视网膜多巴胺D2R在明亮光照抑制近视中的作用机制研究

Myopia prevalence is increasing and in severe cases it markedly reduces the quality of life and even leads to blindness. In order to identify novel therapeutic measures to inhibit this process, the underlying pathogenic mechanisms of this disease require additional clarification. Exposure to outdoor light intensity is one of the one known factors that delays the onset of myopia. This effect is associated with modulation of retinal dopamine levels. Our previous studies showed that activation of either dopamine D1 receptor (D1R) or D2 receptor (D2R) receptor subtypes has opposite effects on refractive error development and myopia in mice. Activation of D1R on ON-bipolar cells and horizontal cells contributes to suppressing myopia inhibition induced by bright light. As D2R is also expressed in the retina, we hypothesize that D2R activation by bright light also contributes to suppressing myopia. Accordingly, we will delineate the role and underlying mechanism of D2R involvement in controlling this process. This project addresses this question with three linked specific aims by determining: 1) the effects of selective D2R agonist and antagonist pairs on light-induced suppression of myopia; 2) the distribution of D2R expression in specific retinal neuronal cell types; 3) if D2R activation on specific retinal neuronal cell types occurs during form deprivation-induced myopia and bright light suppression of this effect. By identifying the role and effects of selective activation of D2R on specific retinal neuronal cell types during form deprivation myopia and bright light-induced inhibition of this process, we expect to provide a needed foundation for analyzing the role of a dynamic balance between D1R/D2R activation in myopia development. The results from this endeavor will clarify which types of visual signaling modulation affect refractive development.

近视是重大的社会卫生问题，明确其发病机制极具现实意义。户外活动抑制近视，而明亮光照是其发挥作用的关键因素。多巴胺参与调控屈光发育，其合成释放受光照调控，提示其可能介导明亮光照的近视抑制作用。申请人前期发现视网膜多巴胺D1受体（D1R）以及D2受体（D2R）均参与屈光发育调控，但作用相反；明亮光照促进视网膜多巴胺的合成并激活D1R是其抑制近视的作用机制之一。由此提出假设：视网膜D2R参与调控明亮光照对近视的抑制作用。本研究拟通过：1）利用D2R激动剂和拮抗剂明确D2R是否参与该过程；2）明确D2R在不同亚型视网膜神经元上的分布；3）明确在明亮光照以及近视过程中不同亚型神经元D2R的激活，进而明确D2R参与该过程的视网膜机制。最后结合D1R和D2R的作用机制，为明确两者间动态平衡调控屈光发育的机制提供基础，也有助于明确参与近视形成的具体视网膜信号通路，从而进一步阐明近视以及光照抑制近视的机制。

为了明确明亮光照是否通过调控D2R抑制近视的发生发展，以及D2类受体通过视网膜哪些类型神经元功能发挥屈光调控作用。本研究首先通过腹腔注射D2类受体激动剂奎比罗，明确D2类受体激动对屈光发育以及对明亮光照对近视的抑制作用的影响。3周大小鼠被随机分为：普通光照组（NL，100-200lux），明亮光照组（BL，2500-5000lux），普通光照+形觉剥夺（NL+FD），明亮光照+形觉剥夺（BL+FD），普通光照+奎比罗（NL+Q），明亮光照+奎比罗（BL+Q）， 普通光照+形觉剥夺+奎比罗（NL+FD+Q），明亮光照+形觉剥夺+奎比罗（BL+FD+Q）。光照、FD和腹腔奎比罗注射均持续四周，奎比罗腹腔注射每天一次，早上9点左右进行注射。实验前后进行屈光度和眼轴检测，屈光度通过红外偏心验光仪进行测量，眼轴通过步进式光学相干断层扫描仪进行检测。其中NL、BL、NL+FD、BL+ FD组视网膜表达c-fos的细胞数量通过免疫荧光进行定量分析。然后通过单细胞测序和，明确D2R在视网膜神经元中的分布。6只3周大小鼠视网膜制成细胞悬液，经10X Chromium systerm（10X Genomics）进行单细胞测序后，通过GSEA分析，分析D2类受体中D2R和D4R在视网膜不同亚型神经元中的分布。然后通过D2-EGFP小鼠进行免疫荧光验证。研究发现明亮光照能够使正常视觉状态下的小鼠眼屈光状态向着远视的方向进展，并部分抑制FDM的进展。选择性D2类受体激动剂Quinpirole不但能够阻断明亮光照对FDM的抑制作用，还能够促进普通光照条件下FDM的进展。这些结果提示D2受体通路可能参与了明亮光照对近视的抑制作用。D2类受体中D2R主要表达在GABA能无长突细胞上，而D4R主要表达在甘氨酸能无长突细胞上，提示其对屈光发育的作用是复杂的，需要进一步的研究。