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<meta charset="utf8"></head><body>改进的血液采样技术以评估小鼠的肠道微生物代谢物

<ol><li>vander Hee, B., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+regulation+of+host+physiology+by+short-chain+fatty+acids%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">Microbial regulation of host physiology by short-chain fatty acids.</a> Trends Microbiol. 29 (8), 700-712 (2021).</li>
<li>Koh, A., De Vadder, F., Kovatcheva-Datchary, P., Bäckhed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+dietary+fiber+to+host+physiology%3A+Short-chain+fatty+acids+as+key+bacterial+metabolites%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">From dietary fiber to host physiology: Short-chain fatty acids as key bacterial metabolites.</a> Cell. 165 (6), 1332-1345 (2016).</li>
<li>Vinolo, M. A. R., Rodrigues, H. G., Nachbar, R. T., Curi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+inflammation+by+short+chain+fatty+acids%5BTitle%5D)+AND+%22Nutrients%22%5BJournal%5D)">Regulation of inflammation by short chain fatty acids.</a> Nutrients. 3 (10), 858-876 (2011).</li>
<li>Layden, B. T., Angueira, A. R., Brodsky, M., Durai, V., Lowe, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short+chain+fatty+acids+and+their+receptors%3A+new+metabolic+targets%5BTitle%5D)+AND+%22Transl+Res%22%5BJournal%5D)">Short chain fatty acids and their receptors: new metabolic targets.</a> Transl Res. 161 (3), 131-140 (2013).</li>
<li>den Besten, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+short-chain+fatty+acids+in+the+interplay+between+diet%2C+gut+microbiota%2C+and+host+energy+metabolism%5BTitle%5D)+AND+%22J+Lipid+Res%22%5BJournal%5D)">The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism.</a> J Lipid Res. 54 (9), 2325-2340 (2013).</li>
<li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elevated+gut+microbiome-derived+propionate+levels+are+associated+with+reduced+sterile+lung+inflammation+and+bacterial+immunity+in+mice%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Elevated gut microbiome-derived propionate levels are associated with reduced sterile lung inflammation and bacterial immunity in mice.</a> Front Microbiol. 10, 159(2019).</li>
<li>Liu, Q., Tian, X., Maruyama, D., Arjomandi, M., Prakash, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+immune+tone+via+gut-lung+axis%3A+Gut-derived+LPS+and+short-chain+fatty+acids%E2%80%99+immunometabolic+regulation+of+lung+IL-1%CE%B2%2C+FFAR2%2C+and+FFAR3+expression%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Lung immune tone via gut-lung axis: Gut-derived LPS and short-chain fatty acids’ immunometabolic regulation of lung IL-1β, FFAR2, and FFAR3 expression.</a> Am J Physiol Lung Cell Mol Physiol. 321 (1), L65-L78 (2021).</li>
<li>De Vadder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbiota-generated+metabolites+promote+metabolic+benefits+via+gut-brain+neural+circuits%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits.</a> Cell. 156 (1-2), 84-96 (2014).</li>
<li>Dalile, B., Van Oudenhove, L., Vervliet, B., Verbeke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+short-chain+fatty+acids+in+microbiota%E2%80%93gut%E2%80%93brain+communication%5BTitle%5D)+AND+%22Nat+Rev+Gastroenterol+Hepatol%22%5BJournal%5D)">The role of short-chain fatty acids in microbiota–gut–brain communication.</a> Nat Rev Gastroenterol Hepatol. 16 (8), 461-478 (2019).</li>
<li>Sun, M., Wu, W., Liu, Z., Cong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbiota+metabolite+short+chain+fatty+acids%2C+GPCR%2C+and+inflammatory+bowel+diseases%5BTitle%5D)+AND+%22J+Gastroenterol%22%5BJournal%5D)">Microbiota metabolite short chain fatty acids, GPCR, and inflammatory bowel diseases.</a> J Gastroenterol. 52 (1), 1-8 (2017).</li>
<li>Cummings, J. H., Pomare, E. W., Branch, H. W. J., Naylor, C. P. E., MacFarlane, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short+chain+fatty+acids+in+human+large+intestine%2C+portal%2C+hepatic+and+venous+blood%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Short chain fatty acids in human large intestine, portal, hepatic and venous blood.</a> Gut. 28 (10), 1221-1227 (1987).</li>
<li>Macfarlane, G. T., Gibson, G. R., Cummings, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+fermentation+reactions+in+different+regions+of+the+human+colon%5BTitle%5D)+AND+%22J+Appl+Bacteriol%22%5BJournal%5D)">Comparison of fermentation reactions in different regions of the human colon.</a> J Appl Bacteriol. 72 (1), 57-64 (1992).</li>
<li>Neis, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distal+versus+proximal+intestinal+short-chain+fatty+acid+release+in+man%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Distal versus proximal intestinal short-chain fatty acid release in man.</a> Gut. 68 (4), 764-765 (2019).</li>
<li>Yamashita, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+acetate+on+lipid+metabolism+in+muscles+and+adipose+tissues+of+type+2+diabetic+Otsuka+Long-Evans+Tokushima+Fatty+%28OLETF%29+Rats%5BTitle%5D)+AND+%22Biosci+Biotechnol+Biochem%22%5BJournal%5D)">Effects of acetate on lipid metabolism in muscles and adipose tissues of type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) Rats.</a> Biosci Biotechnol Biochem. 73 (3), 570-576 (2009).</li>
<li>Ríos-Covián, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+short+chain+fatty+acids+and+their+ink+with+diet+and+human+health%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Intestinal short chain fatty acids and their ink with diet and human health.</a> Front Microbiol. 7, 185(2016).</li>
<li>Jaworska, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vivo+method+for+evaluating+the+gut-blood+barrier+and+liver+metabolism+of+microbiota+products%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">An in vivo method for evaluating the gut-blood barrier and liver metabolism of microbiota products.</a> J Vis Exp. (140), e58456(2018).</li>
<li>Ashcroft, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+to+assess+arteriovenous+differences+across+the+liver+and+hindlimb+muscles+in+mice+following+treadmill+exercise%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol to assess arteriovenous differences across the liver and hindlimb muscles in mice following treadmill exercise.</a> STAR Protoc. 4 (1), 101985(2023).</li>
<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+sample+collection+in+small+laboratory+animals%5BTitle%5D)+AND+%22J+Pharmacol+Pharmacother%22%5BJournal%5D)">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li>
<li>Cheever, A. W., Warren, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Portal+vein+ligation+in+mice%3A+Portal+hypertension%2C+collateral+circulation%2C+and+blood+flow%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Portal vein ligation in mice: Portal hypertension, collateral circulation, and blood flow.</a> J Appl Physiol. 18, 405-407 (1963).</li>
<li>Bloemen, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short+chain+fatty+acids+exchange+across+the+gut+and+liver+in+humans+measured+at+surgery%5BTitle%5D)+AND+%22Clin+Nutr%22%5BJournal%5D)">Short chain fatty acids exchange across the gut and liver in humans measured at surgery.</a> Clin Nutr. 28 (6), 657-661 (2009).</li>
<li>Hoyles, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbiome%E2%80%93host+systems+interactions%3A+Protective+effects+of+propionate+upon+the+blood%E2%80%93brain+barrier%5BTitle%5D)+AND+%22Microbiome%22%5BJournal%5D)">Microbiome–host systems interactions: Protective effects of propionate upon the blood–brain barrier.</a> Microbiome. 6 (1), 55(2018).</li>
<li>Shimizu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Propionate-producing+bacteria+in+the+intestine+may+associate+with+skewed+responses+of+IL10-producing+regulatory+T+cells+in+patients+with+relapsing+polychondritis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Propionate-producing bacteria in the intestine may associate with skewed responses of IL10-producing regulatory T cells in patients with relapsing polychondritis.</a> PLoS One. 13 (9), e0203657(2018).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Propionate+protects+against+lipopolysaccharide-induced+mastitis+in+mice+by+restoring+blood%E2%80%93milk+barrier+disruption+and+suppressing+inflammatory+response%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Propionate protects against lipopolysaccharide-induced mastitis in mice by restoring blood–milk barrier disruption and suppressing inflammatory response.</a> Front Immunol. 8, 1108(2017).</li>
<li>Lucas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-chain+fatty+acids+regulate+systemic+bone+mass+and+protect+from+pathological+bone+loss%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Short-chain fatty acids regulate systemic bone mass and protect from pathological bone loss.</a> Nat Commun. 9 (1), 55(2018).</li>
<li>Xie, C., Wei, W., Zhang, T., Dirsch, O., Dahmen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+of+systemic+and+hepatic+hemodynamic+parameters+in+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Monitoring of systemic and hepatic hemodynamic parameters in mice.</a> J Vis Exp. (92), e51955(2014).</li>
<li>Sänger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrahepatic+vascular+anatomy+in+rats+and+mice+-+Variations+and+surgical+implications%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Intrahepatic vascular anatomy in rats and mice - Variations and surgical implications.</a> PLoS One. 10 (11), e0141798(2015).</li>
<li>Phillips, B. J., Mirzaie, M., Turco, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Portal+Vein+Injuries%3A+A+Review%5BTitle%5D)+AND+%22J+Emerg+Trauma%22%5BJournal%5D)">Portal Vein Injuries: A Review.</a> J Emerg Trauma. 2 (2), 4(2017).</li>
</ol>

<table><tbody><tr><td>2,2,2 Tribromoethanol, 97% (Avertin)</td><td>Sigma Aldrich&amp;nbsp;</td><td>T48402-25G</td><td>Anesthetic agent&amp;nbsp;</td></tr><tr><td>Buprenorphine Hydrochloride Injection 0.3 mg/mL</td><td>PAR Pharmaceutical&amp;nbsp;</td><td>NDC 42023-179-05</td><td>Analgesic agent</td></tr><tr><td>Dressing Forceps</td><td>Miltex&amp;nbsp;</td><td>6-100</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Graefe Forceps</td><td>Roboz</td><td>RS-5136</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Hepatin sodium 1000 USP units/mL</td><td>Hikma</td><td>NDC 0641-0391-12</td><td>Blood sample syringes/tubes heparinization</td></tr><tr><td>Prolene 7-0</td><td>Ethicon</td><td>8696G</td><td>Portal vein ligature</td></tr><tr><td>Scissors</td><td>F.S.T</td><td>14058-11</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Student Halsted-Mosquito Hemostats</td><td>F.S.T&amp;nbsp;</td><td>91308-12</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Surgical tape&amp;nbsp;</td><td>3M Transpore</td><td>1527-1</td><td>Mouse limbs fixation</td></tr><tr><td>U-100 Insulin Syringe 28G1/2</td><td>EXEL</td><td>26027</td><td>Blood sample collection&amp;nbsp;</td></tr></tbody></table>

sequential blood collection from inferior vena cava followed by portal vein to evaluate gut microbial metabolites in mice

使用上述方法，我们可以在同一只小鼠中依次采集 IVC 和 PV 的血液样本，成功率超过 95%。采集的平均血样体积为 IVC 为 0.25 mL，PV 为 0.35 mL。
使用气相色谱-质谱法 （GC-MS），我们测量了粪便、PV 血液和 IVC 血液中 SCFA 的浓度，从而能够追踪乙酸盐 （C2）、丙酸盐 （C3） 和丁酸盐 （C4） 的吸收和转运从结肠隔室，它们由发酵肠道细菌产生，进入体循环。
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<meta charset="utf8"></head><body>作者聚焦:通过短链脂肪酸分析实现肠道微生物组-肺组织通讯

从下腔静脉连续采集血液，然后进行门静脉采集以评估小鼠的肠道微生物代谢物

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous ...

sequential-blood-collection-from-inferior-vena-cava-followed-portal

Research

JoVE Journal

Immunology and Infection

Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice

2.5K 次观看.  University of California San Francisco and San Francisco General Hospital. 我们实验室研究的范围是了解肠道微生物组和肺组织之间的通讯，以及肠道微生物组的成分如何直接影响肺部免疫和炎症反应。我们专注于特定代谢物（即短链脂肪酸）的影响。短链脂肪酸，如乙酸盐、丙酸盐和丁酸盐被认为在影响实验小鼠的不同疾病过程中发挥作用。这些包括 2 型糖尿病的脂质代谢、多软骨炎、病理性骨质流失、过敏性哮喘...

视频: 作者聚焦:通过短链脂肪酸分析实现肠道微生物组-肺组织通讯

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

小鼠全身和肝脏血流动力学参数的监测

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

科研

医学

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

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

一种评估微生物群产品肠血屏障和肝脏代谢的体内方法

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

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 ...

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

使用真空采血系统和眼镜放大镜从非麻醉小鼠的尾静脉中采集改良血液

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

真空采血系统和眼镜放大镜从非麻醉小鼠尾静脉中修饰采血

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

行为学

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

通过小鼠锁骨下静脉穿刺采集血液

Here, we present a protocol to take blood samples from the subclavian vein of mice.

小鼠锁骨下静脉穿刺采血

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

具有稳定的类人肠道微生物组和人体免疫系统的双重人源化 BLT 小鼠模型

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

具有稳定的人类状古特微生物群落和人类免疫系统的双人性化BLT小鼠模型

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

生物化学

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

使用标准化肠袢模型对小鼠肠道通透性和中性粒细胞跨上皮迁移进行功能评估

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

免疫与感染

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

小鼠门静脉离体肝脏灌注

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

离体 小鼠通过门静脉进行肝灌注

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

Murine Fecal Isolation and Microbiota Transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

小鼠粪便分离和微生物群移植

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

探索棕色脂肪组织中的代谢物交换

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

作者聚焦:阐明脂肪组织代谢和疾病预防的新途径 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Facial Vein Venipuncture for Murine Blood Collection

This paper presents an optimized protocol for murine blood collection via facial vein venipuncture using a lancet, offering a rapid, minimally invasive, and cost-effective alternative to other common techniques such as tail vein and retro-orbital sampling. This technique is particularly advantageous for studies requiring repeated sampling, such as mouse phenotyping, serum cytokine analysis, and diabetes research. The protocol is efficient, requires minimal specialized equipment, and is well-suited for high-throughput cellular analysis in mice. Our primary application is the phenotyping of transgenic, congenic, and reporter mice using flow cytometry, demonstrated here with antibody staining and tdTomato reporter mice. The paper outlines the complete procedure, from animal preparation to sample analysis. Key steps include blood collection with a 3 mm lancet, cell lysis, staining, and flow cytometric profiling. Facial vein venipuncture enhances collection efficiency while supporting ethical research practices. This practical and adaptable method is suitable for a wide range of experimental protocols involving murine blood sampling and cellular analysis.

Here, we present a protocol for an optimized blood collection technique in murine subjects through facial vein venipuncture using a lancet. We use this technique alongside flow cytometric assessment to characterize cellular phenotypes of transgenic, congenic, and reporter strains. It is also suitable for serum cytokine analysis and diabetes studies.

用于小鼠采血的面部静脉穿刺

This paper presents an optimized protocol for murine blood collection via facial vein venipuncture using a lancet, offering a rapid, minimally invasive, ...

从下腔静脉连续采集血液，然后进行门静脉采集以评估小鼠的肠道微生物代谢物

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全身及肝脏血流动力学参数的监测小鼠

微生物肠道血屏障及肝脏代谢的体内评价方法

真空采血系统和眼镜放大镜从非麻醉小鼠尾静脉中修饰采血

小鼠锁骨下静脉穿刺采血

具有稳定的人类状古特微生物群落和人类免疫系统的双人性化BLT小鼠模型

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

离体小鼠通过门静脉进行肝灌注

小鼠粪便分离和微生物群移植

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换

用于小鼠采血的面部静脉穿刺

从下腔静脉连续采集血液，然后进行门静脉采集以评估小鼠的肠道微生物代谢物

Transcript

Summary

Explore More Videos

全身及肝脏血流动力学参数的监测小鼠

微生物肠道血屏障及肝脏代谢的体内评价方法

真空采血系统和眼镜放大镜从非麻醉小鼠尾静脉中修饰采血

小鼠锁骨下静脉穿刺采血

具有稳定的人类状古特微生物群落和人类免疫系统的双人性化BLT小鼠模型

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

离体 小鼠通过门静脉进行肝灌注

小鼠粪便分离和微生物群移植

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

用于小鼠采血的面部静脉穿刺

离体小鼠通过门静脉进行肝灌注

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换