这项研究得到了公共福利和 #38 的支持; 通过由教育、科学和技术部 (NRF-2014M3C8A5030612) 资助的韩国国家研究基金会 (NRF) 的安全研究项目, 并得到了中杨、ChaSun, 医学博士和宇 Sa 纪念基金。

此处描述的所有方法均已获得韩国大学医学院国际动物护理与使用委员会 (IACUC) 的批准. #160; 出生后6周, 实验者可诱发 enthansasia 和 #160; 收集各种样本, 如大鼠的皮肤和血清. 
 1. 新生儿辣椒素治疗 AD 的诱导 <ol> <li> 要准备5毫克/毫升的辣椒素溶液, 在80毫升锥形管中加入50毫克的辣椒素到1毫升的吐温1和100% 毫升的15乙醇. </li>
	<li> 添加8毫升生理盐水到锥形管。用力摇动管, 直到没有分离的图层可见 </li> <li> 将辣椒素溶液 (10-#181; L/g) 注入到皮下组织, 在新生儿大鼠颈部中线和背部。
	<ol> <li> 在出生后12ĥ至48小时之间提供辣椒素 (关键). 
		注: 如果注射延迟超过几天, 皮炎将不会发展, 或只会持续几个星期。如果注射过早, 会因剧烈疼痛而发生呼吸窘迫. </li>
		<li> 为了防止新生鼠因辣椒素注射液引起的呼吸道骤停, 推动新生儿的胸部, 以鼓励呼吸或保持新生儿在氧气室, 直到呼吸正常. 和 #160, 因为辣椒素引起的疼痛和 #160;劝阻呼吸, 增加全身麻醉和 #160; #160;p ossibly 会使呼吸困难恶化。因此, 实验者不应该使用 anesthesics 来增加老鼠的存活率. </li>
	</ol> </li> </ol> 2。AD 大鼠皮肤瘙痒和皮炎的评价 <ol> <li> 计数自发划伤行为的瘙痒测量 <ol> <li> 准备塑料室 (20 cm x 30 cm x 20 cm), 配有一面镜子, 以允许全面覆盖查看和孔, 让老鼠呼吸. </li>
		<li> 正确设置和调整数字摄像机, 以便同时记录大鼠的镜像视图和前视图. </li>
		<li> 将老鼠放在塑料室里, 用重物遮住房间的天花板, 以防打开. </li>
		<li> 记录1小时的大鼠自发行为, 播放视频片段, 并计算划伤行为的次数. </li>
	</ol> </li> <li> 使用评分系统评估皮肤损害 <ol> 将老鼠置于感应室. <li>
		<li> 将氧气流量计调至2升/分钟流量, 并将异氟醚蒸发器调整为3% 进行麻醉. </li>
		<li> 识别大鼠耳部和其他部位皮炎的程度和严重性. </li>
		<li> 定义皮肤损害程度为 0.25 cm 2 的单位大小和皮肤病变的严重程度, 按以下 表 1 : 严重度指数; 皮炎评分乘以程度和严重性 (见皮炎评分在强类 = "xfig" > 图 1B ). </li>
	</ol> </li> </ol> 3。气体 FA 对动物的暴露 <ol> <li> 吸入室的制备 <ol> <li> 准备一个亚克力玻璃盒 (55 厘米 x 40 cm x 35 厘米), 它的两侧有两个插座. </li>
		<li> 警告。对于 FA 气体的循环, 将出水口放在高于进口的位置, 使出水口的尺寸大于进口. </li>
		<li> 警告。对于内部气体浓度的测量, 在出口的另一侧, 钻三小通道, 可以关闭. </li>
		<li> 将管道连接到两个插座。将排气管放在建筑物的通风罩或外侧, 以排放气体, 并将其它出口管连接至医用氧气调节器. </li>
		<li> 在亚克力玻璃盒内放置一个传统的鼠笼. </li>
		<li> 在出生后一周内将老鼠放在笼子里5周 (2 小时/天, 5 天/周). </li>
		<li> 尽可能紧密地密封亚克力玻璃盒以防止漏气. 
		注: 密闭密封时, 箱盖应含橡胶。用保鲜膜包住密封腔. </li>
	</ol> </li> <li> 使用医用氧气调节器生成气体 fa <ol> 将5克 fa 溶解为1升水. <li>
		<li> 将 0.5% FA 溶液放在医用氧气调节器的加湿器瓶中. </li>
		<li> 将入口管连接到调节器后, 打开调节器并将流量设置为5升/分. </li>
		<li> 调整溶液浓度和流速以确定燃烧室内气体的浓度. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergy+and+hypersensitivity.+Nature+versus+nurture+in+allergy+and+hypersensitivity.">Allergy and hypersensitivity. Nature versus nurture in allergy and hypersensitivity.</a> Curr Opin Immunol. 15 (6), 603-608 (2003).</li><li>Novak, N., Bieber, T., Leung, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+mechanisms+leading+to+atopic+dermatitis.">Immune mechanisms leading to atopic dermatitis.</a> The J Allergy Clin Immunol. 112 (Suppl 6), S128-S139 (2003).</li><li>Back, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronically+relapsing+pruritic+dermatitis+in+the+rats+treated+as+neonate+with+capsaicin;+a+potential+rat+model+of+human+atopic+dermatitis.">Chronically relapsing pruritic dermatitis in the rats treated as neonate with capsaicin; a potential rat model of human atopic dermatitis.</a> J Dermatol Sci. 67 (2), 111-119 (2012).</li><li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">Animal models of atopic dermatitis.</a> J Invest Dermatol. 129 (1), 31-40 (2009).</li><li>Han, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-Induced+Aggravation+of+Pruritus+and+Dermatitis+Is+Associated+with+the+Elevated+Expression+of+Th1+Cytokines+in+a+Rat+Model+of+Atopic+Dermatitis.">Formaldehyde-Induced Aggravation of Pruritus and Dermatitis Is Associated with the Elevated Expression of Th1 Cytokines in a Rat Model of Atopic Dermatitis.</a> PLOS ONE. 11 (12), e0168466 (2016).</li><li>Xu, B., Aoyama, K., Takeuchi, M., Matsushita, T., Takeuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+cytokine+mRNAs+in+mice+cutaneously+exposed+to+formaldehyde.">Expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde.</a> Immunol Lett. 84 (1), 49-55 (2002).</li><li>Kim, J. Y., Jeong, M. S., Park, K. Y., Seo, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggravation+of+atopic+dermatitis-like+symptoms+by+consecutive+low+concentration+of+formaldehyde+exposure+in+NC/Nga+mice.">Aggravation of atopic dermatitis-like symptoms by consecutive low concentration of formaldehyde exposure in NC/Nga mice.</a> Exp Dermatol. 22 (3), 219-221 (2013).</li><li>Laske, N., Niggemann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+severity+of+atopic+dermatitis+correlate+with+serum+IgE+levels?.">Does the severity of atopic dermatitis correlate with serum IgE levels?.</a> Pediatr Allergy Immunol. 15 (1), 86-88 (2004).</li><li>Fujii, Y., Takeuchi, H., Sakuma, S., Sengoku, T., Takakura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+2,4-dinitrochlorobenzene-induced+chronic+dermatitis+model+in+rats.">Characterization of a 2,4-dinitrochlorobenzene-induced chronic dermatitis model in rats.</a> Skin Pharmacol Physiol. 22 (5), 240-247 (2009).</li><li>Sugimoto, T., Xiao, C., Ichikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+primary+neuronal+death+induced+by+capsaicin+and+axotomy+involves+an+apoptotic+mechanism.">Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism.</a> Brain Res. 807 (1-2), 147-154 (1998).</li><li>Saito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+skin+inflammation+induced+by+repeated+exposure+of+toluene,+xylene,+and+formaldehyde+in+mice.">Characterization of skin inflammation induced by repeated exposure of toluene, xylene, and formaldehyde in mice.</a> Environ Toxicol. 26 (3), 224-232 (2011).</li><li>Jung, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+exposure+induces+airway+inflammation+by+increasing+eosinophil+infiltrations+through+the+regulation+of+reactive+oxygen+species+production.">Formaldehyde exposure induces airway inflammation by increasing eosinophil infiltrations through the regulation of reactive oxygen species production.</a> Environ Toixcol Pharmacol. 24 (2), 174-182 (2007).</li></ol>

作者没有什么可透露的。

本文所描述的用于诱导大鼠 ad 样症状的协议可用于研究 ad 病理生理学, 并作为药物测试的筛选工具。该仪器的气体 FA 接触辣椒素治疗大鼠可用于评估的影响, 接触各种气体的不同动物模型。
新生儿辣椒素治疗 AD 模型依赖于比其他特应性模型更简单的过程, 如 DNCB 治疗模型9和 epicutaneous 敏化模型4, 并且比 NC/衙鼠标4更经济?...

为了探讨甲醛 (fa) 吸入对特应性皮炎 (ad) 进展的影响, 我们建立了一个用于 ad 诱导的协议, 并开发了一种用于慢性 fa 的常规循环室。
AD 是一种慢性炎症性皮肤病, 归因于遗传易感性与环境触发的复杂相互作用1,2。需要一个合适的动物模型来研究 AD 的临床前状态。在环境因素的影响下阐明 AD 的发病机制是最重要的。此外, 为了更好地研究环境诱因对广告进展的影响, 必须区分个体环境因素和量化暴露程度。
我们展示了一种新的大鼠 AD 诱导的新生儿辣椒素治疗模型。此 ad 模型3与其他 ad 模型 (如 epicutaneous 敏化、基因工程 ad 模型以及 ad4的自发鼠标模型) 所引起的 ad 模型具有某些优点。首先, 该模型在3周的时候开发出类似于 ad 的症状, 与婴儿 ad 相对应, 并且比其他自发的小鼠模型 (如 NC/衙鼠标) 更早。在某些情况下, 与基因工程广告模型不同, ad 模型的症状会在20周内自发消退和复发。此外, 模型中的 AD 样症状的诱导过程比 epicutaneous 敏化模型容易, 这与诱导过程中的过敏性接触性皮炎更相似。
我们通过使用丙烯酸玻璃腔和传统的医用氧调节器5 , 介绍了 fa 接触动物模型的新方法, 研究了 fa 吸入对婴幼儿期 AD 发育的影响。调节器允许我们通过调节流速和溶液浓度来使动物接触到恒定的气体浓度。以前的研究已经报告了几种方法暴露 FA 动物模型。徐et al.描述了 FA 解决方案直接应用于皮肤6的曝光方法。另一项最近的研究讨论了使用专门的挥发性溶剂发生器, 模型 49127的 FA 气体暴露的协议。与以往的研究方法相比, 该方法更易于配置, 除 FA 外, 还可应用于其他解决方案。

<table width="776">
	<tbody>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Medical Oxygen Regulator</td>
			<td style="width:159px;">Yuyao Jiahua Medical</td>
			<td style="width:129px;"></td>
			<td style="width:257px;">manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Tube</td>
			<td style="width:159px;"></td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Acrylic Chamber</td>
			<td style="width:159px;">Taejin Acryl</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Plastic Chamber</td>
			<td style="width:159px;">Taejin Acryl</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Paraformaldehyde, powder</td>
			<td style="width:159px;">Sigma</td>
			<td align="right">158127</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Wrap filme</td>
			<td style="width:159px;">Uniwrap</td>
			<td></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Capsaicin</td>
			<td style="width:159px;">Sigma</td>
			<td>M2028</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Tween80</td>
			<td style="width:159px;">Sigma</td>
			<td>P4780</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Digital Video Camera</td>
			<td style="width:159px;">Sony</td>
			<td>HDR-CX380y</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Isoflurane</td>
			<td style="width:159px;">JW pharmaceutical</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
	</tbody>
</table>

effects of exposure of formaldehyde to a rat model of atopic dermatitis induced by neonatal capsaicin treatment

特应性皮炎长期复发瘙痒湿疹和盛行于世界各地, 特别是在发达国家。遗传和环境因素之间的复杂相互作用已知在特应性皮炎的病理生理学中起重要作用。然而, 我们仍然缺乏这一疾病的发病机制的详细情况。因此, 建立适当的动物模型来阐明特应性皮炎的进展是非常重要的。此外, 调查空气污染物等环境因素对特应性皮炎的影响, 扩大了对该病的认识。在这里, 我们描述了一种诱导新生辣椒素治疗大鼠特应性皮炎的方法, 以及一项将甲醛浓度恒定暴露在大鼠体内以揭示其对婴幼儿特应性皮炎发展影响的协议时期.这些协议已成功地应用于一些实验, 并可用于其他物质。

7周龄的辣椒素治疗 ad 大鼠模型的 ad 样皮肤炎症在图 1A中介绍。在视频 1中显示了辣椒素治疗的 AD 大鼠模型的抗刮伤行为。新生儿辣椒素治疗引起皮炎和严重抓伤行为的大鼠3周龄, 和 AD 样症状持续了几个星期 (图 1B, C)。虽然程度不那么严重, 一些大鼠遭受复发性皮炎在16周的年龄后, 早期的广告样症状?...

Watch this Scientific Journal Video about Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment at JoVE.com

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

我们在此描述了新生儿辣椒素治疗诱导大鼠特应性皮炎的方法, 并探讨了甲醛吸入对过敏性皮炎的影响。

甲醛暴露对大鼠新生辣椒素致过敏性皮炎模型的影响

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

effects-exposure-formaldehyde-to-rat-model-atopic-dermatitis-induced

Research

JoVE Journal

Immunology and Infection

9.5K 次观看.  Korea University. 我们在此描述了新生儿辣椒素治疗诱导大鼠特应性皮炎的方法, 并探讨了甲醛吸入对过敏性皮炎的影响。

视频: Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

取得高纯度弓形虫</em一个不连续的氯化铯梯度>卵囊

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

科研

免疫与感染

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

在极少数情况下，表皮转谷氨酰胺酶IgA和组织转谷氨酰胺酶2抗体的识别恒河猴皮炎

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒:三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

用于阴道内艾滋病毒暴露和艾滋病毒感染的人性化 NOG 小鼠

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

革兰阴性细菌败血症的新生儿成像模型

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...