Delayed onset of brain edema and mislocalization of aquaporin-4 in dystrophin-null transgenic mice

Cerebral water accumulation was studied during induction of brain edema in dystrophin-null transgenic mice (mdx-βgeo) and control mice. Immunofluorescence and immunoelectron microscopic analyses of dystrophin-null brains revealed a dramatic reduction of AQP4 (aquaporin-4) in astroglial end-feet surrounding capillaries (blood–brain barrier) and at the glia limitans (cerebrospinal fluid–brain interface). The AQP4 protein is mislocalized, because immunoblotting showed that the total AQP4 protein abundance was unaltered. Brain edema was induced by i.p. injection of distilled water and 8-deamino-arginine vasopressin. Changes in cerebral water compartments were assessed by diffusion-weighted MRI with determination of the apparent diffusion coefficient (ADC). In dystrophin-null mice and control mice, ADC gradually decreased by 5–6% from baseline levels during the first 35 min, indicating the initial phase of intracellular water accumulation is similar in the two groups. At this point, the control mice sustained an abrupt, rapid decline in ADC to 58% ± 2.2% of the baseline at 52.5 min, and all of the animals were dead by 56 min. After a consistent delay, the dystrophin-null mice sustained a similar decline in ADC to 55% ± 3.4% at 66.5 min, when all of the mice were dead. These results demonstrate that dystrophin is necessary for polarized distribution of AQP4 protein in brain where facilitated movements of water occur across the blood–brain barrier and cerebrospinal fluid–brain interface. Moreover, these results predict that interference with the subcellular localization of AQP4 may have therapeutic potential for delaying the onset of impending brain edema.

本研究针对肌营养不良蛋白缺陷型转基因小鼠（dystrophin-null transgenic mice, mdx-βgeo）与对照小鼠在脑水肿诱导过程中的脑积水情况开展研究。对肌营养不良蛋白缺陷型小鼠脑组织的免疫荧光与免疫电镜分析显示，其毛细血管（血脑屏障，blood–brain barrier）周围及胶质界膜（脑脊液-脑界面，cerebrospinal fluid–brain interface）处的星形胶质细胞终足内，水通道蛋白4（aquaporin-4, AQP4）的表达量显著降低。免疫印迹实验结果显示总AQP4蛋白丰度未发生改变，提示AQP4蛋白存在定位异常。研究通过腹腔内（intraperitoneal, i.p.）注射蒸馏水与8-脱氨基精氨酸加压素诱导小鼠脑水肿，采用弥散加权磁共振成像（diffusion-weighted MRI）检测表观扩散系数（apparent diffusion coefficient, ADC），以此评估脑组织水腔室的动态变化。造模最初35分钟内，肌营养不良蛋白缺陷型小鼠与对照小鼠的ADC值均较基线水平下降5%~6%，提示两组小鼠的细胞内积水初始阶段进程一致。在此时间点后，对照小鼠的ADC值出现突发性快速下降，于52.5分钟时降至基线的58%±2.2%，且所有小鼠均在56分钟前死亡。肌营养不良蛋白缺陷型小鼠则经历了一段明显的延迟期，直至66.5分钟时ADC值才降至基线的55%±3.4%，随后全部小鼠死亡。上述结果表明，在水跨血脑屏障与脑脊液-脑界面进行易化转运的脑组织中，肌营养不良蛋白是维持AQP4蛋白极性分布的必要条件。此外，本研究结果提示，干预AQP4的亚细胞定位或可为延缓脑水肿发作提供潜在治疗策略。