| Background and ObjectiveSubarachnoid hemorrhage(SAH) is characteristic of high mortality and recognized as one of the persistent global health problems. Although SAH can be induced by various factors, the induction of SAH by brain aneurysm rupture accounted for 75%~85% of total. The outbreak of SAH may cause drowsiness, confusion, focal neurological deficits, paralysis and even coma. These secondary neurologic injuries seriously threaten health and quality of life of patients. Study of the pathological mechanism for complications of SAH would be benefit for the prognosis and disease treatment for SAH patients. Previous studies showed that the reason for neurological deficits induced by SAH is the occurrence of brain pathological mechanisms, such as hydrocephalus, endothelial cells and neuronal apoptosis, brain edema, blood-brain barrier lose abnormal brain regulatory mechanisms, thrombosis and so on. Among these, brain edema and hydrocephalus have prolonged effects on neurological damage, during which, the role of aquaporins(AQPs) is of great importance. AQP1 and AQP4 are the two dominant AQPs in central nervous system. Studies have shown that AQP1 and AQP4 are relevant to brain tumors, cerebral edema, brain trauma, stroke and AQP4 knockout-caused hydrocephalus. Although, previous studies showed the feedback and compensatory mechanisms of AQPs in the formation of hydrocephalus, the most studies were basedon AQP4 gene knockdown rat, congenital hydrocephalus or the model of hydrocephalus. To data, dynamic changes of AQP1 and AQP4 responding to SAH in regular mice(non-gene knockdown of AQP1 or AQP4) and the correlations between dynamic change of AQP1 or AQP4 and cerebral edema or hydrocephalus have not been reported.In addition, under a variety of physiological or pathophysiological conditions, protein levels of AQP1 and AQP4 can be regulated. Although evidence indicates that multiple mechanisms are involved in regulation of AQP1 and AQP4 expression, but the specific mechanism is not fully elucidated. Extracellular signal-regulated kinase(ERK) comprises ERK1 and ERK2 and belongs to one of the mitogen-activated protein kinase(MAPK) signaling pathway. Its phosphorylation activation state plays an important role in the regulation of brain injury, astrocytes expansion and blood brain barrier dysfunction. Moreover, in other nerve cells, there is a certain relationship between ERK1/2signaling pathway activation and expression of AQP1 and AQP4. However, it is not clear whether the regulatory role of in AQP1 or AQP4 by ERK1/2 is involved in the cerebral edema or hydrocephalus induced by SAH.Therefore, the present study aims to explore the dynamic expression of AQP1 and AQP4 during cerebral edema and hydrocephalus in model of subarachnoid hemorrhage of regular mice. Further, we also detected the regulatory role of ERK1/2 in expression of AQP1 and AQP4 during SAH complicated cerebral edema.Part? Role of AQP4 and AQP1 in acute phase of cerebral edema in mice with subarachnoid hemorrhageMaterials and Methods 1. The male adult Kunming mice(28-32 g weight) were randomly divided into 3groups: normal control group, sham group and SAH group. The SAH group was subdivided into 8h post-SAH, 16 h post-SAH, 24 h post-SAH and 3d post-SAH group. The mouse model of SAH was induced by injecting autologous arterial blood into cisterna magna. The mice in normal control group experienced the same operation but with no treatment. The mice in sham group experienced the same operation and were infused with saline solution. 2. The neurobehavioral function was evaluated in eight mice of each group, then brain water content was examined by the wet/dry method. Evans blue dye extravasation was used to measure the permeability of blood brain barrierin eight mice of each group. Real time-q PCR and Western bot were performed to detect the expression of AQP4 in cerebral cortex and periventricular area, as well as the expression of AQP1 in choroid plexus and periventricular area in eight mice of each group. 3. Statistical analysis: SPSS 13.0 was used for statistical analysis of the data. Data were expressed as mean ± SD. All data were subjected to one-way ANOVA. Differences between experimental groups were determined using q test. Statistical significance was inferred at P < 0.05.Results 1. The SAH mice at different post-SAH time points(8h, 16 h, 24 h and 3d) had reduced neurobehavioral function compared with Sham group. The neurobehavioral function in SAH mice was worst at 8h post-SAH, and was gradually restored with the increase of postoperative time. The content of water in brain and the brain barrier permeability in SAH mice at different post-SAH time points(8h, 16 h, 24 h and 3d) were both significantly elevated than that in Sham group(P<0.05), and the highest values were observed at 24 h post-SAH. The content of water in brain and the brain barrier permeability in SAH mice at 3d post-SAHwere decreased compared with mice at 24hpost-SAH. 2. Real time PCR and western blot results showed that cortical and periventricular AQP4 m RNA and protein expression levels in SAH mice at differentpost-SAH time points(8h, 16 h, 24 h and 3d) were upregulated compared with that in Sham group. The AQP4 expression level was gradually elevated with the increasing post-SAH time, and the highest expression of AQP4 was observed at 24 h post-SAH. Cortical and periventricular AQP4 expression in m RNA and protein levels was down-regulated in SAH mice at 3d post-SAHcompared with the mice at 24 h post-SAH. 3. Real time PCR data demonstrated that choroid plexus AQP1 m RNA expression in SAH mice at different post-SAH time points(8h, 16 h, 24 h and 3d) was downregulated compared with that in Sham group. The AQP1 m RNA expression level was gradually decreased with the increasing post-SAH time, and the lowest level was observed at 24 h post-SAH. Choroid plexus AQP1 m RNA expression level was upregulated in SAH mice at 3d post-SAHcompared with the mice at 24 h post-SAH. Western blot results showed that choroid plexus AQP1 expression in protein level was slightly decreased at 8h, 16 h and 24hpost-SAH. The periventricular AQP1 m RNA and protein expression levels were upregulated in SAH mice at different post-SAH time points(8h, 16 h, 24 h and 3d) compared with that in Sham group, and its expression was gradually increased with the increasing post-SAH time with the highest level at 24 h post-SAH. Periventricular AQP1 expression in m RNA and protein levels was downregulated in SAH mice at 3d post-SAHcompared with the mice at 24 h post-SAH.Part ? Role of AQP4 and AQP1 in chronic phase of hydrocephalus in mice with subarachnoid hemorrhageMaterials and Methods 1. The SAH model was divided into non-hydrocephalus group andhydrocephalus group. The mice in the two groups were subdivided into 1w post-SAH, 2w post-SAH and 4w post-SAH group. The mouse model of SAH was induced by injecting autologous arterial blood into cisterna magna. 2. The neurobehavioral function was evaluated in eight mice of each group, then brain water content was examined by the wet/dry method. Evans blue dye extravasation was used to measure the permeability of blood brain barrier in eight mice of each group. Real time-q PCR and Western bot were performed to detect the expression of AQP4 in cerebral cortex and periventricular area, as well as the expression of AQP1 in choroid plexus and periventricular area in eight mice of each group. 3. Statistical analysis: SPSS 13.0 was used for statistical analysis of the data. Data were expressed as mean ± SD. All data were subjected to one-way ANOVA. Differences between experimental groups were determined using q test. Statistical significance was inferred at P < 0.05.Results 1. The neurobehavioral functions of SAH mice with or without hydrocephalus had no statistical differences among the groups at different post-SAH time points(1w, 2w and 4w)(P>0.05). With the increase of post-SAH time, the values of water content in brain and brain barrier permeability were not altered in SAH mice without hydrocephalus while these values were gradually increased in SAH mice with hydrocephalus. Compared with non-hydrocephalus group, the hydrocephalus group had reduced neurobehavioral function(P<0.05) and elevated water content in brain(P<0.05). The values of brain barrier permeability had no statistical difference between these two groups at 1w post-SAH(P>0.05), while the values were significantly increased in hydrocephalus group compared with non-hydrocephalus group(P<0.05). 2. Real-time PCR data showed that periventricular and cortical AQP4 m RNA expression levels in non-hydrocephalus group had no statistical differences among the groups at different post-SAH time points(1w, 2w and 4w), while its expression inhydrocephalus group was gradually upregulated with the increase of post-SAH time. Compared with non-hydrocephalus group, the hydrocephalus group had similar level of periventricular and cortical AQP4 m RNA expression at 1w post-SAH(P>0.05) and increased levels at 2w and 4w post-SAH(P<0.05). Western blot demonstrated that there was no difference in periventricular AQP4 protein expression between non-hydrocephalus group and hydrocephalus group at 1w and 2w post-SAH, while cortical AQP4 protein was slightly decreased in hydrocephalus group. At 4w post-SAH, both periventricular and cortical AQP4 protein expression levels were upregulated in hydrocephalus group compared with non-hydrocephalus group(P<0.05). 3. Real-time PCR data showed that choroid plexus and periventricular AQP1 m RNA expression levels in non-hydrocephalus group had no statistical differences among the groups at different post-SAH time points(1w, 2w and 4w)(P>0.05), while its expression in hydrocephalus group was gradually upregulated with the increase of post-SAH time. Compared with non-hydrocephalus group, the hydrocephalus group had lower choroid plexus and periventricular AQP1 m RNA levels at 1w and 2w post-SAH and higher levels at 4w post-SAH(P<0.05). Western blot demonstrated that choroid plexus and periventricular AQP1 protein expression was decreased in hydrocephalus group compared with non-hydrocephalus group at 1w post-SAH. Choroid plexus and periventricular AQP1 protein expression had no statistical difference between these two groups at 2w post-SAH. Choroid plexus AQP1 protein expression was in similar level in these two groups at 4w post-SAH while periventricular AQP1 protein expression was higher in hydrocephalus group.Part ? Role of AQP4 and AQP1 in different degree of hydrocephalus in mice with subarachnoid hemorrhageMaterials and Methods 1. According to the severity of hydrocephalus in mice with 4w post-SAH, the mice were divided into mild group, moderate group and severe group. The mouse model of SAH was induced by injecting autologous arterial blood into cisterna magna. The mice with 4w post-SAH and without hydrocephalus were as the control. 2. Severity of SAH was graded from mild(0.20-0.40), moderate(0.41-0.60) to severe(>0.60) based on magnetic resonance imaging(MRI). The neurobehavioral function was evaluated in eight mice of each group, then brain water content was examined by the wet/dry method. Evans blue dye extravasation was used to measure the permeability of blood brain barrier in eight mice of each group. Real time-q PCR and Western bot were performed to detect the expression of AQP4 in cerebral cortex and periventricular area, as well as the expression of AQP1 in choroid plexus and periventricular area in eight mice of each group. 3. Statistical analysis: SPSS 13.0 was used for statistical analysis of the data. Data were expressed as mean ± SD. All data were subjected to one-way ANOVA. Differences between experimental groups were determined using q test. Statistical significance was inferred at P < 0.05.Results 1. The SAH mice with hydrocephalus were grouped into mild, moderate and severe groups based on the value of ventricle to brain diameter. Compared with control group, SAH mice with different degrees of hydrocephalus showed decreasedneurobehavioral function(P<0.05) and increased values of water content in brain and brain barrier permeability(P<0.05). With the increasing severity of hydrocephalus, the neurobehavioral function was gradually reduced and the values of water content in brain and brain barrier permeability were gradually elevated. 2. Real-time PCR and western blot results showed that periventricular and cortical AQP4 expression in m RNA and protein levels were upregulated in SAH mice with different degrees of hydrocephalus compared with control group. The AQP4 expression was gradually elevated with the increasing severity of hydrocephalus. 3. Real-time PCR and western blot results showed that choroid plexus and periventricular AQP1 expression in m RNA and protein levels were upregulated in SAH mice with different degrees of hydrocephalus compared with control group. The AQP1 expression was gradually elevated with the increasing severity of hydrocephalus.Part ? The potential mechanism by AQP4 and AQP1 in acute phase of subarachnoid hemorrhageMaterials and Methods 1.The mice were divided into sham group, SAH plus U0126 group and SAH plus vehicle group. In SAH plus U0126 group, U0126 was given as a final dose of 0.20 ?g U0126/kg body weight in isotonic saline with 0.1% DMSO and was administered intracisternally at 8h post-SAH, 16 h post-SAH and 24 h post-SAH. The mice were killed at 72 h post-SAH for following measurements. In SAH plus vehicle group, the mice were treated as the same with that in the SAH plus U0126 group but was given vehicle, which was 0.1% DMSO in isotonic saline. 2.The neurobehavioral function was evaluated in eight mice of each group, thenbrain water content was examined by the wet/dry method. Evans blue dye extravasation was detected to measure the permeability of blood brain barrier in eight mice of each group. Real time-q PCR and Western bot were performed to detect the expression of ERK?AQP1?AQP4 in periventricular area. 3. Statistical analysis: SPSS 13.0 was used for statistical analysis of the data. Data were expressed as mean ± SD. All data were subjected to one-way ANOVA. Differences between experimental groups were determined using q test. Statistical significance was inferred at P < 0.05.Results 1. Western blot data showed that periventricular p ERK protein expression was upregulated in SAH mice at different post-SAH time points(8h, 16 h, 24 h and 3d) compared with Sham group(P<0.05). The protein expression of p ERK was gradually elevated with the increase of post-SAH time, and the highest level was observed at 24 h post-SAH. Its expression in SAH mice at 3d post-SAHwas decreased compared with mice at 24 h post-SAH. Peason correlation analysis showed that periventricular p ERK protein expression was positively correlated with AQP1 and AQP4 protein expression, respectively(r=0.384, P<0.05; r=0.749, P<0.05). 2. Western blot results showed that periventricular protein expression levels of p ERK, AQP1 and AQP4 were up-regulated in SAH mice compared with Sham group(P<0.05). Their levels were decreased with the injection of ERK inhibitor U0126 at 8h, 16 h, 24 h time points in SAH mice(P<0.05). 3. SAH mice had the significant decrease of neurobehavioral function and the significant increase of content of water in brain and brain barrier permeability compared with Sham group(P<0.05). After the injection of ERK inhibitor U0126 at 8h, 16 h, 24 h time points, the neurobehavioral function was significantly increased and content of water in brain and brain barrier permeability were significantly decreased(P<0.05) than that in SAH mice without the treatment of ERK inhibitor.Conclusion 1. Double cisterna magna injection method was used to establish SAH mouse model.Mice at acute stage(8h, 16 h, 24 h and 3dpost-SAH) had different degrees of brain damage compared with Sham group as the most serious damage was observed at 24hpost-SAH. SAH mice at chronic stage(1w, 2w and 4w post-SAH) with hydrocephalus had more seriousbrain damage compared with SAH mice without hydrocephalus, and the most serious damage was appeared at 4w post-SAH. 2. In SAH miceat acute stage, the highest expression levels of periventricular and cortical AQP4 were observed at 24hpost-SAH. The highest and lowest levels of choroid plexus and periventricular AQP1 expression were also observed at 24hpost-SAH, respectively. In SAH mice at chronic stage, periventricular and cortical AQP4 expression in hydrocephalus group was upregulated starting from 2w post-SAH, and choroid plexus and periventricular AQP1 expression was upregulated starting from 4w post-SAHcompared with non-hydrocephalus group. In addition, it also demonstrated that AQP4 and AQP1 expression levels were elevated with the increasing severity of hydrocephalus. 3. Periventricular p ERK protein expression was positively related to the AQP1 and AQP4 protein expression in SAH mice at acute stage, respectively. ERK inhibitor U0126 significantly downregulated periventricular AQP1 and AQP4 expression. |
PDF Full Text Request