这里描述的所有方法都已得到兽医政府当局 (Landesamt Landwirtschaft, Lebensmittelsicherheit 和 Fischerei, Vorpommern, LALLF M V) 的批准。
1. 动物的术前护理和准备
<ol><li>到达动物设施后, 将动物 (C57Bl/6) 分成相似大小的组, 将每个组保持在一起, 并在整个实验中保持组恒定。 注意: 使用相同性别的动物。用雄性小鼠得到了实验结果。 注意: 如果使用雄性动物, 笼群在7周的年龄开始, 这样就可以建立一个层次结构, 从而最大限度地减少实验期间攻击性行为的风险。</li>
	<li>手术前至少一周, 切换到高能 (> 14 兆焦耳/千克) 和高蛋白 (> 20%) 饲料含有所有必需的微量元素和维生素 (详情, 见材料清单)。 注: 确保所有小鼠在执行手术时重量至少25克。</li>
	<li>通过腹腔注射氯胺酮 (87 毫克/千克 ip) 和盐酸甲苯噻嗪 (13 毫克/千克 ip) 诱导麻醉和镇痛。等到老鼠容忍机械刺激, 如脚趾间距, 没有马达反应。</li>
	<li>将麻醉鼠标固定在位于操作台上的热垫上的仰卧位上, 保证在操作过程中稳定的定位, 避免全身热量的巨大损失。 注: 热衬底应具有36°c 到40°c 的表面温度;手术室温度应为21摄氏度。</li>
</ol>2. 远端结肠造口术
<ol><li>剃掉腹部的头发。在开始手术前, 使用酒精70% 和碘伏消毒手术现场三次。将操作现场悬垂, 保证无菌条件。</li>
	<li>执行15毫米中位剖腹手术通过切割腹部肌肉和腹膜沿 linea, 从而减少失血。</li>
	<li>使用两个迈克迪贝克非创伤性钳, 小心拉盲肠, 末端回肠, 和上升和横结肠从腹膜腔。 注意: 小心严格限制肠道的机械操作, 以防止对肠系膜结构的损伤。</li>
	<li>识别盲肠极, 上升结肠和小肠 (图1a 和 1b)。 注意: 正确识别上升结肠是正确放置结肠造口的基础。在回盲区解剖不明确的情况下, Peyer 斑块的存在识别小肠, 而形成的凳子的出现则是结肠的特点。</li>
	<li>使用标尺确定未来结肠造口的位置。它应该被放置在20毫米远端的回盲阀的远端结肠造口。</li>
	<li>在右上象限的腹壁上再做3毫米切口。通过这个切口提取先前识别的结肠段, 形成一个循环, 小心不要扭曲循环。</li>
	<li>小心通过乙状结肠22口径灵活的静脉注射套管。注意不要损伤肠系膜血管结构。</li>
	<li>将肠道恢复到腹膜腔。</li>
	<li>用简单的缝线和可吸收缝合 (例如, polyglactin 910 或 polyfil 4-0 1/2 c) 将软管的两端固定在皮肤上。</li>
	<li>在关闭剖腹手术前, 用腹腔注射0.5 毫升0.9% 生理盐水进行液体复苏。</li>
	<li>用可吸收缝合 (如polyglactin 910 或 polyfil 4-0 1/2 c) 闭合腹膜和肌肉层, 并进行连续缝合。用可吸收缝合 (如 polyglactin 910 或 polyfil 4-0 1/2 c), 用连续缝合法关闭皮肤。</li>
	<li>通过使用细剪刀执行小计横断来打开形象化冒号循环。避免所有的肠系膜损伤。不要完全横过结肠。</li>
	<li>使用 monofil, 可吸收缝线 (如polydioxanone 或 monofil 6-0 3/8) 修复每个结肠造口, 使用三张全厚针缝合腹膜和皮肤。传入回路, 这是一个功能性的末端结肠造口, 和传出循环, 这是一个粘液瘘, 在这一点上明显分离 (图 1c)。 注: 操作时间应少于20分钟, 以限制流体和热损失。</li>
	<li>完成手术后, 根据制造商的指示, 在超声波浴中使用无醛消毒液消毒仪器。</li>
</ol>3. 假操作 (Colotomy)
<ol><li>执行步骤1.1。到2.4。</li>
	<li>使用标尺确定未来的 colotomy 位置。colotomy 应定位与回盲阀的距离为实验组的结肠造口。</li>
	<li>用细剪刀打开结肠至少2/3 的周长。</li>
	<li>使用 monofil, 可吸收缝线 (例如, polydioxanone, monofil 6-0 3/8 s), 用单层、全厚度中断缝线闭合 colotomy。 注: 实验外科医生的手术时间应少于20分钟, 限制液体和热损失。</li>
	<li>执行步骤2.10。, 2.11。和2.14。</li>
</ol>4. 术后护理
<ol><li>把动物送回笼子里去。提供一个良好的气氛, 37 摄氏度 (如红外线灯), 直到老鼠完全清醒。然后, 保持老鼠在温度和湿度调节的环境 (21 °c; 30% 10% 相对湿度)。允许免费获得食物和饮用水。为促进流体吸收, 提供额外的水浸泡动物饲料。</li>
	<li>当动物表现出对机械刺激的反应时, 注射0.1 毫克/千克体重丁丙诺啡, 开始术后镇痛。小心避免呼吸压抑。</li>
	<li>用1毫克/毫升曲马多补充饮用水, 在术后第一周继续镇痛。</li>
	<li>为了补偿由于流动性降低而减少的液体摄入量, 在第一个术后的一周内在笼子里供应一个固体饮料垫。</li>
	<li>在第一周, 每第二天在第一个月的剩余时间, 每隔三天, 在第二个月, 使用佳中描述的疾病严重性评分, 衡量动物的体重, 并评分动物行为。6。</li>
</ol>

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所有作者都宣称他们没有相互竞争的兴趣。

本协议所提出的小鼠模型可靠地再现了人 dc 的组织病理学特征 ( 例如, 在炎症性肠段粘膜下层淋巴滤泡的发育, 隐窝缩短, 以及减少杯状细胞数)。除了这个优势, 这个模型是由一个非常相似的触发因素引起的, 并呈现出一个中等到轻度的严重性的临床过程, 正如在大多数受影响的人的情况。
为获得重现性结果和可接受的围手术期致死率, 需要一些显微外科技术的实践。然而, 结肠造口和假手术 (colotomy) 可以执行非常低的围手术期死亡率 (< 10%)。一些操作步骤是非常重要的: 1) 积极识别结肠和末端回肠。在回盲区解剖不清楚的情况下, 结肠可以通过存在 Peyer 的补丁和回肠末端来识别。2) exteriorizing 结肠造口回路时, 保证肠系膜不扭曲, 避免肠系膜缺血和机械肠梗阻。3) 用软管穿刺肠系膜时, 确保选择无血管区, 从而预防出血和肠系膜缺血。4) 当在腹壁上创建小切口时, 检查是否有足够的止血。如有必要, 可使用热凝固停止肌肉血管出血。5) 确保结肠环至少在其周长的2/3 处开放, 后壁充分形象化, 从而保证大便通道完全堵塞到远端肠段。6) 确保结肠造口术的两端都能很好地固定在皮肤上, 以避免由于粪便流不能排出而导致感染风险增高或机械性肠梗阻的开放性通道。正常情况下, 所有手术程序都可以不用放大镜或手术显微镜进行。一般而言, 这些设备的使用需要进一步的培训。
即使手术过程的复杂性是适度的, 也有许多必要的手术和围手术期的问题, 以成功终止实验的可重现的结果。
它是必不可少的长期生存与永久性结肠造口, 老鼠有一个完整的盲肠。与回盲阀相邻的近端 colostomies 的实验组显示, 术后一周的死亡率为 100%, 原因是不可逆转的体重损失 (未显示数据)。因此, 最重要的是选择结肠造口部位至少20毫米远端从 Bauhin 的阀门。这种观察可能是由于啮齿动物消化的生理。从近端结肠到盲肠的逆行转运发生在这些物种中。盲肠是由细菌菌群 occurs8 合成养分的地方。正常的 MCV 和妇幼保健的价值在长期随访表明, 动物与远端 colostomies 不遭受严重的维生素缺乏。此外, coprophagy 是啮齿动物在自然环境中营养行为的重要组成部分。在建立模型时, 我们高度关注的是, 由于存在永久性结肠造口, 扰动 coprophagy 可能导致营养不足 (如维生素 B)。然而, 虽然这是一个问题, 在获得营养的条件是有限的, 以前已经表明, coprophagy 失去它的营养价值在实验条件下, 自由过剩的水和营养均衡的食物9.在实验条件下, 优化术后营养摄入的条件至关重要。我们使用了蛋白质含量高、蛋白质品质优化、能量密度提高的动物饲料。动物在手术前至少两周适应这种饮食习惯。术后, 给有利于食物和液体摄入提供了libidum的水浸泡饲料。
在实验性和假组中利用同种性别的小鼠是至关重要的。性已经表明, 不仅对全身炎症的过程, 而且对肠道炎症的影响, 在各种化学诱发的结肠炎模型10,11,12。一般情况下, 雄性动物会产生更明显的疾病, 炎症更大, 更隐窝损伤, 再生少, 促炎性细胞因子水平增高, 体重减轻10。使用雄性动物的另一个好处是更好的年龄匹配的术前体重, 从而更好地补偿最初的术后减肥期。然而, 如果要解决特定的问题 (例如性激素对 DC 表现的影响), 雌性小鼠可以在《议定书》中使用。使用雄性老鼠需要更多的努力来维持稳定的住房条件。雄性小鼠更倾向于积极的相互作用导致伤害和死亡。雄性小鼠与一个显性男性生活在既定的层次上。一旦团队中的层次结构建立起来, 持续的战斗就不那么常见了13,14。由于这个原因, 我们在动物设施的老鼠到达前至少两周就形成了实验组, 直到实验结束, 这些小组才得以维持。一般来说, 实验组的5到7只动物被保存在一个笼子里。维持稳定的社会和住房条件对于尽量减少实验动物的攻击行为至关重要。住房条件已经显示影响体重增加以及免疫参数15,16,17。
对化学诱导和基因改良的结肠炎模型的易感性在小鼠菌株18,19,20之间有很大差异。本议定书是建立在 C57Bl/6 小鼠, 一个小鼠菌株与明确的遗传背景和良好的免疫系统21,22,23显示中间的强炎症反应在葡聚糖硫酸钠诱导的结肠炎模型20和被认为是一个典型的 Th1 菌株23。当需要更严重的疾病活动或 Th2 环境时, 可以考虑使用其他老鼠菌株。
总之, 我们的议定书是评估各种免疫细胞类型、细胞因子、趋化因子和其他信号分子在 DC 发病中的作用的宝贵工具。大量的基因修饰小鼠模型可与 C57BL/6 背景结合, 并可与我们的模型 (例如, 小鼠缺乏 IL-17, IL-10, 趋化因子 CXCL13, 因子受体 CXCR5 和 CCR7, sphingosine-1-phosphate受体 4)。在 C57BL/6 背景下, 同类系小鼠菌株的可用性在很大程度上促进了转移实验, 从而确定了不同细胞类型在 DC 病因学中的作用。最后, 该模型提供了机会, 以评估当地干预 (如修改当地微生物和局部抗炎治疗) 对黏膜免疫的影响和非受影响的肠段和系统性免疫平衡。

近年来, 大量的非传染性结肠炎实体不同于典型的炎症性肠病 (IBDs;i., 克罗恩病或结肠炎溃疡性皮肤) 已临床和病理的特点在人类。导致这些结肠炎形式的发展的病理生理学机制部份地不完全地被了解, 因为适当的动物模型是稀缺的。分流性结肠炎是最近被描述的实体之一。虽然这个词在1980年由 Glotzer1创造了, 相似的表型的最初的描述在1972年被给了 Morson2。该病在50% 至91% 的转移肠患者中发展, 其临床强度变化3,4。鉴于美国每年约有12万口结肠造口患者的发病率, 这一疾病实体构成了一个重要的健康问题。
制定这项议定书的总目标是提供一个小鼠 dc 模型, 它依赖于类似于人类 DC 中所看到的结肠炎触发, 再现人类疾病的主要病理学特征。与其他小鼠结肠炎模型相比, 我们模型中的结肠炎诱导不需要转基因动物 (例如, IL-7 转基因小鼠, n-钙粘蛋白显性阴性小鼠, 或 TGFβ鼠), 应用化学刺激性物质 (例如, 葡聚糖硫酸盐钠 (DSS) 诱发结肠炎, 或硝基苯磺酸 (TNBS) 诱发结肠炎), 或在免疫缺陷小鼠 (如 CD45RB高转移的特定细胞群的转移结肠炎模型) (综述, 见5)。与其他模型相比, 我们的 dc 模型的完整免疫系统允许评估在 dc 发展中所涉及的免疫机制。粘膜炎症对被排除的肠段的限制, 允许评估其对胃肠道其他部位粘膜免疫的影响, 对肠道其他免疫室的免疫平衡 (例如,, Peyer 的补丁和 mesenteriale 淋巴结), 以及整个生物体的免疫平衡。最后, 我们的模型构成了一个适当的工具来研究控制局部炎症刺激的机制, 这是由于当地微生物和消化抗原的正常当地环境的变化。

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			<td height="21" style="height:21px;width:216px;">Operation material</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">heat underlay, 6 watt</td>
			<td style="width:136px;">ThermoLux, Witte + Sutor GmbH</td>
			<td style="width:119px;">461265</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethanol 70% (with methylethylketon)</td>
			<td style="width:136px;">Pharmacie of the University Hospital Greifswald</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Wooden applicators, small cotton head onesided, wooden stick, 150 mm length</td>
			<td style="width:136px;">Centramed GmbH, Germany</td>
			<td style="width:119px;">8308370</td>
			<td style="width:167px;">for disinfection of operation field</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissor</td>
			<td style="width:136px;">Aesculap (BRAUN)</td>
			<td style="width:119px;">BC064R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atraumatic DeBakey forceps</td>
			<td style="width:136px;">Aesculap (BRAUN)</td>
			<td style="width:119px;">OC021R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle holder</td>
			<td style="width:136px;">Aesculap (BRAUN)</td>
			<td style="width:119px;">BM012R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vasofix Safety i.v. cannula 22G</td>
			<td style="width:136px;">Braun Melsungen AG, Germany</td>
			<td style="width:119px;">4268091S-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VICRYL Plus 4-0 violet braided; V-5 17 m 1/2c; 70 cm</td>
			<td style="width:136px;">ETHICON, Inc.</td>
			<td style="width:119px;">VCP994H</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PDS II 6-0 monofil; V-18 13 mm 3/8c; 70 cm</td>
			<td style="width:136px;">ETHICON, Inc.</td>
			<td style="width:119px;">Z991H</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BD Plastipak 1 mL (Syringes with needle with sterile interior)</td>
			<td style="width:136px;">BD Medical</td>
			<td style="width:119px;">305501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Triacid-N (N-Dodecylpropan-1,3-diamin)</td>
			<td style="width:136px;">ANTISEPTICA, Germany</td>
			<td style="width:119px;">18824-01</td>
			<td style="width:167px;">disinfection of surgical instruments in ultrasonic bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medications</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ketamine 10%, 100 mg/mL (ketamine hydrochloride)</td>
			<td style="width:136px;">selectavet, Dr. Otto Fischer GmbH</td>
			<td style="width:119px;">9089.01.00</td>
			<td style="width:167px;">87 mg/kg i.p.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xylasel, 20 mg/mL (xylazine hydrochloride)</td>
			<td style="width:136px;">selectavet, Dr. Otto Fischer GmbH</td>
			<td style="width:119px;">400300.00.00</td>
			<td style="width:167px;">13 mg/kg i.p.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl 0.9%</td>
			<td style="width:136px;">Braun Melsungen AG, Germany</td>
			<td style="width:119px;">6697366.00.00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Buprenovet 0.3 mg/mL (buprenorphine)</td>
			<td style="width:136px;">Bayer, Germany</td>
			<td style="width:119px;">PZN: 01498870</td>
			<td style="width:167px;">0.1 mg/kg s.c.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tramal Drops, 100 mg/mL (tramadol hydrochloride)</td>
			<td style="width:136px;">Gr&#252;nenthal GmbH, Germany</td>
			<td style="width:119px;">10116838</td>
			<td style="width:167px;">1 mg/mL drinking water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ceftriaxon-saar 2 g (ceftriaxone)&#160;</td>
			<td style="width:136px;">Cephasaar GmbH, Germany</td>
			<td style="width:119px;">PZN: 08844252</td>
			<td style="width:167px;">25 mg/kg body weight i.p.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">metronidazole 5 mg/mL</td>
			<td style="width:136px;">Braun Melsungen AG, Germany</td>
			<td style="width:119px;">PZN: 05543515</td>
			<td style="width:167px;">12.5 mg/kg body weight i.p.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Food</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ssniff M-Z Ereich</td>
			<td style="width:136px;">ssniff, Germany</td>
			<td style="width:119px;">&#160;V1184-3</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Solid Drink Dehyprev Vit BIO, pouches</td>
			<td style="width:136px;">TripleATrading, the Netherlands</td>
			<td style="width:119px;">SDSHPV-75</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nikon Eclipse Ci-L</td>
			<td style="width:136px;">Nikon Instruments Europe BV, Germany</td>
			<td style="width:119px;"></td>
			<td>light microscopy</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">VetScan HM 5&#160;</td>
			<td style="width:136px;">Abaxis, USA</td>
			<td style="width:119px;">770-9000</td>
			<td style="width:167px;">Veterinary hematology analyzer of 50 &#181;l venous EDTA-blood</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bandelin SONOREX (Ultrasonic bath)</td>
			<td style="width:136px;">Bandelin electronics, Germany</td>
			<td style="width:119px;">RK 100 H</td>
			<td style="width:167px;">disinfection of surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">NIS-Element BR4 software</td>
			<td style="width:136px;">Nikon Instruments Europe BV, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GraphPad Prism Version 6</td>
			<td style="width:136px;">GraphPad Software, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine distal colostomy, a novel model of diversion colitis in c57bl/6 mice

分流性结肠炎 (DC) 是一种常见的临床情况发生在肠段的病人排除在粪便流由于分流肠。这种疾病的病因仍然不明确, 但似乎有别于传统的炎症性肠病, 如克罗恩病和溃疡结肠炎。研究旨在破译导致这种疾病发展的病理生理学机制, 由于缺乏合适的小鼠模型而受到严重阻碍。该协议生成了小鼠 dc 模型, 有利于研究免疫系统在 dc 发育过程中的作用及其与微生物的相互作用。在这个模型使用 C57BL/6 动物, 结肠远端部分被排除在粪便流通过创造一个远端结肠造口, 触发轻度到中度炎症在排除的肠段, 并再现人类的标志性病变的发展DC 具有适度的全身炎症反应。与大鼠模型相比, 大量的基因修饰的小鼠模型在 C57BL/6 的背景下可用。这些动物与我们的模型相结合, 可以使个体细胞因子、趋化因子或生物活性分子受体 (如白细胞介素 (IL)-17) 发挥潜在作用;IL-10, 趋化因子 CXCL13, 趋性因子受体 CXCR5 和 CCR7, sphingosine-1-phosphate 受体 4) 被评估在 DC 的发病机制。在 C57BL/6 背景下, 同类系小鼠菌株的可用性在很大程度上促进了转移实验, 从而确定了不同细胞类型在 DC 病因学中的作用。最后, 该模型提供了机会, 以评估当地干预 (如修改当地微生物或局部抗炎治疗) 对黏膜免疫的影响和非受影响的肠段和对系统性免疫平衡。

手术在实验性 (结肠造口) 和假 (colotomy) 组均耐受良好。手术和围手术期处理正确执行时, 围手术期死亡率不应超过10%。在术后第一周, 实验性和假组均有明显的体重下降。假组的动物通常在第四术后的一天达到其体重最低点, 但在实验组的一天后发生。体重减轻在实验组更明显, 达到21.7% 的初始体重。相比之下, 假动物失去了10.8% 的初始体重 (图 2)6。术后第一周体重降至最低点后, 两个实验组的体重均持续上升, 但实验组的坡度较慢。严重肠道炎症的体征和症状 (即血流量或液体肠运动均不发生在实验性或假组6)。术后头60天总死亡率在结肠造口组约 40%, colotomy 组约10%。大多数死亡发生在术后第一周 (实验组 61%, 66% 人在假组)。死亡原因如图 3所示。
小鼠远端结肠造口术的形成主要是淋巴细胞性结肠炎的发生, 再现了人 DC 的特点。这些改变随着肠道排斥时间的延长而增加。隐窝长度在被排除的肠段中明显缩短。这一缩短达到统计学意义后14天的粪便分流 (图 3a)。在被排除的肠段30天后, 杯状细胞的隐窝长度减少 (图 3b)。14天后, 在被排除的肠段内的墓穴中, 绝对杯状细胞数也明显减少 (图 3c)。DC 的标志性病变, 粘膜淋巴滤泡的发育, 需要较长的大便偏差时间。虽然淋巴滤泡的数量在两周内可以观察到, 但两个月后的差异变得显著 (图 5 a c)。前面描述的所有病理学变化比被排除的肠段的近端区域更明显。典型的中性渗透作为急性炎症的征兆通常没有被观察 (图 5 d e)。
作为局部肠道炎症的系统性反应的标志, 在手术后的14天内, 结肠造口动物的中性粒细胞计数明显增加。这种差异维持到观察期结束 (60 天)。60天后结肠转移动物的血小板计数略有增加 (图 6)。结肠造口组术后14天血红蛋白水平和红细胞压积减少, 与假组比较。
结果表明, 啮齿类动物的维生素缺乏导致平均红细胞量减少 (MCH) 和平均红细胞血红蛋白 (MCV) 值7。在我们的模型中, 我们看到两个参数在14和30天后的初始增加。在更长的随访之后, MCV 和 MCH 都返回正常值 (图 7)。这表明远端结肠造口术在长期随访中不会导致临床上严重的维生素缺乏。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57616/57616fig1.jpg"> 图 1: 盲肠区域的解剖和外科手术。(a) 术后解剖学的图形化表现。(b) 盲肠的地形和解剖地标。(c) 结肠造口的图形化表现。图 1a已从佳等6修改。<a href="/files/ftp_upload/57616/57616fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57616/57616fig2.jpg"> 图 2: 体重发展。体重以术前体重的百分比显示。结肠造口和假 (colotomy) 动物体重曲线显示, 结肠造口组的术后早期体重损失明显高于假组 (p < 0.001)。值是指平均每组21-26 只动物的标准误差 (21 只动物接受 colostomies; 26 只动物在假组中)。这个数字已从佳等6修改。<a href="/files/ftp_upload/57616/57616fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57616/57616fig3.jpg"> 图 3: 死亡原因。并发症表现为全死因死亡率的百分比, 并在饼图 ((a) 结肠造口组 (b) 假组) 中加以说明。术后并发症由尸检决定。浪费综合征被定义为连续体重损失大于33% 的初始体重, 没有其他发现在尸检。尸检诊断为肠梗阻和吻合口瘘。气孔并发症被定义为 peristomal 脓肿和黏膜分离。其他并发症包括剖腹手术切口裂开, 盲肠缺血, 尸检不清楚的病例。两组患者术后并发症分布有显著性差异 (p = 0.021, 用费舍尔精确试验分析了多达 6 x 6 个应急表)。结肠造口组有39只动物, 假组29例。这个数字已从佳等6修改。<a href="/files/ftp_upload/57616/57616fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57616/57616fig4.jpg"> 图 4: 隐窝长度和杯状细胞数量。用直肠石蜡切片在周期性酸性席夫碱染色后, 确定了窖长和杯状细胞数。(a) 在转移性结肠炎 (DC) 的动物直肠中, 隐窝长度明显减少。(b) 土窖的杯状细胞承载区域的长度被测量并设置为与整个墓穴长度的比值。术后三十和六十天, DC 组内隐窝长度的杯状细胞的百分比降低。(c) 与假组相比, DC 动物的杯状细胞数明显减少。图 (a) 通过 (c) 显示每组5至9只动物的手段和标准误差 (结肠造口14和30天, 假14天: n = 8; 结肠造口60天: n = 5; 假30和60天: n = 9);∗p < 0.05;∗∗p < 0.01。这个数字已从佳等6修改。<a href="/files/ftp_upload/57616/57616fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57616/57616fig5.jpg"> 图 5: 淋巴滤泡和炎症浸润。(a) 被转移的冒号和假动物的淋巴滤泡被计数在直肠的石蜡切片染色苏木精和伊红。该图显示了每组5至9只动物的方法和标准误差 (结肠造口14和30天, 假14天: n = 8; 结肠造口60天: n = 5; 控制30和60天: n = 9), ∗p < 0.05;∗∗p < 0.01。(b) 和 (c) 结肠造口 (b) 和假 (c) 动物的直肠部分的典型例子, 术后60天染有苏木精和伊红。一个突出的淋巴卵泡 (∗) 存在于结肠造口小鼠的黏膜中。刻度条代表100µm. (d) 和 (e) 在石蜡切片上进行了氯乙酸酯酶反应染色, 以检测中性粒细胞颗粒。在转运直肠的横断面上, 在结肠造口组 (d) 和假动物 (e) 术后60天内均观察到急性炎症中性浸润。刻度条代表100µm。这个数字已从佳等6修改。<a href="/files/ftp_upload/57616/57616fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57616/57616fig6.jpg"> 图 6: 中性粒细胞和血小板计数。用兽医血液分析仪测定静脉血标本的血计数。(a) 结肠造口组与假动物术后60天相比, 中性粒细胞计数明显增加。(b) 术后60天内, 结肠造口组的血小板计数略有升高。该图显示了每组5至9只动物的方法和标准误差 (结肠造口14和30天, 假14天: n = 8; 结肠造口60天: n = 5; 假30和60天: n = 9), ∗p < 0.05;∗∗p < 0.01。<a href="/files/ftp_upload/57616/57616fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57616/57616fig7.jpg"> 图 7: 红细胞参数。用兽医血液分析仪测定静脉血标本的血计数。(a) 结肠造口组与假组相比, 血红蛋白和 (b) 红细胞压积显著降低。(c) 结肠造口组与假组相比, 术后平均红细胞容积 (MCH) 显著升高14和30天, 但在60天后恢复正常。(d) 平均红细胞血红蛋白 (MCV) 值在30天后升高, 但在结肠造口组术后60天后不再与假动物相比。该图显示了每组5至9只动物的方法和标准误差 (结肠造口14和30天, 假14天: n = 8; 结肠造口60天: n = 5; 假30和60天: n = 9), ∗p < 0.05;∗∗p < 0.01。<a href="/files/ftp_upload/57616/57616fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice at JoVE.com

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

小鼠远端结肠造口为人分流型结肠炎提供了鼠模型, 一种主要的淋巴细胞结肠炎在结肠段被排除从粪流。

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

murine-distal-colostomy-novel-model-diversion-colitis-c57bl6

Nanyang Technological University

Research

JoVE Journal

Medicine

12.8K 次观看.  Universitätsmedizin Greifswald. 小鼠远端结肠造口为人分流型结肠炎提供了鼠模型, 一种主要的淋巴细胞结肠炎在结肠段被排除从粪流。

视频: Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

科研

医学

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

灵活的结肠镜检查在小鼠使用经过验证的内镜评分系统来评估结肠炎和结肠直肠肿瘤的严重程度

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型:在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...