Nerve Growth Factor and Transient Receptor Potential (TRPV1) Channel in Diabetic

描述（由申请人提供）：糖尿病周围神经病变（DPN）是1型糖尿病常见的慢性并发症，以热、触觉和疼痛感觉的逐渐丧失为特征。周围感觉丧失，再加上微血管功能受损，DPN可导致无法愈合的溃疡并导致截肢。本研究的目的是确定DPN中热感觉改变的机制。一种假说认为DPN是由神经生长因子（NGF）对背根神经节（DRG）神经元的营养支持改变引起的。我们认为NGF可能通过激活NADPH氧化酶超氧化物生成系统来调节热传感器蛋白，瞬时受体电位通道（TRPV1）。因此，我们认为糖尿病早期NGF的增加介导了热超敏反应，而随着疾病进展，NGF水平降低或感觉神经损伤导致感觉丧失。我们将根据概述的具体目标来检验这一假设。目的1。为了确定NGF是否调节体内热感觉、TRPV1和NADPH氧化酶亚基的表达。本实验旨在测试体内给药NGF是否会促进痛觉过敏，以及是否会增加TRPV1和NADPH氧化酶亚基的表达。小鼠将通过足底注射给予NGF， 24小时后进行镇痛测试。收集小鼠和NGF处理后的DRGs和后爪皮肤，检测TRPV1和NADPH氧化酶mRNA和蛋白的表达。目标2。确定糖尿病患者热敏性的改变是否与NGF、TRPV1以及NADPH氧化酶亚基的表达和活性的变化有关。使用双转基因1型糖尿病小鼠模型，我们将测量DRGs和皮肤制剂裂解物中NGF的水平，然后使用生化和分子生物学技术测量TRPV1和NADPH氧化酶的表达。初步研究表明，DRG神经元和后爪皮肤样本中TRPV1水平的升高与痛觉过敏反应呈正相关。目标3。测定NGF或胰岛素治疗是否能恢复糖尿病小鼠正常的热敏性以及TRPV1和NADPH氧化酶亚基的正常表达。基于NGF缺乏产生1型糖尿病小鼠的低痛觉表型特征的假设，我们将给这些动物注射NGF或胰岛素（以维持足够的血糖控制），并确定它是否恢复正常的热感觉。正常血糖控制动物和糖尿病动物的热阈值将在给药前和给药后测定，给药前和给药后分别给药、NGF和胰岛素或这些药物的组合。本研究获得的数据将更好地理解NGF和TRPV1受体在控制DPN热感觉中的作用。

DESCRIPTION (provided by applicant): Diabetic peripheral neuropathy (DPN) is a common chronic complication of type 1 diabetes, characterized by progressive loss of thermal, tactile and pain sensations. The loss of peripheral sensations, coupled with impaired microvascular function in DPN can contribute to non-healing ulcers and lead to amputations. The goal of this study is to determine the mechanism(s) involved in altered thermal sensation in DPN. One hypothesis states that DPN results from altered nerve growth factor (NGF) trophic support to dorsal root ganglion (DRG) neurons. We believe that NGF regulates a thermal sensor protein, transient receptor potential channel (TRPV1), presumably by activating the NADPH oxidase superoxide generating system. Accordingly, we propose that an increase in NGF early in diabetes mediates thermal hypersensitivity, while low NGF levels or damage to sensory nerves as the disease progresses leads to sensory loss. We will test this hypothesis based on the specific aims outlined. Aim 1. To determine whether NGF regulates thermal sensation, the expression of TRPV1 and NADPH oxidase subunits in vivo. This aim will test if in vivo administration of NGF promotes hyperalgesia and whether this increases TRPV1 and NADPH oxidase subunit expression. Mice will be administered NGF by intraplantar injections, followed by analgesic testing 24 h later. DRGs and hindpaw skin obtained from vehicle and NGF treated mice will be collected to determine TRPV1 and NADPH oxidase mRNA and protein expression. Aim 2. To determine if altered thermal sensitivity in diabetes is associated with changes NGF, TRPV1 and the expression and activity of NADPH oxidase subunits. Using a double transgenic mouse model of type 1 diabetes we will measure the levels of NGF in lysates of DRGs and skin preparations, followed by measuring TRPV1 and NADPH oxidase expression using biochemical and molecular biology techniques. Preliminary studies indicate increased levels of TRPV1 in DRG neurons and hind paw skin samples correlates positively with the hyperalgesic response. Aim 3. To determine whether NGF or insulin therapy restores normal thermal sensitivity and the normal expression of TRPV1 and NADPH oxidase subunits in diabetic mice. Based on the hypothesis that a deficiency in NGF produces the hypoalgesic phenotype characteristic of type 1 diabetic mice, we will administer NGF or insulin (to maintain adequate glycemic control) to these animals and determine if it restores normal thermal sensation. The thermal thresholds of euglycemic controls and diabetic animals will be determined prior to and following administering either vehicle, NGF and insulin or a combination of these agents. The data obtained from this study would provide a better understanding of the role of NGF and the TRPV1 receptor in the controlling thermal sensations thermal in DPN. PUBLIC HEALTH RELEVANCE: The proposed project will focus on understanding some of the changes in nerve cell function, which lead to loss of pain perception in patients with diabetes, a complication termed diabetic peripheral neuropathy. We believe that the information gained by this study could contribute significantly to our appreciation of this disease and contribute to the development of novel drug therapies.