SERT KO rats are a model of sex specific visceral pain

描述（由申请人提供）：本项目将测试假设5-羟色胺（5-HT，血清素）和雌激素信号相互作用，以增加初级传入神经元供应结肠的兴奋性。兴奋性增加是由导致雌性大鼠内脏高敏感性的离子通道表达变化引起的。这一假设将是 使用雄性和雌性5-羟色胺转运体（SERT）敲除（KO）大鼠进行测试，我们提出这是性别特异性内脏高敏感性的独特动物模型。内脏痛的潜在病理生理学尚不清楚，部分原因是缺乏可进行机制和干预研究的动物模型。然而，已发表的数据表明，5-HT信号的改变可能在人类中发挥作用。此外，内脏疼痛在女性中比男性更常见，这表明5-HT和性别在内脏疼痛的发生中存在相互作用。将在3个具体目标中检验总体假设。具体目标1将检验以下假设：野生型（WT）和SERT KO大鼠结肠中5-HT的体外细胞外可用性增加，且肠嗜铬（EC）细胞的5-HT释放不受SERT KO影响。电流测定法将测量粘膜附近对机械和化学粘膜刺激的反应。将使用免疫组织化学（IHC）和蛋白质印迹技术验证SERT缺失，并评估WT和SERT KO大鼠肠道中含5-HT的EC细胞。将评估多巴胺和去甲肾上腺素转运蛋白以及有机阳离子转运蛋白的IHC定位，以确定其在SERT KO大鼠中的表达是否增加。具体目标2将检验雌激素与5-HT相互作用导致雌性SERT KO大鼠内脏高敏感性的假设。对结直肠球囊扩张的内脏反应将用于测量有和无雌激素替代的完整和卵巢切除雌性WT和SERT KO大鼠的内脏敏感性。在具体目标3中，将研究在短期原代培养中维持的结肠投射感觉神经元的功能特性。这些研究将检验以下假设：当在体外使用全细胞膜片钳方法进行研究时，来自雌性而非雄性SERT KO大鼠的结肠投射感觉神经表现出兴奋性增加。兴奋性的增加被认为是由于5-HT和雌激素对感觉神经元的相互作用，导致河豚毒素抗性钠通道的上调。这些研究将表明，在雌性SERT KO大鼠中增加的5-HT可用性改变了内脏敏感性，正如在女性人类肠易激综合征患者中发生的那样。数据表明，SERT KO大鼠是研究导致内脏高敏感性的肠道感觉神经供应变化的模型。 公共卫生相关性：与肠道动力障碍相关的性别相关内脏痛影响了多达20%的美国人口。拟议的研究将试图建立一个动物模型的性别相关的内脏疼痛，可用于确定的病理生理机制负责这种常见的疾病。该动物模型也可用于开发治疗内脏疼痛的新药。

DESCRIPTION (provided by applicant): This project will test the hypothesis that 5-hydroxytryptamine (5-HT, serotonin) and estrogen signaling interact to increase excitability of primary afferent neurons supplying the colon. Increased excitability is caused by changes in ion channel expression that result in visceral hypersensitivity in female rats. This hypothesis will be tested using male and female serotonin transporter (SERT) knockout (KO) rats, which we propose is a unique animal model of gender specific visceral hypersensitivity. The underlying pathophysiology of visceral pain is unclear and this is partly due to a lack of animal models where mechanistic and interventional studies can be conducted. However, published data indicate that alterations in 5-HT signaling may play a role in humans. In addition, visceral pain i more common in women than in men, suggesting that there are interactions between 5-HT and gender in the genesis of visceral pain. The overall hypothesis will be tested in 3 specific aims. Specific aim 1 will test the hypothesis that there is increased extracellular availability of 5-HT n vitro in the colon of wild type (WT) and SERT KO rats and that 5-HT release from enterochromaffin (EC) cells is unaffected by the SERT KO. Amperometry will measure 5-HT near the mucosa in response to mechanical and chemical mucosal stimulation. Immunohistochemical (IHC) and Western blot techniques will be used to verify SERT deletion and to assess 5-HT-containing EC cells in the gut of WT and SERT KO rats. IHC localization of the dopamine and norepinephrine transporters and organic cation transporters will be assessed to determine if their expression increases in SERT KO rats. Specific aim 2 will test the hypothesis that estrogen interacts with 5-HT to cause visceral hypersensitivity in female SERT KO rats. The visceromotor response to colorectal balloon distention will be used to measure visceral sensitivity in intact and ovariectomized female WT and SERT KO rats with and without estrogen replacement. In Specific Aim 3, the functional properties of colon projecting sensory neurons maintained in short term primary culture will be studied. These studies will test the hypothesis that colon projecting sensory nerves from female but not male SERT KO rats exhibit increased excitability when studied using whole cell patch clamp methods in vitro. The increased excitability is proposed to be due to interactions between 5-HT and estrogen on sensory neurons that lead to upregulation of tetrodotoxin-resistant sodium channels. These studies will show that increased 5-HT availability in female SERT KO rats alters visceral sensitivity as occurs in female human irritable bowel syndrome patients. The data would indicate that the SERT KO rat is a model for studying changes in the sensory nerve supply of the gut that leads to visceral hypersensitivity. PUBLIC HEALTH RELEVANCE: Gender-related visceral pain associated with gut motility disturbances affects up to 20% of the U.S. population. The proposed studies will attempt to establish an animal model of gender-related visceral pain that can be used to identify the pathophysiological mechanisms responsible for this common disorder. The animal model could also be used to develop new drug treatments for visceral pain.