Name: assessment of perigenital sensitivity and prostatic mast cell activation in a mouse model of neonatal maternal separation
Uploaded: 2015-08-13T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation at JoVE.com

The authors would like to thank Janelle Ryals, Rachel Supple, and Frank Wang for technical assistance. This work was supported by NIH grants R03 DK080182 (JAC), R01 DK099611 (JAC), R01 DK103872 (JAC), Center of Biomedical Research Excellence (COBRE) grant P20 GM104936 (JAC), start-up funds and core support from the Kansas Institutional Development Award (IDeA) P20 GM103418, core support from the Kansas IDDRC P30 HD002528, and The Madison and Lila Self Fellowship Program (ANP).

在这个协议中所描述的所有实验符合美国国立卫生研究院指南根据由堪萨斯大学医学中心机构动物护理和使用委员会的大学指定的准则。 1.新生儿母婴分离（NMS） <ol><li>日常监测大坝怀孕产仔胎。 注：出生的垃圾被认为是P0的一天。 </li><li>在P1，从家里取出笼坝并放入一个干净的二手集装箱。 </li><li>擦干净，戴手套的手之间的少数床上用品，以保持家笼的气味。 </li><l...

这个协议提供了方法用于使用新生儿应力，在网管系统的形式，以诱导症候表示的CP / CPPS的成年雄性小鼠。作为成年人，网管小鼠显示显著perigenital机械异常性疼痛，以及对肥大细胞脱粒的前列腺组织中的证据。使用NMS的是一种新颖的方法来开发的CP / CPPS的，因为它复制早期生活压力即常报道患有慢性盆腔疼痛12-14，临床前模型以及掺入一种非侵入性的诱导，如相对于更常用的，chemically-?...

慢性盆腔疼痛本身不是一种疾病，而是与经历的患者诊断为肠易激综合征（IBS）正在进行的自发和/或诱发疼痛，间质性膀胱炎/膀胱疼痛综合征（IC / PBS），慢性阴部疼痛相关的术语，或慢性前列腺炎/慢性骨盆疼痛综合征（CP / CPPS）。这些综合征常常伴发与共享许多特征在于它们具有无相关的病理或鉴定潜在的病因，虽然免疫系统，中枢神经系统和外周神经系统内的功能障碍已被证明对这些病症1的维护和发展作出贡献-3。慢性骨盆疼痛更可能呈现与症状的其他非盆腔相关功能性疼痛病症和心境障碍，包括焦虑，抑郁，和惊恐障碍4-6，这已经与下丘脑的改变的功能相关的垂体 -肾上腺（HPA）轴7-10。暴露于早期生活应激或创伤是用于开发HPA异常和相关慢性疼痛综合症10,11和，作为这种显著危险因素，患者的官能骨盆疼痛病症一个显著子集报告已经经历了不利的童年事件如虐待或忽视12-14。 新生儿母婴分离（NMS），其中涉及删除从他们的时间在断奶前一段在设定时间内大坝幼崽的啮齿动物模型已被用于在过去的二十年里，研究早期生活压力的结果。在一般情况下，网络管理系统已被证明能增加HPA轴活化，并产生焦虑样行为，通过直接影响基因表达的下丘脑内，以及扰乱从边缘结构15-18的下游调节。正确的HPA轴功能的干扰已被证明对提高大肠癌19-22贡献</sup&gt;和阴道显示的啮齿类动物模型NMS 16敏感性，但尽管产后膀胱炎23-25 ​​的长期影响广泛的特性，早期生活压力的影响已经在很大程度上消失在未学泌尿生殖器官。因此，下面的研究将描述如何在小鼠中进行NMS和后来评价perigenital机械灵敏度和肥大细胞浸润/活化在前列腺来验证使用雄性网管小鼠作为CP / CPPS的临床前模型。 所有的诊断慢性盆腔疼痛性疾病的，CP / CPPS可能是最公认和特点综合症，尽管有大约14％的26终生患病率和估计4400美元（每年门诊费用两倍，腰痛或类风湿关节炎27）。患者在会阴，直肠，前列腺，阴茎，睾丸，和/或腹部28，遇到一个喜CP / CPPS报告疼痛gher程度的心理压力比对照组病人29，和目前常用的有或症状被诊断为伴发慢性盆腔疼痛或情绪障碍5,29-31。复发性感染，漏上皮，神经性炎症，和自身免疫都被推测为CP的电位基本原因/ CPPS 2,32,33，以及肥大细胞活化和脱颗粒34。从人带有CP / CPPS表达前列腺分泌物增加了肥大细胞类胰蛋白酶和神经生长因子（NGF）水平34，以及后来的研究证实，类胰蛋白酶和羧肽酶A（CPA3），肥大细胞活化的标志物，也增加了尿CP / CPPS患者35。为肥大细胞的发生和维护CP / CPPS的潜在作用已经动物研究对这一综合征的一大焦点迄今。最常用的啮齿动物模型用于研究的CP / CPPS是一种实验性自身免疫性前列腺炎（EAP）的模型根通过皮下注射前列腺抗原在完全弗氏佐剂，这导致不同程度的取决于所使用34,36-39物种和株前列腺发炎产生的。肥大细胞浸润和活化/脱粒已被证明能增加以下诱导的EAP 34,35,40的。转基因小鼠缺乏既肥大细胞34或类胰蛋白酶受体PAR2 35不发展前列腺战术敏感度以下EAP，不像野生型小鼠的EAP。虽然这种临床前模型复制了许多人的CP / CPPS的特点，诱导方案并不表示人的条件，其中有一个不同的病因，往往不涉及直接的炎症，感染，或前列腺损伤。 早期生活压力对CP / CPPS在人类发展的影响已经在很大程度上未经调查去了哪里;然而，一项研究胡等人 41，证明了我ñ谁报的童年身体，情感史，和/或性虐待是显著更容易出现症状提示CP / CPPS的。此外，他们发现，既疼痛和泌尿分数增加患者的身体和情感滥用史。我们已经证实，在雌性C57BL / 6小鼠同网管范例产生阴道过敏和异常的基因表达在两个阴道和膀胱暗示功能失调HPA轴输出16。这方面的证据结合CP / CPPS患者与其他合并症42，其中IC / PBS和情绪障碍，已经更清楚地显示出早期生活应激暴露12-14链接呈现的高患病率，提供了理由使用网管模型小鼠研究CP / CPPS。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 female mice&#160;</td>
			<td>Charles River</td>
			<td>027</td>
			<td>Timed or untimed pregnant females should be monitored daily for in-house birth.</td>
		</tr>
		<tr>
			<td>2L glass beaker(s)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS10002L</td>
			<td>Each NMS litter will be held in the same beaker throughout the 21 day separation period.</td>
		</tr>
		<tr>
			<td>VWR Forced Air Incubator, Basic</td>
			<td>VWR International</td>
			<td>414005-124</td>
			<td>The incubator should be held at 33&#176;C and 50% humidity.</td>
		</tr>
		<tr>
			<td>Touch Test Sensory Evaluator, Kit of 20 (Semmes-Weinstein Von Frey Aesthesiometer for touch assessment)</td>
			<td>Stoelting</td>
			<td>58011</td>
			<td>The following monofilaments should be used for perigenital sensitivity assessment: 1.65 g, 2.36 g, 2.83 g, 3.22 g, 3.61 g, 4.08 g, 4.31 g, and 4.74 g.</td>
		</tr>
		<tr>
			<td>Animal Enclosure (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>435</td>
			<td>Be sure to place a heavy object on top of the individual, acrylic animal enclosures to prevent mice from escaping.&#160;</td>
		</tr>
		<tr>
			<td>Mesh Stand for mice and rats (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>410</td>
			<td>Place stand on a stable table top for comfortable access.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>Dilute the 10x PBS stock to 1x PBS.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>A 4% paraformaldehyde solution is needed to intracardially perfuse mice and postfix tissue in preparation for mast cell staining.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td>Cryoprotect fixed tissue in 30% sucrose solution at 4&#176;C overnight.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>246654</td>
			<td>Chill heptane on dry ice to freeze tissue.</td>
		</tr>
		<tr>
			<td>Standard Cryomold&#160;</td>
			<td>VWR International</td>
			<td>4557</td>
			<td>To assist in freezing prostate tissue prior to cryostat sectioning</td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>12 x 125 mL</td>
		</tr>
		<tr>
			<td>VWR Superfrost Plus Micro Slide</td>
			<td>VWR International</td>
			<td>48311-703</td>
			<td>75 x 25 x 1 mm</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Sigma-Aldrich</td>
			<td>198161</td>
			<td>Certified by the Biological Stain Commission. Suitable for use as a metachromatic stain for mast cells.</td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Biology Grade, Fisher Bioreagents; 95% and 100% solutions will be needed to dehydrate stained cryosections before mast cell visualization.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Acidified NaCl solution should be made fresh before staining cryosections.</td>
		</tr>
		<tr>
			<td>GeneMate Sterile 50 mL Centrifuge Tubes&#160;</td>
			<td>BioExpress</td>
			<td>C-3394</td>
			<td>Disposable tubes used to mix toulidine blue elements or dip slides into 1xPBS.</td>
		</tr>
		<tr>
			<td>Coplin staining dish for 10 slides, with ground glass cover</td>
			<td>Fisher Scientific</td>
			<td>08-815</td>
			<td>Hold up to 10 standard 3 x 1 in. (75 x 25mm) slides back-to-back. Use separate dishes for Toluidine Blue working solution, 95% EtOH, 100% EtOH, and xylene.</td>
		</tr>
		<tr>
			<td>Xylenes (Histological)</td>
			<td>Fisher Scientific</td>
			<td>X3P</td>
			<td>Once dehydrated, tissue is cleared with xylene.</td>
		</tr>
		<tr>
			<td>Microscope Slide Boxes, 100-Place</td>
			<td>VWR International</td>
			<td>82003</td>
			<td>Boxes can be used for storage and transportation of slides.</td>
		</tr>
		<tr>
			<td>Fisherbrand Microscope Slide Box</td>
			<td>Fisher Scientific</td>
			<td>22-363-400</td>
			<td>These slide boxes are typically small enough to fit inside a cryostate.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Glycerol is a traditional mounting medium.</td>
		</tr>
		<tr>
			<td>Mounting Medium, Richard-Allan Scientific</td>
			<td>VWR International</td>
			<td>4111</td>
			<td>Mounting medium firmly bonds the coverslip to the slide.</td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Rectangular, No. 1</td>
			<td>VWR International</td>
			<td>48393-106</td>
			<td>A coverslip is placed over the dehydrated and cleared tissue to protect the sample.</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>The Advanced Automated Research Microscope Eclipse 90i, used by our lab, has been discontinued and replaced Eclipse Ni-E.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

assessment of perigenital sensitivity and prostatic mast cell activation in a mouse model of neonatal maternal separation

慢性前列腺炎/慢性骨盆疼痛综合征（CP / CPPS）有14％的终生患病，是50岁以下的泌尿外科最常见的诊断为男性，但它是最不被理解和研究慢性盆腔疼痛症。慢性盆腔疼痛报告显著子在经历了早期的生活压力或滥用，它可以显着地影响到下丘脑 - 垂体 - 肾上腺轴（HPA）的运作和监管。肥大细胞活化，这已被证明是增加两个尿中并表达的CP / CPPS患者的前列腺分泌物，部分是由HPA轴的下游激活调节。新生儿母婴分离（NMS）已使用了二十多年研究的早期生活压力的结果在啮齿类动物模型，包括改变HPA轴和内脏敏感性。在这里，我们提供使用网管作为CP / CPPS的雄性C57BL / 6小鼠的临床前模型中的详细协议。我们描述的方法用于执行网管，评估perigenital机械异常性疼痛，和肥大细胞活化的组织学证据。我们还提供证据表明，早期的心理压力可以让男性泌尿生殖系统的小鼠长期影响。

小鼠经历NMS显示指示CP / CPPS的行为证据。当具有渐变一系列的von Frey单丝8周龄网管小鼠中测试时相比幼稚同行（n = 4时）显示perigenital机械异常性疼痛（5例， 图2）。这是证明作为显著减少（P &lt;0.01;学生t检验）在引起了积极的行为反应的机械痛阈，记录为轻快加加速度或跳跃响应于单丝应用或舔或咬行为朝向单丝。 这暴露于NMS小鼠也显示CP / CPPS的组织学?...

Watch this Scientific Journal Video about Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation at JoVE.com

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

我们正在测量perigenital机械灵敏度和肥大细胞活化在雄性C57BL / 6小鼠中进行的早期生活应激范式的前列腺 - 以诱导的慢性前列腺炎/慢性骨盆疼痛综合征的临床前模型新生儿母婴分离，。

的Perigenital敏感性和前列腺肥大细胞活化新生母婴分离的小鼠模型评估

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