Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium.
Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium.
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DOI：
10.1016/j.juro.2013.01.048
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发表时间：
2013-08
期刊：
影响因子：
--
通讯作者：
Osborne PB
中科院分区：
文献类型：
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作者：
O'Mullane LM;Keast JR;Osborne PB
最近的证据表明，尿路上皮充当化学、机械或热刺激的感觉传感器，向神经末梢和膀胱壁中的其他细胞发出信号。在完整膀胱中研究神经尿路上皮通讯的细胞和分子基础并不容易，这导致我们建立了一种共培养背根神经节（DRG）感觉神经元和膀胱尿路上皮细胞的方法。通过酶处理和机械解离分离从成年雌性 Sprague-Dawley 大鼠解剖的 DRG 和膀胱中获得的感觉神经元和尿路上皮细胞。将它们一起或单独铺在胶原蛋白包被的基质上，并在角质形成细胞培养基中培养 48-72 小时。进行逆行示踪剂标记以识别用于功能测试的膀胱传入。与神经元单一培养物相比，与尿路上皮细胞共培养的神经元的神经突生长和复杂性有所增加。酪氨酸激酶抑制剂 K252a 降低了尿路上皮细胞的生长促进作用，但 TrkA-Fc 对 NGF 信号传导的上游抑制没有作用。尿路上皮细胞的 Fura-2 钙成像显示对 ATP (100μM) 和 TRPV4 (4α-PDD，10μM) 激活有反应，但对 TRPV1（辣椒素，1μM）、TRPV3（法呢基焦磷酸，1μM）或 TRPA1（芥子油，100μM）没有反应。相比之下，共培养的神经元被除法呢基焦磷酸之外的所有激动剂激活。共培养为研究泌尿系统动物模型中的神经泌尿道上皮相互作用提供了一种新的方法。我们的结果表明，神经元特性不仅在尿路上皮存在的情况下得以维持，而且神经突的生长通过不依赖 NGF 的机制得到增强。
Recent evidence suggests the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli that signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder, which led us to establish a method of co-culturing dorsal root ganglion (DRG) sensory neurons and bladder urothelial cells. Sensory neurons and urothelial cells obtained from DRG and bladders dissected from adult female Sprague-Dawley rats were isolated by enzyme treatment and mechanical dissociation. These were plated together, or separately, on collagen-coated substrate and cultured in keratinocyte medium for 48–72 h. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to neuronal monocultures. The growth-promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor, K252a, but upstream inhibition of NGF signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to ATP (100μM) and activation of TRPV4 (4alpha-PDD, 10μM), but not TRPV1 (capsaicin, 1μM), TRPV3 (farnesyl pyrophosphate, 1μM), or TRPA1 (mustard oil, 100μM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Our results suggest that neuronal properties are not only maintained in the presence of urothelium but that neurite growth is potentiated by an NGF-independent mechanism.