National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, Incheon, Korea.
Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon, Korea.
Andrology. 2017 Sep;5(5):964-970. doi: 10.1111/andr.12387. Epub 2017 Aug 14.
Penile erection is a neurovascular phenomenon that requires well coordinated and functional interaction between penile vascular and nervous systems. In order to provide a useful tool to examine pathologic changes in the erectile tissue, mainly focusing on penile neurovascular dysfunction, we established the technique to determine the differential distribution of endothelial cells, smooth muscle cells, pericytes, and nerve fibers in the mouse penis using immunohistochemical staining with three-dimensional reconstruction. Immunofluorescent staining of penile tissue was performed with antibodies against CD31 (an endothelial cell marker), smooth muscle α -actin (SMA, a smooth muscle cell marker), NG2 (a pericyte marker), or βIII-tubulin (a neuronal marker). We reconstructed three-dimensional images of penile vascular or neurovascular system from stacks of two-dimensional images, which allows volume rendering and provides reliable anatomic information. CD31-positive endothelial cells, SMA-positive smooth muscle cells, and NG2-positive pericytes were evenly distributed and composed sinusoidal or venous wall. However, the endothelial layer of the cavernous artery or dorsal artery was mainly covered with smooth muscle cells and rarely associated with pericytes. The reconstructed three-dimensional images clearly visualized typical wavy appearance of nerve fibers that evenly innervate to cavernous sinusoids, cavernous artery, dorsal vein, and dorsal artery. We observed a significant decrease in CD31-positive endothelial cells, NG2-positive pericytes, and βIII-tubulin-positive nerve fibers in the penis of diabetic mice compared with those in normal condition. Our protocol for immunofluorescent staining with three-dimensional reconstruction will allow a better understanding of the penile neurovascular anatomy and may constitute a standard technique to determine the efficacy of candidate therapeutics targeting therapeutic angiogenesis or neural regeneration.
阴茎勃起是一种神经血管现象，需要阴茎血管和神经系统之间的良好协调和功能相互作用。为了提供一种有用的工具来检查勃起组织的病理变化，主要集中在阴茎神经血管功能障碍上，我们建立了使用三维重建的免疫组织化学染色来确定小鼠阴茎中内皮细胞、平滑肌细胞、周细胞和神经纤维的差异分布的技术。使用针对 CD31（内皮细胞标志物）、平滑肌α-肌动蛋白（SMA，平滑肌细胞标志物）、NG2（周细胞标志物）或 βIII-微管蛋白（神经元标志物）的抗体对阴茎组织进行免疫荧光染色。我们从二维图像堆栈重建阴茎血管或神经血管系统的三维图像，这允许体积渲染并提供可靠的解剖信息。CD31 阳性内皮细胞、SMA 阳性平滑肌细胞和 NG2 阳性周细胞均匀分布并构成窦状或静脉壁。然而，海绵体动脉或背动脉的内皮层主要覆盖有平滑肌细胞，很少与周细胞相关。重建的三维图像清楚地可视化了均匀地支配海绵窦、海绵体动脉、背静脉和背动脉的神经纤维的典型波浪状外观。与正常情况相比，我们观察到糖尿病小鼠阴茎中的 CD31 阳性内皮细胞、NG2 阳性周细胞和 βIII-微管蛋白阳性神经纤维明显减少。我们的三维重建免疫荧光染色方案将有助于更好地了解阴茎神经血管解剖结构，并可能构成一种标准技术，以确定针对治疗性血管生成或神经再生的候选治疗药物的疗效。
