这项工作得到了印度政府生物技术部向新德里国家免疫学研究所提供的核心赠款的支持。巴塔查尔吉博士目前的讲话是胃肠病学、肝病和营养科, 洛杉矶儿童医院。

本议定书所载程序已由新德里国家免疫学研究所机构动物伦理委员会批准。审批的串行参考编号为 IAEC#319/13.
注: 一般手术程序有很好的资源20和针对啮齿动物手术的特定协议21。对于那些第一次做动物外科手术的人来说, 建议在对动物进行手术前, 广泛地练习使用假的程序。
1. 准备
<ol><li>在试验之前, 保持无菌磷酸盐缓冲盐水 (PBS) 或盐水准备好。</li>
	<li>组装一个包括剪刀, 锯齿钳, 组织钳, 棉花, 棉花芽, 尼龙螺纹, 和不同的微型针持有人的手术套件。高压釜的手术套件。如果免疫受损的点头, 应特别小心。协议中包含了 "免疫" 鼠标。</li>
	<li>执行完整的实验程序, 从准备到手术结束, 在生物安全类 I 型橱柜。</li>
	<li>重点头。手术前6-8 周大鼠。在这项研究中使用了体重在14-18 克之间的老鼠。</li>
	<li>用剪毛机刮上中央和有疑的腹部区域的小鼠。将脱毛膏均匀涂抹在整个区域, 用刮刀彻底除去修剪过的毛发。2-5 分钟后, 用一块湿不育棉轻轻地去除毛发。</li>
	<li>将鼠标放在异氟醚腔内, 并解锁氧气缸的阀门。保持氧流的速率为4升/分和异氟醚汽化4% 诱导麻醉。<ol>
<li>通过轻柔的脚趾捏, 确保鼠标被正确地麻醉。</li>
	</ol>
</li>
	<li>在生物安全柜内放置一个手术板。把动物放在手术板上, 这样鼠标的腹部就会朝上, 老鼠的前部被放在与异氟醚和氧气供应相连的鼻锥内。</li>
	<li>减少异氟醚汽化至 2%, 并保持整个手术过程。</li>
	<li>对老鼠的皮肤进行消毒, 用70% 乙醇浸泡的无菌棉擦拭消毒。</li>
</ol>2. 手术方法
<ol><li>
部分肝切除术
	<ol>
<li>在胸骨下方的皮肤上进行横向切口, 垂直于剑突过程, 并与胸腔平行, 在直操作剪刀的帮助下。</li>
		<li>用镊子或无菌湿棉小贴士轻轻地将与腹部肌肉层相连的皮肤与皮肤和腹部肌肉层分开。用无菌棉花小贴士浸泡皮区, 避免脱水。</li>
		<li>通过在剑突下方的腹膜层进行横切口, 使左叶的面积平滑。使用两个湿润的棉花小贴士来暴露和举起肝脏的左叶。</li>
		<li>将棉花尖放在切口的腹部一侧, 再将另一条棉花尖放在隔膜侧。轻轻按下放置在隔膜上的尖端, 并给出一个滑动推的另一尖端, 以解除肝脏左叶。</li>
		<li>通过在微钳或棉花小窍门的帮助下, 通过在左叶上滑动一条带环的尼龙线, 并将环滑向靠近脐部的左叶底部。轻轻地向下推尼龙线循环到底部的左叶。</li>
		<li>用显微外科针架和微钳将尼龙线的两端系在左叶顶部。在另一侧增加两个结节。</li>
		<li>用剪刀把绑的裂片解剖出来。不要试图切断非常接近线程。如果手术时间超过5分钟, 保持腹膜腔和器官湿润与无菌 PBS, 以避免因液体流失而脱水。</li>
		<li>用4-0 线线缝合法缝合腹膜。随后, 通过不连续缝合, 尽快关闭皮肤。</li>
	</ol>
</li>
	<li>
细胞移植 注: 根据李et(C57BL/6-Tg) 所描述的程序, 荧光蛋白表达肝细胞与转基因 gfp 小鼠 (30 Scha/J) 分离。22和沈et al。23与单核细胞 (24 ) 分化的人源肝样细胞 (NeoHep) 也用于移植。然而, 从任何其他来源获得的细胞也可以在议定书中使用。<ol>
<li>在 Iscove 修饰 Dulbecco 的培养基 (IMDM) 的大约50µL 中悬浮20万个可行细胞, 并将其吸入1毫升胰岛素注射器, 30G 针。把注射器放在冰上, 把它放凉。</li>
		<li>放置鼠标的方式, 左侧面部分朝上执行手术的人。确定脾区, 横向解剖周围的忧郁症地区的皮肤, 其次是通过腹膜层的短切口, 只是为了暴露脾脏。</li>
		<li>轻轻抬起脾脏, 并在两个 PBS 湿棉花小贴士的帮助下, 把它放在腔外。</li>
		<li>一只手用两根棉花尖小心翼翼地握住脾脏, 将注射器的针头精确地垂直于脾脏。轻轻刺穿脾脏, 将针慢慢地推入体内;针不应该比2毫米更深。</li>
		<li>慢慢推下注射器的活塞, 将细胞注入脾脏。移植后, 保持注射器针稳定, 并从脾脏中慢慢取出, 以避免出血或细胞丢失。</li>
		<li>用棉尖将脾脏放回腹膜腔后, 通过连续缝合4-0 条线缝合腹膜层。用同样的缝线缝合皮肤间断。避免使用伤口夹来闭合皮肤;相反, 用4-0 缝合线关闭它。伤口剪辑限制了鼠标的自然运动, 经常剪辑变得松散, 迅速出来。</li>
	</ol>
</li>
</ol>3. 术后护理
<ol><li>关闭皮肤后, 使用无菌棉尖, 用碘溶液 (betadine) 擦拭缝合部位的周围环境。</li>
	<li>注射一剂量抗生素头孢噻肟为600毫克/千克体重 (通常12毫克头孢噻肟100µL 生理盐水/小鼠) 腹腔使用1毫升注射器。</li>
	<li>每日剂量止痛 Meloxicam 为1毫克/千克体重 (通常12µg Meloxicam 100 µL 生理盐水/小鼠) 到动物腹腔, 在手术后三天。</li>
	<li>手术完成后, 停止异氟醚气体的流动, 把鼠标放回单独通风的笼子里。</li>
</ol>4. Euthanization 和刻画
<ol><li>实验结束后 (1 天10天后手术), 弄死小鼠根据机构动物伦理准则。</li>
	<li>收集血液由末期流血的动物, 刺穿眼球末端丛。</li>
	<li>从血液中分离出血清。</li>
</ol>

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作者没有什么可透露的。

部分肝切除术是一种研究肝再生的方法, 据报道过度肝切除是模拟 AHF 模型。在 AHF 动物模型中, 啮齿动物, 特别是小鼠, 是研究最多的模型。为了获得小鼠肝脏损伤模型, 多达70% 的肝切除已报告具有良好的存活率25,26。然而, 在裸和其他 immunodeficient 鼠, 70% 例肝切除被报告为致命, 动物死亡24小时内27。
米切尔和 Willenbring28展示了一种可重复和良好耐受的方法, 2/3 小鼠部分肝切除。点头。免疫缺陷小鼠, 我们获得了100% 的生存率时, 肝切除被限制到切除左肝叶, 这是接近30% 的总肝脏质量。根据对裸鼠的观察27, 我们观察到在点头时肝切除的百分比会进一步增加。老鼠的生存率会急剧下降。此外, 其余肝叶中肝细胞增殖1天30% 部分肝切除证实了本程序在移植和植入研究中的应用。
在最近的一篇论文中, 艾哈迈德 S.U. et 。29显示了在 immunodeficient 小鼠肝内移植肝细胞癌的程序。他们展示了移植肿瘤细胞的程序, 包括脾脏在内的各种器官, 并进行肝切除以促进肝内植入。
在米切尔28和艾哈迈德29报告的过程中, 由于手术中通常首选较小的切口, 因此有机会进行更小的切口。有证据显示30 , 较小长度的手术切口会导致压力荷尔蒙分泌减少, 如皮质醇和儿茶酚胺。此外, 我们发现, 涉及一个更大的切口的程序需要更高水平的技能, 并更难以重现, 相比的程序有两个较小的切口。
在本文中, 我们已经描述了一个程序, 其中要求最小切口暴露的左叶的肝脏, 并在执行30% 肝切除后, 另一小切口, 以暴露脾脏的细胞被植入。本程序不需要任何专门的外科手术技能, 并可在5-7 分钟内完成。此外, 我们没有发现任何证据显示任何形态学或解剖异常的其余肝脏肿块, 如组织学研究证明。此外, 6-8 周大的免疫受损小鼠在手术过程中没有缺血或坏死。部分肝切除术后肝损伤的诱导是由高浓度的肝脏酶、ALT 和 PHOSin 小鼠血清所证实的。由于门静脉系统比其他静脉系统具有更高的可进入性, 因此选择了在其他静脉通路上进行细胞移植的脾路线。我们已经证明了移植的肝细胞从转基因 GFP 小鼠和 NeoHep 是分化的肝细胞样细胞的人的起源, 在部分 hepatectomized 点头。动物。该过程不限制用于移植的细胞类型。
但是, 此过程不会在鼠标中创建 AHF 条件, 因为只执行30% 部分肝切除。这种有限的肝切除为肝脏中存在的肝细胞提供了增殖潜能, 从而为植入移植肝细胞提供了更多的机会。它只说明了肝细胞的迁移和植入, NeoHep 从脾脏到肝脏, 并没有对肝脏造成额外损害的移植程序。
总之, 这个过程很简单, 可以很容易地被实践和掌握, 以获得可重现的结果。在肝损伤或肝再生研究中, 几种细胞源 (干细胞或肝样细胞) 的再生潜能可以很容易地用这种手术方法来评价。

目前, 肝细胞移植是一种替代全器官移植治疗严重肝病患者。据信, 它可以把病人移植到全身器官,1。除了同种异体肝细胞2外, 异体肝细胞3和干细胞 (4 ) 的肝细胞也正在动物模型中进行研究。在这一背景下, 受体移植细胞的归巢和植入电位是急性肝功能衰竭 (AHF) 细胞治疗的重要标准。
为研究肝细胞或肝样细胞的移植5, AHF 是由手术的6或药理7程序在动物模型中创建的, 其次是移植细胞。通过药理试剂制作 AHF 动物模型, 许多 hepatotoxins 如 d-乳糖8、乙酰氨基酚9、四氯化碳10、硫乙酰胺11、刀豆蛋白 12、脂多糖13已使用、等。从这个列表中, 每个试剂为 AHF 生成一组独特的功能, 但不幸的是, 没有单一试剂模仿人类 AHF。此外, hepatotoxins 诱发的 AHF 时间长, 使动物处于慢性应激状态, 难以获得重现性结果。
另一方面, 部分肝切除术 (phx-空港) 的手术方法是技能依赖性的, 在培养所需技能后, 可重现性结果容易获得。仅以手术干预诱发 AHF, 需要切除超过70% 的肝脏;然而, 70% 的肝切除术仍可用于研究肝脏移植细胞的植入和增殖, 以分析其在肝损伤期间的治疗能力14。肝细胞移植术后经腹膜切除后, 经腹腔15、尾静脉16、肝静脉17或脾18。目前, 肝静脉输液和肝细胞脾移植是首选的程序, 因为它们更容易繁殖。
在本文中, 我们已经描述了30% 部分肝切除在点头的程序。(点头。CB17-Prkdcscid/J) 肝左叶切除的小鼠。其次是移植 20万 GFP 表达小鼠 (C57BL/6-Tg (不列颠哥伦比亚) 30 scha/J) 肝细胞以及人类起源 NeoHep19在脾脏。这个过程导致肝脏移植细胞的植入。这个程序是最不具侵入性和轻微疼痛的技术。

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</table>

intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice

部分肝切除术是研究肝再生和细胞基础疗法在各种病理条件下的作用的一种多功能和可再生的方法。部分肝切除也促进移植细胞的植入和增殖, 加速新生血管和细胞向肝脏迁移。在这里, 我们描述了一个简单的协议, 执行30% 肝切除和移植细胞在脾脏的非肥胖糖尿病/严重联合 immunodeficient 点头。(点头。CB17Prkdc的/J) 鼠标。
在这个过程中, 做了两个小切口。第一切口是暴露和切除肝脏的左叶, 另一小切口被做暴露脾脏为脾移植细胞。这个程序不需要任何专门的外科手术技能, 它可以在5-7 分钟内完成, 减轻压力和疼痛, 更快的恢复, 更好的生存。我们已经展示了从绿色荧光蛋白 (gfp) 中分离出的肝细胞移植, 表达小鼠 (转基因 C57BL/6-Tg (不列颠哥伦比亚) 30 scha/J), 以及人源性肝细胞 (NeoHep) 在部分 hepatectomized 点头。老鼠。

30% 部分肝切除术后肝细胞增殖:用免疫组化 (IHC) 染色 Ki-67 细胞增殖标记物, 观察30% 肝切除术后剩余肝脏中肝细胞的增殖。一天后肝切除, 小鼠被安乐死, 其余肝叶切除, 石蜡切片获得。切片染色的 Ki-67 抗体, 其次是标签与辣根过氧化物酶 (HRP) 共轭二级抗体。以二氨基联苯胺为基质, 用于开发褐色色素以识别染色细胞。细胞核被用苏木精染色, 并在20X 物镜下观察。图 1显示了肝脏部分的代表性 IHC 图像。约13% 的细胞 (13.66 0.317, N=3) 是 Ki-67 阳性, 这证实了30% 肝切除促进肝细胞在小鼠肝脏的增殖。
解剖学研究:点头肝的代表形象。老鼠, 肝切除术后10天显示在图 2中。该图像证实, 其余的肝脏是健康的, 没有明显的异常。
在第一个小时的手术中存在移植的细胞:对移植后2小时内荧光蛋白阳性肝细胞的存在进行了流动细胞分析, 证实了移植细胞的归巢。
在酶消化2小时的 GFP-肝细胞移植后, 从宿主小鼠的脾和肝中获得单细胞悬浮液。使用流式细胞仪估计移植的 GFP 阳性细胞的百分比。从相应的 FSC-SSC 点图中选择了一个散射门, 以消除碎片和双峰细胞。该地块的象限门是由在没有移植细胞的控制小鼠的脾脏和肝脏中获得的细胞悬浮强度的背景 fluorochrome 产生的。脾脏中发现了大约1.7% 个 gfp 阳性肝细胞, 移植后2小时内未发现 gfp 肝细胞。代表性数据显示在图 3中。
免疫组化:术后十天, 对小鼠进行了安乐死, 右外叶、右内侧叶和肝左内侧叶切除, 并进行了低温切片, 获得5µm 切片。这些部分然后被检查为被移植的细胞的归巢和植入。图 4显示了对抗 GFP (red) 进行免疫组化 (IHC) 染色的代表性图像, 用于识别表达小鼠肝细胞的 GFP (面板A、 B和C);与抗人白蛋白 (红色), 以及抗人类连接蛋白 32 (绿色), 以识别肝细胞样细胞 (NeoHep) 的人类起源 (面板D, E, F, 和G)。在两种情况下, 细胞核的复染为 4 ', 6-diamidino 2-苯基吲哚 (DAPI, 蓝色)。在这些图像面板中, 一些表达肝细胞的嫁接 GFP (面板a、 B和C) 和 NeoHep (面板D、 E、 F和G) 明显可见。
肝脏分泌酶的生化分析:为了检查手术后肝脏的功能, 对不同的肝分泌酶进行了生化分析。图 5中的条形图表示健康点头的血清中天门冬氨酸转氨酶 (AST)、丙氨酸转氨酶 (ALT) 和碱性磷酸酶 (磷) 的平均值。小鼠和不同组的部分 hepatectomized 小鼠。从图中清楚地看出, 1 天的肝切除术后, 与健康小鼠相比, AST、ALT 和磷酶的水平显著增加。
ALT 和 AST 酶水平恢复正常, 而磷水平在非移植小鼠术后十天内保持高位。然而, 在移植小鼠10天后, 所有三种酶的水平都下降到正常。
肝脏组织学研究:对 hepatectomized 和移植小鼠的肝脏标本进行进一步的组织学分析, 研究手术后的解剖变化。图 6显示了有代表性的明亮场图像, 苏木精和伊红 (H 和 E) 染色肝切片的健康小鼠无手术, 1 天后部分肝切除和10天后部分肝切除和 NeoHep 移植。在这些图像中, 1 天的部分肝切除术后观察到轻度围胆管纤维化或结缔组织增生引起的肝脏损伤, 手术后十天没有发现异常。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56018/56018fig1.jpg"> 图 1: 点头的 Ki-67 染色的代表图像。肝切除术1天后肝脏切片 30%.切片染色 Ki-67 抗体, 辣根过氧化物酶 (HRP) 共轭二次抗体, 和民建联 (褐色) 作为 HRP 基质, 核复染的苏木精 (蓝色)。指示箭头指出了一些 Ki-67 阳性 (褐色) 细胞。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56018/56018fig1large.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/56018/56018fig2.jpg"> 图 2: 重新生成点头的肝叶。老鼠术后10天.A: 尾状叶 (尾状的过程), B: 右外侧叶, C: 右内侧裂片, D: 左内侧裂片, E: 心脏, F: 左外侧叶在肝切除后剩余, 和 G: 尾状叶 (乳突)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56018/56018fig2large.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/56018/56018fig3.jpg"> 图 3: 荧光蛋白阳性小鼠 heptatocytes 在脾脏和肝脏中的百分比。老鼠.上面的面板显示一个年龄匹配的控制鼠标的细胞剖面, 其中没有细胞移植。下部的面板显示2小时后, GFP 肝细胞移植 hepatectomized 鼠细胞剖面。图的 y 轴表示在对数刻度上以任意单位测量的 GFP (FITC 通道) 的荧光强度, x 轴表示线性尺度上任意单位的正散射 (FSC)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56018/56018fig3large.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/56018/56018fig4.jpg"> 图 4: 移植的 GFP 在再生宿主肝中表达小鼠肝细胞和 NeoHep 的归巢.部分 hepatectomized 点头的肝脏部分的代表性图像。免疫荧光蛋白阳性小鼠肝细胞移植 (A C), 显示细胞植入和归巢。小组 A: 核 (蓝色: DAPI), 小组 B: 抗 GFP (红色: Alexa 594), 小组 C: 合并的图象。面板 (D G) 显示部分 hepatectomized 点头的肝脏部分。NeoHep 移植的老鼠。小组 D: 核 (蓝色: DAPI), 小组 E: 人的抗连接蛋白 32 (绿色: alexa 氟 488), 小组 F: 人的抗白蛋白 (红色: Alexa 氟 594) 和小组 G: 合并的图象。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56018/56018fig4large.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/56018/56018fig5.jpg"> 图 5: 对小鼠血清中不同肝分泌酶的生化分析.柱条代表不同酶的平均值 (AST/SGOT, ALT/SGPT, 磷)。术后的时间点显示在 X 轴上。在对照组, 有年龄匹配点头。未进行手术的小鼠。误差线表示平均值的标准错误, N=3 和 * 指示p< 0.05, NS 表示p> 0.05 中的非意义。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56018/56018fig5large.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/56018/56018fig6.jpg"> 图 6: 小鼠肝组织切片的病理学研究.有代表性的明亮的现场显微图像的苏木精和伊红 (H 和 E) 染色肝组织部分点头。20X 物镜下的小鼠。小组 a 显示肝脏部分从健康点头。同一年龄的老鼠没有任何手术。小组 B 显示肝脏部分1天后部分肝切除术。在这个图像中, 箭头表明轻度胆道纤维化或结缔组织增生区。C 组显示肝脏部分十天后部分肝切除和细胞移植。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56018/56018fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

我们已经描述了一个协议, 执行部分肝切除 (phx-空港) 和细胞移植通过脾脏点头。(点头。CB17-Prkdc有缺陷的/J) 鼠标。在本议定书中, 切口是为了揭露和切除肝脏的左叶, 其次是另一个切口的脾移植细胞。

肝细胞部分肝切除术后脾移植。小鼠

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

intrasplenic-transplantation-hepatocytes-after-partial-hepatectomy

Nanyang Technological University

Research

JoVE Journal

Medicine

13.9K 次观看.  National Institute of Immunology. 我们已经描述了一个协议, 执行部分肝切除 (phx-空港) 和细胞移植通过脾脏点头。(点头。CB17-Prkdc有缺陷的/J) 鼠标。在本议定书中, 切口是为了揭露和切除肝脏的左叶, 其次是另一个切口的脾移植细胞。

视频: Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

科研

医学

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

颈椎异位心脏移植小鼠

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

猪自体移植肝细胞外细胞外细胞基因治疗

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

通过结合70%部分切除术和对乙酰氨基酚，生成急性肝衰竭大鼠模型

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

矫形肝移植大鼠模型中动脉重联后并发症减少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...