这项工作得到了英国心脏基金会 (PG/13/60/30406) 和伯明翰大学的支持。

所有动物程序均经动物福利伦理审查机构和英国内政部批准 (英国, 项目许可 40/3745)。小鼠在 C57BL/6 背景, 8-12 星期老, 19-25 g 身体重量, 两个性别使用。
1. 动物麻醉
<ol>
	<li>将一只老鼠放入感应室, 并由5% 异氟醚与100% 氧气混合诱导麻醉。使用流量为0.5 升/分. 等待, 直到鼠标完全停止移动, 其呼吸频率变得罕见。</li>
	<li>将鼠标从腔室中取出, 用电动剪刀刮掉腹部。尽可能彻底地去除腹部的毛发, 避免腹腔的污染。</li>
	<li>将鼠标置于仰卧位, 并将鼠标的鼻子放入与麻醉系统相连的圆锥中。将异氟醚的百分比降低到 2-3% (确切的百分比可能与动物略有不同)。确保动物睡觉时的呼吸速率比平时低, 但要避免喘气。如果发生喘息, 将异氟醚的百分比降低0.5%。
	<ol>
		<li>检查踏板反射, 以确认正确的麻醉和开始手术, 只有当它是阴性的。将兽医软膏涂抹在眼睛上以防止干燥。</li>
	</ol>
	</li>
	<li>将 anti-bactericidal 溶液 (1% 必) 涂抹在老鼠的腹部上, 用不育的棉花芽以某种方式擦拭该区域, 不包括手术部位的电位污染 (例如, 以圆形方式内而外)。重复3次。</li>
</ol>
2. 下腔静脉狭窄和小鼠恢复的应用
注: 所有的仪器以及材料 (棉花芽, 纱布, 生理盐水等) 与手术区域接触必须是无菌的。操作者必须在手术前擦洗, 并在操作过程中佩戴无菌手套和礼服。显微镜手柄应用无菌箔包裹。
<ol>
	<li>在手术前和整个实验期间给予小鼠反的治疗。例如, 使用丁丙诺啡0.1 毫克/千克皮下30分钟手术前, 然后每12小时, 直到动物被安乐死。由于这种方法的血栓形成类似于无菌炎症, 避免使用抗炎药物作为前。</li>
	<li>用不育的褶皱盖住老鼠, 在褶皱处做个洞, 露出手术部位。使用剪刀, 沿腹中线, 1.5 厘米从胸骨下切开切口。使用棉花芽取出的肠道到 left-hand 一侧的鼠标和覆盖他们的无菌纱布浸泡在温暖 (37 ° c) 盐水, 以避免干燥。</li>
	<li>从下腔静脉与左肾静脉融合的位置, 确定下腔静脉及其侧支 (可能有0、1或 2) 在尾端方向。结扎所有可见的侧分支具7-0 惰性普理灵缝合尽可能接近下腔静脉。
	<ol>
		<li>试着不要直接用钳子夹住旁边的树枝, 而要把它们用钳子握在手里, 一手把它们围起来, 然后用钳子在树枝下做一条地道。不要关闭后面的分支。</li>
	</ol>
	</li>
	<li>用钳子夹住周围的组织, 拉下下腔, 在下腔和主动脉之间的小面积上产生张力, 在下腔静脉与左肾静脉之间的夹角为确切。考虑到几乎不可能将主动脉与下腔静脉分离, 甚至低于2-3 毫米 (在尾部方向)。</li>
	<li>在你的右手上用钳子尖端的发散动作在主动脉和下腔之间制造一个洞。记住, 运动越多, 破坏船只的几率就越大。理想情况下, 尽量减少运动次数到3-5。</li>
	<li>准备一块7-0 的惰性普理灵缝合约2厘米长。把它放在老鼠的腹腔内, 在操作者的 left-hand 一侧的附近的下腔。</li>
	<li>把下腔拉上来再离开在主动脉和下腔静脉之间的孔中插入你的正确的钳子贴士, 这样提示就会出现在下腔静脉的另一侧。进行缝合并将其拉回孔 (图 3A)。</li>
	<li>用缝线做一个临时结, 但不要把它关上。将30G 针 ("间隔") 弯曲为挂钩, 并立即将其关闭 (图 3B)。</li>
	<li>卸下分隔符 (图 3C)。</li>
	<li>用棉花芽将肠回到腹膜腔, 并均匀地分布在腹腔内。</li>
	<li>闭合腹膜与6-0 乔缝合使用连续的循环。关闭第一个和最后一个循环作为一个结。</li>
	<li>用金属钉缝合皮肤。</li>
	<li>用0.5 毫升温热 (37 ° c) 的葡萄糖盐水皮下注射鼠标以恢复机体的液体平衡。</li>
	<li>将鼠标放入一个温暖的 (25 ° c) 房间或室内的单个笼子里, 把食物和凝胶水放进笼子里。观察直到鼠标完全恢复 (一般, 大约1小时)。</li>
</ol>

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作者没有什么可透露的。

在这里, 一项协议的下腔静脉狭窄, 模拟血流畸变, 一个主要的触发因素 DVT, 提出。下腔静脉狭窄在 2-6 h6,8,23 (鲽鱼 A, 未发布数据, 2016) 内诱导 65-100% C57BL/6 小鼠的血栓发育。该方法的一个主要限制是血栓大小的变异性24 (图 4), 这是观察后, 短期 (6h) 和长期 (48 小时) 下腔静脉狭窄。这种变化的原因 (由于使用了相同的条件, 如间隔, 麻醉等) 仍然晦涩难懂, 但人们可以推测, 小鼠之间的解剖变化 (例如, 下腔静脉的宽度, 两侧和背部的数量和位置分支) 作为这一现象的基础。血栓大小的变异性使血栓形成的患病率 (小鼠血栓的百分比) 的主要结果。血栓的患病率可以用一个应急表和费舍尔的精确测试来比较。其中一个实验是一个例外, 当减少血栓大小与相同的血栓流行后, 注射 podoplanin 抑制抗体被观察到7。
该方法在研究静脉血栓形成时特别有用。它允许调查血管壁的早期事件, 如招募细胞, 最终导致血栓形成。药物的亲或血栓作用可以用这个模型来评估, 血栓形成率是一个主要的读数。48 h 的狭窄适用于显露一个血栓表型, 而 6 h 狭窄可以使用, 如果血栓表型是期望的。也可进行血栓及周围静脉壁的组织学分析。
侧枝是否应结扎或左专利的问题仍然是开放的。一组表明, 结扎侧支不增加血栓的大小和位置的一侧分支接近1.5 毫米的下腔静脉结扎网站显着损害血栓发展25。侧支的闭合可能导致血管内皮损伤, 同时也增加手术时间26。在我们的手, 缺乏侧支关闭显著减少血栓形成率 (下至 10-30% 后 48 h 狭窄;鲽鱼, 未发表的), 因此我们结扎所有可见的侧枝。
理想情况下, littermate 控制应作为小鼠, 即使在相同的背景, 但从不同的来源, 可能有轻微不同的血栓形成率。如果研究了 DVT 本身对任何参数 (如生物化学) 的影响, 应使用假操作动物。假手术的小鼠接受相同的手术, 但在下腔静脉周围的结扎是松散闭合的, 没有产生狭窄。
本议定书中最常见的错误 (关键步骤) 是努力将主动脉和下腔分离, 而不是精确地在血管之间的夹角, 但稍低, 这通常会导致出血。当大量出血发生时, 小鼠的良好恢复变得不太可能, 建议停止实验并安乐动物。正常情况下, 小鼠恢复良好, 在笼子内移动, 并没有大幅减肥。我们建议在20克以上的雄性和雌性中使用小鼠, 并在手术结束前将动物 (特别是雄性) 放在单独的笼子里, 以避免打架和受伤。据报道, 另一个 (电解) 模型的 DVT, 雄性小鼠产生较大的血栓比女性27。对我们的数据进行分析并没有发现男女小鼠的血栓形成率有显著差异 (未发表)。因此, 鼓励研究人员在两个性别的小鼠上使用下腔静脉狭窄模型进行实验。
拆线和/或主食可能不会被排除, 特别是在长时间的实验 (一周和更长)。因此, 应每天至少检查两次小鼠的缝合完整性, 并应特别注意在笼床上出现血迹。
值得注意的是, 与其他动物模型一样, 下腔静脉狭窄在翻译成人方面有其局限性。例如, 小鼠 IVCs 没有瓣膜, 而人类 DVT 则在静脉瓣膜内发育。此外, 人类有垂直脊柱定向与肌肉泵是一个重要的机制, 加速血液流动的静脉。相反, 小鼠有水平的脊柱定向, 没有肌肉泵的作用, 支持血液回流到心脏。在将鼠标数据转换为人类疾病时, 应考虑这些限制因素。

深静脉血栓 (DVT) 是在深静脉的血栓的发展, 通常 (但不仅是) 腿部。在与肺动脉栓塞的连词 (PE; 一起选定的静脉血栓栓塞, 职业教育) 它在大约90万美国人年年开发并且代表一个严肃的健康问题和经济问题1,2。PE 是 DVT 的并发症, 当血栓从最初的位置分离出来并到达肺部时, 就会发生, 这可能导致呼吸功能不全和死亡。职业教育的死亡人数超过了艾滋病、乳腺癌和交通事故的死亡率, 合并了3。
造成 DVT 的主要因素, 如癌症或外伤, 是缺乏流动性4,5。这可能是由于外科手术 (特别是骨科)、瘫痪、长途飞行或其他原因造成的。静脉血流依赖于肌肉泵, 因此, 肢体固定的血流会导致血栓形成。本文所描述的方法的目的是重述这样的血流畸变6,7。在下腔静脉部分流限制 (中静脉) 模仿人的静脉瓣膜创造的条件, 结果形成一个类似于人血栓结构的血栓6。血栓的大部分是红色的, 包括红细胞、纤维蛋白和血小板的组成。血栓有一个小的 "白色部分" 丰富的血小板 (图 1), 这类似于 "线的拉·赞恩" 描述的人静脉血栓。血栓的两个部分也包含中性粒细胞8, 这是在将来的血栓的站点被招募的第一个细胞之中6,9。红色部分中性粒细胞排出中性粒细胞胞外陷阱 (网), 而白色部分中性粒细胞似乎缺乏网状8。与人类 DVT 相似, 小鼠血栓形成伴有血浆 D-二聚体水平升高 (图 2)。低分子量肝素 (素), 用于 DVT 预防的病人, 也防止血栓形成的小鼠。这种方法的一个重要优点是缺乏内皮剥脱性9, 这是人 DVT 的特点10。这一特征使下腔静脉狭窄成为一个更具临床相关性的 DVT 模型, 例如, 用氯化铁诱导血栓形成, 这导致内皮剥脱, 其中血栓主要由血小板组成11, 12,13。多组14,15,16, 在下腔静脉完全停滞的模型是首选。与狭窄相比, 在血管中保留残余流量, 停滞的应用完全停止了流动, 从而限制了系统管理的物质对血栓形成部位的可及性。同时, 狭窄和停滞引起血栓形成的机制也不同: 狭窄导致局部炎症的发生 (内皮细胞活化, Weibel Palade 体含量的释放, 免疫干细胞的招募和血小板到血管壁) 导致 "immunothrombosis"6,9,17, 而停滞似乎诱发而形成血栓, 特别是, 组织因子和其他凝血和与纤溶相关的机制18,19,20。因此, 狭窄和停滞模型反映了静脉血栓的发展稍有不同的方面, 虽然他们的机制可能肯定重叠。深静脉血栓的电解质下腔模型 (EIM)21,22仅诱导部分咬合血管壁的血栓。因此, 对不同药物的系统管理对血栓生长的影响进行检测是方便的。这种模式, 但是, 假设中断的下腔静脉壁完整性 (插入针) 和诱导血栓的电流, 使病理生理学相关性这一机制至少有争议。

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stenosis of the inferior vena cava: a murine model of deep vein thrombosis

深静脉血栓 (DVT) 及其破坏性的并发症, 肺栓塞, 是一个严重的健康问题, 高死亡率。静脉血栓形成的机制仍然模糊不清。缺乏机动性 (例如, 手术后或长途飞行) 是导致 DVT 的主要因素之一。缺乏流动性的病理生理学后果是静脉瓣膜血流停滞。在这里, 一个模型被描述为模拟这种流动干扰为血栓驱动因子。在该模型中, 建立了下腔静脉的局部流动限制 (狭窄)。关闭约90% 的下腔腔 48 h 导致血栓的形成与人类的结构相似。相似之处是: i) 血栓体积大部分是红色的, 即红细胞和纤维蛋白, ii) 存在白色部分 (拉·赞恩), iii) non-denuded 内皮单层, iv) 升高的血浆 D-二聚体水平, 和 v) 的可能性, 以防止低分子量肝素血栓形成。限制包括可变大小的血栓和事实, 一定比例的野生型小鼠 (0-35%) 可能不会产生血栓。除了视觉观察和测量, 血栓可能是可视化的非侵入性技术, 如超声, 这使得监测动态血栓的发展。在较短的时间点 (1-6 h), 活体显微镜可用于直接观察事件 (如, 在血管壁上招募细胞) 在血栓形成之前。这个方法由世界各地的几个小组使用, 使得有可能发现 DVT 的基本机制的启动, 并确定潜在的目标, 可能是有益的预防。

本文介绍了一种由流动畸变引起的深静脉血栓形成模型。在下腔静脉狭窄的诱导引起血流的畸变和停滞, 在一段时间后 (在这种情况下, 48 h) 导致血栓的形成, 结构类似于人 DVT 血栓 (图 1a)。在下腔静脉内血栓的存在很容易被直观地检测到 (图 1B)。模型和人 DVT 之间的一个重要相似性是升高的血浆 D-二聚体水平 (图 2)。D-二聚体是纤维蛋白降解的产物, 它在血液中的存在表明一个活跃的血栓过程正在发生。
该模型在图 3-c中的 step-by 步骤中呈现。下腔静脉被仔细地暴露, 在下腔和主动脉之间有一个孔, 缝合 (普理灵 7-0) 通过下腔静脉下的孔。然后, 下腔静脉结扎缝合在一个间隔 (30G 针,图 3D), 在此之后, 分隔符被删除。这一程序允许约90% 关闭的下腔静脉腔离开剩下的10% 专利。这确保血流停滞最终导致血栓形成。
图 4显示了模型的主要缺点, 即可变血栓大小。所有这些血栓都是在相同的起源, 年龄和类似体重的野生型雄性小鼠进行的相同实验中得到的。虽然所有的小鼠产生血栓 (血栓形成率为 100%), 其大小明显不同, 在广泛的范围内。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56697/56697fig1.jpg"> 
图 1:48 h 限制/狭窄后代表血栓(a) 48 小时静脉狭窄后的典型血栓。注意红色 (红细胞丰富) 和白色 (富含血小板的) 部分。(B) 下腔静脉 (左) 或无 (右) 血栓。缩放条形图 = 2 mm<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig1large.jpg" target="_blank">请单击此处查看此图形的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56697/56697fig2.jpg"> 
图 2: 下腔静脉狭窄后血浆 D-二聚体水平升高.小鼠受下腔静脉狭窄。血液被采取在被表明的时间点, 稳定1:9 与3.8% 枸橼酸钠, 血浆由离心 (2300 x g, 5 min) 和 D 二聚体水平决定由 ELISA 试剂盒根据制造商的指示。圆圈指定假手术小鼠, 交叉指定下腔静脉狭窄。<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig2large.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/56697/56697fig3.jpg"> 
图 3:小鼠下腔静脉血栓模型中的流量限制/狭窄.给出了模型的连续阶段。(a) 在下腔静脉和主动脉之间有一个孔, 并通过它在下腔静脉周围进行缝合。(B) 一个结是封闭在一个间隔 (30 克针)。(C)删除分隔符: 外科医生在关闭鼠标前看到的最终视图。虚线黄线划定下腔。班代表左肾静脉。刻度条 = 0.2 毫米 (D) 间隔 (30 克针)。缩放条形图 = 2 mm<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig3large.jpg" target="_blank">请单击此处查看此图形的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56697/56697fig4.jpg"> 
图 4.血栓大小的变异性。 小鼠受48小时静脉狭窄的影响。提出的是在同一实验中获得的血栓。缩放条形图 2 mm<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig4large.jpg" target="_blank">请单击此处查看此图形的较大版本.</a>

Watch this Scientific Journal Video about Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis at JoVE.com

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

我们在这里描述下腔静脉狭窄是深静脉血栓形成的小鼠模型。该模型重述血流限制, 是人类静脉血栓形成的主要诱因之一。

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

stenosis-inferior-vena-cava-murine-model-deep-vein

Research

JoVE Journal

Immunology and Infection

23.7K 次观看.  University of Birmingham. 我们在这里描述下腔静脉狭窄是深静脉血栓形成的小鼠模型。该模型重述血流限制, 是人类静脉血栓形成的主要诱因之一。

视频: Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

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 ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

高频超声监测小鼠瘀血致深静脉血栓形成

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

用于口腔中性粒细胞病评价的鼠期牙周炎的强结合诱导模型

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

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