这项工作是从宾夕法尼亚州卫生署的拨款支持。 米格尔加西亚托斯卡诺博士是坦普尔大学的​​博士后研究员，在这项研究中的阿方马丁Escudero基​​金会资助。 我们要感谢的视频制作Iliya Yordanov和凯文Kotredes。

1。作为人类脓毒症小鼠模型的盲肠结扎穿孔对于此过程的C57BL / 6小鼠（7-9周龄）。 <ol><li> 1:1氯胺酮（75mg/kg）和甲苯噻嗪（按15mg/kg）的解决方案由腹腔注射麻醉鼠标。作为参考，一个重达20克的小鼠注入30μL1:1的解决方案。另外，可以使用麻醉蒸发器吸入异氟醚麻醉的小鼠。 </li><li>剃须鼠标腹部和消毒面积首次申请优碘用70％酒精棉签（1）在下面的图片序列擦拭的解决方案。洗必泰可作为替代消毒剂。可选:无菌的悬垂性，可用于维护该地区的清洁。 </li><li>在无菌条件下，实行1至2厘米的中线剖腹探查术和揭露与毗邻大肠盲肠（2和3）。 </li><li> 6.0丝线缝合（6-0普理灵，8680G;爱惜康）在其下面的回肠盲肠的阀门基地，是紧紧盲肠结扎穿孔一次或两次用19号针头（4和5）在同一一侧盲肠。请注意结扎盲肠结扎点从盲肠末端的距离定义的长度，将决定其严重程度。距离&gt;1厘米生产高档败血症≤1厘米的距离而产生中旬低档败血症。另外请注意，使用方法，这里显示的盲肠穿孔通过，并通过穿刺技术（通过盲肠针）的标准是不同的。这两种方法可靠地产生同样的败血症的结果。然而，通过，并通过技术的一个缺点是，它不便于控制的粪便量，盲肠被​​挤压时挤压。 </li><li>盲肠，然后轻轻挤压，挤出穿孔网站（6）少量的粪便。盲肠是返回到腹腔和腹膜6.0丝线缝合关闭。 （7）。 </li><li>皮肤反射7毫米剪辑（RS - 9258，Roboz手术器械）或米歇尔伤口剪辑（7毫米，RS - 9270）（8）关闭。 </li><li>复苏小鼠注射皮下注射1毫升预热0.9％生理盐水，使用25G针的解决方案。这种液体复苏措施，将引起败血症的高动力的阶段。这关键的一步是在讨论"部分进一步描述。 </li><li>可选:皮下注入丁丙诺啡（为0.05mg /公斤体重）或手术后镇痛的曲马多（20mg/kg体重）。被告知，这些阿片类药物的可以抑制呼吸和运动，这些影响可以被曲解为败血症的迹象。 </li><li>动物被暂时放在加热垫，或接触到的150W的红外线取暖灯或者立即返回到一个笼子里，直到他们从麻醉中恢复过来。恢复时间从30分钟至1小时。 </li><li>免费提供食物和水（水凝胶），放在笼底。 </li><li>小鼠进行监测，每12小时为一到两周的生存，或在不同的时间点不同的参数分析安乐死。 </li></ol>深水动物实验设计控制，将遵循不结扎和穿刺剖腹探查技术。 6至12小时后，手术过程中小鼠将成为昏昏欲睡和发展的发烧，立毛，腹泻，huddling，和不适，所有的败血症症状。非常严重的败血症小鼠可以勉强移动死前表现出体温的急剧下降。在这一步小鼠应安乐死，避免长时间的痛苦和磨难。 <img src="/files/ftp_upload/2860/2860fig0.jpg" alt="图0" /> 2。最常见的分析参数为了评估程序的结果，不同的参数，可以分析在器官，细胞提取物或体液。样品可以在不同时间点收集从3个小时的外科手术后一周。 <ol><li>小鼠的存活率。 </li><li>血清，腹腔及器官提取的细胞因子和趋化因子。 <ol><li> 白细胞介素6:生产和单核细胞，树突状细胞，巨噬细胞，B细胞，T细胞，粒细胞，肥大细胞和多细胞类型释放。在急性期反应和炎症反应中起着重要作用。 </li><li> 肿瘤坏死因子-α:Pleiotrophic起着核心作用，在炎症和细胞凋亡的细胞因子，。公司生产的单核细胞，巨噬细胞，中性粒细胞，树突状细胞和成纤维细胞。 </li><li> nterleukin -1β:由单核细胞，NK细胞，树突状细胞，B细胞和T细胞的产生。引起发热和急性期蛋白的合成。 </li><li> 白细胞介素10:促进吞噬细胞的摄取和Th2反应，但抑制antigEN介绍和Th1细胞促炎症反应。 </li><li> 白细胞介素10:促进吞噬细胞的摄取和Th2反应，但抑制抗原提呈和Th1细胞促炎症反应。 </li><li> 单核细胞趋化蛋白-1（也被称为CCL2）:新兵单核细胞，记忆性T细胞，树突状细胞和组织损伤和感染的网站。 </li><li> KC（CXCL1）:巨噬细胞和血管内皮细胞产生。鼠标KC是一个强有力的中性粒细胞的诱食和激活。 </li><li> RANTES的:一个单核细胞趋化。它可以chemoattract未刺激的CD4 + / CD45RO +记忆性T细胞和刺激的CD4 +和CD8 + T细胞与NA VE和记忆体的表型。 </li><li> 干扰素γ:由Th1细胞，细胞毒性T细胞，树突状细胞和自然杀伤细胞（NK）分泌的。提高在巨噬细胞抗原提呈和催化活性，并抑制Th2细胞的活性。促进白细胞迁移所需的附着力和约束力，促进NK细胞活性。 </li><li> 高流动性B组- 1蛋白在单核细胞是核因子κB激活提前糖化终产物（RAGE）的细胞受体结合，激活在败血症的后期阶段释放促炎介质。 </li></ol></li><li>作为衡量中性粒细胞浸润的髓过氧化物酶测定机关。髓过氧化物酶（MPO）是一个最丰富的中性粒细胞粒目前的过氧化物酶。这是一个存储azurophilic颗粒的中性粒细胞的溶酶体蛋白。 MPO有血红素色素，从而导致富含中性粒细胞的分泌物，脓和粘液的一些形式，如绿色。 </li><li>细菌器官和血液流加载，作为衡量每毫升菌落形成单位的数量。 </li></ol> 3。代表性的成果电过程最初是在C57BL / 6小鼠品系。我们测试的几个参数来调节通过改变结扎的长度和厚度的针， 如在图1和2所示的严重性败血症。在这两个因素中，结扎的长度似乎比针的厚度，以改变％的生存更有效。 如图1所示，增加结扎的长度超过1cm挑起与小鼠≤1厘米结扎相比，增加100％的死亡率。增加针的厚度也下降，从100％（使用22G针）的％生存到55％（使用19G针）与两个穿孔。我们还测试了在C57BL / 6和129SvJ小鼠中电的影响，以确定是否显示不同的小鼠品系相似或不同的CLP诱导的脓毒症的易感性。图3显示，在相同条件下，129SvJ老鼠比C57BL / 6表示％，死亡率增加，更容易感染。 <img src="/files/ftp_upload/2860/2860fig1.jpg" alt="图1" /> 图1。动物的生存盲肠结扎长度的影响 。电在C57BL / 6小鼠盲肠结扎使用两个不同长度的。这种单一的参数显着影响，因为与结扎的面积在不到3天死亡1厘米以上的所有组中的动物时，一组动物有一个约1厘米的结扎面积相比，动物的生存。 （N = 8）。 <img src="/files/ftp_upload/2860/2860fig2.jpg" alt="图2" /> 图2。针上生存的厚度的影响。电C57BL / 6小鼠使用两个大小不同的针，19G和22G。与盲肠穿孔使用19G针的小鼠表现出55-60％的生存。与此相反，小鼠盲肠穿孔使用22G针显示100％的生存，虽然他们经历了在第3-4天，炎症的典型症状消失后4天（N = 8）。 <img src="/files/ftp_upload/2860/2860fig3.jpg" alt="图3" /> 图3。比较脓毒症易感性之间的两个不同的小鼠品系 。CLP模型129SvJ和C57BL / 6相同的条件下进行。在评估期结束后129SvJ应变％的存活率为25％，比在C57BL / 6株少，说明一个更高的易感性多种微生物感染引起的炎症。 （N = 6）。

<ol>
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没有利益冲突的声明。<br /&gt;动物实验，根据实验动物护理和使用指南"国家卫生研究所，动物护理和使用坦普尔大学委员会批准。

在这里，我们详细显示如何执行电在小鼠模型，并调节强度等级。 相比其他动物模型的败血症，中电可以执行在任何不同的年龄和性别的小鼠品系。它是一个相对容易和便宜的外科手术。在这种模式下，强度等级有直接影响生存的百分比。结扎的长度，厚度的穿刺针和数量等参数，可以控制调节的严重性/电死亡率。因此，获得戏剧性的变化中电严重性，用户可以改变结扎的?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>氯胺酮</td><td> Ketaset </td><td> 0856-2013-01 </td><td></td></tr><tr><td>甲苯噻嗪</td><td> AnaSed </td><td> NADA＃139-236 </td><td></td></tr><tr><td>胰岛素注射器29G </td><td>埃克塞尔</td><td> 26028 </td><td></td></tr><tr><td>普理灵6-0丝线缝合， </td><td>爱惜康</td><td> 8680G </td><td></td></tr><tr><td> 19G和25G针</td><td>屋宇署</td><td> 305186 </td><td></td></tr><tr><td>剃须刀</td><td>一般供应</td><td>一般供应</td><td></td></tr><tr><td>红外线加热灯</td><td>一般供应</td><td>一般供应</td><td></td></tr><tr><td>米歇尔伤口剪辑7毫米</td><td> Roboz手术INSTR </td><td> RS - 9270 </td><td></td></tr><tr><td>耳挂面膜</td><td> Fisher Scientific则</td><td> 19-130-4181 </td><td></td></tr><tr><td>解剖剪刀</td><td> Roboz手术INSTR </td><td> RS - 6702 </td><td></td></tr><tr><td>优碘的解决方案</td><td> VWR </td><td> 63410-992 </td><td></td></tr><tr><td>手术钳</td><td> Roboz手术INSTR </td><td> RS - 5135 </td><td></td></tr><tr><td> 70％异丙醇垫</td><td> Fisher Scientific则</td><td> 22-031-350 </td><td></td></tr><tr><td>丁丙诺啡</td><td>贝德福德实验室</td><td> 55390-100-10 </td><td></td></tr><tr><td>曲马多</td><td> Sigma - Aldrich公司</td><td> 42965 </td><td></td></tr></tbody></table>

cecal ligation puncture procedure

人力败血症的特点是密集和大规模的感染，没有在本地主机中设置的全身反应。目前，在十大死亡原因，在美国重症监护病房 1中，败血症的行列。在败血症，有可能重叠的两个既定的血流动力学阶段。初始阶段（高动力）被定义为大规模生产的炎性细胞因子和活性氧的巨噬细胞和中性粒细胞，影响血管通透性导致低血压，心功能，导致代谢的变化，最终导致组织坏死和器官功能衰竭。因此，最常见的原因的死亡率是急性肾损伤。第二阶段（hypodynamic）是一个消炎的过程，涉及改变单核细胞抗原提呈，减少了淋巴细胞增殖功能和细胞凋亡增加。这种状态称为免疫抑制或免疫抑制大幅增加nocosomial感染和最终死亡的风险。这些病理生理过程的机制没有得到很好的特点。因为败血症的两个阶段，可能会导致不可逆的和不可弥补的损害，它是必不可少的，以确定患者的免疫和生理状态。这是许多治疗药物失败的主要原因。同一药物在不同的败血症阶段，可能是治疗或其他有害或有没有效果 2,3。了解在各级败血症关键是有一个合适的和全面的动物模型，再现了疾病的临床过程。这一点很重要的特点败血症过程中发生的病理生理机制，控制潜在的治疗药物测试模型条件。 为了研究人类败血症研究者的病因有不同的动物模型。临床使用最广泛的的模型是盲肠结扎穿孔（CLP）。电模式，使粪便材料释放进入腹膜腔，产生一个加剧的多种微生物感染引起的免疫反应的盲肠穿孔。这种模式符合人类生存条件，是临床相关。在人类，小鼠进行液体复苏的中华第一（初）高动力的阶段，在时间的推移到第二次（晚）hypodynamic阶段。此外，细胞因子的配置文件是类似人类败血症看到那里是增加淋巴细胞凋亡（4,5检讨） 。由于涉及多个重叠的败血症机制，研究人员需要一个合适的脓毒症模型控制的严重性，以取得一致的和可重复性的结果。

Watch this Scientific Journal Video about Cecal Ligation Puncture Procedure at JoVE.com

Cecal Ligation Puncture Procedure

作为一个有价值的工具，为研究人类败血症盲肠结扎穿刺的小鼠模型。

盲肠结扎穿刺过程

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the ...

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54.6K 次观看.  Temple University. 作为一个有价值的工具，为研究人类败血症盲肠结扎穿刺的小鼠模型。

视频: Cecal Ligation Puncture Procedure

The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL varies between 2.5 and 20% 2-5. Over the years, many strategies aimed at lowering the incidence of anastomotic leakage have been examined6, 7. 
The cause of AL is probably multifactorial. Etiological factors include insufficient arterial blood supply, tension on the anastomosis, hematoma and/or infection at the anastomotic site, and co-morbid factors of the patient as diabetes and atherosclerosis8. Furthermore, some anastomoses may be insufficient from the start due to technical failure.
Currently a new device is developed in our institute aimed at protecting the colorectal anastomosis and lowering the incidence of AL. This so called C-seal is a biofragmentable drain, which is stapled to the anastomosis with the circular stapler. It covers the luminal side of the colorectal anastomosis thereby preventing leakage.
The C-seal is a thin-walled tube-like drain, with an approximate diameter of 4 cm and an approximate length of 25 cm (figure 1). It is a tubular device composed of biodegradable polyurethane. Two flaps with adhesive tape are found at one end of the tube. These flaps are used to attach the C-seal to the anvil of the circular stapler, so that after the anastomosis is made the C-seal can be pulled through the anus. The C-seal remains in situ for at least 10 days. Thereafter it will lose strength and will degrade to be secreted from the body together with the gastrointestinal natural contents.
The C-seal does not prevent the formation of dehiscences. However, it prevents extravasation of faeces into the peritoneal cavity. This means that a gap at the anastomotic site does not lead to leakage.
Currently, a phase II study testing the C-seal in 35 patients undergoing (colo-)rectal resection with stapled anastomosis is recruiting. The C-seal can be used in both open procedures as well as laparoscopic procedures. The C-seal is only applied in stapled anastomoses within 15cm from the anal verge. In the video, application of the C-seal is shown in an open extended sigmoid resection in a patient suffering from diverticular disease with a stenotic colon.

The C-seal is a biofragmentable drain protecting the stapled colorectal anastomosis. In this video, details of the manufacturing, deployment and use are shown.

C密封:一个Biofragmentable漏漏电保护吻合结直肠吻合

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL ...

科研

医学

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

免疫与感染

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

导尿在反刍动物的肠袢

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

生物学

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

脓毒症诱导免疫抑制的切卡结扎和穿刺和鼻内感染双重模型的设计

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Here we present a protocol to surgically create &#39;intestinal ligated loops&#39; in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains&#39; virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

麻醉特异性病原体游离鸡结扎肠环模型研究荚梭菌毒力

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as ...

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.

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

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 ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

在盲肠结扎和穿刺诱导的脓毒症小鼠模型中评估可靠的生物标志物

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

Laparoscopic Non-Mesh Cerclage Pectopexy for Pelvic Organ Prolapse

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore apical support for treating POP. Sacrocolpopexy and pectopexy are indicated for apical prolapse. Using a synthetic mesh in these techniques increases success by enhancing apical support. However, the implantation of synthetic mesh is associated with mesh-related complications. In addition, the exorbitant cost of synthetic mesh and lack of universal access limit the popularity of these procedures. The current study develops a unique technique known as laparoscopic non-mesh cerclage pectopexy (LNMCP), in which permanent cervical cerclage sutures are embedded in the round ligament until the iliopectineal ligament. The iliopectineal ligament was sutured, resulting in a firm cervical suspension. The procedure was successfully performed in 16 cases in the hospital. The surgical duration was 67.8 min &plusmn; 15.5 min, and the blood loss was 73.1 mL &plusmn; 51.1 mL. No procedural complications were seen. LNMCP is associated with an objective success rate of 100% and a subjective success rate of 93.8%. LNMCP for patients with apical prolapse obviates the need for a mesh, thereby avoiding complications associated with mesh erosion and reducing medical costs. In addition, it is easy to perform even in resource-poor areas without access to synthetic mesh.

The present pilot study describes the development of laparoscopic non-mesh cerclage pectopexy to treat pelvic organ prolapse. The procedure can be used to prevent any complications associated with the use of mesh.

腹腔镜非网片环扎胸肌固定术治疗盆腔器官脱垂

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore ...

Surgical Technique for Lumbar Spinal Catheter Insertion in Pigs Enabling Continuous Access to the Thecal Sac in a Terminal Setup

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine and human central nervous systems (CNS). However, accessing the cerebrospinal fluid (CSF) in larger pig breeds by conventional lumbar puncture techniques can be challenging due to an oblique orientation of the spinal spinous processes and a limited interlaminar space. Accordingly, an open surgical procedure for inserting a lumbar spinal catheter for continuous CSF sampling at the L4/L5 level in pigs is thoroughly described in this work. After positioning the pig and identifying the anatomical landmarks, a dorsal midline surgical incision is made to expose the spinous processes. By advancing the introducer needle, the spinal catheter is inserted inside the thecal sac of the spinal canal while leaving the bone structures of the spine intact. This method allows continuous infusion into or sampling from the porcine thecal sac with minimal bleeding or CSF leakage. The procedure is simple, time-efficient, and reproducible across different experimental setups, offering significant potential for various pre-clinical studies, including pharmacokinetic research, surgical training, and spinal cord injury models.

We present a technique for inserting a lumbar spinal catheter at the L4-L5 level in a 3-month-old Danish Landrace pig as part of a terminal research protocol, enabling continuous infusion or CSF sampling from the thecal sac.

猪腰椎导管插入手术技术，可在终端设置中连续进入鞘囊

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine ...