| Brain edema is a common complication associated with focalischemia in stroke and increases the risk of brain herniation anddeath. Experimentally, reperfusion following focal cerebralischemia exacerbates the level of brain edema. Changes inblood-brain barrier permeability are responsible for the brain edemaassociated with reperfusion after cerebral ischemia. Astrocytes areintermediately positioned between neurons and brain vessels, bothin contact with their stellate extensions, and therefore occupy a keysignaling position between these two important players. Astrocytesare in contact with endothelial cells of capillary vessels, which formthe blood-brain barrier where important transports take place. Workin astrocyte-endothelial co-cultures has demonstrated that calciumsignals can be communicated between astrocytes and endothelialcells in a bidirectional way, making use of both paracrine ATPsignaling and gap junctions. Gap junctions are ubiquitousthroughout the central nervous system. Gap junctions are the sites ofdirect cell-to-cell communication, facilitating the exchange ofchemical and electrical signals between cells. In this experiment, weinvestigate the influence of gap junction blockade on blood-brainbarrier (BBB) permeability at different time points of reperfusionafter middle cerebral artery occlusion (MCAO) and discuss thepossible mechanism of the gap junctional influence on the change inpermeability of BBB in ischemia.In the test laser scanning confocal microscope(LSCM) wasused to investigate the change of connexin(Cx)43 levels anddistribution. The MCAO/R model was induced using intraluminalsuture technique first described by Longa with a little modification.To identify the influence of the function of astrocytic gap junctionon focal ischemia stroke, octanol, the specific blocker for gapjunctions was used in an intervention study. A total of 60 Wistar ratswere divided into 4 groups: the sham-operation group, control group,octanol-treatment group and DMSO vehicle control group. Controlgroup were divided further into senven subgroups, I2hR1h(ischemia for 2 hours and reperfusion for 1 hour),I2hR3h, I2hR6h,I2hR12h, I2hR24h, I2hR48h and I2hR72h. There were 6 rats in eachgroup. Praxiology changes and neurologic impairment wereevaluated by Longa five-grade scoring standard. We observed thechanges in permeability of BBB by measuring the amount of EB inbrain. Evans Blue(EB, 2% in saline , 4ml/kg) was injected throughfemoral vien 1 hour before killing the rats. After the animals weredecapitated, the brains were removed carefully and cut into 3mmslices. Coronal blocks were next divided into right and lefthemispheres. Samples were weighed and placed in 50%trichloroacetic acid solution. Following homogenization andcentrifugation, the extracted dye was diluted with (1:3), and itsfluorescence was determined (excitation at 620 nm and emission at680 nm) with a luminescence spectrometer. Calculations were basedon external standards in the same solvent (100～1000ng/ml). Thetissue content of EB was quantified from a linear standard curvederived from known amounts of the dye and was expressed pergram of tissue. Octanol-treatment group and DMSO vehicle controlgroup were done at the point of the peak of permeability of BBB.With the same method ,we measure the amount of EB in brain. Tocompare the amount of EB with the same point of group's, weinvestigate the influence of octanol on BBB permeability.After operation, animals of all groups except the sham-operatedone displayed obvious manifestation of neurologic impairment.Cx43 disribute extensively between cells in the brain, especiallyaround the vessels. The Cx43 expression formed into bigger plagueand remained linear disposition in the penumbra after reperfusionsubsequent to cerebral ischemia. At 3h of reperfusion after cerebralischemia for 2h, the permeability of BBB began to increase, reachedthe peak at 24h of reperfusion and was still elevated at 72h, andafterwards began to decrease. Octanol group was done at 24h ofreperfusion after cerebral ischemia. The amount of EB of octanolgroup was significantly lower than of corresponding operationcontrol group.From the study we can conclude that the permeability of BBBafter reperfusion subsequent to cerebral ischemia is graduallyincreased in 24h. Cx43 expression is concentrated around vessels inbrain. The Cx43 form into bigger plague and the function maybestrengthen after reperfusion. Gap junction might aggravate thedisruption of BBB. Octanol, the specific blocker of gap junctions,could effectively prevent the permeability of BBB from increasingand has a protective effect on BBB. |
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