Restraint stress-induced neurogenic bladder inflammation

描述(申请人提供)：在没有感染的情况下，膀胱通常是亚急性或慢性炎症的部位，例如间质性膀胱炎(IC)，这是一种主要发生在女性的痛性膀胱疾病。IC患者的尿频和盆腔疼痛症状通常在围产期和应激下恶化。膀胱肥大细胞增多症伴肥大细胞活化在IC中已有报道。我们还发现，急性束缚应激引起大鼠膀胱肥大细胞的激活，这一过程依赖于神经肽神经降压素(NT)和P物质(SP)，因为辣椒素处理的啮齿动物不存在耗尽其SP含量的感觉神经纤维，NT受体拮抗剂SR48692也抑制了这一过程。此外，用雌二醇预处理膀胱可通过激活膀胱肥大细胞上的高亲和力雌激素受体来增强SP的刺激作用。最近的研究表明，感染嗜神经性伪狂犬病病毒的肥大细胞缺陷小鼠不会发生膀胱炎。肥大细胞位于血管周围，靠近神经突起，可在过敏刺激、直接神经刺激和急性束缚应激反应中分泌许多血管活性、促炎和神经敏化分子。促肾上腺皮质激素释放激素(CRH)在应激状态下从下丘脑释放出来，激活下丘脑-垂体-肾上腺(HPA)轴。然而，CRH及其结构相关的尿皮质激素(UCN)也在具有促炎作用的外周释放。CRH和UCN诱导的大鼠皮肤肥大细胞活化和血管通透性增加，均可被CRH中和血清或CRH受体拮抗剂antalarmin所抑制。CRH或急性应激诱导的皮肤血管通透性在W/W v肥大细胞缺陷小鼠中缺失，但在它们/对照组中存在，表明它是肥大细胞依赖的。急性应激也会引发大鼠膀胱肥大细胞的激活，而这种激活可被NT受体拮抗剂阻断。近年来发现IC患者尿液中促炎症细胞因子白介素6(IL-6)和肿瘤坏死因子-α(TNF-α)升高。我们假设膀胱中的急性应激释放(CRH)和/或(UCN)直接或通过SP或NT导致肥大细胞激活、血管通透性增加和促炎分子的表达。我们建议使用正常和遗传缺陷雌性小鼠来研究急性应激和CRH/UCN对以下方面的影响：(1)膀胱肥大细胞和尿路上皮核因子-kappaB(NF-kappaB)的激活，以及留置尿管收集的尿中组胺、IL-6和TNF-α的水平；(2)~(99)TcGp渗出法测定膀胱血管通透性；(3)W/W肥大细胞缺陷小鼠、CRH基因敲除小鼠和它们/对照组的血管通透性、尿液介质释放以及NF-kappaB的激活；(4)CRH/UCN膀胱内注射可诱导小鼠膀胱肥大细胞和尿路上皮细胞核因子-kB的活化，以及组胺、IL-6和TNF-α的分泌。这些研究将帮助我们了解急性应激如何触发膀胱肥大细胞激活，从而导致血管通透性增加和促炎分子释放。我们的发现可能与IC的病理生理学有关，并可能提出新的治疗方法。

DESCRIPTION (provided by applicant): The urinary bladder is often the site of subacute or chronic inflammation, in the absence of infection, as in interstitial cystitis (IC), a painful bladder disorder occurring mostly in women. Symptoms of urinary frequency and pelvic pain commonly worsen perimenstrually and under stress in IC. Bladder mastocytosis with mast cell activation has been documented in IC. We also showed that acute immobilizationstress in rodents induced bladder mast cell activation, a process that was dependent on the neuropeptides neurotensin (NT) and substance P (SP), as it was absent in rodents treated with capsaicin to deplete sensory nerve fibers of their SP content and was also inhibited by the NT receptor antagonist SR48692. Moreover, pretreatment of bladder with estradiol increased the stimulatory effect of SP, by activating high affinity estrogen receptors that we have identified on bladder mast cells. It was recently shown that bladder inflammation could not occur in mast cell deficient mice infected with the neurotropic pseudorabies virus. Mast cells are located perivascularly close to nerve processes and may secrete many vasoactive, proinflammatory and neurosensitizing molecules in response to allergic triggers, as well as by direct nerve stimulation and by acute immobilization stress. Corticotropin releasing hormone (CRH) is released from the hypothalamus under stress and activates the hypothalamic-pituitary-adrenal (HPA) axis. However, both CRH and its structurally related urocortin (Ucn) are also released in the periphery where they have proinflammatory effects. CRH and Ucn induced rat skin mast cell activation and increased vascular permeability, both of which were inhibited by pretreatment with neutralizing antiserum to CRH or the CRH-receptor (CRH-R) antagonist, antalarmin. CRH or acute stress-induced skin vascular permeability was absent in W/W v mast cell deficient mice, but was present in their +/+ controls indicating it is mast cell dependent. Acute stress also triggered rat bladder mast cell activation that was blocked by a NT-receptor antagonist. The proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosisfactor-alpha (TNF-alpha) were recently shown to be elevated in urine of IC patients. We are hypothesizing that acute stress releases (CRH) and/or (Ucn) in the bladder leading, directly or through SP or NT, to mast cell activation, increased vascular permeability and the expression of proinflammatory molecules. We propose to use normal and genetically deficient female mice to investigate the effect of acute stress and CRH/Ucn on: (1) bladder mast cell and urothelial Nuclear Factor kappa B (NF-kappaB)activation, as well as the levels of histamine,lL-6 and TNF-alpha in the urine collected from an indwelling catheter; (2) bladder vascular permeability quantitated by 99Technetium-gluceptate (99Tc) extravasation; (3) Vascular permeability, urine mediator release, as well as NF-kappaB activation in W/W v mast cell deficient mice, as well as in CRH knock-out mice and their +/+ controls; (4) mouse bladder mast cell and urothelial NF-KB activation, as well as secretion of histamine, IL-6 or TNF-alpha induced by intravesical administration of CRH/Ucn. These studies will help us understand how acute stress triggers bladder mast cell activation leading to increased vascular permeability and proinflammatory molecule release. Our findings may be relevant to the pathophysiology of IC and may suggest new therapeutic approaches.