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><li>加的深度为1 - 2厘米家笼寝具到干净，适当标记的2升玻璃烧杯中。添加约一半的任何附加富集寝具材料，是存在于家笼，例如 nestlet，皱纸，向玻璃烧杯中。 </li><li>轻轻放置所有幼仔的从单个窝，单独地在同一烧杯中。 </li><li>立即将在孵化器将烧杯在33℃，湿度50％保持180分钟。 </li><li>从二级容器中取出大坝，并返回到她家笼。回到家笼其适当的住房的位置在动物饲养中。 </li><li>重复此步骤，对每个垃圾进行网管，使用干净的手套和二级容器每窝/大坝。 </li><li>在180分钟间隔周期的结束，因为他们被拆除返回以相同的顺序将窝对他们的家笼中。 </li><li>检索是当天早些时候分离第一胎的家笼。从笼子中取出大坝并放入一个干净的二手集装箱。 </li><li>从孵化器中删除第一烧杯中，擦了一把干净的戴手套的手之间的床上用品，以保持家笼的气味，同时处理的幼崽。 </li><li>轻轻移动幼仔，分别从烧杯中的家笼。 </li><li>返回任何铀浓缩和床上用品留在烧杯中家笼。 </li><li>返回大坝从二级容器的家笼，并在动物饲养中返回到其适当的住房的位置。 </li><li>冲洗烧杯，用水，并将其返回到培养箱中。使用相同的烧杯整个分离步骤的每个垃圾。 </li><li>重复此步骤，对每个垃圾经历相同的顺序网管系统，因为他们被放置在孵化器。 </li><li>重复整个过程每窝经受网管每天通过P21。 </li><li>通过将同性别的同窝在一起，干净笼子完整的食物和水对断奶P22 NMS和天真的幼崽。幼稚幼仔应出生和收容在相同的条件网管小鼠下，但经历没有额外的处理的正常畜​​牧业任务之外。 </li></ol> 2. Perigenital机械感<ol><li>冯弗雷设备安装<ol><li>把老鼠的试验室，让他们适应环境30分钟，而留在家里的笼子。 </li><li>通过将吸收性焊升高丝网顶表（79厘米×28厘米），它提供了足够的空间，以允许研究者以从下侧接近而不惊人的小鼠，约55厘米的高度之下准备的测试区域。 </li><li>将多达12只，分别下清晰，穿孔塑料室（11厘米×5厘米×3.5厘米）的丝网桌子上。放置在腔室顶部上的重物，以防止小鼠逸出。 </li><li>适应的小鼠前逃避阈值评估到屏幕上的另外30分钟。 </li></ol></li><li> Perigenital机械感度评估<ol><li>执行使用了一组标准分级的von Frey单丝43的上下方法。使用以下单丝:1.65克，2.36克2.83克，3.22克，3.61克，4.08克，4.31克和4.74克。 <ol><li>握住3.22克单丝的基极与暴露在缩回单灯丝。 注:某些型号可能没有缩回单丝。 </li><li>位于所述探针，使得单丝被垂直定向并且当鼠标警报，不动的，而它的体重均匀地分布后爪之间将其应用到左或右阴囊侧，避免了中线，直至有轻微的弯曲在灯丝观察。 </li><li>保持在适当位置的单丝为10秒或直至动物显示了积极的响应。 注意:阳性反应被认为是一个轻快加加速度或跳跃响应于单丝应用或舔或朝向单丝咬行为。如果10秒单丝的应用程序在移动鼠标时不显示这些行为，单丝必须遵循最小1分钟长的休息时间重新测试。如果小鼠既不移动也不是10秒单丝应用期间出现任何上面列出的行为，它被认为是一个否定的响应。 </li><html><li>记录的反应，无论是正面或负面的，在实验室笔记本电脑或笔记本电脑，并在所有使用3.22克单丝剩余小鼠重复这一过程。 </li><li>使用下一个合适的单丝再次测试所有的老鼠。注意:如果鼠标展示在该3.22克单丝的否定响应，应用3.61克单丝以相同的方式，记录的响应。如果鼠标展示在3.22克单丝的积极响应，应用2.83克单丝和记录的响应。 </li></ol></li><li>继续测试使用下一个更大或更小的单丝各小鼠，在适当时进行额外的4个应用第一肯定响应观察后。 </li><li>回到老鼠的笼子。 </li><li>使用日志单位最终冯弗雷单丝中试系列应用价值中，Chaplan 等43所描述来计算50％G撤出门槛。 </li></ol></li></ol>标题"&gt; 3。酸化甲苯胺蓝肥大细胞染色<ol><li>组织处理<ol><li>从已经心内灌注冰-冷的4％低聚甲醛45小鼠解剖前列腺组织44。后缀的组织在4％多聚甲醛进行1小时，在RT，然后cryoprotect在30％的蔗糖在1×PBS中，在4℃CO / N。 </li><li>制备庚烷的小（30毫升）浴，并将其放置在干冰上。 </li><li>在冲洗1X PBS前列腺组织和干燥使用轻型擦拭。 </li><li>将前列腺组织进入冷的庚烷，直到它被冻结。 </li><li>立即将冷冻的前列腺组织中含有干冰上安装媒体和地方cryomold直到冻结。 </li><li>商店cryomolds在-20℃。 </li><li>横向切割前列腺组织成7使用低温恒温器微米厚的冰冻切片。 </li><li>放置8 - 12横跨组织的长度，到带正电的显微镜载玻片非串行冰冻切片。 </li><li>商店完成载玻片在-20℃直到进一步处理。 </li></ol></li><li>准备酸化甲苯胺蓝<ol><li>加入1克甲苯胺蓝O操作百毫升70％的乙醇和旋涡，直到在溶液中以制备1％的储液。原液可以储存在室温下长达三个星期。 </li><li>加入1克NaCl至100毫升的水在烧杯放在搅拌盘的顶部。 </li><li>调整用12 M盐酸直至0.5的pH范围的NaCl溶液的pH - 1.0实现。 </li><li>通过组合8毫升的甲苯胺蓝原液和32毫升1％的NaCl溶液中制备0.25％甲苯胺蓝工作溶液。吸液管上下以确保甲苯胺蓝原液的总掺入和混合使用涡流。该解决方案的工作必须做出新的，用后丢弃。 </li></ol></li><li>染色前列腺冷冻切片<ol><li>除去被从冷冻处理的幻灯片，并允许平衡至室温约30μm研究。 </li><li>通过单独浸渍幻灯片约1秒到50毫升锥形管中含有的1×PBS的足够体积，以确保在载玻片上的组织切片将被完全淹没洗组织切片。允许幻灯片空气干燥的纸巾。 </li><li>在含有足够0.25％的工作甲苯胺蓝溶液的玻璃染色缸地方滑动，以确保在载玻片上的组织切片将被完全淹没并孵育10 - 15分钟，同时在一个平台上摇杆设定为每分钟15转。 </li><li>浸幻灯片1X PBS约1秒至洗出多余的甲苯胺蓝。 </li><li>放置在一个滑动箱或机架滑轨以这样的方式，该滑动是在其两侧以允许排水。 </li><li>干的炉中的幻灯片，在37℃下进行至少2小时或O / N在室温。 </li><li>脱水幻灯片迅速使用酒精，使载片醇曝光之间完全干燥。干片由排在纸巾上一个第二擦背部和边缘用轻型擦拭。 <ol><li>浸泡在95％乙醇的幻灯片约1秒，并允许完全干燥。重复此步骤1 - 10次，直到组织比干燥更紫蓝色。 </li><li>沉浸在100％EtOH中的幻灯片大约1秒，并允许以完全干燥。重复此步骤1 - 10次，直到组织是黑暗中蓝色，而不是紫色。 </li></ol></li><li>通过将在100％二甲苯幻灯片3分钟固定的污渍。晾干。 </li><li>盖玻片的载玻片与甘油，PBS或二甲苯基永磁安装介质。沥干多余的介质，并储存在室温。 </li></ol></li><li>量化肥大细胞<ol><li>可视化的甲苯胺蓝染色的组织切片，在20X放大倍率下使用光学显微镜（ 图2）。 </li><li>算的可视化区域之内的本肥大细胞的总数和非脱颗粒和脱颗粒的肥大细胞区分。 40X尊离子可能是必要的，以确认在​​一些肥大细胞造粒状态。 注:非脱颗粒的肥大细胞表现出与没有或微弱的核轮廓和/或细胞周围没有颗粒挤压密实异染性。脱颗粒的肥大细胞显示出不太激烈异染，并有内细胞质和细胞边界以外的核和/或游离的颗粒的一个明显的清晰轮廓。 </li><li>继续评估当前组织切片的整体的非脱颗粒和脱颗粒的肥大细胞的总数。重复上至少有7个额外的单独章节此过程中，跨越长度的每个组织。 </li><li>根据以下等式计算脱颗粒（DG）的总的肥大细胞的百分比为每个组织/小鼠:（DG肥大细胞/总肥大细胞）×100。 </li></ol></li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>Pregnant C57BL/6 female mice&amp;nbsp;</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>2 L 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&amp;deg;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.&amp;nbsp;</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&amp;deg;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&amp;nbsp;</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&amp;nbsp;</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.&amp;nbsp;&amp;nbsp;</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

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

assessment-perigenital-sensitivity-prostatic-mast-cell-activation

Research

JoVE Journal

Medicine

9.2K 次观看.  University of Kansas Medical Center. 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，。 

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

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

使用方法比较物种调查父亲的反应神经生物学

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

科研

神经科学

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

细菌性前列腺炎小鼠模型的测量触觉异常

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

免疫与感染

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

使用妊娠期心理社会压力的Murine模型作为母亲和婴儿精神障碍的翻译相关范式

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

行为学

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

医学

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

使用聚（I:C）生成妊娠中期母体免疫激活的可重复模型，以研究后代的易感性和恢复力

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

<meta charset="utf8"></head><body>为早期产后疼痛研究创建早产大鼠模型

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

<meta charset="utf8"></head><body>用于疼痛研究的早产大鼠模型

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

用于疼痛研究的早产大鼠模型

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...