目的：对小鼠伊文思蓝静脉推注联合视网膜铺片的技术进行优化，以提高视网膜铺片染色实验的准确性和可重复性。方法：C57BL/6雄性小鼠尾静脉注射10g/L(1%)伊文思蓝0.3mL后体内分别循环10、20min，处死后摘取眼球，4%多聚甲醛分别固定20、40、60min，将不同体内循环时间及不同组织固定时间对于视网膜血管的走形、分布及渗漏的影响进行了优化比较。尾静脉注射失败后，通过腹腔注射1%伊文思蓝0.3mL，体内循环3h，固定60min进行补救，观察视网膜血管的走形、分布及渗漏。同时将尾静脉注射后视网膜血管的走形、分布及渗漏与腹腔注射后的结果进行效果比较，确定最佳的体内循环时间以及视网膜铺片的条件。结果：尾静脉注射后，与体内循环20min，固定20或40min条件下视网膜血管情况相比，伊文思蓝体内循环10min，固定60min，视网膜血管走行、血管分支清楚，血管渗漏较少。尾静脉注射失败后，腹腔注射补救措施结果显示视网膜血管走行、各级血管形态清晰。腹腔注射伊文思蓝体内循环3h与尾静脉注射伊文思蓝体内循环10min相比，视网膜血管的走形、分布的效果无明显差别。结论：优化伊文思蓝静脉推注联合视网膜铺片的技术，可提高视网膜铺片染色实验的准确性和可重复性，为相关视网膜血管病变的研究提供借鉴方法。

AIM: To optimize the technique of intravenous injection of Evans blue and retinal preparations in mice, improving the accuracy and repeatability of staining experiment of retinal preparations.METHODS: C57BL/6 male mice were intravenous injected with 10g/L(1%)Evans Blue 0.3mL and circulated in vivo for 10 or 20min, and the eyes were removed after sacrificed and fixed in 4% paraformaldehyde for 20, 40 or 60min. When failure of intravenous injection, the experiment was remediated by intraperitoneal injection of 1% Evans Blue 0.3mL, circulated in vivo for 3h and fixed for 60min to observe morphology, distribution and leakage of the retinal vessels. Besides, we compared the morphology, distribution and leakage of the retinal vessels after intravenous injection with those after intraperitoneal injection to determine the optimal conditions for in vivo circulation time and retinal preparations.RESULTS: After intravenous injection, compared to the retinal vascular condition under 20min in vivo circulation time of Evans blue and 20 or 40min of fixation, with 10min of in vivo Evans blue circulation and 60min of fixation, the morphology of retinal vascular was more intact with less retinal vascular leakage, and the vascular branches are clear. When intravenous injection failed, remediated results from intraperitoneal injection showed that the morphology and distribution of retinal vessels were intact. There was no significant difference in morphology, distribution and leakage of the retinal vessels after 3h of intraperitoneal Evans blue circulation compared to 10min intravenous Evans blue circulation.CONCLUSION: This experiment optimizes the protocol, improves the accuracy and reproducibility of retinal preparations, and provides a reference for the study of related retinal vascular diseases.

国家自然科学基金项目(No.81773795); 陕西省科技资源开放共享平台资助项目(No.2020PT-003)