway. Within certain extent, there is greater effect onp38MAPK pathway with the more amount of SB203580.