Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
J Pediatr Surg. 2010 Dec;45(12):2403-7. doi: 10.1016/j.jpedsurg.2010.08.039.
The piebald lethal mouse with a deletion of endothelin-B receptor gene (EDNRB) is a model for Hirschsprung's disease (HD), whereas the SOX10 gene is vital for the development of intestinal neural crest-derived cells. Recently, we created a SOX10 transgenic mouse with intestinal neural crest-derived cells visible with enhanced green fluorescent protein (VENUS), that is, SOX10-VENUS(+)/EDNRB(sl/sl) to investigate intestinal innervation in HD.
SOX10-VENUS(+)/EDNRB(sl/sl) (n = 30) were compared with wild-type littermates as controls (EDNRB(s/s), n = 30). Mice were killed on days 3, 7, or 12 of age. The entire colorectum was excised, fixed with 4% paraformaldehyde, and examined using fluorescence microscopy alone without staining.
In normoganglionic colorectum from controls, a grid network of nerve fibers/glial cells was visualized that connected smoothly with extrinsic nerve fibers running along the colorectal wall. In aganglionic colorectum from SOX10-VENUS(+)/EDNRB(sl/sl) mice, there was no grid network and more extrinsic nerve fibers than controls that invaded the colon wall becoming elongated with branching fibers. Normoganglionic colon from controls and SOX10-VENUS(+)/EDNRB(sl/sl) mice appeared the same. Innervation patterns did not change over time.
This is the first time for abnormal enteric innervation in aganglionic colon in a model for HD to be visualized without staining.
内皮素-B 受体基因（EDNRB）缺失的花斑致死小鼠是先天性巨结肠（HD）的模型，而 SOX10 基因对于肠道神经嵴衍生细胞的发育至关重要。最近，我们创建了一种具有肠道神经嵴衍生细胞的 SOX10 转基因小鼠，这些细胞可通过增强型绿色荧光蛋白（VENUS）显现，即 SOX10-VENUS(+)/EDNRB(sl/sl)，以研究 HD 中的肠神经支配。
将 SOX10-VENUS(+)/EDNRB(sl/sl)（n = 30）与野生型同窝仔鼠作为对照（EDNRB(s/s)，n = 30）进行比较。在 3、7 或 12 日龄时处死小鼠。取出整个结肠直肠，用 4%多聚甲醛固定，并用荧光显微镜检查，无需染色。
在对照的正常神经节结肠直肠中，可见到网格状的神经纤维/神经胶质细胞网络，这些网络与沿结肠直肠壁运行的外源性神经纤维平滑连接。在 SOX10-VENUS(+)/EDNRB(sl/sl) 小鼠的无神经节结肠直肠中，没有网格状网络，而且外源性神经纤维比对照组更多，这些纤维侵入结肠壁并变得拉长，分支纤维增多。对照的正常神经节结肠直肠和 SOX10-VENUS(+)/EDNRB(sl/sl) 小鼠的结肠直肠看起来相同。神经支配模式没有随时间而改变。
这是首次在 HD 模型的无神经节结肠直肠中可视化异常肠神经支配，而无需染色。
