作者希望感谢安迪 Poczobutt 和 Jori 博士 Leszczynski 对这份手稿的宝贵帮助和编辑。 得到国家心脏、肺和血液研究所 (NHLBI RO1 HL078929、HL014985 和 RO3 HL095439) 和退伍军人事务部 (成绩审查) 提供的赠款的支持。

在本手稿中所述实验中遵循了所有适用的国际、国家和 (或) 机构指南, 以照顾和使用动物 (小鼠)。
该方法使用 FVBN 成年小鼠, 年龄 16-20 周, 发现是最佳的目的, 本研究。1天包括步骤 1-5 和天2包括步骤 6-7 (图 1)。
1. 设备准备
<ol><li>如果使用氯胺酮/甲苯噻嗪作为麻醉药 (如建议的那样), 请确保有充足的无菌、一次性注射器和针头供应。如果使用异氟醚作为麻醉剂, 检查氧气罐和异氟醚的液位, 以确保在开始试验前有足够的供应。同时, 组装 nosecone 呼吸电路并将其连接到感应箱;在呼吸回路上附加新的炭罐。通过打开氧气并确定第二阶段读数大约 50 psi 来准备感应箱。</li>
	<li>把暖气垫打开37摄氏度。</li>
	<li>为手术准备直肠温度探头。</li>
</ol>2. 鼠标准备
这一步骤包括麻醉, 脱毛和定位 (成年 FVBN 小鼠-年龄 16-20 周)。
<ol><li>称量老鼠并记录重量。</li>
	<li>麻醉老鼠。<ol>
<li>管理氯胺酮和甲苯噻嗪 IP (分别为 80-100 毫克/千克和 7.5-16 毫克/千克)。然而, 建议以较低剂量的氯胺酮和甲苯噻嗪 (分别为30毫克/千克和6毫克/千克) 开始。</li>
		<li>用大约 0.1-0.25 倍的剂量氯胺酮/甲苯噻嗪在整个手术中保持麻醉。氯胺酮和甲苯噻嗪是这项研究中选择的麻醉剂, 因为观察到更多的重现性和生存能力。其他研究中所选择的麻醉剂药物可以被发现是不同的。 如果使用异氟醚, 将鼠标放在感应腔内, 将异氟醚打开至 5%, 直到鼠标完全失去知觉为止。然后使用鼻 nosecone 设置在 1.5-2.5% 异氟醚在整个过程的其余部分。</li>
	</ol>
</li>
	<li>每 2-3 分钟通过脚趾捏来监测小鼠, 检查是否有适当的麻醉深度。</li>
	<li>剃掉鼠标的腹颈区域。</li>
	<li>将鼠标放在预热垫上的仰卧位置。用胶带将鼠标的爪子和脚固定在手术表面。</li>
	<li>将人工泪膏放在眼睛上, 以防手术时干燥。</li>
</ol>3. 手术细节
<ol><li>在颈静脉的右腹颈上做一个1厘米的切口。</li>
	<li>应用一或两滴利多卡因 (1-2%) 进入切口区域疼痛管理和促进血管舒张。等待2分钟, 以使利多卡因生效。</li>
	<li>通过钝夹层暴露和隔离右颈内静脉。用4-0 缝线将静脉栓起来, 用止血轻轻缩回血管的延髓端。用细剪刀切开一个洞, 大约3毫米以下或尾鳍到领带, 大约一半通过静脉直径。</li>
	<li>将 PV-1 聚氯乙烯导管从末端标记为1.5 厘米, 并将其插入颈静脉内, 使用血管扩张, 并将导管螺纹至该血管的尾端, 约为1.5 厘米。</li>
	<li>将导管安全地栓在船尾部 (切口下面), 用4-0 丝。在步骤3.3 中, 将血管的延髓部分与导管的外侧绑在一起, 该缝合线的松尾用于栓住延髓颈静脉。</li>
	<li>用4-0 丝将皮肤松散地放回导管周围, 以帮助防止身体发热和组织干燥。</li>
	<li>将导管连接到含有血气生理盐水 (10 U/毫升) 的注射器上, 并冲洗导管。</li>
</ol>4. 注射
<ol><li>注射埃文斯蓝溶液 (50 ul 的30毫克/毫升溶液0.9% 正常, unheparinized 生理盐水, 或约50毫克/千克) 入颈静脉导管, 其次是少量的血气盐水冲洗线。</li>
	<li>2分钟后, 注入物质 P (100 ul 0.3 微米溶液0.9% 正常, unheparinized 生理盐水, 或 1 nmol/千克), 其次是少量的血气盐水冲洗线。P 物质通过内皮层增强血浆蛋白的渗出;在这个协议中, 它通常会导致增加大约1.5 倍的等离子渗出值, 使血浆渗出值更容易测量。</li>
	<li>注射物质 P 后等待18分钟。在这段时间, 埃文斯蓝色染料将平衡和流通。</li>
	<li>终止实验18分钟后注射物质 P (20 分钟后, 埃文斯蓝注射液) 通过牺牲鼠标与颈椎脱位。这是可能的老鼠可以直接 cervically 脱臼, 而不首先给予过量的麻醉剂, 因为老鼠可能仍然很好麻醉从手术。然而, 如果有必要, 麻醉药氯胺酮/甲苯噻嗪, 异氟醚, 或戊巴比妥的过量可能会给, 其次是颈椎脱位。</li>
</ol>5. 分离器官
<ol><li>切开小鼠的胸腔和重力灌注 (从高度大约 51 cm 或 20 ") 心脏和血管与50毫升50毫米柠檬酸钠, pH 3.5。pH 值3.5 推测保留埃文斯蓝对白蛋白的约束力。</li>
	<li>用解剖手术刀 (如膀胱、肾脏、胃、肝脏、胰腺、近端或远端结肠、回肠、十二指肠、侧面皮肤、耳朵、尾部、心脏和/或肺部) 切除 1-5 相关器官 (组织), 并除去任何残留物, 如果目前。</li>
	<li>在室温 (RT) 磷酸盐缓冲生理盐水 (PBS; 1.44 克的 Na2HPO4, 0.24 克的2PO4, 8.0 克氯化钠和 0.20 g 的氯化钾在 1 L, pH 7.4) 冲洗器官。</li>
	<li>用纸巾将器官涂抹, 将每一个器官切成两半, 然后将每半部分 (湿重) 分别称量。</li>
	<li>在150摄氏度的烘干烤箱中, 在48小时内烘干。</li>
	<li>将其他组织的一半, 在一个一致的体积 (最多200µL) 的甲酰胺在离心管48小时 (最多72小时), 以提取埃文斯蓝色。</li>
</ol>6. 组织 OD 的测量
<ol><li>从离心管中取出50µL 的埃文斯蓝注入的甲酰胺 (经过 48-72 小时的 RT 孵化), 放入96井聚苯乙烯板的一个井中。小心不要转移组织件连同甲酰胺。</li>
	<li>安置50µL 新, 纯净的甲酰胺入每二个空的井96井板材为空白。</li>
	<li>测量并记录在吸光度板读取器上的96井板的每个井的 OD620 。620毫微米是埃文斯蓝色的吸光度最大值。</li>
</ol>7. 等离子体渗出量的计算
<ol><li>称48小时内在烤箱中的干燥组织的一半。</li>
	<li>计算每个单独的鼠标所关心的特定器官的湿重/干重比, 从该组织一半的湿重开始 (在步骤5.4 中获得), 除以该组织一半的干重 (在步骤7.1 中获得)。</li>
	<li>计算在甲酰胺中组织一半的干重 (g), 方法是将组织一半的湿重除以湿: 特定器官的干重比 (在步骤7.2 中计算), 然后再将其放在甲酰胺 (步骤5.4 中获得)。</li>
	<li>计算修正的 OD620值。从96井板的每个实验井 (包含埃文斯蓝注入的甲酰胺从每个组织, 获得在步骤 6.3) 的 od620值开始, 减去空白井 OD620值 (平均 od620值两个含纯甲酰胺的井, 在步骤6.2 中制备, 在步骤6.3 中从每个实验值中获得620的值。</li>
	<li>通过将被校正的 OD620 (在步骤7.4 中计算) 除以组织一半在甲酰胺中的干重来计算等离子渗出值 (在步骤7.3 中计算)。血浆渗出量将为620/克干重。</li>
	<li>分析数据并表示为平均值的扫描电镜. 统计比较组的 t 检验或单向分析方差和 Scheffé的多比较测试 (lycofs01.lycoming.edu), 酌情。</li>
</ol>

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作者没有什么可透露的。

如上文所述, 研究血浆渗出可能最终导致新的知识的原因或新的方法来抑制或治疗血浆渗出。在目前的手稿中, 使用了埃文斯蓝染料, 成功地使用了等离子渗出协议。虽然这些数据显示了一个假设, 即新经济可以保护血管对等离子渗出, 这是一个次要目标, 目前, 主要目标是提出一个优化的协议, 可以很容易地使用在平均实验室为血浆渗出的未来研究。本文所报告的协议的重点是适应未来的研究与不同...

器官中的血管泄漏是指在器官中毛细血管静脉内皮的间隙产生的渗出或血浆渗漏。这种血浆渗出或增加的血管通透性, 可能产生的某种类型的炎症反应, 可能会有严重的后果。因此, 重要的是, 这一现象, 其原因, 调制器和后果, 被研究和理解, 同样, 调查人员有良好的工具和协议, 以研究他们。内皮间隙可以通过一些刺激产生, 但通常是由肽神经递质和/或 tachykinins 在内皮的作用产生的。这一过程的主要自然发生的调解人之一, 导致增加血浆渗出, 是 undecapeptide 速激肽神经肽, 物质 P1。
利用埃文斯蓝染料的白蛋白结合特性研究和测量血管通透性或血浆渗出的方法已经开发出来, 通常以其准确性、简单性、经济性、安全性以及允许从多个组织一次测定血浆渗出量, 如果需要2,3,4,5,6,7,8,9.这个埃文斯蓝色的协议, 以评估血浆渗出在 FVBN 小鼠的器官使用所有这些, 但增加了一些重要的修改, 使它一般有用和适应未来的研究, 涉及的平均实验室, 进行或将对与血浆渗出或血管通透性相关的因素进行重要研究。本协议将 P 物质引入到 1 nmol/千克的小鼠体内, 使血浆的渗出性增强1.5 倍。这增加了协议的灵敏度, 从而导致更容易观察和获得的结果。其他影响渗透性的因素, 如各种其他多肽、化学物质或某些形式的毒性损伤, 可根据需要由其他实验室使用或研究。本协议采用颈静脉注入法, 对埃文斯蓝和 P 物质进行系统的介绍, 需要进行末端手术。然而, 颈静脉注射5,7,10, 即使在考虑必要的终端手术技术, 更容易掌握和导致生产更一致的结果比其他静脉注射, 包括尾静脉注射4,9。虽然埃文斯蓝可能是由复古眶静脉窦注射提供, 但文献中没有发现使用这种方法的埃文斯蓝色。然而, 对于尾静脉注射, 重现性掌握这种技术的高度专业知识和实践, 大大限制了它的使用成功的埃文斯蓝色注射。相比之下, 我们的协议中所描述的选择性颈静脉注射方法, 提供了一个技术上可获得的解决方案。一个关键的程序, 以灌注鼠标的静脉, 执行后, 在牺牲的埃文斯蓝色灌注鼠标, 删除过剩的埃文斯蓝色染料, 并已在本协议标准化。先前描述的灌注方法已经过仔细检查和修改, 以获得目前的程序。此处描述的其他修改都是优化的、直接的和廉价的。
埃文斯蓝染料的方法存在一些重要的局限性。例如, 低灵敏度有时与此方法可能会阻止一些额外的病理和组织学检查的组织从埃文斯蓝注射动物。然而, 这些和其他限制导致了其他方法和模型的发展, 然而, 仍然使用埃文斯蓝色。用荧光 (而不是视觉范围) 光谱法测量埃文斯蓝可以提高方法的灵敏度。此外, 还开发了埃文斯蓝染色组织的荧光显微术, 以便观察更明显的部位11的血管渗漏。此外, 全身成像和扫描的活动物以前注射埃文斯蓝12允许调查埃文斯蓝浓度的持续方式, 而不是在一个特定的时间点的实验。然而, 这种方法需要适当的成像设施, 可能是非常昂贵的。对埃文斯蓝进行的修改, 并在体外模型中进行, 如在细胞培养或小鸡胚模型13 (CAM) 也被描述。这些模型由荧光和活体14显微镜监测, 并允许定量的血管通透性变化随着时间的推移, 但可能会提出问题,在体内条件的准确建模, 也可能是昂贵。
有其他方法来确定和量化血管渗漏或渗透性, 这并不涉及埃文斯蓝的管理。这些方法可能使用适当的荧光分子 (如白蛋白或荧光素), 或 isotopically 标记或其他标记的分子, 对活动物 (或细胞培养或胚 (CAM) 模型13, 其次是无创影像学 (PET 扫描, MRI, 活体显微镜, 全身扫描) 或侵入性成像 (荧光显微镜)3,12,15。虽然这些技术可能比其他埃文斯蓝色方法提供一些优势, 但它们也有缺点, 其中可能包括其相当复杂、必要的专门知识、资源和高昂的货币成本。
Neprilysin16 (酶酶, 也称为 CD10, 夫人, 或 Enkephalinase) 已被建议参与抑制血浆渗出, 至少部分, 通过酶代谢和失活的内源性物质 P。因此, 在细胞表面酶的组织中, P 物质的作用可能会有衰减, 大概是酶活性的影响。
最初, 我们用这种改良的埃文斯蓝协议对 P 诱导的血浆渗出进行了试验, FVBN 野生型 (小波) 和抗结蛋白 (KO) 小鼠。从这些初步研究中推测, 在 P 增强血浆渗出物中的抗结蛋白参与, 我们描述了这些和进一步的实验, 其中涉及新经济在血浆渗出的作用。然而, 这篇手稿的重点不是在血浆渗出, 而是血浆渗出实验本身的作用。文中的结果代表了通过使用此修改后的协议可以获得的结果类型。埃文斯蓝法测量血浆渗出已被优化和修改, 如下面详细描述 FVBN 小鼠。

<table border="0" cellpadding="0" cellspacing="0" style="width:687px;" width="686">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane</td>
			<td style="width:167px;">Vet One</td>
			<td style="width:119px;">200-070</td>
			<td style="width:185px;">inhaled anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ketamine</td>
			<td style="width:167px;">Vet One</td>
			<td style="width:119px;">200-055</td>
			<td>injectable anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">xylazine</td>
			<td style="width:167px;">Lloyd Laboratories</td>
			<td style="width:119px;">139-236</td>
			<td>injectable anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">syringes (10,3 &#38; 1 cc)</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">309604, 309657, 309659</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">needles (20G1,23G1 &#38; 26G1/2)</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">305178, 305193, 305111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane induction chamber</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:119px;">941443</td>
			<td>1 Liter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nosecone breathing circuits&#160;</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:119px;">RC2</td>
			<td>Rodent Circuit Controller 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">oxygen tank</td>
			<td style="width:167px;">Airgas</td>
			<td style="width:119px;">UN 1072</td>
			<td>100% medical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heating pad</td>
			<td style="width:167px;">CWE Inc.</td>
			<td style="width:119px;">TC-1000</td>
			<td>temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rectal temperature probe</td>
			<td style="width:167px;">CWE Inc.</td>
			<td>10-09012</td>
			<td>mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">balance (for rodents)</td>
			<td style="width:167px;">Ohaus</td>
			<td style="width:119px;">CS 2000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>15000-00</td>
			<td style="width:185px;">Vannas Spring scissors 3mm straight blade (cutting vessels)&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11151-10</td>
			<td style="width:185px;">Graefe extra fine forceps (isolating mouse vessels)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-hemostats</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>13009-12</td>
			<td style="width:185px;">Halstead-mosquito hemostats (blunt dissect, hold tissue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools -suture drivers</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>12502-12</td>
			<td style="width:185px;">Olsen-Hegar suture drivers (suturing)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11627-12</td>
			<td style="width:185px;">Adson-Brown alligator forceps (tissue grasping suturing, rat)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14110-15</td>
			<td style="width:185px;">Mayo tough cut scissors 15 cm (surgery, dissection, bones, rat)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>18025-10</td>
			<td style="width:185px;">suture tying forceps (used for Millar cath)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14078-10</td>
			<td style="width:185px;">Lexer Baby scissors straight (surgery, mouse)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11254-20</td>
			<td style="width:185px;">Dumont #5 fine-tip forceps (rat vessels, dissection)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14082-09</td>
			<td style="width:185px;">Dissector scissors 12 mm (surgery, rat mouse)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11051-10</td>
			<td style="width:185px;">10 cm Graefe forceps (tissue grasping, rat mouse)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11251-35</td>
			<td style="width:185px;">Dumont 5/45 forceps (introducer for vessels)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-retractors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>17012-11</td>
			<td style="width:185px;">Weitlaner retractors 2/3 tooth (rat surgical)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11294-00</td>
			<td style="width:185px;">Dumont #4 forceps (vessel isolation rats, mice)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11297-00</td>
			<td style="width:185px;">Dumont #7 forceps (tissue grasping, dissection)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-scissors</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>14058-11</td>
			<td style="width:185px;">tough cut iris scissors (mouse dissection, bones)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11009-13</td>
			<td style="width:185px;">serrated, curved Semken forceps (tissue grasping, mouse rat)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-hemostats</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>13003-10</td>
			<td style="width:185px;">Hartman curved hemostats (blunt dissect, hold tissue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">surgical tools-forceps</td>
			<td style="width:167px;">Fine Science tools</td>
			<td>11006-12</td>
			<td style="width:185px;">Adson serrated forceps (tissue grasping)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">clippers</td>
			<td style="width:167px;">Oster</td>
			<td style="width:119px;">A5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tape</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:119px;">159015G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">artificial tear ointment</td>
			<td style="width:167px;">Akorn Inc</td>
			<td style="width:119px;">13985-600-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lidocaine</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:119px;">0409-4277-01</td>
			<td>2% injectable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyvinyl catheters</td>
			<td style="width:167px;">Tygon</td>
			<td style="width:119px;">PV-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Evans blue</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Substance P</td>
			<td style="width:167px;">Bachem&#160;</td>
			<td style="width:119px;">H-1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heparin</td>
			<td style="width:167px;">Sagent Pharmaceuticals</td>
			<td style="width:119px;">25201-400-10</td>
			<td>1000 U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saline solution</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:119px;">0409-7138-09</td>
			<td>0.9% sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phenobarbital&#160;</td>
			<td style="width:167px;">Vortech</td>
			<td style="width:119px;">0298-9373-68</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium citrate</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">BP327-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">P4417-50TAB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kimwipes for blotting</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">06-666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">formamide</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">47670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microbalance</td>
			<td style="width:167px;">Denver Instrument</td>
			<td style="width:119px;">APX-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microfuge tubes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polystyrene 96 well plate</td>
			<td style="width:167px;">Becton Dickenson</td>
			<td style="width:119px;">351172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">absorbance plate reader</td>
			<td style="width:167px;">BioTek</td>
			<td style="width:119px;">Synergy 2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gels</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>3450014&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">protein molecular weight standard</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>1610374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protran supported nitrocellulose</td>
			<td style="width:167px;">Amersham (GE)</td>
			<td style="width:119px;">10600015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gel box</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>1658005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween20</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:119px;">P-1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">S271-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary NEP polyclonal antibody&#160;</td>
			<td style="width:167px;">R &#38; D Systems</td>
			<td style="width:119px;">AF1182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">doxycycline chow</td>
			<td style="width:167px;">Teklad (HARLAN)</td>
			<td>TD.130750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FVB/NJ wild type mice</td>
			<td style="width:167px;">Jackson</td>
			<td style="width:119px;">001800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">secondary antibody (goat anti-rabbit)</td>
			<td style="width:167px;">ZyMed</td>
			<td style="width:119px;">81-6120</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL solution-Western Lightening Plus</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:119px;">NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">film</td>
			<td style="width:167px;">Pierce</td>
			<td style="width:119px;">34091</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized evans blue protocol to assess vascular leak in the mouse

血管渗漏, 或血浆渗出, 有许多原因, 可能是一个严重的后果或炎症反应的症状。这项研究最终可能导致新的知识, 关于抑制或治疗血浆渗出的原因或新方法。重要的是, 研究人员有适当的工具, 包括最好的方法, 以研究血浆渗出。在本文中, 我们描述了一个协议, 使用埃文斯蓝色染料方法, 以评估血浆渗出的器官 FVBN 小鼠。该协议是故意简单, 尽可能大的程度, 但提供高质量的数据。埃文斯蓝染料的选择主要是因为它是容易的平均实验室使用。我们使用这个协议提供证据和支持的假说, 即酶 neprilysin 可能保护血管, 以防止血浆渗出。然而, 本议定书可在实验上使用, 并易于适应于其他品系的老鼠或其他物种, 在许多不同的器官或组织, 研究可能涉及其他因素, 在理解, 预防或治疗血浆渗出。该协议已从现有的协议中得到了广泛的优化和修改, 并结合了可靠性、易用性、经济性和材料和设备的一般实用性, 使该协议优于一般实验室用于从器官中定量血浆渗出。

在图 1中, 显示了该过程的示意图, 发现这会导致 FVBN 小鼠器官中最可靠和最一致的 P 物质诱导的血浆渗出值。这一过程通常需要两天的时间, 至少48小时的等待。如果对所有的实验进行比较, 这是可能的, 更广泛地传播出来。例如, 在1天隔离器官后, 这些器官可以在液氮中闪光冷冻, 储存在-80 摄氏度;在仔细地解冻器官 (在几天到1星期之内), 他们可以被?...

Watch this Scientific Journal Video about An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse at JoVE.com

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

本文描述了一种经济的、优化的、简单的协议, 它使用埃文斯蓝色染料方法来评估 FVBN 小鼠器官中的血浆渗出物, 这些细胞可以适应其他菌株、物种和其他器官或组织的使用。

一种优化的埃文斯蓝协议对小鼠血管泄漏的评估

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

an-optimized-evans-blue-protocol-to-assess-vascular-leak-in-the-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

26.1K 次观看.  University of Colorado Denver. 本文描述了一种经济的、优化的、简单的协议, 它使用埃文斯蓝色染料方法来评估 FVBN 小鼠器官中的血浆渗出物, 这些细胞可以适应其他菌株、物种和其他器官或组织的使用。

视频: An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

科研

医学

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

小鼠膈肌功能诊断超声成像

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

腔内药物输送到鼠标动静脉瘘内皮

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

套筒技术在主动脉移植小鼠模型中的应用-教学视频

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

抗疟药的紫外线-Vis和裸眼测定优化的格里斯反应

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

研究血管医疗器械血细胞相容性和宿主细胞活化的体外血流循环模型

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.