麦承认支持这项工作从国立卫生研究院和国家癌症研究所资助编号-1R15CA152985 01A1。这个项目也得到了奖学金计划和迈阿密大学学者暑期的迈阿密大学博士学位本科机会迈阿密大学本科教学科研奖励计划。

在视频中进行，如下所述的程序已获机构动物护理和使用委员会 (IACUC) 在迈阿密大学。
 1.制备及刺激试验 <ol><li>建立两个不同的区域，为外科手术、 手术台和术后表。舞台这两个领域与所有材料和必要的用具。
	<ol><li>阶段的操作表排气罩下。把桌子布置与设备允许程序的连续和不受阻碍地性能的方式。</li>
		<li>建立一个术后的表在同一个房间里和附近的主表的操作。保持这两个表作为整个过程的无菌环境。</li>
	</ol></li> <li>以下的地方用品在手术台上: 一个玻璃罐盖、 一 15 毫升管、 手术剪刀、 70%乙醇、 两个泡沫板、 两个钳、 两个 1 毫升 21 计注射器，两个 50 毫升管、 一个离心管一挤压瓶一对一个低温瓶、 四个手术垫、 保龄球、 一个配药的消毒湿巾，尖锐物品容器。</li>
	<li>在术后表上放置以下物品: 一个分析天平，一个杜瓦 L 月 4 日的液态氮，浅全口杜瓦瓶、 浮动的微细管架、 一把手术剪刀、 两个钳、 四个低温瓶和一个配药的无菌湿巾。</li>
	<li>一个 50 毫升和 75%卷一 15 毫升管装满福尔马林。</li>
	<li>在每个角落里，用一根针贴上一个手术垫到近似 30 厘米 × 30 厘米泡沫板作为夹层板。在操作过程中使用的剩余的四个针脚。准备将器官转移到术后表手术垫小泡沫板。 
	警告: 异氟烷 （99.9%) 是一种有毒的化学，和应使用在排气罩以确保最高安全级别的从清除废物的麻醉气体 8。有关研究人员开放 drop 方法用异氟醚的使用相关的风险的其他信息可以发现在一篇文章由泰勒和穆 8.</li>
	<li>将一个手术垫放入玻璃罐，用几滴异氟醚 （99.9%) 浸泡和上方以防止直接接触的异氟醚与鼠标放置一张纸巾。同样，使用手术垫线剩余管和浸泡用几滴异氟醚和附加垫置于顶端，以防止直接接触鼠标和异氟醚。</li>
	<li>倒进浅的宽嘴里杜瓦瓶直到最高填充线达到液态氮。</li>
	<li>为剥离至麻醉选择鼠标的地方分庭，即 玻璃罐用垫浸透几滴异氟烷 （99.9%) 盖上盖子，~ 1 分钟 
	注意: 此时间而有所不同从老鼠。一旦鼠标是无意识的删除它从分庭，然后将其放置在操作盘。</li>
	<li>东方鼠标，它躺在腹侧和与它的头朝外的科学家。将内管内衬与异氟醚 （99.9%)，滴几滴手术垫头部放和执行刺激测试脚夹以确保鼠标是对刺激反应迟钝。
	<ol><li>如果这个测试失败，并且鼠标响应脚夹点测试，请重复步骤 1.8。</li>
	</ol></li></ol> 2。最初的切口、 心脏穿刺，和安乐死 <ol><li>引脚鼠标到外科泡沫板的四肢和湿用 70%乙醇鼠标腹侧。</li>
	<li>用钳夹附近的尿道口的毛皮和轻微向上拉。用通过腹腔从尿道开放，中线开始和结束在下巴的手术剪刀做一个切口。
	<ol><li>在起始点附近的初始切口，抢一侧的毛皮的镊子和使另一个切口用剪刀沿对角线方向向下和向背爪子。</li>
		<li>重复此步骤以同样的方式在另一侧. 
		注意: 毛皮可以牵制打造一个更广泛的开头，但不是必要。</li>
	</ol></li> <li>找到心并清除心包，是周围的心，以避免堵塞针头的囊。
	<ol><li>钳子抓住心包膜，然后用剪刀剪。通过仔细地将针头插入心脏跳动执行心脏穿刺法，慢慢开始收回柱塞。</li>
		<li>最佳采血，使用针的柱塞模仿心脏的泵血动作，避免太快绘图。 
		注: 通常约 1 毫升的血可以收集。</li>
		<li>完成后采血，消除血液离心管和注射器的锐器盒内处置。</li>
		<li>后心脏穿刺口，实施安乐死删除附件连接心脏。 
		注: 肝素抗凝血剂，未添加到在此过程中心脏穿刺法，可以使血液凝结在这特定的研究中，血清集合之前注射器。但是，如果研究者想要防止血液凝固，血浆收集，肝素可以添加到前心穿刺注射器。</li>
	</ol></li> <li>如果这项研究涉及基因分型的鼠标，用剪刀剪耳朵的一部分并将放入离心管的基因型核查。</li></ol>
 3.胰腺提取 <ol><li>定位鼠标左侧的胃。轻轻地 （以避免撕裂） 开始使用两个钳，从胃部和十二指肠分隔胰腺。 
	注: 分离胰腺从胃和肠道时, 它是非常重要，轻轻地用镊子指导器官隔离的胰腺组织，不粉碎或撕裂胰腺的镊子。
	<ol><li>继续分离胰腺从小肠空肠和回肠部分，最后由大肠盲肠。</li>
	</ol></li> <li>在盲肠，复位钳，继续沿着剩余结肠直肠走向胰腺分离。 
	注: 在这一点上，它是方便剪切和删除的部分从胃里到该地区紧接直肠结肠。</li>
	<li>定位胰腺和附加脾。滑向右边的鼠标胰腺。用钳要完全分离胰腺和附加广义的脾脏分隔的胰腺和胸腔内的剩余联系。</li>
	<li>删除胰腺和展开检查。离开附加到用于识别目的胰腺脾。
	<ol><li>删除所有结缔组织、 脂肪和肠系膜组织从胰腺。 
		注: 这种组织是白的颜色，因此可以很容易区分，是粉色的颜色的胰腺组织。这是特别重要，如果整个胰腺需要被删除。例如，如果胰腺需要加以权衡和比较体重或动物群体之间。在 Ptf1a cre / +;LSL Kras G12D / + 小鼠模型，特别是在较旧的月份，硬纤维胰腺组织可能存在。在这种情况下，必须进行仔细去除的胰腺为肠道可能在肿瘤组织中交错式。在先进的情况下，异常脾和肝组织可能也会出现。</li>
	</ol></li> <li>如果需要，在这一点上删除其他的器官。</li></ol>
 4.数据收集和存储 <ol><li>解压后的器官，将样品移至术后区域保存。</li>
	<li>权衡每个器官和将它们放置到他们各自的低温瓶。 
	注: 沿机智h 质量的每个示例中，任何违规行为应记录以备将来参考。</li>
	<li>一旦重质器官放入液氮为管理单元冻结。</li>
	<li>后冻结的管理单元，存储长期储存在-80 ° C 器官。</li>
	<li>地方福尔马林保存样品在台阶顶上一夜之间，和第二天早上从福尔马林更改他们的解决方案，为 70%乙醇。 
	注意: 这些样品应储存在 4 ° C 的长期存储。</li>
	<li>为长时间存放，冻结在-80 ° C.血液和耳朵冲床。血清收集从血液，使血液凝结为 30 分钟，然后离心它。删除使用吸管的血清部分，然后存储在-80 ° C.</li>
</ol> 5。清理一下 <ol><li>Sanitize 所有解剖工具与消毒湿巾。帽管内衬异氟醚浸泡手术垫。用新鲜的手术垫替换手术垫泡沫板上。处置的部分鼠标不收集设施每 &#39; s 动物处置政策。</li>
</ol>

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作者没有透露。

重要的意义，现有的方法 虽然其他非正式的视频鼠标夹层动脉瘤存在，视频本文提供专业质量第一、 同行评审、 直观示范的所有详细的步骤所需的提取和收获的小鼠胰腺的解剖10.正在主要器官代谢活性和胰岛素的胰腺生产、 夹层和收获的胰腺允许保存的生理特性11。通过隔离胰腺，可能对样品进行未来的分析。这个程序允许从其他组织在相同的时间框架内同一机体内部的相互作用的研究与比较。
这项技术的局限性 此过程的最大限制是终止老鼠的生命，从而防止纵向集合和采样的多个组织样本从同样一只老鼠。为了分析有关年龄、 性别或其他量词的趋势，必须执行横断面的人口，我们已经为我们的胰腺癌代谢生物标志物的研究。本议定书的另一个局限是无法暂停程序。一旦启动的狗狗会被安乐死，程序必须在其整体进行。
议定书 》 中的关键步骤 用捏后的爪刺激测试执行关键是鼠标的要确保鼠标得到人道的待遇。如果鼠标不响应这种刺激，然后程序可能会按计划执行。然而，鼠标应该显示经过刺激试验的不良的反应，鼠标应返回到麻醉室为额外的时间和重复，直到对刺激试验的反应不观察12测试期。
同样，终端心脏穿刺法，其后在移走接驳至心终端血液样本收集因为安乐死第二种方法确保鼠标的人文牺牲后，立即。为了确保有效的抽血，科学家应当使用打气的动作是类似的心跳的鼠标，注射器允许的最大集合的血液分析。
修改和故障排除 切换到 70%乙醇溶液从福尔马林机关准备进行组织学分析所需的嵌入过程的机关。不同的存储解决方案可能需要应科学家选择与机关执行其他的实验。在分析之前，它是重要限制任何潜在的解冻的器官储存在-80 ° C 冰箱里保持器官的完整性。
使用 Ptf1acre / +;LSL KrasG12D / +小鼠模型最小化的非胰腺原发性肿瘤和疾病13发生。因此，它是重要的是注意是明显对胰腺或其他器官在解剖和分析组织样品的收集过程中的违规。
未来的应用程序 收获的小鼠胰腺的解剖允许多种类型的分析，对同一样品进行。最受欢迎的这些包括但并不仅限于荧光显微镜、 苏木精和曙红组织学、 免疫组化，质谱，核磁共振光谱学6，7，14,15.可以用上述16技术研究疾病如糖尿病、 胰腺炎、 胰腺癌。

鼠标已成为人类胰腺癌1，2重要动物模型。在 Ptf1acre / +;LSL KrasG12D / +小鼠模型，大鼠肉瘤 (K-Ras) 癌基因激活只在胰腺，导致胰腺，胰腺上皮内肿瘤 (PanINs)，这一进展被称为癌前病变引发的克里斯汀对胰腺导管腺癌，通常所说的 PDACs3。这个鼠标模型系统提供了一个人类胰腺癌4，5，与 PanINs 出现在首五个月的生活和频繁地进展 PDAC 内的附加优势的最佳动物模型单 44，5，而胰腺癌最频繁地出现在人类身上 60-70 岁的年龄。
由 Ptf1a 淋巴结清扫术胰腺提取cre / +;LSL KrasG12D / +小鼠不同年龄允许详细纵向组织学检查癌症发展的胰腺，从最早的 PanIN 阶段通过进展到 PDAC3，4,5.在年龄从五到十五个月收获胰腺可也用于准备组织提取物表征在胰腺4代谢在从健康到病变组织的过渡过程中发生的全球变化6，7。
本文介绍了一个完整的视觉指南执行鼠标胰腺提取所需的步骤并提供指导方针的胰腺，进一步分析存储。本指南将同样宝贵，为个人进行研究其他胰腺的疾病，包括 I 型糖尿病，和应该是特别有用的学生和研究人员的新研究涉及收获的小鼠胰腺使用清扫术代谢轮廓分析或组织学分析。

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
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			<td height="21" style="height:21px;width:216px;">Glass Jar&#160;</td>
			<td style="width:288px;">Corning</td>
			<td style="width:119px;">3140-150</td>
			<td style="width:167px;">The glass jar used in the video has been discontinued.&#160; This is its replacement.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lid of Glass Jar</td>
			<td style="width:288px;">Corning</td>
			<td style="width:119px;">9985-150</td>
			<td>The glass jar lid used in the video has been discontinued.&#160; This is its replacement.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 mL Falcon Tubes</td>
			<td style="width:288px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:216px;">Floating Microtube Rack</td>
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			<td height="21" style="height:21px;width:216px;">Cryogenic Vial 1.2 mL, Sterile</td>
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			<td height="21" style="height:21px;width:216px;">Isothesia (Isoflurane)&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Liquid Nitrogen</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse Kras Strain</td>
			<td style="width:288px;">The Jackson Laboratory</td>
			<td style="width:119px;">OO8179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse Cre Strain</td>
			<td style="width:288px;">MMRRC</td>
			<td style="width:119px;">OOO435-UNC</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of the mouse pancreas for histological analysis and metabolic profiling

我们一直在调查胰腺特异性转录因子、 1a-cre 重组酶;脂氧合酶-停止-脂氧合酶-克里斯汀大鼠肉瘤、 甘氨酸对天冬氨酸在 12 密码子 (Ptf1acre / +;LSL-KrasG12D / +) 鼠标应变作为人类胰腺癌模型。我们当前的研究的目标是找出新型代谢生物标记物的胰腺肿瘤。我们进行代谢过程的尿液、 粪便、 血液，和胰腺组织提取物，以及胰腺期癌的组织学分析。小鼠胰腺不是定义良好的实质器官，像人类，而是弥漫性分布的软组织不容易标识由个人熟悉鼠标内部解剖或有很少或没有表演经验的个人鼠标器官解剖。这篇文章的目的是提供一个详细的逐级直观示范指导新手在小鼠胰腺切除的解剖。这篇文章应该向学生和研究人员新需要收获的小鼠胰腺剥离术治疗代谢轮廓分析或组织学分析的研究尤为重要。

图 1显示的操作环境区的概述和图 2显示了岗位操作区域。虽然此设置提供最小数量的设备和分期，个人可以选择改变了这一点，最适合个人需要。议定书 》 应根据实验的具体需要进行优化。此过程以终止的一只老鼠，需要适当的狗狗会被安乐死9生活的方式进行。当研究者是准备好了时，鼠标放入麻醉室与异氟醚浸泡垫 (图 3)。
鼠标是无意识的一旦删除鼠标并将其背侧板上。应施行脚趾夹点的程序，以确保鼠标是响应疼痛 (图 4)。应用 70%乙醇消毒初始切口区。终端抽血必须在胰脏切除，以确保充足的血液检索之前首先，进行。血液搬迁之前应去除心包膜，以防止堵塞的开放 21 G 针。完成终端血后绘制、 心分离作为一种辅助方法的安乐死和胰腺然后删除。
开始通过定位胃、 胰脏切除手术 (图 5) 为提供一个良好的起点。注: 极端小心切除术后的胰腺，是微妙而脆弱的组织，并因此应与温柔的力量执行所有操作。使用镊子，由开始轻轻地从胃胰腺开始解剖和继续胰腺组织分开工作从胃到十二指肠、 空肠和回肠 ( (GI) 胃肠外衬图 6)。一旦达到了盲肠，容易拆卸的胰腺被通过复位钳，这样一个钳持有盲肠和其他钳用来继续胰腺分离大肠 (图 7)。删除后从大肠，胰腺放在右边的鼠标和任何剩余的附件是切断（图 8)。
应检查散开胰腺和任何异常应记录 (图 9)。在 Ptf1acre / +;LSL KrasG12D / +鼠标应变，胰腺可能包含硬的瘤 (图 10)。其他机构也应检查的潜在的转移。一旦胰腺已被删除，它应该权衡和记录重量。胰腺的一部分应该是管理单元-在液氮冷冻供将来代谢分析分析或其他测试和胰腺部分应放在福尔马林为未来的组织学分析。图 11显示各机关的初始存储从用于清扫收集在以后分析。机关由清扫收集并存储为进一步的研究将取决于个别研究者的目标。
可以用于组织和血液样本，进行组织学分析和代谢过程。胰腺组织的组织学分析示例如图 12所示。代谢轮廓分析可以进行管理单元冷冻组织样本和血液样本。胰腺组织提取物的亲水性和疏水性组件代表核磁共振光谱 (NMR) 光谱所示图 13A及13b 条，分别。收集关于血清样品制备从血液在末梢血画过程的时间收集代表核磁共振谱如图 14所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55647/55647fig1.jpg"> 
图 1: 分期的营业面积。正确的工具和解剖的操作条件的总体布局。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55647/55647fig2.jpg"> 
图 2: 分期手术后地区。正确的工具和术后的程序的操作条件的总体布局。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55647/55647fig3.jpg"> 
图 3: 麻醉室。通过异氟醚麻醉的适当环境。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55647/55647fig4.jpg"> 
图 4: 刺激考试。不被遭受刺激试验对小鼠前初始的切口，以确保任何疼痛或不适。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55647/55647fig5.jpg"> 
图 5: 开始清除胰腺。鼠标以指示的胰腺，由钳指示位置的初始萃取的方向。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig5large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55647/55647fig6.jpg"> 
图 6: 胰腺提取沿肠道。隔离的胃肠道胰腺的过程。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig6large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55647/55647 alt="Figure 7" class="xfigimg" src=""></img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55647/55647>fig7.jpg"/ > 
图 7: 在盲肠胰脏切除。重新定位钳一次盲肠被到达。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig7large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55647/55647fig8.jpg"> 
图 8: 胰脏切除手术。在右边的鼠标胰腺的地方。任何剩余的附件应削减完全删除胰腺。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig8large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55647/55647fig9.jpg"> 
图 9: 胰腺检查。胰腺，多有附加脾宗正从鼠标中移除之后。脾由垂直箭头和胰腺水平箭头所示。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig9large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/55647/55647fig10.jpg"> 
图 10: 胰腺检查。显示正在审查从鼠标删除后胰腺肿瘤的附加脾与胰腺。脾由垂直箭头和胰腺水平箭头所示。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig10large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/55647/55647fig12.jpg" style="opacity: 0.9; font-size: 14px;">
图 11: 存储取出器官。适当的存储的器官和样品收集，准备长期存储和未来分析。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig11large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/55647/55647fig11.jpg" style="opacity: 0.9;"> 
图 12: 胰腺组织的组织学分析。苏木精和伊红染色图像从胰腺组织。A） 正常胰腺组织从 Ptf1acre /-;LSL KrasG12D /-鼠标控制。B) PanIN 组织从胰腺的 Ptf1acre / +;LSL KrasG12D / +研究鼠标。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig12large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/55647/55647fig13.jpg"> 
图 13: 代谢谱分析。一维质子核磁共振谱 (NMR) 光谱的胰腺组织提取后组织均匀化和遭受氯仿/甲醇提取物 A） 亲水性和 B） 疏水相成分。核磁共振谱收购了 850 兆赫，适合在代谢分析分析中使用。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig13large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/55647/55647fig14.jpg"> 
图 14: 代表核磁共振氢谱对血清。末梢血画过程所采集的血液可以用于代谢分析分析。这个光谱演示一个典型一维质子 850 兆赫核磁共振谱收集从末梢血画样品获得的血清。<a href="//ecsource.jove.com/files/ftp_upload/55647/55647fig14large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

Watch this Scientific Journal Video about Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling at JoVE.com

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

本文视频提供了详细的演示了从一只老鼠成功删除胰腺解剖组织学分析和代谢过程所需的程序。

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

dissection-mouse-pancreas-for-histological-analysis-metabolic

Universidad San Sebastian

Research

JoVE Journal

Medicine

36.2K 次观看.  Miami University. 本文视频提供了详细的演示了从一只老鼠成功删除胰腺解剖组织学分析和代谢过程所需的程序。

视频: Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

人类蛋白质组分析玻璃体元素剖析

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

科研

医学

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

全玻璃体液清扫Vitreodynamic分析

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

应用改良袖套技术的小鼠胰腺移植模型

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

小鼠尿的解剖和外植体培养对囊附着的作用分析

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...