改良脑灌注固定方法在组织透明技术中的效果评估

Evaluation of improvement of cerebral perfusion fixation in the tissue optical clearing technique

组织透明技术能够在不破坏组织整体结构的前提下观察其内部的三维结构信息,目前常用的方法有多种,但在操作复杂性、荧光兼容性以及组织形变等方面各有优缺点,因此开发新型透明技术势在必行。在长期试验中我们发现组织灌注固定的充分性在组织透明中非常重要,本文以优化组织透明效果为出发点,采用改进的双侧颈总动脉灌注技术灌注固定大鼠和兔脑组织,进行HE染色和免疫荧光试验,然后对剩余部分脑组织切片进行透明处理,检测透光率。结果显示,与传统经心脏灌注技术相比,双侧颈总动脉灌注技术需要的灌注液和固定液可节省50%以上;脑组织切片透光率更高,近红外波长区域检测结果经统计分析,具有显著性差异(P<0.05);同时,与传统灌注技术相比,脑组织HE染色和免疫荧光效果也较好,红细胞数量显著减少(P<0.05),组织灌注固定更充分。

Biomedical samples can be made transparent by using tissue optical clearing techniques without destroying the overall structure. However, the currently reported transparent techniques have their own advantages and disadvantages in operational complexity, fluorescence compatibility, and tissue deformation. Therefore, it is necessary to develop a new optical clearing technique. In long-term experiments, we have found that the adequacy of tissue perfusion fixation is very important in optical clearing techniques. To improve tissue transparency, we carried out experimental studies. The improved bilateral common carotid artery perfusion technique was used to perfuse and fix the brain tissues of rats and rabbits, then part of the brain samples were obtained for HE staining and immunofluorescence, and the remaining brain tissue sections were used to detect light transmittance. The results showed that saline and fixative were saved by more than 50% in the experimental group. Their transmittance of brain tissue sections was better and the statistical analysis was significantly different in the near-infrared band(P<0.05). At the same time, compared with those of the control group, the HE staining and immunofluorescence of the experimental group were also better, the number of red blood cells was significantly reduced, the perfusion fixation in the tissues was more sufficient(P<0.05).