这项工作是由运营补助KJ从加拿大自然科学和工程研究理事会（NSERC）和克罗恩和加拿大结肠炎基金会（CCFC）的支持。 VM由CFRI奖学金和GB从加拿大卫生研究院（CIHR）的毕业生奖学金支持。 BAV是儿童肠道和肝脏疾病儿科IBD的研究和加拿大研究主席在儿科胃肠病学和KJ基金会（儿）主席是儿童和儿童与家庭研究所（CFRI）临床科学家奖励计划所支持的高级临床科学家，加拿大英属哥伦比亚大学。

涉及动物主题程序遵循机构动物护理和使用委员会（IACUC）在英属哥伦比亚大学概述动物护理指引。 在此协议中提到的程序将用于大鼠和小鼠进行描述，而代表性的结果将从SD大鼠呈现。 1。 DNBS和直肠内给药的制备<ol><li>为DNBS大鼠直肠滴注，新鲜制备15-30毫克DNBS/250微升50％乙醇中。对于小鼠，准备2-6毫克DNBS/100微升50％乙醇。注意:优化的确切DNBS的浓度在每个设施来进行给药。雄性Sprague-Dawley大鼠，小鼠和C57BL / 6小鼠是优选用于诱导DNBS结肠炎，然而其他小鼠和大鼠品系都可以使用。小鼠和大鼠只用50％乙醇处理，可作为对照组却给出乙醇的影响是轻微的或不存在，可以OMIt为纳入本组的研究12。 </li><li>附加1ml注射器给19号针头，其具有聚乙烯导管（PE-90 - 大鼠或PE-50 - 小鼠）固定到其末端。 </li><li>轻轻麻醉在加热垫的大鼠或小鼠休息（ 例如，通过经鼻锥给予1.5％异氟醚，动物应该还是有眨眼和吞咽反射和显示定时呼吸）。适用撕裂凝胶它的眼睛，避免眼睛干燥。 </li><li>轻轻涂抹用指尖压力施加到动物的后端，以消除任何粪便可存在于远端结肠。 </li><li>轻轻插入导管直肠，与导管达到大约8 cm肛门近端大鼠或小鼠3-4厘米。注入少量溶液的同时插入导管，以润滑，允许更容易地插入。注:如果同时插入导管任何感觉到阻力，不要继续插入，因为这可能导致穿孔肠壁。取出导管并尝试重新插入轻轻，再次润滑冒号作为诉讼。 </li><li>慢慢地注入250（只）或100（小鼠）微升DNBS的，或等体积或50％乙醇的控制。注射DNBS后，放置90秒的动物头低位，以避免DNBS的损失。 </li><li> DNBS给药后，给动物喂食8％的蔗糖水0.2％的盐水，以防止在第一周脱水。 </li><li>仔细观察动物，每天监测体重下降和脱水或痛苦的实验期间的迹象。注:减肥可迅速接近最大允许的减肥廉署建议。因为这大部分是从脱水而得，体重减轻应该被用作用于管理乳酸林格氏液来限制脱水的指标。请务必与健康问题的适当设施兽医咨询。 </li></ol> 2。合作llecting组织和评估DNBS攻击小鼠组织学损伤<ol><li>在与DNBS攻击后所需的时间点，通过与异氟醚麻醉室补充足够的氧气过量安乐死的动物。观察动物直至不再上升或下降的胸部被看见，并且没有可触知的心跳。确认动物已被正确地缺乏应对脚趾捏或角膜的感人，其次是颈椎脱位处死。 </li><li>打开腹腔，取出整个大肠。纵向打开肠;宏观粘膜溃疡应观察从大鼠结肠（2-3厘米的小鼠）的前端约5-6厘米近端。注:去掉大肠后，如果需要评估的宏观损伤分数。从对照组动物相应的解剖位置收集结肠段。注:个人得分的组织进行宏观损伤分数应蒙蔽到各治疗组的身份。严重程度和溃疡的程度（0-10），总结与分数缺席或腹泻的存在（0或1），粘连（0，1或2）和最大结肠壁厚:使用以下或类似标准得分的组织切片以毫米为单位11。 </li><li>收集0.5厘米部分近端的最大宏观损伤的部位。急速冷冻在液氮中10.5厘米部到后评估的髓过氧化物酶（MPO）活性（中性粒细胞浸润的测量），并放置在10％中性缓冲福尔马林中用于组织学分析的其他部分。注意:快速冷冻组织中髓过氧化物酶测定法可以储存在-80℃下长达1个月。 </li><li>允许在步骤2.3收集组织学分析的组织以福尔马林中固定过夜，然后洗净用70％乙醇。在石蜡中嵌入组织，并切成所需厚度（3-5微米），染色用苏木精和伊红（HE）进行组织学评分。 </li&gt; <li>有两个观察者评分从各动物在光学显微镜下至少3的组织切片，以确定组织学损伤评分为每个组。注:个人得分的组织进行组织学损伤分数应失明的治疗组的身份。炎症性细胞浸润的程度（0 =不存在，3 =透壁），粘膜结构的损耗（0 =不存在，3 =重度），存在或不存在隐窝脓肿（0-1）:利用以下或类似标准得分的组织切片以及杯状细胞耗竭（0-3）。确定组织学损伤评分为这些分数11的总和。 </li></ol> 3。上DNBS表演髓过氧化物酶（MPO）含量挑战肠组织<ol><li>检测MPO活性在步骤2.3中获得的0.5厘米结肠段。均匀化在MPO匀浆缓冲液的组织（0.5％十六烷基三甲基溴化铵在50mM磷酸钾缓冲液（pH 6.0）），得到50毫克结肠segmeNT /毫升匀浆缓冲液悬浮。 </li><li>在10000等分的匀浆溶液为1毫升，离心分离机，在4℃下进行2分钟×克。 </li><li>混合100μl的各等分试样的2.9毫升50含有邻dianisidinedihydrochloride的0.167毫克/毫升和0.0005％过氧化氢在反应杯mM磷酸盐缓冲液（pH 6.0）。 </li><li>测量吸光度，用分光光度计在460nm处的速率。注意:MPO活性定义为酶的降解的H 2 O 2 1微摩尔/分钟至H 2 O在室温下的数量，然后用每组织毫克单位。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在这个协议中描述的DNBS模型是可以用来研究IBD的各个方面是有价值的，廉价的，重现性结肠炎模型。当给药直肠内给大鼠或小鼠，二硝基苯磺酸诱导的炎症和组织损伤的结肠相当程度，类似人类克罗恩病中的它的各种组织学特征，包括多形核细胞的浸润透和主要的NF-κB依赖Th1细胞的活化期。 该模型已被用于研究认为对IBD的影响，如紧张，抑郁，和饮食，以及肠神经系统受累?...

炎性肠疾病（IBD）的特征是慢性复发性炎症的胃肠（GI）道，导致腹泻，体重减轻和腹部疼痛的症状。溃疡性结肠炎（UC）和克罗恩氏病（CD）是IBD的两种主要形式，并且可以由炎症的位置，在胃肠道内加以区别。在UC患者中，炎症通常涉及直肠和连续了冒号在不同程度上受影响的只是肤浅的粘膜延伸。与此相反，光盘可以影响任何部分在胃肠道的，虽然它主要影响回肠和盲肠。 CD往往表现为透壁炎症常与肉芽肿有关，导致stricturing（fibrostenotic）和/或穿透（瘘管）的疾病。虽然鸡传染性法氏囊病的病因仍不清楚，但大家均认为鸡传染性法氏囊病是多因素的，包括互动的主机之间的免疫系统，遗传易感性和responSES环境和微生物因素。 迄今为止，已经提出了不同型号的IBD中显示UC和CD的特性不同的临床，组织学和免疫反应。最常用的模型包括转基因小鼠（IL-2，IL-10，SAMP / YIT），感染诱发的模型（ 柠檬酸杆菌啮齿类柠檬酸杆菌 ， 鼠伤寒沙门氏菌 ），过继转移模型（CD45 + RB高，CD62L +细胞转入SCID小鼠）和化学诱导的结肠炎模型（葡聚糖硫酸钠（DSS），三硝基苯磺酸（TNBS）和二硝基苯磺酸（DNBS））。由于其低成本和快速起效的疾病的，化学的模型被认为是非常宝贵的IBD的各方面的研究。每个列出的化学性结肠炎模型都有优点和局限性在临床，免疫学和组织病理学的相关性IBD的某些方面。 DSS是用来诱发大肠杆菌最常见的化学方法之一那朵在啮齿类动物1。 3-10％DSS（分子量:42 kDa的）的给药小鼠的饮用水7-10天可诱发症状，结肠炎的症状，包括体重减轻，腹泻带血，结肠缩短，粘膜溃疡和中性粒细胞浸润。这个模型在药物筛选研究，以及探讨上皮再生，对粘膜的动态平衡先天免疫的影响，以及炎症，促进肠道发育不良和腺癌发展中的作用机制尤其有用。然而有一些缺点，在DSS模式，包括变异在DSS的需要引起结肠炎在不同的动物设施的浓度，以及不一致的水吸收的小鼠，从而不一致暴露于DSS，导致变化的程度，范围和分布粘膜损伤和溃疡的结肠。所有这些特点导致结果的异质性，并限制各研究FR比较结果的能力OM不同的研究小组。 一种替代的DSS模型是结肠炎的半抗原诱导的DNBS或TNBS模型。这款机型采用的黏膜增敏剂DNBS或三硝基苯磺酸，稀释于不同浓度的乙醇直肠滴注。乙醇的管理是一个先决条件，打破结肠黏膜屏障以允许DNBS或TNBS渗入固有层。二硝基苯磺酸/ TNBS然后将haptenize局部结肠和肠道微生物蛋白质成为免疫原性的，从而引发宿主的先天和适应性免疫反应。在一般情况下，这种模式是有严重，有时带血腹泻，体重减轻和肠壁增厚但是症状变化取决于所用啮齿动物的种类，以及时机，剂量和暴露于DNBS或TNBS在使用的程度相关联的研究。大鼠和小鼠之间存在着重要的区别应该指出，与购买和板较低的成本，以及洛韦的好处ř体重，以减少处理每个动物的成本在体内 。这应该是设置针对结肠炎的更加迅速和严重的过程中小鼠，其中较脆弱和敏感的动物可能会很快达到一个人性化的终点。 肠道炎症最初起因于乙醇诱导损伤肠上皮细胞，从而增加上皮通透性，微生物渗入到粘膜，宿主蛋白质的haptenization，这一切导致中性粒细胞浸润，巨噬细胞和Th1型T淋巴细胞的到受损粘膜。相比于二硝基苯磺酸，三硝基苯磺酸被认为是由于其在与碱，如氢氧化钠和氢氧化钾接触，可以造成爆炸的危险高度氧化性的化学危险品。因此DNBS目前被视为化学在三硝基苯磺酸的首选诱发结肠炎。在啮齿动物中，二硝基苯磺酸结肠炎被认为是最方便的方法之一，研究福卢翼鸡传染性法氏囊病相关疾病的修饰符: <ol><li>抑郁症和结肠炎的再活化:它已经表明，压力，焦虑和抑郁在IBD患者常常与疾病复发相关联。二硝基苯磺酸结肠炎是一个合适的模型来研究抑郁症及其后果的作用对结肠炎的活化小鼠。该方法通常包括初始诱导结肠炎通过DNBS，其次是结肠炎的留下的小鼠6-8周的分辨率。小鼠，然后给予抑郁症引起药物如利血平，或嗅觉bulbectomy诱发抑郁其次是通过挑战与DNBS 2的subcolitic剂量测试他们对结肠炎激活。 </li><li>压力和活化结肠炎:压力是一直与IBD的发病另一种常见的环境因素。有越来越多的证据机构，表明慢性应激和起病UC和CD的症状在啮齿动物之间有很强的关联š以及在人类和非人类灵长类动物3。二硝基苯磺酸结肠炎是研究结肠炎的应力相关的活化在小鼠和大鼠的良好模型。该方法通常使用在如上述的抑郁症，除了代替抑郁症类似的方式，将动物暴露于应激如声波和束缚应激4。 </li><li>神经源性炎症:许多研究已经在肠神经系统（ENS）的结构和功能，如从动物模型和从IBD患者的组织样品中看到描述任一瞬时的或永久的改变。 DNBS是一个很好的模型，探讨炎症在ENS 5的影响，同时研究去甲肾上腺素能和胆碱能神经通路6。 </li><li>损伤修复机制:在IBD的发病机制，宿主衍生的损伤介质如活性氧簇（ROS），一氧化氮（NO），细胞间粘附分子3（ICAM-3），和P-选择素都被示出为p躺在肠上皮破坏作用。 DNBS是研究引起的上调这些介质以及由使用选择性的药物或抑制剂的7,8调控机制修复损伤的良好模型。 </li><li>透壁炎症:除了DNBS的上述应用中，该模型也可以用于研究在肠道的透壁炎症，在CD患者中找到一个经典特征。既DNBS和TNBS诱发的结肠炎与淋巴细胞的显著浸润有关，进入结肠黏膜使得这些模型来研究T细胞依赖的免疫机制，特别有用。 </li></ol>在与DSS的模型比较，DNBS和TNBS诱导的结肠炎的优点包括成本低，结肠炎的快速发展（通常需要1-3天以示重现性溃疡和发炎）和远端结肠一致的局部破坏。但其缺点是对于一个要求更高水平的技术知识，对二硝基苯磺酸/ TNBS剂量优化，以及需要麻醉直肠给药。 在此方法的论文中，我们研究了不同浓度的N-6和n-3上使用的植物油红花油（SO）的改变结肠黏膜响应DNBS诱导的结肠炎大鼠多不饱和脂肪酸的效果和低芥酸菜子油（CO）的和鱼油（FO）。它已被证明是n-6和n-3脂肪酸是肠道炎性疾病的重要介质，通过其作为细胞膜phosopholipids 9酰基部分的作用。在对比中的n-6脂肪酸的促炎细胞电位，n-3脂肪酸的酸在足够高的摄取是潜在有效的抗炎剂。为n-3脂肪酸的抗炎作用是直接通过更换花生四烯酸作为花生酸类基板的介导，通过改变proinfl的抑制花生四烯酸代谢和间接ammatory基因表达和细胞信号传导。在n-3脂肪酸也引起的resolvins，家庭中的抗炎介​​质。摄入大量的n-3脂肪酸与在生产中促炎细胞花生酸，细胞因子，趋化因子，活性氧和粘附分子的表达的减少有关。在这项研究中，大鼠自由进食饲料相同的所有营养物质，除了脂肪酸，具有作为从脂肪，20％的二氧化硫，20％CO，18％的鱼油加2％红花油（FO）10-11％的一个能量。作为每日能量的百分比，则SO饮食提供15％的亚油酸（LA），与&lt;0.06％的α-亚麻酸（ALA）和无二十碳五烯酸（EPA）或二十二碳六烯酸（DHA）时，CO饮食有4.2洛杉矶％和1.9％的ALA与不含EPA或DHA和FO的饮食提供1.4％的EPA，4.9％的DHA，0.32％LA和0.12％的ALA。脂类饮食引发三周后，将小鼠给药直肠内DNBS或50％乙醇和处死5天后。该INFLammatory反应是减肥，组织学损伤评分和组织髓过氧化物酶活性的考核评估。

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1 mL syringe</td>
			<td style="width:121px;">BD Biosciences</td>
			<td style="width:119px;">309659</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">19 gauge needle</td>
			<td>BD Biosciences</td>
			<td>305187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing PE-50</td>
			<td>BD Biosciences</td>
			<td>427517</td>
			<td>for intra-rectal administration in mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing PE-90</td>
			<td>BD Biosciences</td>
			<td>427519</td>
			<td>for intra-rectal administration in rat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNBS</td>
			<td>MP Bio</td>
			<td>150959</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tear gel</td>
			<td>Novartis</td>
			<td>63601662596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Formalin</td>
			<td>Fisher</td>
			<td>5F93-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Fisher</td>
			<td>A-962.4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hexadecyltrimethyl-ammonium bromide</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>H5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium phosphate buffer solution</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>79628-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">O-dianisidine dihydrochloride</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>D3252</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">30% H2O2</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>31642</td>
			<td>hydrogen peroxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrophotometer</td>
			<td>BIORAD</td>
			<td>Benchmarker Plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Microscope</td>
			<td>Zeiss</td>
			<td>Axio Image.Z1</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Data analysis software program </td>
			<td>GraphPad Software</td>
			<td style="width:119px;"> GraphPad Prism Software Version 4.00 </td>
			<td><a href="http://www.graphpad.com/">San Diego California USA, www.graphpad.com</a></td>
		</tr>
	</tbody>
</table>

dnbs/tnbs colitis models: providing insights into inflammatory bowel disease and effects of dietary fat

炎性肠疾病（IBD），包括克罗恩病和溃疡性结肠炎，一直与遗传基础相关联，以及最近宿主免疫应答的微生物和环境代理。二硝基苯磺酸（DNBS）诱导的结肠炎允许一个学习的IBD相关的环境触发诸如压力和饮食，潜在的疗法的效果，其作用机制底层肠道炎症及粘膜损伤的发病机理。在本文中，我们研究了在结肠粘膜的炎症反应的大鼠DNBS诱导的结肠炎的饮食的n-3和n-6脂肪酸的影响。所有大鼠饲喂相同日粮除了不同类型的脂肪酸[红花油（SO），低芥酸菜子油（CO），或鱼油（FO）]三周之前暴露于直肠内DNBS。直肠内给予乙醇对照组大鼠持续了5天的学习体重增加，而，DNBS处理的大鼠饲喂脂肪的饮食都洛吨重量FO和CO喂养大鼠48小时，大鼠经72小时受够展现显著的减肥效果。经过72小时后DNBS体重增加恢复，5天后DNBS，对FO组有较高的体重比SO或CO基团。结肠部分收集后5天DNBS处理呈局灶性溃疡，隐窝破坏，杯状细胞耗竭，以及急性和慢性炎症的细胞不同的严重程度之间饮食组的粘膜渗透。该SO喂食组表明最严重的损害随后的CO，和FO送入这表明组织损伤的温和度组。同样，结肠髓过氧化物酶（MPO）活性，中性粒细胞活性的标志是显著高于SO其次是二氧化碳喂养大鼠，具有显著降低MPO活性FO喂养大鼠。这些结果证明了使用二硝基苯磺酸诱导的结肠炎，如在该协议中概述，以确定饮食在IBD的发病机制的影响。

大鼠（的Sprague-Dawley）或小鼠（C57BL / 6）与二硝基苯磺酸的挑战通常会导致瞬时体重减轻，腹泻，有时与存在于粪便血。然而，因素如遗传，饮食和肠道菌群可以修改易感性二硝基苯磺酸诱导的结肠炎。因此，包括在实验动物应来自同一机构，在本地衍生或从一个动物供应商订购的。所有的动物都应该为减肥而苦恼结肠炎期间遭受症状的程度进行认真的监测。 在图1-4</st...

Watch this Scientific Journal Video about DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat at JoVE.com

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

二硝基苯磺酸/ TNBS诱导的结肠炎提供了一种替代，并研究炎性肠病的病理生物学廉价的方法。在本文所讨论的协议概述DNBS的成功应用诱导结肠炎小鼠和大鼠，并允许一个深入研究宿主介导的肠道反应。

二硝基苯磺酸/ TNBS结肠炎模型:提供洞察炎症性肠病和膳食脂肪的影响

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

dnbstnbs-colitis-models-providing-insights-into-inflammatory-bowel

Research

JoVE Journal

Medicine

47.1K 次观看.  BC Children's Hospital. 二硝基苯磺酸/ TNBS诱导的结肠炎提供了一种替代，并研究炎性肠病的病理生物学廉价的方法。在本文所讨论的协议概述DNBS的成功应用诱导结肠炎小鼠和大鼠，并允许一个深入研究宿主介导的肠道反应。 

视频: DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed&#160;the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min).
A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p &#60;0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm&#160;and control halitosis in humans and animals. Even though human and animal species present some differences,&#160;this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

膳食补充剂的口臭犬管理疗效

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

胃食管反流与小鼠手术模型

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

体外肠囊评估黏膜通透性在胃肠道疾病模型

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...