局部亚低温对大鼠脑出血后水通道蛋白4表达的影响

目的观察局部亚低温对大鼠自体血注入法脑出血模型水通道蛋白4(AQP-4)mRNA及蛋白质表达的影响,探讨局部亚低温减轻脑出血后水肿的可能机制。方法雄性W istar大鼠240只,随机分为脑出血(ICH)组和脑出血加局部亚低温(ICH+H)组。每组分为对照、脑出血后6、24、72 h,5、7 d共6个亚组,ICH+H组于注血后给予4 h的局部亚低温治疗,各亚组分别进行血脑屏障(BBB)通透性、脑水含量的检测以及应用逆转录(RT)-PCR及W estern印迹对AQP-4进行测定。结果ICH组大鼠脑组织水含量、BBB通透性以及AQP-4 mRNA表达的增加始于脑出血后6 h,AQP-4蛋白质表达的增加始于脑出血后24 h,均至72 h达高峰(P<0.01)。AQP-4 mRNA及蛋白质表达的变化与BBB通透性的变化呈正相关(r=0.78和r=0.76)。ICH+H组的脑组织水含量和BBB通透性在各时间点与ICH组相比明显下降,而AQP-4 mRNA吸光度值(A)在脑出血后48 h与ICH组相比开始较低,在72 h,由ICH组的1.25±0.03降至1.04±0.02(P<0.01),AQP-4蛋白质A值在各时间点与ICH组相比明显下降,在72 h,由ICH组的0.77±0.08下降至0.25±0.04(P<0.01)。结论脑出血后BBB完整性的破坏可以导致AQP-4表达的上升。局部亚低温可以抑制脑出血后AQP-4 mRNA和蛋白质表达的增加。局部亚低温可能既通过减轻BBB完整性的破坏在转录水平抑制AQP-4 mRNA表达的增加,也可以在翻译水平直接抑制AQP-4蛋白质的表达,来减轻脑出血后的脑水肿形成。

Effect of local mild hypothermia on expression of aquaporin-4 following intracerebral hemorrhage in rats

OBJECTIVE: To examined the effect of local mild hypothermia on the expression of aquaporin-4 (AQP-4) following intracerebral hemorrhage (ICH) in rats and clarified the mechanism of hypothermia on brain edema formation following ICH. METHODS: Two hundreds and forty male Wistar rats were randomly divided into two groups: the intracerebral hemorrhage (ICH) group, in which autologous arterial blood were stereotaxically injected into right caudate nucleus; the local mild hypothermia (ICH + H) group, in which the rats were given 4 h local mild hypothermia after the injection of blood. Each group was divided into 6 subgroups: control, 6 h, 24 h, 72 h, 5 d and 7 d after operation; Brain water content was determined by dry-wet weight method and the permeability of BBB was measured by Evans-Blue extravasation. RT-PCR and Western blot were respectively used to evaluate AQP-4 mRNA and protein expression. RESULTS: In ICH group, compared with control, ICH significantly increased BWC, the permeability of BBB and the expression of AQP-4 mRNA, all began at 6 h and peaked at 72 h (P < 0.01), the increased protein expression of AQP-4 began at 24 h and also peaked at 72 h (P < 0.01). AQP-4 expression positively correlated, both at the mRNA and the protein level, with the permeability of BBB (r = 0.78 and r = 0.76 respectively). In ICH + H group, compared with ICH group, the elevation of BWC, BBB permeability and AQP-4 protein expression were strongly attenuated at all time point by hypothermia treatment (P < 0.01), while AQP-4 mRNA levels demonstrated a modest attenuation from 48 h. At 72 h, AQP-4 mRNA optical density (A) decreased from 1.25 +/- 0.03 (ICH group) to 1.04 +/- 0.02 (P < 0.01), AQP-4 protein expression (A) decreased from 0.77 +/- 0.08 (ICH group) to 0.25 +/- 0.04 (P < 0.01). CONCLUSIONS: This study indicates that BBB breakdown can increase the expression of AQP-4; local mild hypothermia can significantly reduce brain edema formation after ICH by suppressing the elevation of AQP-4 protein expression; Inhibition of BBB breakdown and the elevation of AQP-4 protein expression with local mild hypothermia appear to contribute to brain protection in this model.