Name: an in vivo method for evaluating the gut-blood barrier and liver metabolism of microbiota products
Uploaded: 2018-10-20T14:00:00
Description: Watch this Scientific Journal Video about An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products at JoVE.com

这项工作得到了波兰科学和高等教育部的支持, 钻石赠款号: DI2017 009247。

根据指令2010/63 欧盟对用于科学目的的动物进行保护, 并经华沙当地生物伦理委员会批准, 对雄性京都大鼠进行了实验。
1. 插入 Intraintestinal 管理的线路
注意:在这里, 我们建议使用导管 intracolonic 管理标记。它可以通过口服或灌胃在消化道的不同级别 (如胃或十二指肠) 进行修改。切记使用一次性手术服, 包括手术服、兜帽和手套, 并确保遵...

<ol>
	<li>Camilleri, M., Madsen, K., Spiller, R., Greenwood-Van Meerveld, B., Verne, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+barrier+function+in+health+and+gastrointestinal+disease.">Intestinal barrier function in health and gastrointestinal disease.</a> Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society. 24 (6), 503-512 (2012).</li><li>Keita, A. V., Soderholm, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+barrier+and+its+regulation+by+neuroimmune+factors.">The intestinal barrier and its regulation by neuroimmune factors.</a> Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society. 22 (7), 718-733 (2010).</li><li>Farhadi, A., Banan, A., Fields, J., Keshavarzian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+barrier:+an+interface+between+health+and+disease.">Intestinal barrier: an interface between health and disease.</a> Journal of Gastroenterology and Hepatology. 18 (5), 479-497 (2003).</li><li>Jaworska, 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=Hypertension+in+rats+is+associated+with+an+increased+permeability+of+the+colon+to+TMA,+a+gut+bacteria+metabolite.">Hypertension in rats is associated with an increased permeability of the colon to TMA, a gut bacteria metabolite.</a> PloS one. 12 (12), e0189310 (2017).</li><li>Fujii, H., Nakai, K., Fukagawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+oxidative+stress+and+indoxyl+sulfate+in+progression+of+cardiovascular+disease+in+chronic+kidney+disease.">Role of oxidative stress and indoxyl sulfate in progression of cardiovascular disease in chronic kidney disease.</a> Therapeutic Apheresis and Dialysis: Official Peer-Reviewed Journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy. 15 (2), 125-128 (2011).</li><li>Tomasova, L., 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=Intracolonic+hydrogen+sulfide+lowers+blood+pressure+in+rats.">Intracolonic hydrogen sulfide lowers blood pressure in rats.</a> Nitric Oxide: Biology and Chemistry. 60, 50-58 (2016).</li><li>Brahe, L. K., Astrup, A., Larsen, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+butyrate+the+link+between+diet,+intestinal+microbiota+and+obesity-related+metabolic+diseases?.">Is butyrate the link between diet, intestinal microbiota and obesity-related metabolic diseases?.</a> Obesity Reviews: An Official Journal of the International Association for the Study of Obesity. 14 (12), 950-959 (2013).</li><li>Ufnal, M., 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=Trimethylamine-N-oxide:+a+carnitine-derived+metabolite+that+prolongs+the+hypertensive+effect+of+angiotensin+II+in+rats.">Trimethylamine-N-oxide: a carnitine-derived metabolite that prolongs the hypertensive effect of angiotensin II in rats.</a> The Canadian Journal of Cardiology. 30 (12), 1700-1705 (2014).</li><li>Huc, T., Nowinski, A., Drapala, A., Konopelski, P., Ufnal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+and+indoxyl+sulfate,+gut+bacteria+metabolites+of+tryptophan,+change+arterial+blood+pressure+via+peripheral+and+central+mechanisms+in+rats.">Indole and indoxyl sulfate, gut bacteria metabolites of tryptophan, change arterial blood pressure via peripheral and central mechanisms in rats.</a> Pharmacological Research. 130, 172-179 (2018).</li><li>Ufnal, M., Pham, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut-blood+barrier+permeability+-+A+new+marker+in+cardiovascular+and+metabolic+diseases?.">The gut-blood barrier permeability - A new marker in cardiovascular and metabolic diseases?.</a> Medical Hypotheses. 98, 35-37 (2017).</li><li>Le Ferrec, E., 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=In+vitro+models+of+the+intestinal+barrier.+The+report+and+recommendations+of+ECVAM+Workshop+46.+European+Centre+for+the+Validation+of+Alternative+methods.">In vitro models of the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European Centre for the Validation of Alternative methods.</a> Alternatives to Laboratory Animals: ATLA. 29 (6), 649-668 (2001).</li><li>Bohets, H., 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=Strategies+for+absorption+screening+in+drug+discovery+and+development.">Strategies for absorption screening in drug discovery and development.</a> Current Topics in Medicinal Chemistry. 1 (5), 367-383 (2001).</li><li>Grootjans, J., 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=Non-invasive+assessment+of+barrier+integrity+and+function+of+the+human+gut.">Non-invasive assessment of barrier integrity and function of the human gut.</a> World Journal of Gastrointestinal Surgery. 2 (3), 61-69 (2010).</li><li>Bielinska, 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=High+salt+intake+increases+plasma+trimethylamine+N-oxide+(TMAO)+concentration+and+produces+gut+dysbiosis+in+rats.">High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats.</a> Nutrition. 54, 33-39 (2018).</li><li>Clarke, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+Ussing+chamber+studies+of+mouse+intestine.">A guide to Ussing chamber studies of mouse intestine.</a> American journal of physiology. Gastrointestinal and Liver Physiology. 296 (6), G1151-G1166 (2009).</li><li>Denno, D. M., 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=Use+of+the+lactulose+to+mannitol+ratio+to+evaluate+childhood+environmental+enteric+dysfunction:+a+systematic+review.">Use of the lactulose to mannitol ratio to evaluate childhood environmental enteric dysfunction: a systematic review.</a> Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 59 Suppl 4, S213-S219 (2014).</li><li>Lugea, A., Salas, A., Casalot, J., Guarner, F., Malagelada, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+hydrophobicity+of+the+rat+colonic+mucosa+is+a+defensive+barrier+against+macromolecules+and+toxins.">Surface hydrophobicity of the rat colonic mucosa is a defensive barrier against macromolecules and toxins.</a> Gut. 46 (4), 515-521 (2000).</li><li>Bloemen, J. G., 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=Short+chain+fatty+acids+exchange+across+the+gut+and+liver+in+humans+measured+at+surgery.">Short chain fatty acids exchange across the gut and liver in humans measured at surgery.</a> Clinical Nutrition. 28 (6), 657-661 (2009).</li><li>Huc, 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=Colonic+hydrogen+sulfide+produces+portal+hypertension+and+systemic+hypotension+in+rats.">Colonic hydrogen sulfide produces portal hypertension and systemic hypotension in rats.</a> Experimental Biology and Medicine. 243 (1), 96-106 (2018).</li></ol>

直接,在体内, 测量 GBB 渗透率的方法保持胃肠系统中的 closetophysiological 条件 (保留肠道血流), 几乎不受肝肾功能的影响。
这项技术的关键步骤是插入门导管。这必须在同一时间轻轻和果断地完成。轻微, 短出血可能发生从正确执行的门静脉穿刺;但是, 当针头插入容器时, 它会停止。持续出血表明门静脉穿孔。为便于导管插入, 门静脉应充分暴露。exteriorizing 肠后, 肠系膜?...

肠道血屏障 (GBB), 也被称为肠道屏障, 是一个复杂的多层系统, 分离肠道腔与血液, 以限制有害化合物的通过, 同时允许营养素的吸收1。它由三主要层组成: 粘液层、上皮和固有。
许多因素可能影响 GBB 的完整性和功能2。已经表明, GBB 在胃肠道和肠外疾病, 包括心血管和代谢疾病3, 这可能导致肠道细菌代谢物的通过增加到血液4的紊乱。肠道细菌代谢物的渗透增加可能影响整个机体的功能。例如, 最近的研究显示, 细菌代谢产物, 如吲哚, H2S, 短链脂肪酸 (欢迎) 和三甲胺 n-氧化物, 在循环系统功能5,6,7 显著影响,8,9。最后, 有人建议, 增加的 GBB 渗透率可以作为心血管和代谢疾病的标志, 与形态学和功能改变在肠道10。因此, 跟踪细菌代谢物的肠道门血-肝系统血液通路可能对基础和临床科学有兴趣。
常用的实验方法对 GBB 通透性进行体外检测, 使用切除肠段、粘膜片段或人工膜11、12。这些方法的准确性受到损害的事实, GBB 的正常运作需要恒定的肠道血流。另一方面, 可用的活体方法是基于对外源标记13的尿液或外周血浓度的评估。但是外源性化合物的外周血和尿液浓度受肾脏功能的影响,即肾小球滤过率和管状排泄, 以及肝脏代谢,即第一次代谢。两个参数在独立于 GBB 函数的研究对象之间可能有显著差异。
本文介绍了用门静脉采血法直接测定大鼠 GBB 通透性的方法。这种在体内的方法保留肠道血流, 几乎不受肝脏和肾脏功能的影响。所描述的方法并不常用, 可能是因为一些方法上的困难。我们详细描述了门静脉导管和下腔静脉在肝静脉汇合上方的插管。从门静脉和下腔静脉的血液取样允许评估 GBB 通透性和肝脏清除, 以及跟踪胃肠门血-肝系统血液通路的兴趣分子, 如肠道细菌代谢物或药物。我们还介绍了在我们的实验室测试的方法的几个应用。这些包括对 tma 的结肠通透性的评估, 肠道细菌代谢物, tma 的肝清除率的评价, 和欢迎的肠门血-肝-系统血通路的评价。
为了评估肠血屏障通透性, 应遵循以下协议步骤, 按顺序: 1 (插入 intraintestinal 管理线), 3 (门静脉导管), 4 (门静脉血取样), 6 (肠道通透性标记的管理), 4。
为了评估肝脏清除和肠道门血-肝系统的血液通路, 应遵循以下协议步骤, 按顺序: 1 (插入 intraintestinal 行政部门的行), 2 (下腔静脉导尿), 3 (门静脉导管), 4 (门静脉血采样), 5 (下腔静脉采血), 6 (胃肠通透性标记的管理), 4, 5, 7 (肝间隙的计算).

<table border="0" cellpadding="0" cellspacing="0" style="width:1131px;" width="1131">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Needle OD: 9 mm</td>
			<td style="width:259px;">Becton Dickinson&#160; S.A.</td>
			<td style="width:119px;">301300</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Polyethylene catheter ID: 0.025&#34;, OD: 0.040&#34;&#160;</td>
			<td>Scientific Commodities, Inc.</td>
			<td>#BB520-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Polyethylene catheter ID: 0.012&#34;, OD: 0.025&#34;&#160;</td>
			<td>Scientific Commodities, Inc.</td>
			<td>#BB520-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-Flex Tubing,Opaque White 1/50&#34;ID x 1/12 &#34; OD&#160;&#160;</td>
			<td>Cole-Parmer Instrument Co.</td>
			<td>06424-59</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pediatric Foley catheter (size 10F or 8F)&#160;</td>
			<td>Sigmed</td>
			<td>0000 80305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Surgical ligatures 3/0</td>
			<td>Yavo Sp. Z o.o.&#160;</td>
			<td>P48JE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbable surgical sutures - Polyglactine 910 4/0</td>
			<td>KRUUSE Polska Sp. Zo.o.</td>
			<td>152336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Tissue glue - Loctite 454Cyanoacrylate Adhesive</td>
			<td>Loctite&#160;</td>
			<td>1370127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone iodine</td>
			<td>EGIS Pharmaceuticals PLC</td>
			<td>4449 11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Heparin - Heparinium WZF&#160;</td>
			<td>WZF Polfa S.A.</td>
			<td>02BK0417</td>
			<td>Dilute 10 times with physiological saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Glycerin 86%</td>
			<td>Laboratorium Farmaceutyczne Avena</td>
			<td>5.90999E+12</td>
			<td>Serves as a lubricant in colon catheterization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Xylocaine 2%</td>
			<td>AstraZenca&#160;</td>
			<td>9941342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Urethane</td>
			<td>Sigma-Aldrich (Merck)&#160;</td>
			<td>U2500-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Trimethylamine solution 45%</td>
			<td>Sigma-Aldrich (Merck)&#160;</td>
			<td>92262-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Syringes 2 mL</td>
			<td>B.Braun Melsungen AG</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:568px;">Saline 250 mL</td>
			<td>Fraesenius Kabi Polska Sp. Z o.o.</td>
			<td>15LL707WL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors, straight, length 115 mm, 4 1/2 &#34;blunt ends</td>
			<td>Braun</td>
			<td>NS-010-115-PKM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artery forceps type Micro-Adson bent, length 140 mm 5 1/2 &#34;</td>
			<td>Braun</td>
			<td>KN-008-140-ZMK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anatomic forceps, lenght 95 mm, 3 3/4&#34; sharp 0.7x0.55</td>
			<td>Braun</td>
			<td>PO-001-007-ZMK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Scissors type Vannas,&#160; straight, lenght 85 mm, 3 3/8 &#34; the length of the blades 6 mm</td>
			<td>Braun&#160;</td>
			<td>NO-010-085-PMK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Towel clamps type Backhouse, lenght 130 mm, 5 1/8&#34;&#160;</td>
			<td>Braun</td>
			<td>HO-128-130-PMK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holders, lenght 150 mm, 6&#34; t=0.4 1/2&#160;</td>
			<td>Braun&#160;</td>
			<td colspan="2">IM-927-150-PZMK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delicate Scissors, lenght 110 mm , straight, 4 3/8&#8221; sharp&#160;</td>
			<td>Braun</td>
			<td>NO-052-110-PMK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anatomic forceps, lenght 95 mm, 3 3/4&#34; sharp</td>
			<td>Braun</td>
			<td>PO-022-001-PMK</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo method for evaluating the gut-blood barrier and liver metabolism of microbiota products

肠道血屏障 (GBB) 控制养分、细菌代谢物和药物从肠道腔到血液的通道。GBB 完整性在胃肠道、心血管和代谢疾病中受到干扰, 这可能导致生物活性化合物 (如肠道细菌代谢物) 更容易进入血液。因此, GBB 的通透性可能是肠道和肠外疾病的标志物。此外, 细菌代谢物的渗透率增加可能影响整个机体的功能。
研究 GBB 通透性的常用方法是在体内进行。这些方法的准确性是有限的, 因为 GBB 的功能取决于肠道血流。另一方面, 通常使用的活体方法可能有偏见的肝脏和肾脏的表现, 因为这些方法是基于评估尿液或/和外周血浓度的外源标记。在这里, 我们提出了一种直接测量大鼠 GBB 通透性的方法, 采用基于门静脉采血的活体法, 可保持肠道血流, 几乎不受肝肾功能的影响。
聚氨酯导管插入门静脉和下腔静脉刚好高于肝静脉汇合处。血液在基线和被选择的标记的管理后被取样到胃肠道的所需部分。在这里, 我们提出了一些应用的方法, 包括 (1) 结肠通透性对 tma, 肠道细菌代谢物的评价, (2) tma 肝清除率的评价, 和 (3) 肠道门静脉血-肝-外周血通路的评价细菌衍生的短链脂肪酸。此外, 该协议还可用于跟踪药物的肠道吸收和肝脏代谢或门静脉血压测量。

成功测定了大鼠 GBB 通透性和肝清除率。我们已经证明高血压大鼠与血压正常大鼠相比, 其结肠通透性增加到 TMA (图 2)4。在另一项研究中, 我们发现高盐摄入不会影响 TMA 的 GBB 通透性和肝间隙 (图 3)14。
测量粪便、门血和外周血中欢迎的浓度, 我们...

Watch this Scientific Journal Video about An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products at JoVE.com

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

营养物质、微生物代谢物和药物进入血液循环的途径由肠道血屏障 (GBB) 控制。我们描述了一种直接测量体内 GBB 通透性的方法, 与常用的间接方法相比, 它几乎不受肝肾功能的影响。

微生物肠道血屏障及肝脏代谢的体内评价方法

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

肠道血屏障是一个多层系统，控制营养物质、细菌代谢物、药物和其他外源化合物从肠道流明进入血液的通道。正如我们所了解的，屏障的完整性可能在胃肠道以及心血管和一些代谢疾病中受到损害，这可能导致生物活性化合物（如肠道细菌代谢物）更容易进入血液，并可能影响整个生物体的功能。因此，肠道血屏障的渗透性可能是肠道和肠道疾病的标记。在这段视频中，我们直接测量了大鼠在将测试物质给消化道后使用门户血液采样的肠道血液屏障渗透率。我们详细描述了入口静脉和劣质静脉的分类，就在肝静脉汇合的上方。从入口静脉和劣质静脉的血液采样不仅能够评估肠道血液屏障的渗透性，还可以评估肠道细菌代谢物或药物等有关分子的肝清除和吸收途径。这种方法几乎不受肝肾功能的影响，并保留了肠道血流量，这是一个强大的优势，在体内和体外常用的方法。动物应在手术前通宵禁食。在全身麻醉期间执行所有程序。实验后动物被安乐死。我们的程序从插入行开始，用于训练线管理。在这里，我们建议在分色中管理标记。它也许被各级消化道的行政部门修改。例如，胃或十二指肠。然而，在将标记法管理到胃肠道上部时，考虑到近呼吸道的可变速度和可能与酶和胃酸的相互作用。使用小儿Foley导管大小10F或8F作为结肠导管。标记导管以指示将插入结肠的零件大约 8 厘米。在将导管插入结肠之前，检查肛门区域和直肠中粪便的计数。如果凳子存在，通过按摩直肠区域来清空直肠。沿导管放润滑剂。接下来，用润滑剂润湿了阿努斯及其周围环境。将导管用导管穿过外部肛门括约肌。进行缓慢的前进、后退和循环运动。在插入导管时，通过腹部眼皮继续检查导管的位置。下一步是劣质的维纳卡瓦导管。您可以在肠道渗透性研究中跳过此过程。尝试感受股动脉上的脉搏，在脉搏明显的地方纵向切割皮肤约两厘米。解剖筋膜和肌肉，以可视化神经血管束。接下来，从神经血管束解剖股骨静脉。先是神经，然后是股动脉，然后是静脉。在解剖包时要小心，因为股骨静脉的几个小分支很容易损坏。接下来在股骨静脉上放两个连字。还不要打结。用针架抓住近线连结的末端。然后小心地将连结末端与支架向上拉，以关闭静脉的近端部分。等到静脉里满是血，绑住大肠结。接下来，使用显微手术剪刀在结和近结字之间的静脉上做一个小切口。然后插入导管使用钳子或针与弯曲的端，因为我们在视频中做。刺穿静脉，使用针头的弯曲尖端作为导管的导轨。插入导管时松开近向连结。插入导管约六至七厘米，具体取决于动物的大小，将导管的近端放在肝静脉汇合上方的劣质静脉中。实验后应确认导管的正确位置。用两个单手术结将导管固定在股骨静脉中。也系上近线连字。试图通过注射器抽血检查导管的附度。用 0.3 毫升的肝素盐水冲洗导管。用两层单针关闭手术伤口。现在，我们进入门户静脉导管。准备门户导管。它由针头、柔性聚氨酯导管、柔性聚乙烯导管尖端、插头和连结体组成。手术的第一步是进行中线腹腔切除术。准备操作场后，将皮肤从胸骨的西腓纵向切到肚脐。沿着白线切割腹壁的肌肉。接下来，在 y 形状中展开切口，使西腓软骨介于两个切口之间。下一步是门户静脉解剖。首先，用盐水润湿拭子。然后外化 cecum、 升序和横向结肠， 和小肠循环.把肠子放在左边，露出肠子的根部。用湿润的纱布覆盖肠道，防止它们干燥。使入口静脉小心地将肝叶移动到两侧或向上，用湿润的拭子朝隔膜移动。在这里，我们可以看到门户静脉和上肠静脉。本地化门户静脉中未覆盖的一部分，并在门户静脉下传递连字。为了保护组织免受损伤，请用生理盐水溶液润湿连结。用钳子将连结的末端夹住，轻轻拧紧以稳定容器。将导管连结的较长部分穿过入口静脉的自由部分，然后拉扯，使导管位于入口静脉旁边。将针头插入上肠静脉，在上肠静脉和门户静脉交界处以下三毫米。以 30 度角握住针头，进入静脉后，减小角度，将针头几乎水平地与入口静脉平行推进，长度约为 6 至 7 毫米。在插入针头的地方涂抹一两滴组织胶水。取出覆盖肝脏的棉签。之后把肠子放回腹腔。用加热的盐水溶液滋润肠道，用湿润的无菌纱布盖住。检查导管的附度，并用肝素盐水冲洗导管。静脉血自发地回流到导管中。五分钟后检查肠道的颜色和围体运动，以确保适当的肠血流保持。最后，用三层缝线关闭腹腔，将导管的外侧部分外化肚脐周围。要从入口静脉收集血液，请打开入口导管塞，让血液自由流动。使用注射器和钝针，收集不超过0.7毫升的血液。用肝素盐水冲洗导管，然后关闭导管塞。在这里，我们提出了示范性门户血液采样方案，用于肠道渗透性评估。来自劣质维纳卡瓦的血液样本是肝清除和吸收研究所必需的。从劣质的静脉中采集血液，与从入口静脉取血时完全一样进行。在这里，我们提出了一个示范性的血液采样方案，用于肝脏清除测量和跟踪选定化合物的肠道-门户血液-肝脏-系统血液通路。在我们的研究中，我们使用细菌代谢物，三乙胺作为渗透性的标志。然而，许多其他物质，包括类渗透标记也可以使用。各种细菌和代谢物和肠道也可用于治疗吸收途径或肝间隙。拆下导线，使用足够体积的无菌水充气气球，通常为一毫升，但在插入前检查气球的大小。气球直径不应超过一厘米。将大鼠头向下放置，以尽量减少从结肠流出的施用溶液的风险。使用排水口和结肠导管慢慢管理测试物质。我们使用三甲胺的剂量为每公斤100毫克，体重溶解在0.7毫升盐水中。十分钟后，将导管气球放气。肝间隙理解为肝萃取，可由门户血液浓度和劣质维纳卡瓦血浓度的差异或低劣的维纳卡瓦和门户血液浓度的比例来表示。我们成功地测量了大鼠三甲胺的肠血屏障渗透性和肝清除性。以下是与高血压大鼠相比，高血压大鼠对TMA的结肠渗透性有所提高的代表性结果。在另一项研究中，我们研究了高盐摄入量是否影响三乙胺的肠道血屏障渗透性和肝脏清除性。三甲胺的分气内注射导致门户血液TMA浓度显著增加。增加和肝间隙的大小在各组之间相似。我们还跟踪了短链脂肪酸的吸收途径。我们测量了粪便、门户血和外周血的浓度。在这段视频中，我们描述了一个全面的方案，包括直接测量体内的肠血屏障渗透性，与常用方法相比，它不受肝脏和肾脏功能的影响，肝间隙测量，第三，评价外源化合物的吸收途径。提出的技术可以调整到其他实验目的。例如，门户血压测量或药物给药直接到门户静脉。

Research

JoVE Journal

Medicine

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

科研

医学

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Subnormothermic的技术<em&gt;前体内</em&gt;肝脏灌注的存储，评估和修复边际移植肝

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

An Improved Method for Rapid Intubation of the Trachea in Mice

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

用于评估椎弓根螺钉的麻醉，手术和收获方法<em&gt;体内</em&gt;猪腰椎模型

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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

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

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

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