| Section one The expression of aquaporin 4 after fluid percussion brain injury in ratsObjective: To observe the regular pattern of aquaporin4 (AQP4) and its mRNA expression after fluid percussion brain injury in the rat; Discussion on relationship of AQP4 expression and brain edema after fluid percussion brain injury.Methods: 64 adult male SD rats, weighting 250g-350g, were randomly divided into 3 groups: Normal Control group(NC group) (8 rats) which were not given any treatment. Sham Operation group(SO group) (8 rats) which were given general anesthesia and craniotomy without a shock. Brain Injury group(BI group) (48 rats) which were given general anesthesia, craniotomy and a shock.At 1st hour, 6th hour, 12th hour, 24th hour, 3rd day and 7th day after being shocked in BI group, after operation in NC group and SO group, 8 rats were randomly selected in each group. After anesthesia by 10% chloral hydrate, animal brains were taken out and 3 mm thick coronal slices were harvested in the brain injury area. The examples were fixed in 4% paraformaldehyde fixative 24 hours or more. After conventionaly dehydrated and embedded into paraffin, the examples were cut into 4μm thick sections for Hematoxylin-Eosin (HE) staining and AQP4 was detected by immunohistochemistry staining. About 100mg brain tissue were taken from the front of the injury area to measure the brain water content by dry-wet weight method. The brain tissue behind the injury area were divided into two parts to detect AQP4 protein by Western Blot and AQP4mRNA expression by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) separately. Results:1 Brain water contentBI group compared with NC group, there was not significant difference at 1st hour (P>0.05). At 6th hour, brain water content ascended slightly (79.11±1.276), but rosed significantly higher to 12th hour (80.26±1.473,P<0.05), up to its peak from 24th hour to 3rd day(82.74±1.097, 82.04±1.694, P<0.05), at 7th day it becamed lower (80.72±1.866, P<0.05).2 ImmunohistochemistryThe results showed that AQP4 mainly expressed in the deep section of the cerebral cortex, leptomeninges, glia cell membrane and foot processes, nerve cell membrane, choroid plexus and ependyma and so on. In BI group, AQP4 expression began to increase at 6th hour, continued to increase at 12th hour(638.80±190.27, P<0.05), and reached its peak at 24th hour (944.20±101.84), being significantly higher than NC group (P<0.05). At 7th day, AQP4 expression decreased (538.00±118.88),but was still higher than NC group (P<0.05).3 Western BlotAQP4 protein expression in BI group began to increase at 6th hour(0.336±0.017), continued to rise at 12th hour (0.488±0.029, P<0.05), reached its peak at 24th hour (0.762±0.013, P<0.05), and reduced at 7th day (0.525±0.016), but being still higher than the NC group (P<0.05).4 RT-PCRIn BI group, AQP4mRNA expression began to increase at 6th hour (0.626±0.054), continued to increase at 12th hour (0.677±0.048, P<0.05), reached its peak from 24th hour to 3rd day (0.764±0.077, 0.758±0.057, P<0.05), and decreased at 7th day (0.723±0.057), being still higher than NC group (P<0.05).Conclusions:1 AQP4 and its mRNA expression have a tendency to increase in edemous brain after fluid percussion brain injury in the rat.2 Brain edema and AQP4 expression were positively correlated after fluid percussion brain injury in the rat.3 The correct regulation of AQP4 expression in a timely manner after brain injury may be a new idea in the treatment of cerebral edema.Section two Effects of different doses of mannitol on aquaporin 4 expression after fluid percussion brain injury in ratsObjective: Study on effects of different doses of mannitol on AQP4 and its mRNA expression after fluid percussion brain injury in rats; Discussion of the intervenient affect of different doses of mannitol on brain edema, and to provide a theoretical basis for screening the best dose of mannitol in clinic.Methods: 208 adult male SD rats, weighting 250g-350g, were randomly divided into 6 groups: Normal Control group(NC group) (8 rats) which were not given any treatment; Sham Operation group(SO group) (8 rats) which were given general anesthesia and craniotomy without a shock. Brain Injury group(BI group) (48 rats) which were gived general anesthesia, craniotomy and a shock. Animals in Mannitol group, according to different doses (0.5g/kg, 1.0g/kg, 2.0g/kg), were randomly divided into 3 sub-groups: Mannitol 1 group(M1 group), Mannitol 2 group(M2 group) and Mannitol 3 group (M3 group), each group is composed of 48 animals, which were injected mannitol into the tail vein every 6 hours after being shocked.At 1st hour, 6th hour, 12th hour, 24th hour, 3rd day and 7th day after being shocked in BI group, after operation in NC group and SO group, 8 rats were randomly selected in each group. After anesthesia by 10% chloral hydrate, animal brains were taken out and 3 mm thick coronal slices were harvested in the brain injury area. The examples were fixed in 4% paraformaldehyde fixative 24 hours or more. After conventionaly dehydrated and embedded into paraffin, the examples were cut into 4μm thick sections for Hematoxylin-Eosin (HE) staining and AQP4 was detected by immunohistochemistry staining. About 100mg brain tissue were taken from the front of the injury area to measure the brain water content by dry-wet weight method. The brain tissue behind the injury area were divided into two parts to detect AQP4 protein by Western Blot and AQP4mRNA expression by RT-PCR separately.Results:1 Brain water contentM1, M2 and M3 group compared with BI group respectively at the corresponding time point, it was slightly lower than BI group at 1st hour (P>0.05), close to or slightly higher than BI group after 6th hour (P>0.05), significantly higher at 12th hour (81.91±0.630, 82.07±0.846, 82.38±0.871, P<0.05), continued to rise at 24th hour (85.62±0.918, 85.77±1.075, 86.00±0.962, P<0.05), reached its peak at 3rd day (86.18±0.817, 86.21±1.375, 86.50±0.767, P<0.05). Until 7th day, brain water content becamed lower (84.26±1.006, 84.51±0.660, 84.67±1.125), which was still higher than BI group (P<0.05). There was no significant difference of brain water content between M1, M2 and M3 group (P>0.05).2 ImmunohistochemistryM1, M2 and M3 groups compared with BI group respectively, AQP4 protein expression had not statistically significance of at 1st hour(P>0.05). From 6th hour to 12th hour, it increased significantly (968.00±145.25, 1003.4±169.91, 1058.6±218.26, P<0.05), reached peak from 24th hour to 3rd day (1424.0±538.98, 1443.8±464.83, 1473.4±450.34, P<0.05), and decreased at 7th day (794.20±320.26, 833.60±165.34, 870.20±314.22), but being still higher than BI group (P<0.05). There was no significant difference of AQP4 protein expression between M1, M2 and M3 group (P>0.05).3 Western BlotM1, M2 and M3 group compared with the BI group respectively, AQP4 protein expression had not statistically significance of at 1st hour (P>0.05). It became slightly higher at 6th hour (P>0.05), rosed significantly higher than the BI group at 12th hour(0.573±0.026, 0.598±0.025, 0.607±0.033, P<0.05), reached its peak from 24th hour to 3rd day (0.906±0.022, 0.928±0.015, 0.930±0.016, P<0.05), and decreased at 7th day (0.641±0.049, 0.663±0.021, 0.666±0.027), which being still higher than the BI group (P<0.05). There was no significant difference of AQP4 protein expression between M1, M2 and M3 group (P>0.05).4 RT-PCRM1, M2 and M3 group compared with BI group respectively, AQP4mRNA was slightly higher than BI group at 6th hour, significantly increased at 12th hour (0.782±0.024, 0.801±0.059, 0.820±0.019, P<0.05), reached its peak form 24th hour to 3rd day (0.928±0.111, 0.932±0.130, 0.942±0.132, P<0.05), and decreased at 7th day, which being still higher than BI Group (P<0.05). There was no significant difference of AQP4mRNA between M1, M2 and M3 group (P>0.05).Conclusions:1 Mannitol can promote AQP4 and its mRNA expression, and alleviate brain edema in early stage and enhance brain edema in later stage after fluid percussion brain injury in rats.2 The effect on brain edema and AQP4 expression have not significant difference after adiministration of high, moderate, low doses of mannitol. Also because of its side effects, we suggest to apply low-dose mannitol at short-term in the early stage after brain injury. |
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