这项工作部分通过国家卫生研究院（DK097335和T32AI007260）和爱荷华大学斯蒂德家庭儿科系得到支持。

所有动物程序均由爱荷华大学机构动物护理和使用委员会（协议#8041401）批准。所有动物都安置在爱荷华大学批准的实验室动物护理评估和认证协会（AALAC）中。所有小鼠都是野生型菌株C57Bl/6J。
1. 在怀孕小鼠中建立FEMI
<ol><li>LPS 准备<ol>
<li>使用从 大肠杆菌 O55:B5（库存浓度 2 毫克/毫升）提取的 LPS。</li>
		<li>稀释 LPS 库存浓度 1:100 与无菌盐水的工作浓度为 20μg/mL。</li>
	</ol>
</li>
	<li>母体 LPS 注射<ol>
<li>在妊娠日e15注入怀孕的水坝。这个时间点是大约75%，通过阴郁的怀孕，使这个模型发展类似于人类怀孕的早期三个月，这是当大多数早产时，由于胆囊炎发生。</li>
		<li>注射前立即称量怀孕小鼠，以确定适当的LPS配给。</li>
		<li>使用以下公式计算工作浓度的剂量:5 μL x 克体重 （gbw），总剂量 LPS 为 100 μg/kg。对于对照动物，使用等量的正常盐水注射。</li>
		<li>每次注射前，涡流 LPS 解决方案在高位 15 秒内三次。</li>
		<li>将LPS音量绘制成1毫升注射器。</li>
		<li>用擦伤技术约束怀孕的老鼠。保持在后部重新任职的位置，并执行注射。<ol>
<li>插入一个30米8毫米针斜面在腹部的右下象限（以避免膀胱和腹部血管）在30\u201240°的角度。插入约1/4至1/2的针的长度。</li>
			<li>在注射前拉回注射器柱塞以确保负压。如果存在负压，则继续注射。</li>
			<li>注射后，监测小鼠约30分钟，然后返回笼子，在怀孕的剩余时间。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 分娩和照顾后代，以及肠道收获
<ol><li>通常通过e20阴道分娩交付幼崽。 注意:此模型确实具有预期的剂量依赖胎儿损失率，可在 图 1 中看到，并在以下结果中讨论。</li>
	<li>允许幼崽与母亲呆在一起，并接受脂肪饲料。</li>
	<li>在收获日，通常是产后第14天（P14），按照机构动物护理和使用委员会的议定书，通过子宫颈脱位对幼崽实施安乐死。</li>
	<li>使用剪刀和钳子，通过皮肤和腹膜，在腹部的中线下做一个垂直切口，整个腹部长度。用剪刀将小肠从胃切除到粪便，用钳子切除肠子。</li>
	<li>分离并保留小肠的1/3（人类肠的分节代表），丢弃近端小肠、粪便和结肠。</li>
	<li>用剪刀将氦部分分成两半。</li>
	<li>将近半部分放在RNA稳定解决方案中，以供以后的RNA定量。</li>
	<li>将解剖剂的一半放在 10% 中性缓冲形式中，用于幻灯片准备。</li>
</ol>3. 肠道损伤评分
<ol><li>部分石蜡嵌入组织到5 μm厚片，并安装在玻璃滑梯上。 注意:我们将标本发送到石蜡嵌入、分割和安装到幻灯片上的石蜡核心。</li>
	<li>根据标准程序去除幻灯片。</li>
	<li>根据标准程序，使用血氧林和异丙素的污渍部分。</li>
	<li>得分部分在3分尺度内肠损伤，如前所述42，43。<ol>
<li>使用光显微镜，评估两个单独的失明研究者在3点尺度上对全身肠道损伤进行评估，评估静脉的完整性和与地下室膜43 的分离（补充图1）。肠道损伤最好按放大20倍和数孔0.50进行评估。</li>
		<li>分配 0 分来描述正常的粘膜。</li>
		<li>分配一个分数1描述轻度损伤，其中包括亚皮鲁恩哈根的空间的发展，真空或副皮提升仅限于拉米纳丙丙或villi的提示。</li>
		<li>分配一个2分来描述严重的伤害，表示上皮提升和蒸发大于一半的维利，维利失真，或粘膜溃疡和拉米纳丙酸的解体。</li>
	</ol>
</li>
</ol>4. 帕内思和杯细胞的量化
<ol><li>去亲和后，组织部分的污渍滑动从步骤2.8与阿尔西亚蓝/周期酸希夫污渍，以表示杯和帕内思细胞，如先前描述的44，45根据以下步骤。 注:虽然阿尔西亚蓝/周期酸希夫污渍不是特定于帕内思或杯状细胞，在我们的经验，失明经验丰富的研究者有等效的细胞定量使用这种污渍相比，细胞靶向抗体，与显着较少的背景染色46。</li>
	<li>脱水、污渍和脱水滑梯如下。<ol>
<li>两次在二甲苯中浸入滑梯 10 分钟。 注意:二甲苯应用于烟气罩。</li>
		<li>用100%埃托冲洗。</li>
		<li>淹没幻灯片在100%EtOH 3分钟，然后在90%EtOH 3分钟，其次是70%EtOH 3分钟，最后淹没幻灯片在50%EtOH为3分钟。</li>
		<li>在自来水下清洗5分钟。 注意:将该部分远离自来水，以防止组织样本丢失。</li>
		<li>使用标准咖啡滤镜过滤阿尔西恩蓝色污渍溶液。</li>
		<li>污渍在阿尔西亚蓝色污渍中滑动 15 分钟，然后在自来水下清洗 2 分钟。</li>
		<li>在200mL的双蒸馏水中稀释1毫克周期酸。在此解决方案中浸入幻灯片 5 分钟。然后在自来水下洗1分钟。</li>
		<li>用希夫的试剂污渍 10 分钟。在自来水下清洗5分钟。</li>
		<li>用血氧林将滑梯染色1分钟，然后在自来水下清洗2分钟。</li>
		<li>将它们浸入酸醇（1 mL 盐酸混合在 99 mL 中，70% EtOH）中 1 分钟。</li>
		<li>浸入斯科特的自来水（自来水中NaHCO3 浓度0.1%）1分钟，然后在自来水下清洗1分钟。</li>
		<li>脱水滑梯。<ol>
<li>在 70% EtOH 中浸入每个幻灯片 10 次，然后在 90% EtOH 中浸 10 次，在 100% EtOH 中浸 10 次。</li>
			<li>在 100% EtOH 中浸入幻灯片 10 分钟，然后在新鲜的二甲苯中浸入两次，每次 3 分钟。</li>
		</ol>
</li>
		<li>在标本上放置一滴安装的介质，并在标本上放置盖子。</li>
	</ol>
</li>
	<li>杯细胞计数<ol>
<li>使用光显微镜，数数杯细胞（补充图2）。对于每一块肠道组织，计算杯细胞和500上皮细胞的数量，并表达杯细胞比作为每100个上皮细胞的比例。杯形细胞最好按放大20倍和数值光圈0.5计算。</li>
	</ol>
</li>
	<li>帕内思细胞计数<ol>
<li>使用光显微镜，数数帕内思细胞（补充图2）。对于每一块肠道组织，表达为每个肠道密码的Paneth细胞的比例。每片肠道组织数100个肠道密码。Paneth 细胞最好按 20x-60 倍的放大倍数和数值光圈 0.50-1.30 计算。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

胆管炎影响2\u20124%的期限和25\201230%的早产8，9。然而，胆管炎的影响可以延长很久的出生，因为它已被证明对胎儿和新生儿有显著的影响10，11，12，13，14，15，16。重要的是，胆...

胆汁膜在哺乳动物怀孕中起着不可或缺的作用。它们包括具有多种功能的巧克力和氨骨。它们包围和保护胎儿，促进母体和胎儿隔间1之间的对羟基苯甲烷信号，并在胆汁膜内创建局部反馈回路，这可能参与启动隔膜1。目前对膜的了解表明，氨基安尼翁提供结构屏障功能，而胆汁提供免疫缓冲，主要保护发育中的胎儿免受母体免疫系统2。这些膜的炎症被称为胆囊炎。从历史上看，临床胆管炎的诊断是在孕产妇发烧加上一个或多个胎儿或产妇临床发现3，4后作出的。然而，虽然这个定义在临床上是有用的，但它缺乏精确性使得胆管炎的研究具有挑战性。2015年，为了澄清诊断结果，尤尼斯·肯尼迪·施莱佛国家儿童健康与人类发展研究所的专家小组研讨会将胆管炎定义为宫内炎症或感染，或两者兼有（三I）3。这一澄清是重要的，因为虽然微生物诱发感染是子宫/羊水炎症的一个重要原因，它发生的频率低于无菌子宫/羊水炎症5，6，7。总的来说，胆管炎仍然是一个严重的公共卫生问题，正如在2\u20124%的定期分娩和25\u201230%的早产8，9。
胆管炎对胎儿和新生儿有显著影响。文献记载，胆管炎与早产症许多发病率增加有关，包括支气管肺发育不良10、脑白质损伤11、心室出血12、早产视网膜病变13，以及疑似和确诊的早期新生儿败血症14、15。由于我们对不成熟的肠道的损伤和修复机制感兴趣，因此必须注意，胆管炎也与后来的坏死性肠球炎（NEC）15、16的发展有关。NEC是一种毁灭性的胃肠道疾病的早产儿，导致一个不良的调节主机反应炎症和随后的肠道坏死17。在美国，NEC每年影响超过4000名婴儿，其中多达三分之一的婴儿死于这种疾病18。NEC的发病机制可能涉及肠道不成熟、不成熟免疫系统调节不良、肠道炎症和细菌转移19的组合，最终导致肠道坏死的最后一条常见途径。重要的是，NEC的发病通常发生在出生后几周，并可能暴露于胆管炎，使得胆管炎与NEC随后的发展之间的机械联系变得模糊不清。胆管炎可能有助于NEC病理生理学的一个潜在机制是通过对母体免疫系统的调节，随后产生强烈的胎儿炎症反应，可能破坏正常的胎儿发育模式21，22，23。
啮齿动物和绵羊24、25、26、27、28、29、30、31、32等多种哺乳动物模型存在。然而，在胆管炎引起的胎儿接触产妇炎症（FEMI）之后，很少有关于肠道发育的数据。为了探讨FEMI与不成熟肠道损伤的后续发展之间的关系，我们采用了脂糖（LPS）诱导的FEMI模型。脂糖是克阴性细菌细胞外表面的主要成分，是多种真核物种（包括人类33）与生俱来的免疫系统的有力刺激剂。产妇LPS注射导致无菌炎症级联，没有活细菌的混淆作用，它是一个成熟的模式诱导早产34，以及急性胆管炎和胎儿炎症反应综合征（FIRS）的模型，这是最严重的形式的胆囊炎24，35。它也已被证明诱导大脑白质和灰质损伤的绵羊模型36和穆林模型37，38，39，40。然而，据我们所知，我们是第一个使用这种模型的胆管炎和FEMI来调查它对胃肠道发育的影响，过去出生，以及调查胆管炎和NEC41，42的后期发展之间的可能机械性联系。

<table>
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>Newcomer supply</td>
			<td>1003A</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl6/J mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin stain</td>
			<td>Leica</td>
			<td>381562</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Sigma</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>2CE-MQVJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid</td>
			<td>ACROS</td>
			<td>H5106</td>
			<td>CAS# 10450-59-9</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermofisher</td>
			<td>Am7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#39;s reagent</td>
			<td>Sigma</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Secor Imager 2400</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-Plex Assay</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine model of fetal exposure to maternal inflammation to study the effects of acute chorioamnionitis on newborn intestinal development

胆管炎是早产的常见诱发剂，与早产的许多发病率有关，包括坏死性肠结肠炎（NEC）。然而，这两种情况之间的机械联系仍有待发现。我们采用了胆囊炎的穆林模型，该模型涉及脂糖（LPS）诱导胎儿接触母体炎症（FEMI）。FEMI的这种模型诱导无菌产妇、胎盘和胎儿炎症级联，这在许多临床胆管炎病例中也存在。虽然存在利用活细菌并更准确地模拟上升感染导致胆管炎的病理生理学的模型，但这些方法可能会对不成熟的肠道发育和相关发育的微生物群产生间接影响。使用此协议，我们已经证明，LPS 诱发的 FEMI 导致妊娠损失和早产的剂量依赖性增加，以及后代正常肠道发育的中断。此外，我们已经证明，FEMI显著增加后代的肠道损伤和血清细胞因子，同时减少杯状细胞和Paneth细胞，这两者都提供了肠道炎症的第一线先天免疫力。虽然类似的LPS诱导FEMI模型已经被用来模拟胆管炎和中枢神经系统随后的异常之间的关联，据我们所知，这个协议是第一个试图阐明胆管炎和后来肠道发育的扰动之间的机械联系，作为胆管炎和NEC之间的潜在联系。

在胚胎第15天接触FEMI会导致妊娠依赖剂量的丧失和早产的剂量依赖率（图1）42。在实验中，我们选择使用 100μg/kg 的 LPS 剂量来尽量减少妊娠损失和早产（早产期和宫内胎儿死亡之间 50% 的损失），同时使胎儿受到严重的炎症性侮辱。
使用这种方法，我们接下来检查了FEMI对后代后续伤害的影响。使用3点组织学量表测量全身肠道损伤，我...

Watch this Scientific Journal Video about A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development at JoVE.com

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

我们开发了一个胆管炎模型，以模拟胎儿接触产妇炎症（FEMI），而不会引起活体并发症，以检查FEMI对后代肠道发育的影响。这允许研究胆管炎后肠道损伤发展的机械原因。

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

a-murine-model-fetal-exposure-to-maternal-inflammation-to-study

Research

JoVE Journal

Medicine

3.7K 次观看.  University of Iowa. 我们开发了一个胆管炎模型，以模拟胎儿接触产妇炎症（FEMI），而不会引起活体并发症，以检查FEMI对后代肠道发育的影响。这允许研究胆管炎后肠道损伤发展的机械原因。

视频: A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

的不可逆和可逆单侧输尿管梗阻小鼠模型

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

干粉和药品的雾化吸入气雾剂递送小鼠使用鼻只曝光系统

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.