| Objective:To explore the effect and possible complications of the 10% hypertonic saline (hypertonic saline, HS) treating ischemic cerebral edema and its corresponding elementary therapeutic mechanism compared with 20% mannitol; To further explore the effect of 10%HS on regulating the expression of AQP4 in the perivascular astrocytes and neuronal apoptosis in the peri-ischemic brain tissue,and to clarify its molecular mechanisms of treating ischemic brain edema and the effct of neurological functionMethods:Part 1 The effect of 10% hypertonic saline and 20% mannitol on cerebral edema and to elementarily explore their mechanisms1. Experimental animals and groups:Healthy adult Sprague-Dawley male rats were randomly divided into four groups:sham operation group, cerebral ischemia +normal saline group (shortname:normal saline group), cerebral ischemia +20%mannitol group (shortname:mannitol group)and cerebral ischemia +10% HS group(shortname:10%HS group). The treatment start after 6h the right of permanent middle cerebral artery occlusion (permanent middle cerebral artery occlusion, PMCAO), In addition, according to duration of treatment, the SD rats were randomly subdivided into four groups:2,6,12h and 18h group. After 6h PMCAO, the normal saline group,mannitol group and 10% HS group,normal saline,20% mannitol or 10% HS were pump via the tail vein according to 0.3ml/h uniform;while the sham-operated group was not interfered with any drugs.2. Measures:①The brain water content (brain water content, BWC) of non-ischemic hemisphere and ischemic hemisphere at different time points in each group;②Ischemic and brain sodium ions content of ischemic hemisphere;③Plasma concentration of Na+,Cl- and plasma osmolality;④The accumulation of mannitol in brain tissue at different treatment time points through autoradiograph;⑤Pathological changes in ischemic brain tissue 18h after treatment in each group by hematoxylin-eosin (HE) staining.Part 2 The study of the molecular mechanism for 10%hypertonic saline to ameliorate cerebral edema and its effect on neurological function1. Healthy adult SD male rats were randomly divided into 3 groups:sham operated group,normal saline group and 10% HS group. The rats in the 10% HS group were further subdivided into three groups according to different treatment times:0,6 and 18 h subgroups. Likewise, at 6 h following MCAO, rats in cerebral ischemic group were also subdivided into three groups correspondingly:0,6 and 18h subgroups following the onset of treatment with normal saline. Rats in sham-operated group were also subdivided correspondingly into three groups:0,6 and 18h subgroups following the onset of treatment with normal saline. At 6h following operation, rats in sham-operated group were not treated with any drugs. Rats in the normal saline group were treated with normal saline (0.3 ml/h). At 6h following PMCAO, rats in the 10% HS treatment group were treated with a continuous i.v. infusion (0.3 ml/h) of 10% HS via the tail vein until the end of the experiment.2. The BWC of the ipsilateral ischemic cerebral hemisphere was analyzed by wet-to-dry ratio at 0,6 and18h in each group,and neuron apoptosis were detected by the TUNEL technique, AQP4(aquaporin-4,AQP4)mRNA were detected by real-time fluorescence quantitative PCR, AQP4 protein were detected by immunoblotting (Western Blot) in peri-ischemic brain tissue of rats at 18h in each group.Data analysis and statistics:All data were handled by the SPSS13.0 statistical software, different statistical methods were applied according to different types of data. Normal distribution measurement data was expressed with mean±standard deviation (x±S). Univariate-factor measurement data was analyzed by one-way ANOVA, but two-factor measurement data was analyzed by two-way classification ANOVA. Multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett's T3 mothod. Bivariate correlation analysis of measurement data analyzed by Pearson correlation analysis. The difference was significant with P<0.05.Results:Part 1 The effect of 10% hypertonic saline and 20% mannitol on cerebral edema and to explore elementarily their mechanisms1. BWC:The difference of the BWC of ischemic hemisphere was significant among the sham operated group, the normal saline group, the 20% mannitol group and the 10% HS group after treatment 2,6,12 and 18h increased significantly (P 2h=0.000,P6h=0.000,P12h=0.000,P18h=0.000). At the same time, The difference of the BWC of ischemic cerebral hemisphere was significant at treatment 2,6,12 and 18h in the sham operated group,the normal saline group,the mannitol group and the 10% HS group respectively; however, so was not in the sham operated group (P =0.271). Compared with the sham operated group, the BWC of ischemic cerebral hemisphere increased in thenormal saline group,the mannitol group and the 10% HS group at the different treatment time analyzed by LSD method(P<0.05). Compared with the normal saline group, the BWC of ischemic cerebral hemisphere deceased in the mannitol group and the 10% HS group at the treatment time of 2h,6h and 12h (the 10% HS group:P2h=0.000,P6h=0.000,P12h=0.000; the mannitol group:P2h=0.018,P6h=0.025,P12h=0.030). But at the treatment time of 18h, the BWC of ischemic cerebral hemisphere had no difference between the mannitol group and the normal saline group (P=0.551), but the difference also were between the normal saline group and the 10% HS group (P=0.000). The BWC of non-ischemic cerebral hemisphere in the normal saline group continued to increase, and compared with 2h, the BWC of non-ischemic cerebral hemisphere increased both 12h and 18h (P12h=0.016,P18h=0.003).The BWC of non-ischemic cerebral hemisphere in the 10% HS group decreased at the treatment time of 6,12,and18h compared with corresponding time in the normal saline group (P6h=0.029,P12h=0.012,P18h=0.000).2. Brain tissues sodium content:Compared with sham operated rats, the brain sodium content in ischemic cerebral hemisphere increased significantly in the normal saline rats, the mannitol rats and the 10% HS rats at the treatment of 2,6,12hand 18h analyzed by LSD method (all P=0.000); it have no significant difference between the 10% HS rats and the normal saline rats, the mannitol rats (P2h=0.154,P6h=0.663, P12h=0.698, P12h=0.063; P2h=0.441,P6h=0.334,P12h=0.598,P12h=0.957 respectively).3. Plasma concentration of Na+, Cl- and plasma crystal osmolality press:Plasma concentration of Na+,Cl- and plasma crystal osmolality press increased significantly in the 10% HS group at different treatment time points in compared with other groups (Pall=0.000).4. The correlation analysis:①There was no significant postive correlation between the BWC of the ischemic cerebral hemisphere or the non-ischemic cerebral hemisphere and brain sodium content in the sham operated group,normal saline group,mannitol group and 10%HS group (Pall>0.055)②There were negative correlation between the BWC and the difference value of plasma sodium concentration and brain sodium content of the ischemic cerebral hemisphere and the non-ischemic cerebral hemisphere in 10%HS group at the treatment of 2,6,12 and 18h (Pall<0.05);but there were no significant negative correlation in the sham operated group, the normal saline group and the mannitol group (Pall>0.05)③There were significant negative correlation the BWC of ischemic cerebral hemisphere and the corresponding plasma concentration of Na+,plasma crystal osmolality press in 10% HS group at the treatment of 2,6,12 and 18h(plasma Na+: r2h=-0.811, P2h=0.05, r6h=-0.865, P6h=0.026, r12h=-0.863, P12h=0.027, r18h=-0.911, P18h=0.012; plasma crystal osmolality press:r2h=-0.81, P2h=0.49, r6h=-0.888, P6h=0.018, r12h=-0.887, P12h=0.018, r18h=-0.915, P18h=0.01)。5. (1) The amount of radioactive of 3H-mannitol in the brain tissue surrounding infarct in the ischemic cerebral hemisphere:the amount of radioactive of 3H-mannitol in the brain tissue surrounding ischemic infarct continuously increased from 2h to 18h after intervened with 3H-mannitol. It increased significantly at 18h in compared with the other intervention time (P<0.05).It indicated that mannitol continues to accumulate in the ipsilateral infarcted region. The amount of radioactive of 3H-mannitol increased in the corresponding non-ischemic cerebral hemisphere from 2h to 18h, and compared with 2h, the amount of radioactive of 3H-mannitol s did not increased at 18h (P=0.064).But compared with 6h, those increased significantly at 12h and 18h (Pall=0.000). Moreover the amount of radioactive of 3H-mannitol in the ischemic cerebral hemisphere were much higher than those in the non-ischemic cerebral hemisphere at 12h and 18h (P12h,=0.045,P18h=0.001)(2) Autoradiograph of brain tissue:a small amount of black silver particles were distributed in the ischemic brain tissue around the infarct in the ischemic cerebral hemisphere after 2h intervened with 3H-mannitol, and continued to increase from 6h to 18h. Compared with 2h,3H-mannitol increased at 12h and 18h (P12h=0.000,P18h=0.000).It suggested that mannitol continues to accumulate in the ipsilateral infarcted region.The black silver particles increased continuously in the corresponding non-ischemic cerebral hemisphere from 2h to 18h, and those increased significantly at 12h and 18h. But the black silver particles in the ischemic cerebral hemisphere were less than those in the non-ischemic cerebral hemisphere at 12h and 18h.6. Pathological features of ischemic brain tissue after 18h treatment:It was seen that cells are scarce and there were many neuronal necrosis in the infarct area, accompanied by cell lysis and softening foci formation, cell structure vague, cell deformation, coloring to enhance,the disappeared nuclear. At the same time, it could be seen that granulocytes were attached and infiltrated to blood vessels walls. Moreover,It was seen that there were cells and interstitial edema, significantly at insubcortical structures and transitional zone of infarct area to non-infarct zone in the normal saline group, the mannitol group and 10% HS group in light microscope.Part 2 The study of the molecular mechanism for 10%hypertonic saline to ameliorate cerebral edema and its effect on neurological function1. The BWC of ipsilateral ischemic hemisphere:The BWC in the ipsilateral ischemic hemispheres increased significantly in the sham operation group,the normal saline group and the 10% HS group at 0,6 and 18 h after treatment respectively (P 0h=0.000,P6h=0.000,P18h=0.000). The BWC in the ipsilateral ischemic hemispheres in the normal saline group increased significantly when compared with that in the sham-operated group at the treatment time of 0,6 and 18h(P oh=0.001,P6h=0.000,P 18h=0.000). but there was no significant difference in the BWC in the ipsilateral ischemic hemisphere at Oh between normal saline group and 10% HS group (P =0.978). Treatment of ischemic rats with 10% HS attenuated the ipsilateral ischemicevoked increase in BWC at 6 and 18 h after treatment (P=0.000).2. Apoptotic neurons in peri-ischemic brain tissue:There was no evidence of immunoreactive NeuN-labeled neurons marked by TUNEL labeling in the sham-operated rats. At 6 and 18 h after treatment, however, NeuN immunoreactive neurons showing TUNEL-labeled nuclei were increased significantly in both the normal saline group and 10% HS group (P6h=0.000,P18h-0.000). The percentage of TUNEL-positive neurons at 6 and 18 h after treatment in peri-ischemic brain tissue in the normal saline group was significantly higher than that in the the sham-operated group(P=0.000). At 6 and 18 h after treatment with 10% HS, NeuN immunoreactive neurons showing TUNEL-labeled nuclei were decreased significantly when compared with normal saline rats (P 6h=0.001,P18h=0.004)3. AQP4 expression in the peri-ischemic brain tissue:APQ4 expression detected by immunofluorescence double staining significantly increased in the peri-ischemic brain tissue in the normal saline group in compared with sham-operated group after 18h treatment, while that was significantly reduced in the 10% HS group. Compared with the sham-operated group, APQ4mRNA and protein expression also continued to increased in the normal saline group, there was a significant difference (P=0.009,P=0.000 respectively). Compared with the normal saline group, AQP4mRNA and protein expression significantly decreased in the 10% HS group, difference were significant (P=0.033,P=0.000 respectively)Conclusions: ①Compared with 20% mannitol, continuous intravenous infusion of an equal volume of 10% HS can be more lasting and effective in treatment of cerebral edema, while it has no significant complications.②It is one of major reasons for 10%HS to reduce cerebral edema more effectively than 20%mannitol that higher sodium concentration gradient was been established between the plasma and brain tissue.③It was one of the reasons that 20% mannitol failed to treat cerebral edema that continuous intravenous injection of 20% mannitol accumulate continuously in the brain tissue and more severely in the ischemic cerebral hemisphere than the non-ischemic cerebral hemisphere.④These results suggest that 10% HS exerts anti-edema effects possibly through downregulation of AQP4 expression in the perivascular astrocytes in the ischemic cerebral edema. At the same time 10% HS may play a neuroprotective role through reducing brain edema and neuronal apoptosis.
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