这项研究得到了国防医学院的资助以及日本卫生、劳动和福利省为研究棘手疾病而提供的健康和劳动科学研究补助金的支持。

实验方案经国防医学院动物研究委员会批准（第16058号）。这些动物被保存在标准实验室周（CLEA日本公司，东京，日本）。实验室动物按照国家卫生研究院的指导方针进行治疗。
1. 淋巴细胞的收集和分离
注:由于淋巴细胞必须是新鲜的，不能储存，因此每次实验都必须从大鼠那里收集淋巴细胞。此外，肠道热带淋巴细胞必须直接从它们循环的胸管中收集。所有程序预计将在 6 小时内完成，所有仪器、手套和表面均清洁。为了使动物保持生理稳定状态，实验中使用的所有盐水和其他液体都应保持温暖。
<ol><li>将大麻管浸入热水（约80-90°C）后，形成一个像肝素曲线（半径约5毫米）一样的制罐管（直径1毫米），并提前几个小时用胶带将其固定在塑料板上。</li>
	<li>麻醉雄性威斯塔大鼠（8-12周），腹腔内注射米达佐兰（2毫克/千克）、多米托（0.15毫克/千克）和贝图尔帕尔（2.5毫克/千克）的混合物，并用剪发器剪下腹部的体毛。剃须后牢固地取出头发，使头发不会进入腹腔。</li>
	<li>在确认手术所需的麻醉深度稳定后，通过脚趾捏捏，从中线到左下部区域水平用手术剪刀切口。小心不要通过观察光下的血管来损坏腹膜内的血管。</li>
	<li>用湿纱布包裹胃部，将胃轻轻地移到身体的右侧，以揭示反胃器官。使用手术剪刀在脊柱直立肌肉和脂肪组织之间插入一个缺口，并用手指将它们直接分离。</li>
	<li>小心地剥去胸管周围的结缔组织。多余的结缔组织会随着年龄的增长而增加。从隔膜的左十字下方露出胸管，直到可见胸管的20毫米长度。使用精密钳子或湿棉棉签轻轻地将胸管和主动脉之间的粘附物分开。<ol>
<li>仔细执行这个过程，因为一些大鼠有动脉从腹部动脉分支穿过胸管或短胸管由于淋巴丛（小胸管像蜘蛛网一样蔓延）的较高位置。避免不必要的接触胸管，以避免诱发水肿。</li>
	</ol>
</li>
	<li>在隔膜左十字下方的胸管上用绳子（3-0丝）涂上连结。胸腔胸管变得分散。</li>
	<li>通过刺穿手术剪刀的尖端，通过发夹弯曲的针管，并在某一点上将管子放到 iliopsoas 肌肉上，在腹部壁上打一个洞（直径 5 毫米）。用含有10U/mL肝素的普通盐水填充罐装管。</li>
	<li>在松开的胸管下放置一根绳子（3-0根丝绸）后，用罐管的锋利边缘刺穿胸管，将其朝向尾巴约5毫米，用罐装管将胸管固定。</li>
	<li>为了提供盐水以防止脱水，使用精密钳子在胃的蚂蚁前壁上创建一个孔（直径3毫米），并通过火焰环将硅管（直径2毫米）传给十二指肠。缝合伤口并维持波尔曼笼中的动物后，开始以3mL/h的流动速度从硅管中将含糖盐水注入每只老鼠的十二指肠。用纸巾盖住整个笼子，让它保暖。</li>
	<li>将制冷管固定在冰上含有6 U/mL肝素、10%胎儿牛血清和RPMI 1640介质（pH 7.4;见材料表）的50mL圆锥管盖的孔中，并在冰上收集胸管淋巴细胞（TDLs）。小心不要将管子的尖端接触到圆锥管的壁上，以防止由于管内纤维蛋白血块的形成而导致的罐管闭塞。淋巴液（约20 mL），包括约1.0 x 108~109淋巴细胞可以在6小时内获得。为了在完全麻醉下获得淋巴细胞，对麻醉平面进行监测，并适当观察淋巴流量。同样的麻醉剂在老鼠被放入鲍尔曼的笼子后大约3小时以同样的剂量添加，以连续接受6小时的深度麻醉。</li>
</ol>2. 淋巴细胞标签与卡盒氟雷西汀二乙酸苏奇米迪尔酯 （CFDSE）
<ol><li>溶解 CFDSE （材料表） 在二甲基硫化物 （DMSO） 到 15.6 mM （500μg 的 CFDSE 溶解在 60 μL 的 DMSO）。</li>
	<li>在RPMI 1640的50 mL中孵化淋巴细胞（1 x 108~109），在37°C下孵化50微升的CFDSE溶液，30分钟，如前10次所述。</li>
</ol>3. 微血管研究实验设置
<ol><li>麻醉接受者大鼠（8-12周）与腹膜注射的混合物米达佐兰（2毫克/公斤），多米托（0.15毫克/公斤）和贝图尔帕尔（2.5毫克/公斤），并通过脚趾捏确认麻醉的深度。在梳理毛皮之前将腹部弄湿，以尽量减少头发污染。然后通过中线切口打开接受者威斯特大鼠的腹部。</li>
	<li>将鼠放在便携式不锈钢板上（约 120 x 300 mm），中心周围有一个长方形孔，上面覆盖着玻璃滑梯（24 x 50 mm）。选择约10厘米的伊利尔段，包括PP进行观察。</li>
	<li>保持肠道尽可能温暖和潮湿与磷酸盐缓冲盐水（PBS）加热到37°C。 浸泡纱布，用于覆盖小肠与PBS。</li>
	<li>同时用2%的异氟化物进行连续麻醉（见材料表），将滑梯放在显微镜的舞台上，并选择合适的区域来观察一些高清病毒穿过血清的PP中的微循环。PP 的大小范围;较大的适合使用 CLSM 观察微循环。小肠在有些地方不直:一个至少2厘米长的直段，没有任何张力是适合观察。</li>
	<li>覆盖相邻的肠道部分，用浸泡在PBS的吸水棉中浸泡。将肠道部分放置在两个轧制的棉球之间，并尽可能将其从大鼠体内放置，以防止它被老鼠的心跳和呼吸振动。</li>
	<li>使用 1 mL 注射器，缓慢地将 （超过 1 分钟） CFSE 标记的 TDLs （1 x 108 细胞） 注射到受体大鼠的血管中。快速静脉注射可能会影响系统循环。</li>
</ol>4. 淋巴细胞的微循环
<ol><li>使用 CLSM 持续监控 PP 微血管中的 TDL，并每隔 30 分钟在计算机上使用延时摄影记录 3 小时。从塞罗萨到PP的 HEV 深度约为 25μm，使对频闪和毛细血管的观测深度达到 30μm。</li>
	<li>将得克萨斯红-德克斯特兰（25毫克/千克）注射到每个受体大鼠的血管中，以弄脏血液，将霍赫斯特33342（5毫克/千克）染色细胞核。</li>
	<li>（可选）要量化淋巴细胞动力学，将粘附于 HEV 的淋巴细胞定义为"粘合淋巴细胞"，将从 HEV 迁移到频闪的淋巴细胞定义为"迁移淋巴细胞"。然后计算每个视野（约0.3毫米2）迁移的平均百分比（迁移淋巴细胞/粘合淋巴细胞+迁移淋巴细胞）。</li>
</ol>

<ol>
	<li>Miura, S., Hokari, R., Tsuzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunity+in+gut+and+lymphoid+cell+trafficking.">Mucosal immunity in gut and lymphoid cell trafficking.</a> Annals of Vascular Diseases. 5, 275-281 (2012).</li><li>Stein, J. V., Nombela-Arrieta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+control+of+lymphocyte+trafficking:+a+general+overview.">Chemokine control of lymphocyte trafficking: a general overview.</a> Immunology. 116, 1-12 (2005).</li><li>Yopp, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine-1-phosphate+receptors+regulate+chemokine-driven+transendothelial+migration+of+lymph+node+but+not+splenic+T+cells.">Sphingosine-1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells.</a> The Journal of Immunology. 175, 2913-2924 (2005).</li><li>Lucke, S., Levkau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+functions+of+sphingosine-1-phosphate.">Endothelial functions of sphingosine-1-phosphate.</a> Cellular Physiology and Biochemistry. 26, 87-96 (2010).</li><li>Bollman, J. L., Cain, J. C., Grindlay, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+the+collection+of+lymph+from+the+liver,+small+intestine,+or+thoracic+duct+of+the+rat.">Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat.</a> The Journal of Laboratory and Clinical. 333, 1349-1352 (1948).</li><li>Bhalla, D. K., Murakami, T., Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculation+of+Intestinal+Lymphoid+Follicles+in+Rat+Peyer's+Patches.">Microcirculation of Intestinal Lymphoid Follicles in Rat Peyer's Patches.</a> Gastroenterology. 81, 481-491 (1981).</li><li>Miura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Demonstration+of+Sequential+Migration+Process+of+Lymphocyte+Subpopulations+in+Rat+Peyer's+Patches.">Intravital Demonstration of Sequential Migration Process of Lymphocyte Subpopulations in Rat Peyer's Patches.</a> Gastroenterology. 109, 1113-1123 (1995).</li><li>Tomura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cellular+movement+in+vivo+with+photoconvertible+fluorescence+protein+"Kaede"+transgenic+mice.">Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice.</a> Proceeding of the National Academy of Sciences of the United States of America. 105, 10871-10876 (2008).</li><li>Schulz, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophy+of+Infected+Peyer's+Patches+Arises+From+Global,+Interferon-Receptor,+and+CD69-independent+Shutdown+of+Lymphocyte+Egress.">Hypertrophy of Infected Peyer's Patches Arises From Global, Interferon-Receptor, and CD69-independent Shutdown of Lymphocyte Egress.</a> Mucosal Immunology. 7, 892-904 (2014).</li><li>Higashiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-Selectin-Dependent+Monocyte+Recruitment+Through+Platelet+Interaction+in+Intestinal+Microvessels+of+LPS-Treated+Mice.">P-Selectin-Dependent Monocyte Recruitment Through Platelet Interaction in Intestinal Microvessels of LPS-Treated Mice.</a> Microcirculation. 15, 441-450 (2008).</li><li>Shirakabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+colitis+through+blocking+lymphopcytes+entry+to+Peyer's+patches+by+sphingosine-1-phosphate+lyase+inhibitor.">Amelioration of colitis through blocking lymphopcytes entry to Peyer's patches by sphingosine-1-phosphate lyase inhibitor.</a> Journal of Gastroenterology and Hepatology. 33, 1608-1616 (2018).</li><li>Cenk, S., Thorsten, R. M., Irina, B. M., Ulrich, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy:+Visualizing+Immunity+in+Context.">Intravital Microscopy: Visualizing Immunity in Context.</a> Immunity. 21, 315-329 (2004).</li><li>Alex, Y. C. H., Hai, Q., Ronald, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+the+Landscape+of+In+Vivo+Immunity:+Insights+from+Dynamic+In+Situ+Imaging+of+Secondary+Lymphoid+Tissues.">Illuminating the Landscape of In Vivo Immunity: Insights from Dynamic In Situ Imaging of Secondary Lymphoid Tissues.</a> Immunity. 21, 331-339 (2004).</li></ol>

作者没有什么可透露的。

在这里，我们描述了收集天真的肠道热带淋巴细胞和观察它们在大鼠PPS中的迁移的协议。这些程序可以揭示淋巴细胞在PP的微血管中如何移动，并使得在正常或用药条件下对它们的动态进行视觉比较成为可能。直接观察这些动态有很多优点，以获得一些药物的免疫修饰的线索，虽然观察期仅限于几个小时。
我们在方法中提到了一些提示。其中，最微妙的程序之一是操作员必须...

佩耶的斑块（PPs）由小肠的拉米纳柱状物中的数百个淋巴状毛囊组成。PPs分为卵泡、间曲区域和位于毛囊下部的细菌中心，其中淋巴细胞受到抗原表现的刺激。没有发泄淋巴血管，抗原通过上皮细胞层从肠道流明侵入拉米纳丙酸。覆盖淋巴卵泡的上皮区域称为卵泡相关上皮，其中专门穿插M细胞吸收粘膜抗原。M细胞从发光侧摄取抗原，然后由支气管细胞捕获抗原，并呈现给通过高内皮静脉（HEV）1的内皮流入PP的天真淋巴细胞。PPs在肠道免疫中起着重要作用，与炎症的早期阶段有关。许多分子相互作用涉及淋巴细胞进入二级淋巴器官（SLOs），包括粘附分子、化疗因子2、3和磷酸-1-磷酸盐4:因此，有许多预期的治疗目标。因此，观察PP内淋巴细胞动力学使我们能够瞥见炎症的早期阶段，并检查几种有希望的药物的用处。
这里的方法侧重于PP中淋巴细胞的迁移，其中包括几个程序（可以进入胸管5， 收集淋巴细胞和注射到收集淋巴细胞后的长期观察）。由于这些程序很复杂，很难确切地看到以前的报告中每个程序是如何执行的，因此我们在这里提到了一些实现成功观察的技巧。例如，将管子插入胸管非常困难，制精的初始成功率不到50%。然而，我们改进了方法，成功率超过80%。我们在此手稿中提到了一些其他提示，这些提示对于成功观测以实现对淋巴细胞在若干条件下的跨内皮迁移进行定量评估是必要的。
在以前的报告中，很难理解随着时间的推移的三维变化，如静脉注射印度墨水染色PP6的血管结构，或显微镜是单焦7。近年来，利用凯德小鼠等一些光可转换荧光蛋白转基因动物的观察方法，澄清了体内的系统细胞运动。另一项研究澄清了CD69从PP9独立关闭淋巴细胞出口。由于其高分析能力，我们使用共分体激光扫描显微镜 （CLSM）。现在，我们可以很容易地获得高分辨率图像，并用它来分析淋巴细胞动力学。
在这份报告中，我们演示了一系列评估PP淋巴细胞迁移的方法。首先，我们展示了收集淋巴细胞的胸管罐装的精制方法。其次，我们改进了观察方法，在微观观测下尽可能保持客观器官，从而能够获得3小时的高质量图像。第三，我们量化淋巴细胞迁移的细胞运动，以评估某些药物的影响。这些修改后的协议将有助于开展粘膜免疫学评估。

<table border="0" cellpadding="0" cellspacing="0" style="width:913px;" width="913">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">A1R+</td>
			<td style="width:213px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:265px;">Comfocal Laser Scanning Microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxyfluorescein diacetate succinimidyl ester</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechest 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Wako Pure Chemical Industries</td>
			<td style="width:119px;">099-06571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium</td>
			<td>GIBCO</td>
			<td style="width:119px;">11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Texas Red&#8211;dextran</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">D1863</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopic observation of lymphocyte dynamics in rat peyer's patches

天真的淋巴细胞在生理条件下从血液循环到淋巴组织，被公认为肠道免疫中的一个重要现象。二级淋巴细胞器官的频谱，如Peyer的斑块（PPs）或中性淋巴结，是天真淋巴细胞感知抗原的地方。天真的淋巴细胞通过血液循环，以达到高内皮静脉，进入PP的入口。据估计，一些免疫调节器会影响淋巴细胞迁移，但对微循环动力学的精确评估非常困难，建立体内淋巴细胞迁移观测方法有助于澄清精确机制。我们改进了从淋巴管中收集淋巴细胞的方法，并观察了大鼠PP中肠道淋巴细胞的详细动态。我们选择了折叠式激光扫描显微镜来观察体内的老鼠PP，并使用延时摄影进行记录。我们现在可以获得清晰的图像，有助于淋巴细胞动力学的分析。

从淋巴中收集淋巴细胞 要为大鼠准备胸管罐头，如 图1 所示，在紧张的胸管中切口，然后将大鼠维持在波尔曼的笼子中，如 图2所示。
当淋巴细胞收集良好时，我们可以获得约20 mL/6 h淋巴液，含有约107~108/mL淋巴细胞。在 TDL 中，70% 的快速 CD4，其中约 90% 的细胞表示 CD62L，这意味着 TDL 的主要组成（+6...

Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

在这里，我们描述了一个精确的方法，收集胸管淋巴细胞和观察肠道热带淋巴细胞在老鼠Peyer的补丁的迁移3小时使用延时摄影。这项技术可以澄清淋巴细胞的动态在炎症条件下是如何受到影响的。

鼠佩耶斑块中淋巴细胞动力学的微观观察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

microscopic-observation-of-lymphocyte-dynamics-in-rat-peyer-s-patches

Research

JoVE Journal

Medicine

3.9K 次观看.  National Defense Medical College. 在这里，我们描述了一个精确的方法，收集胸管淋巴细胞和观察肠道热带淋巴细胞在老鼠Peyer的补丁的迁移3小时使用延时摄影。这项技术可以澄清淋巴细胞的动态在炎症条件下是如何受到影响的。

视频: Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

科研

医学

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

几内亚猪模型心肌损伤后 hiPSC-derived 衍生心肌贴片的植入

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

哮喘鼠模型中的针灸

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

通过在小鼠心脏中应用光遗传学多位点光刺激进行高级心律管理

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

The Microscopic Transcanal Approach in Stapes Surgery Revisited

The microscopic transcanal (aka transmeatal) surgical approach was first described in the 60s, offering a minimally invasive means of reaching the external auditory canal, the middle ear, and epitympanon. Such an approach avoids a retroauricular or endaural skin incision; however, working through a narrow space needs angled microsurgical instruments and specific training in otologic surgery. The transcanal approach restricts the working space; however, it offers a binocular microscopic vision into the middle ear without extended skin incisions and thus, reducing post-operative pain and bleeding. In addition, this minimally invasive approach avoids scar tissue complications, hypoesthesia of the auricle, and potential protrusion of the pinna. Despite its numerous advantages, this method is still not routinely performed by otologic surgeons. Since this minimally invasive technique is more challenging, there is a need for extensive training in order for it to be widely adopted by otologic surgeons. This article provides step-by-step surgical instructions for stapes surgery and reports possible indications, pitfalls, and limitations using this microscopic transcanal technique.

This article describes the microscopic transcanal technique for stapes surgery, providing step-by-step surgical instructions for familiarizing surgeons with this approach.

重新审视镫骨手术中的显微经管入路

The microscopic transcanal (aka transmeatal) surgical approach was first described in the 60s, offering a minimally invasive means of reaching the ...

Microscopic Replantation of Penile Glans Amputation Due to Circumcision

Circumcision using a disposable stapler is becoming quite popular in China. However, improper surgical procedures also bring the risk of penile glans amputation, which is a very rare iatrogenic genital injury. Such complication is conventionally treated by simple hemostasis to achieve self-healing, early gross replantation, or delayed plastic surgery. However, these may lead to obvious unfavorable outcomes such as amputated glans loss, necrosis, malformation healing, or urethral orifice stenosis. In the present study, we adopted microscopic replantation as an emergency approach to achieve the precise anastomoses and anatomic reconstruction of penile glans. The goal of this protocol is to present a detailed emergency management strategy with meticulous surgical skills for the penile glans amputation. The postoperative results showed that the original shape of the glans was perfectly restored with satisfactory cosmetic appearance. The micturition function was completely restored to normal without any obvious complications. There was also no significant reduction in the sensation of amputated glans area. Hence, early meticulous microscopic replantation as soon as possible is an ideal emergency management strategy for the penile glans amputation due to circumcision.

The present protocol describes the emergency management of microscopic replantation of penile glans amputation due to circumcision.

包皮环切术导致截肢的显微镜再植

Circumcision using a disposable stapler is becoming quite popular in China. However, improper surgical procedures also bring the risk of penile glans ...

Microscopic Cyst Resection for the Treatment of Patients Diagnosed with Epididymal Cyst

Epididymal cysts mostly occur in men aged 20-40 years old. Previous reports have covered concerns about postoperative complexes, including postoperative asoedema, hematoma, sustaining pain, and seminal tract obstruction in patients who have undertaken nonmicroscopic epididymal cyst resection or epididymal resection. Nonmicroscopic epididymal cyst surgery is suggested for patients with childbirth plans as a precaution. The treatment of male epididymal cysts via microtechnology is obviously a beneficial option; we took the lead in carrying out microscopic epididymal exploration and cyst resection surgery in China. From September 2017 to April 2021, 41 young and middle-aged male patients diagnosed with epididymal cysts underwent microtechnology treatment in a program titled &#34;microscopic epididymal exploration and cystectomy&#34;. The postoperative follow-up lasted for 3-50 months. The results confirmed that, as microscopic manipulation largely improved visualization of the subtle tissue structures of the epididymis, the cyst could be clearly dissected apart and completely removed intact under the microscope. Bleeding during the operation was significantly reduced (2-3 mL) and wound drainage was not required. According to follow-up data, microscopic treatment significantly reduced the incidence of postoperative scrotal hematoma, edema, and long-term postoperative pain, thereby promising a higher surgical success rate as well as recurrence prevention. Besides, preliminary experience and reflection suggest that microscopic epididymal exploration and cystectomy provide efficient preservation of the epididymal patency through refined treatment, while a better prognosis can be achieved. We recommend that surgery be carried out before the epididymal cyst develops to 0.8 cm in diameter, for fear that a larger epididymal cyst (&#62;0.9 cm in diameter) could cause the complete destruction of all tubules of the ipsilateral epididymis - a more severe case with damage to the testicular output network.

Here, we present a protocol to treat patients diagnosed with an epididymal cyst using microscopic cyst resection. Based on microscopic manipulation, a largely improved visualization of the subtle tissue structures of the epididymis was obtained, so the cyst could be clearly dissected apart and completely removed intact.

显微镜囊肿切除术治疗诊断为附睾囊肿的患者

Epididymal cysts mostly occur in men aged 20-40 years old. Previous reports have covered concerns about postoperative complexes, including postoperative ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

异位灌注异位心脏移植大鼠模型

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

大鼠外套增强大鼠电针期间的悬液固定

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.

作者聚焦：改善电针期间大鼠固定的高效大鼠服装 

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

在大鼠针灸实验中使用大鼠夹克改进悬吊固定

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...