这项工作得到了“Société Française d'Anesthésie et de Réanimation”（法国巴黎）、“Fondation d'entreprises Genavie”（法国南特）、“Fédération française de cardiologie”（法国）、“Agence nationale de la recherche”（20-ASTC-0032-01-hErOiSmE）（法国巴黎）和“Direction Générale de l'Armement”（法国巴黎）的支持。Thomas Dupas 在攻读博士学位期间得到了法国 Direction Générale de l'Armement （DGA） 和 Région des Pays de la Loire 的资助。Antoine Persello 在攻读博士学位期间得到了法国 InFlectis BioScience 的资助。我们感谢 “Agence Nationale de la Recherche” （法国巴黎）、“Direction Générale de l'Armement” （法国巴黎） 和 “Sauve ton coeur” 协会（法国）支持这项工作。我们感谢 UTE IRS-UN 核心设施（SFR Bonamy，南特大学，法国南特）和 IBISA 核心设施 Therassay（法国南特）的协助和技术支持。

根据欧盟指令 2010/63/EU，所有程序均按照区域伦理委员会（协议（协议（#17858 和 #32499，CEEA-Pays de la Loire，France））获得批准和执行。该报告符合当前的 ARRIVE 指南和美国国立卫生研究院 （NIH） 关于实验动物护理和使用指南（NIH Pub. No. 85-23,2011 年修订）。
1. 大鼠的道德状况和一般信息
<ol><li>雄性 Wistar Han 大鼠 （Charles River， Saint-Germain-Nuelles， France） 在 10 周龄时被运送到 Unité Thérapeutique Expérimentale。将大鼠在标准温度（21-24°C），湿度（40%-60%）和12小时光照/黑暗循环下饲养至少1周，光照期从上午07：30开始。</li>
	<li>随意提供食物和水。使用体重为 300-400 g 的 11-13 周龄大鼠进行方案。</li>
</ol>2. 房间设置和准备步骤
<ol><li>打开麻醉站，将加热垫设置为 37.5 °C，并用未消毒的窗帘覆盖。</li>
	<li>对方案所需的手术器械进行消毒：DeBakey 无创镊子 （1）、精细锋利的剪刀或手术刀 （1）、标准图案镊子 （2）、持针器 （1）、Vannas 显微解剖剪刀 （1）、夹子 （1）。</li>
	<li>为颈静脉和股动脉准备导管。<ol><li>对于颈静脉导管，将聚乙烯 （PE） 管 （PE50） 安装在 23 G 针头上，末端剪掉尖端。</li>
		<li>对于股动脉导管，准备步骤 2.3.1 中描述的导管，并将 PE10 管连接到 PE50 管的末端。</li>
	</ol></li>
	<li>准备用于股动脉和颈静脉的压力传感器和导管，用 100 UI/mL 的肝素化乳酸林格溶液填充它。将含有 100 UI/mL 肝素化乳酸林格溶液的 2 mL 注射器放在导管和压力传感器的末端。小心不要出现任何气泡，因为这些气泡会导致信号缺陷。</li>
	<li>打开与压力传感器相关的软件，并根据供应商的说明进行校准。</li>
</ol>3. 为大鼠手术做准备
<ol><li>用感应箱麻醉大鼠（参数：七氟烷 8%，空气流速：1 L/min）。通过踏板反射检查麻醉深度后，将麻醉维持在七氟烷 4%，气流速率为 0.6 L/min。由于这些分子的血流动力学效应，避免了术前使用镇痛药。 注意：从此步骤开始，每 20 分钟通过踏板反射评估麻醉深度。</li>
	<li>称量大鼠，将其置于加热垫上的背侧卧位，然后在腹股沟和颈部区域脱毛。</li>
	<li>使用非无菌纱布垫，用交替的 10% 和 4% 聚维酮碘溶液（每次 3 次）对脱毛区域进行消毒。</li>
	<li>将一滴眼药膏涂抹在动物的眼睛上以防止干燥。</li>
	<li>将大鼠放在手术台上的加热垫上（背侧 decubitus）。放置润滑的直肠探针以控制大鼠的体温。将麻醉维持在七氟烷 4%，气流速率为 0.6 L/min。</li>
	<li>将手套换成无菌手套，将无菌窗帘放在大鼠身上，然后在腹股沟和颈部区域切割。</li>
</ol>4. 颈静脉插管
<ol><li>通过可见的搏动，将颈静脉区域定位在锁骨上方的右下颈部区域。使用 DeBakey 无创镊子，轻轻抓住皮肤，然后用细而锋利的剪刀或手术刀做一个精确的切口。</li>
	<li>将一滴局部麻醉剂（利多卡因 2%）涂抹在切口区域。用标准图案的镊子轻轻切割组织。</li>
	<li>找到颈静脉，然后用标准图案的镊子轻轻地将其从肌肉隔室中释放出来。放置 4/0 丝缝合线并牢固地结扎远端（朝向头部）。使用带有持针器的缝合线拉紧颈静脉。</li>
	<li>在近端（朝向心脏）放置 4/0 缝合线，并在不收紧的情况下准备一个外科医生结。使用 Vannas 显微解剖剪刀在颈静脉上做一个小切口。</li>
	<li>用小镊子小心抓住静脉壁后，用手或标准镊子将 PE50 导管插入颈静脉。在这个阶段，使用无菌纱布垫擦拭最终的血滴，这些血滴可能会从静脉中渗出。</li>
	<li>将导管稍微向前推进（0.5 厘米），并通过抽取少量血液然后重新注射来验证其在静脉中的正确位置（套管中的血液表明它在静脉中）。拧紧先前准备好的结以固定导管。</li>
	<li>将导管留在原位，连接到预装乳酸林格氏液的注射器上，并用湿润的无菌纱布垫覆盖切口区域。</li>
</ol>5. 股动脉插管
<ol><li>使用 DeBakey 无创镊子抓住左腿 Scarpa 三角形的皮肤，并用细而锋利的剪刀或手术刀切开。将一滴局部麻醉剂（利多卡因 2%）涂抹在切口区域。用标准图案镊子轻轻稀释组织。</li>
	<li>找到股骨三联征（动脉、静脉和神经）。将一个镊子穿过三联体下方，并使用第二个标准模式镊子非常轻柔地将动脉与神经和股静脉分开。 注意：动脉较小，呈粉红色，有搏动。这是一个关键的步骤;静脉和动脉很脆弱，很容易破裂。</li>
	<li>放置 4/0 缝合线并在远端（腿侧）结扎股动脉。用持针器缝合，以拉紧股动脉。在近端侧（心脏侧）放置 4/0 缝合线，并准备打结而不闭合。夹住动脉（将夹子放在近端侧缝合线的上游）。</li>
	<li>用 Vannas 显微解剖剪刀横向切开股动脉（应有少量血液从动脉流出）。使用标准型镊子，轻轻抓住动脉壁，用导管（PE10 末端）插管股动脉，用镊子固定。</li>
	<li>轻轻松开夹紧以检查是否泄漏，然后检查压力信号（验证导管是否在股动脉中），并检查血液是否没有回流到导管中（阀门/压力传感器泄漏的迹象）。</li>
	<li>如果信号良好（预期值：收缩压：120 mmHg，舒张压：80 mmHg）并且没有渗漏迹象，则稍微推进导管（0.5 cm）并收紧先前准备好的外科医生结。</li>
	<li>以 100 UI/kg 的浓度对动物进行肝素化，并在切口区域涂抹湿润的无菌纱布垫。手术后，以 0.6 L/min 的空气流速维持 3% 七氟烷的麻醉。</li>
</ol>6. 失血性休克方案（图 1）
<ol><li>第 1 阶段：稳定（5 分钟）。<ol><li>肝素化后，等待 10 分钟，让压力值稳定下来。为基础血流动力学值进行 5 分钟记录。</li>
	</ol></li>
	<li>第 2 阶段：放血（5 分钟）： 注意：此阶段对应于模型的固定体积阶段。<ol><li>使用 1 mL 注射器在 5 分钟 （500 μL/30 s） 内从股动脉抽取 5 mL 血液（预期值：收缩压：45 mmHg，舒张压：30 mmHg）。</li>
		<li>在室温下制备 50% 乳酸林格氏液与 50% 采集血液的混合物。将混合物放入 2 mL 注射器中，并将其放在颈静脉插管的末端。</li>
	</ol></li>
	<li>第 3 阶段：失血性休克（75 分钟） 注意：此阶段是模型的固定压力阶段。<ol><li>将平均动脉压维持在平均 35 mmHg。当平均动脉压超过或等于 38 mmHg 时，通过股动脉抽取 200 μL 血液（n = 12 只大鼠的平均值：10.2 mL）。</li>
		<li>如果平均动脉压降至 32 mmHg 以下，则通过颈静脉注射 200 μL 的 50% 血液-50% 乳酸林格氏液混合物（n = 12 只大鼠的平均值：0.90 mL）。</li>
		<li>在出血性休克期结束时用纱布垫对尾部进行无菌准备后，使用针尖 （26 G） 测量尾部滴血上的外周血乳酸。</li>
	</ol></li>
	<li>第 4 阶段：静脉输液复苏（20 分钟）<ol><li>用 20 mL 注射器在室温 （RT） 下以 10 mL/kg 的浓度在 20 分钟内用乳酸林格氏液复苏（350 g 大鼠的流速为 10.5 mL/h），通过带有注射泵的颈静脉导管。</li>
	</ol></li>
</ol>7. 手术结束和恢复以及术后随访
<ol><li>夹住颈静脉和股动脉并取出导管。结扎血管。仔细检查是否有血液渗出。</li>
	<li>在切口区域添加一滴局部麻醉剂。使用 5-0 无菌缝合线用皮下和皮肤缝线缝合切口区域。用 10% 聚维酮碘溶液消毒。</li>
	<li>使用带有 0.05 G 针头的 0.3 mL 注射器皮下注射丁丙诺啡（0.3 mg/kg，26 mL/kg）。</li>
	<li>等待大鼠从麻醉中撤离，同时监测温度。当它出现醒来的迹象（弧菌移动、爪子运动）时，取下直肠探针，将老鼠放在笼子里的加热垫上。10 分钟后，将老鼠送回他的住宿室。</li>
	<li>每 8 小时皮下注射丁丙诺啡（0.05 mg/kg，0.3 mL/kg）。</li>
	<li>如步骤 6.3 所述，在出血性休克诱导后 16 小时评估呼吸速率、行为、温度和血乳酸。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66523/66523fig01.jpg"> 图 1：混合大鼠出血性休克模型。 使用 BioRender.com 创建 <a href="https://www.jove.com/files/ftp_upload/66523/66523fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
8. 出血性休克诱导后 24 小时
<ol><li>按照步骤 3.1 中的说明麻醉动物。将动物放在手术台上，并按照步骤 3.5 中的说明插入直肠探针。按照步骤 2.3 和 2.4 中针对颈静脉的描述，准备用于颈动脉插管的导管。</li>
	<li>使用 DeBakey 无创镊子抓住皮肤，并用细而锋利的剪刀或手术刀在颈部中间切开一个切口。滴一滴局部麻醉剂（利多卡因 2%）。</li>
	<li>用标准图案镊子轻轻稀释组织。分离唾液腺，然后打开气管肌肉，露出气管环。</li>
	<li>取左颈动脉，用标准镊子将其与神经分离（动物的呼吸极有可能加速）。用 4/0 丝线向远端（头部）结扎颈动脉。</li>
	<li>在近端侧（心脏侧）放置缝合线，并准备一个外科医生结，但不要闭合它。夹住动脉（将夹子放在近端侧缝合线的上游）。</li>
	<li>用 Vannas 显微解剖剪刀切开颈动脉（应有少量血液从动脉流出）。使用标准图案的镊子，轻轻抓住动脉壁以扩大开口，并用提供的导管插管颈动脉，用镊子固定。</li>
	<li>松开并检查压力信号以确认导管位置，并检查血液是否没有回流到导管中（阀门/压力传感器泄漏的迹象）。</li>
	<li>如果信号良好且没有泄漏，则将导管稍微向前推进（0.5 厘米）并收紧并固定先前准备好的外科医生结。</li>
	<li>以 100 UI/kg 的浓度对动物进行肝素化，并在切口上涂抹湿润的无菌敷布。</li>
	<li>手术后，使用 3% 七氟烷以 0.6 L/min 的气流速率维持麻醉。</li>
	<li>等待 10 分钟以记录 24 小时的血流动力学值（预期值：收缩压：120 mmHg，舒张压：60 mmHg）。</li>
	<li>为了评估器官损伤的血浆标志物，从颈动脉抽取 1 mL 血液并以 1600 x g 离心 10 分钟。分装并保存血浆以供进一步分析。</li>
	<li>在 24 小时测量血流动力学值后立即处死大鼠并收集血液。 注意：安乐死的方法应适应之后将收集的特定参数或样本。假手术组仅接受手术（第 1-5 、 7 和 8 节），而不接受失血性休克手术（第 6 节）。实验者将动物随机分配到假休克组和失血性休克组之间。只有实验者知道实验不同阶段的组分配。</li>
</ol>

大鼠血管插管与出血性休克诱导

<ol>
	<li>Cannon, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhagic+Shock.">Hemorrhagic Shock.</a> New Eng J Med. 378 (4), 370-379 (2018).</li><li>Fox, E. E., Holcomb, J. B., Wade, C. E., Bulger, E. M., Tilley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Earlier+endpoints+are+required+for+hemorrhagic+shock+trials+among+severely+injured+patients.">Earlier endpoints are required for hemorrhagic shock trials among severely injured patients.</a> Shock. 47 (5), 567-573 (2017).</li><li>Frink, M., Andruszkow, H., Zeckey, C., Krettek, C., Hildebrand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+trauma+models:+an+update.">Experimental trauma models: an update.</a> J Biomed Biotechnol. 2011, 797383 (2011).</li><li>Hauser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+traumatic,+hemorrhagic+shock.">Preclinical models of traumatic, hemorrhagic shock.</a> Shock. 24, 24-32 (2005).</li><li>Kemming, 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=Can+we+continue+research+in+splenectomized+dogs?+Mycoplasma+haemocanis:+Old+problem+-+new+insight.">Can we continue research in splenectomized dogs? Mycoplasma haemocanis: Old problem - new insight.</a> Eur Sur Res. 36 (4), 198-205 (2004).</li><li>Carroll, R. G., Iamsa, S. G., Pryor, W. H., Allison, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+hemorrhage:+A+clinically+relevant+canine+model+of+hemorrhagic+shock.">Single hemorrhage: A clinically relevant canine model of hemorrhagic shock.</a> Resuscitation. 16 (2), 119-126 (1988).</li><li>Cheung, A. T., Duong, P. L., Driessen, B., Chen, P. C., Jahr, J. S., Gunther, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+function,+oxygenation+and+microvascular+correlation+during+treatment+of+hemorrhagic+shock+with+blood+substitutes.">Systemic function, oxygenation and microvascular correlation during treatment of hemorrhagic shock with blood substitutes.</a> Clin Hemorheol Microcirc. 34 (1-2), 325-334 (2006).</li><li>. Hemorrhage and hemorrhagic shock in swine: A review Available from: <a target="_blank" href="https://apps.dtic.mil/sti/citations/ADA221297">https://apps.dtic.mil/sti/citations/ADA221297</a> (1990)</li><li>Lomas-Niera, J. L., Perl, M., Chung, C. -. S., Ayala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+and+hemorrhage:+an+overview+of+animal+models.">Shock and hemorrhage: an overview of animal models.</a> Shock. 24, 33-39 (2005).</li><li>Redl, H., Bahrami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+animal+models:+baboons+for+trauma,+shock,+and+sepsis+studies.">Large animal models: baboons for trauma, shock, and sepsis studies.</a> Shock. 24, 88-93 (2005).</li><li>Marques-Bonet, T., Cheng, Z., She, X., Eichler, E. E., Navarro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+distribution+of+intraspecific+and+interspecific+sequence+divergence+of+human+segmental+duplications+relative+to+human/chimpanzee+chromosomal+rearrangements.">The genomic distribution of intraspecific and interspecific sequence divergence of human segmental duplications relative to human/chimpanzee chromosomal rearrangements.</a> BMC Genom. 9, 384 (2008).</li><li>Schlag, G., Redl, H. R., Till, G. O., Davies, J., Martin, U., Dumont, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-L-selectin+antibody+treatment+of+hemorrhagic-traumatic+shock+in+baboons.">Anti-L-selectin antibody treatment of hemorrhagic-traumatic shock in baboons.</a> Crit Care Med. 27 (9), 1900 (1999).</li><li>F&#252;l&#246;p, A., Tur&#243;czi, Z., Garbaisz, D., Hars&#225;nyi, L., Szij&#225;rt&#243;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+hemorrhagic+shock:+A+review.">Experimental models of hemorrhagic shock: A review.</a> Eur Surg Res. 50 (2), 57-70 (2013).</li><li>Zingarelli, B., Ischiropoulos, H., Salzman, A. L., Szab&#243;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+by+mercaptoethylguanidine+of+the+vascular+and+energetic+failure+in+haemorrhagic+shock+in+the+anesthetised+rat.">Amelioration by mercaptoethylguanidine of the vascular and energetic failure in haemorrhagic shock in the anesthetised rat.</a> Eur J Pharmacol. 338 (1), 55-65 (1997).</li><li>Gonzalez, R. J., Moore, E. E., Ciesla, D. J., Nieto, J. R., Johnson, J. L., Silliman, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-hemorrhagic+shock+mesenteric+lymph+activates+human+pulmonary+microvascular+endothelium+for+in+vitro+neutrophil-mediated+injury:+the+role+of+intercellular+adhesion+molecule-1.">Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1.</a> J Trauma. 54 (2), 219-223 (2003).</li><li>Hoppen, R. A., Corso, C. O., Grezzana, T. J. M., Severino, A., Dal-Pizzol, F., Ritter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertonic+saline+and+hemorrhagic+shock:+hepatocellular+function+and+integrity+after+six+hours+of+treatment.">Hypertonic saline and hemorrhagic shock: hepatocellular function and integrity after six hours of treatment.</a> Acta Cir Bras. 20 (6), 414-417 (2005).</li><li>Rupani, B., 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=Relationship+between+disruption+of+the+unstirred+mucus+layer+and+intestinal+restitution+in+loss+of+gut+barrier+function+after+trauma+hemorrhagic+shock.">Relationship between disruption of the unstirred mucus layer and intestinal restitution in loss of gut barrier function after trauma hemorrhagic shock.</a> Surgery. 141 (4), 481-489 (2007).</li><li>Cai, B., Dong, W., Sharpe, S., Deitch, E. A., Ulloa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+inflammatory+responses+in+experimental+models+of+hemorrhage.">Survival and inflammatory responses in experimental models of hemorrhage.</a> J Surg Res. 169 (2), 257-266 (2011).</li><li>Esiobu, P., Childs, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+hemorrhagic+shock+for+studying+vascular+hyperpermeability.">A rat model of hemorrhagic shock for studying vascular hyperpermeability.</a> Methods Mol Biol. 1717, 53-60 (2018).</li><li>Sordi, R., Chiazza, F., Patel, N. S. A., Doyle, R. A., Collino, M., Thiemermann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning'+with+low+dose+lipopolysaccharide+aggravates+the+organ+injury+/+dysfunction+caused+by+hemorrhagic+shock+in+rats.">Preconditioning' with low dose lipopolysaccharide aggravates the organ injury / dysfunction caused by hemorrhagic shock in rats.</a> PLoS One. 10 (4), 0122096 (2015).</li><li>Liu, Y., 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=Modified+shock+index+and+mortality+rate+of+emergency+patients.">Modified shock index and mortality rate of emergency patients.</a> World J Emerg Med. 3 (2), 114-117 (2012).</li><li>Sahu, N., 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=Shock+index+as+a+marker+for+mortality+rates+in+those+admitted+to+the+medical+intensive+care+unit+from+the+emergency+department.">Shock index as a marker for mortality rates in those admitted to the medical intensive care unit from the emergency department.</a> Cureus. 12 (4), e7903 (2020).</li><li>Zou, 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=Glucosamine+improves+cardiac+function+following+trauma-hemorrhage+by+increased+protein+O-GlcNAcylation+and+attenuation+of+NF-&#954;B+signaling.">Glucosamine improves cardiac function following trauma-hemorrhage by increased protein O-GlcNAcylation and attenuation of NF-&#954;B signaling.</a> Am J Physiol Heart Circ Phy. 296 (2), H515-H523 (2009).</li><li>N&#246;t, L. G., Marchase, R. B., F&#252;l&#246;p, N., Brocks, C. A., Chatham, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucosamine+administration+improves+survival+rate+after+severe+hemorrhagic+shock+combined+with+trauma+in+rats.">Glucosamine administration improves survival rate after severe hemorrhagic shock combined with trauma in rats.</a> Shock. 28 (3), 345 (2007).</li><li>N&#246;t, L. G., Brocks, C. A., V&#225;mhidy, L., Marchase, R. B., Chatham, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+O-linked+&#946;-N-acetylglucosamine+levels+on+proteins+improves+survival,+reduces+inflammation+and+organ+damage+24+hours+after+trauma-hemorrhage+in+rats.">Increased O-linked &#946;-N-acetylglucosamine levels on proteins improves survival, reduces inflammation and organ damage 24 hours after trauma-hemorrhage in rats.</a> Crit Care Med. 38 (2), 562-571 (2010).</li><li>Patel, N. 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=Inhibition+of+macrophage+migration+inhibitory+factor+activity+attenuates+haemorrhagic+shock-induced+multiple+organ+dysfunction+in+rats.">Inhibition of macrophage migration inhibitory factor activity attenuates haemorrhagic shock-induced multiple organ dysfunction in rats.</a> Front Immunol. 13, 886421 (2022).</li><li>Patel, N. 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=Inhibition+of+Bruton's+tyrosine+kinase+activity+attenuates+hemorrhagic+shock-induced+multiple+organ+dysfunction+in+rats.">Inhibition of Bruton's tyrosine kinase activity attenuates hemorrhagic shock-induced multiple organ dysfunction in rats.</a> Ann Surg. 277 (3), e624-e633 (2023).</li><li>Curcio, L., D'Orsi, L., De Gaetano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+mathematical+models+of+hemorrhagic+shock.">Seven mathematical models of hemorrhagic shock.</a> Comput Math Methods Med. 2021, e6640638 (2021).</li><li>R&#246;nn, T., Lendemans, S., de Groot, H., Petrat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+severe+hemorrhagic+shock+in+rats.">A new model of severe hemorrhagic shock in rats.</a> Comp Med. 61 (5), 419-426 (2011).</li><li>Hussmann, B., Lendemans, S., de Groot, H., Rohrig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+replacement+with+Ringer-lactate+is+detrimental+in+severe+hemorrhagic+shock+but+protective+in+moderate+hemorrhagic+shock:+studies+in+a+rat+model.">Volume replacement with Ringer-lactate is detrimental in severe hemorrhagic shock but protective in moderate hemorrhagic shock: studies in a rat model.</a> Crit Care. 18 (1), 5 (2014).</li><li>Mihara, R., Takasu, A., Maemura, K., Minami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+severe+hemorrhagic+shock+at+a+mean+arterial+pressure+of+40+mmHg+does+not+lead+to+brain+damage+in+rats.">Prolonged severe hemorrhagic shock at a mean arterial pressure of 40 mmHg does not lead to brain damage in rats.</a> Acute Med Surg. 5 (4), 350-357 (2018).</li><li>Vincent, J. -. L., De Backer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+shock.">Circulatory shock.</a> N Engl J Med. 369 (18), 1726-1734 (2013).</li><li>Halbgebauer, R., 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=Hemorrhagic+shock+drives+glycocalyx,+barrier+and+organ+dysfunction+early+after+polytrauma.">Hemorrhagic shock drives glycocalyx, barrier and organ dysfunction early after polytrauma.</a> J Crit Care. 44, 229-237 (2018).</li><li>Chipman, A. M., Jenne, C., Wu, F., Kozar, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contemporary+resuscitation+of+hemorrhagic+shock:+What+will+the+future+hold.">Contemporary resuscitation of hemorrhagic shock: What will the future hold.</a> Am J Surg. 220, 580-588 (2020).</li></ol>

作者声明，他们没有已知的可能影响本文报道的工作的竞争性经济利益或个人关系。支持本研究结果的数据可应合理要求从通讯作者处获得。

在本文中，我们首次描述了基于固定压力和固定体积模型混合的代表性大鼠失血性休克模型。我们证明，电击诱导后 24 小时，我们的模型与血流动力学参数和代谢的改变有关。
由于其复杂的病理生理学，出血性休克的研究需要利用综合动物模型。事实上，体外方法无法模拟这种疾病所涉及的所有途径。在出血性休克方案后唤醒动物是确保更好地复制临床情况的一个步骤。由于唤醒动物的难度，很少有研究包括这个阶段。唤醒动物的罕见研究在短时间内（2 小时或 6 小时）牺牲了它们，这并不能完全反映患者16、18、23、24 的情况。尽管开发了出血性休克模型，但只有少数研究评估了电击诱导后 24 小时的参数（炎症、细胞凋亡、器官功能障碍），从而突出了这种方案的难度 25,26,27。计算机和数学模型的发展彻底改变了研究。已经开发了许多失血性休克的数学模型，但这些模型中的大多数都没有考虑到失血性休克期间体液交换的全部范围，在潜在的临床适用性之前需要改进28。迄今为止，主要挑战之一是开发一种尽可能接近人类病理的动物模型。
文献中描述了大量的出血性休克模型，它们在血管入路、抽血量或目标压力 方面 有所不同13。更一般地说，出血性休克模型可分为 3 组：固定体积出血、固定压力出血和不受控制的出血。固定体积出血的标准化和可重复性是困难的，可以用血容量/体重比来解释，血容量/体重比随大鼠的体重线性降低。固定压力出血被广泛使用，从而解释了设置（目标压力、休克持续时间）因研究而异，因此很难将结果从一个模型转移到另一个模型。同样重要的是要指出，血流动力学损害在出血性休克的病理生理学中起着关键作用，但尚未进行系统评估，这可能会增加研究之间结果的差异。最后，不受控制的出血模型虽然具有临床相关性，但引发了可重复性和伦理问题。为了尽可能协调临床相关性、标准化和可重复性，我们开发了一种具有固定体积和固定压力阶段的混合模型。
在此处描述的模型中，手术后 24 小时温度和呼吸频率不会改变。这可以通过以下事实来解释：手术是在无菌条件下进行的，从而限制了促炎反应。失血性休克被定义为由于与血压下降相关的失血引起的急性循环衰竭。与人类一样，这种出血性休克模型导致平均动脉压降低，主要是由于舒张压降低。有趣的是，如前所述，在这种出血性休克模型中，复苏阶段后的心率保持不变 29,30,31。平均动脉压的下降可能与器官灌注减少有关，导致多脏器功能障碍，这可以通过我们模型中各种血浆标志物（肌酐血症、心肌肌钙蛋白 T、ASAT 和 ALAT）的增加来说明。氧气供应的中断导致厌氧代谢，从而导致乳酸血症增加32。如前所述，这种出血性休克模型导致血乳酸水平升高30。这种增加可能与股动脉水平引起的缺血有关。然而，考虑到假手术组中的动物患有生理性乳酸血症并接受了与出血性休克组相同的外科手术，这种增加似乎与出血性休克方案有关。综上所述，所有这些数据都证实了本研究中描述的方案允许开发一种新的大鼠相关出血性休克模型。
该模型的局限性是使用肝素，肝素对于减少血液与塑料材料（如插管）接触时的自然凝固至关重要。然而，肝素的使用会影响与创伤性失血性休克相关的凝血病33。这项研究涉及 11-13 周龄的健康雄性动物。考虑到性别、年龄和合并症（高血压、糖尿病等）会影响结果，在我们的模型中评估它们的影响是相关的。在该方案中，复苏步骤是通过注射乳酸林格氏液进行的，乳酸林格液是一种可促进凝血病和组织水肿的晶体液34。尽管血液制品的使用是最佳的，但这些血液制品是稀缺且易腐烂的，并且可能很难为整个方案提供足够的大鼠血液库存。血液制品和基于晶体液/胶体的复苏失血性休克模型是两种互补的方法。
该模型的优势是 1） 其高重现性（由结果的低可变性说明），2） 它易于应用（大多数仪器是经典的，血管入路是已知的）和 3） 它的临床相关性，特别是由于动物觉醒和多内脏功能障碍。根据 补充文件 1 中描述的行为评分，设置了限制点。根据附表，如果达到 9 分以上的分数，将讨论牺牲。如果达到 11 分，动物将被系统地实施安乐死。在这项研究中，没有一只动物的分数高于 8，因此，没有一只动物被排除在研究之外。这可以解释为什么此处描述的模型与死亡率比另一项 24 小时研究低 3 倍（16% 对 47%）相关25。
该模型的关键步骤是出血性休克阶段。遵守 32-38 mmHg 的压力范围很重要。事实上，我们观察到，将平均动脉压保持在 32 mmHg 以下会导致动脉压快速突然下降。相反，将压力保持在 38 mmHg 以上并不能提供足够接近临床现实的模型。这些观察结果与其他模型13 中针对的平均动脉压的间隔一致。
总之，我们证明了本研究中详述的大鼠失血性休克模型具有临床相关性，并且可用于通过识别新的生物学参与者/途径来理解病理生理机制，以及通过测试不同的候选分子来确定新的治疗策略。

失血性休克 （HS） 是一种休克状态，其特征是血容量显著流失，导致组织缺氧。HS 是一种复杂的病理学，将血流动力学和代谢变化与促炎和抗炎反应相关联。全世界每年约有 190 万人死于出血及其后果1。目前的护理指南主要涉及静脉输液（补充或不补充血管活性分子）和氧疗。然而，这些治疗是对症的，可能无效，这解释了为什么 HS 相关死亡率仍然很高2。这证明了确定新的分子和细胞机制的重要性，从而证明降低死亡率的治疗方法的重要性。
动物模型允许破译疾病所涉及的病理生理机制并测试新的治疗策略。文献中存在许多出血性休克的动物模型。这些模型不仅在所使用的物种上有所不同，而且在诱导 HS 的方式上也不同（例如，固定压力与固定体积）（表 1、表 2）3,4,5,6,7,8,9,10,11,12,13 .此外，同一类型的模型（例如，出血时间、目标平均动脉压）中的方案也有所不同（表 3）14、15、16、17、18、19、20。考虑到现有出血性休克模型的种类繁多和复制临床情况的复杂性，这种病理学的临床前研究仍然有限。开发可重复、标准化且易于实施的出血性休克模型符合每个人的利益。这将有助于各种研究之间的比较，从而揭示出血性休克的复杂病理生理学。该方案的目的是开发一种新的临床相关的大鼠出血休克模型，使用两个连续的出血阶段，固定体积，然后是固定的低血压阶段。
表 1：用作出血性休克模型的物种 3,4,5,6,7,8,9,10,11,12,13。<a href="https://www.jove.com/files/ftp_upload/66523/Table%201.xlsx" target="_blank">请点击此处下载此表格。</a>
表 2：失血性休克的不同类型13. <a href="https://www.jove.com/files/ftp_upload/66523/Table%202.xlsx" target="_blank">请点击此处下载此表格。</a>
表 3：固定压力方案诱导的大鼠失血性休克实验模型的多样性示例。 出血性休克不同实验模型的参数总结。红色显示的血管是动脉，蓝色显示的血管是静脉。对于复苏，以采血量作为参考（血液：复苏量与休克期间采血量相同;x2：复苏量为休克期间采血量的两倍;x4：复苏量为休克期间采血量的四倍）。MAP： 平均动脉压;RL： 乳酸林格 14,15,16,17,18,19,20。<a href="https://www.jove.com/files/ftp_upload/66523/Table%203.xlsx" target="_blank">请点击此处下载此表格。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>TERUMO</td>
			<td>MDSS01SE</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mL syringe</td>
			<td>TERUMO</td>
			<td>SS*02SE1</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>TERUMO</td>
			<td>MDSS20ESE</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia induction chamber</td>
			<td>TEMSEGA</td>
			<td>HUBBIV4</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Microlance 3 23 G needle</td>
			<td>Becton Dickinson</td>
			<td>300800</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Microlance 3 26 G needle</td>
			<td>Becton Dickinson</td>
			<td>304300</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood pressure transducer</td>
			<td>emka TECHNOLOGIES</td>
			<td>BP_T</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprecare</td>
			<td>Axience</td>
			<td>N/A</td>
			<td>1 mL vial, buprenorphine 0.3 mg/mL</td>
		</tr>
		<tr>
			<td>DE BAKEY, Atraumatic Vascular Forceps</td>
			<td>ALLGAIER instrumente medical</td>
			<td>09-543-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermal Betadine 10%</td>
			<td>Mylan</td>
			<td>N/A</td>
			<td>125 mL bottle</td>
		</tr>
		<tr>
			<td>Fine Forceps - Curved / Serrated</td>
			<td>Fine Science Tools</td>
			<td>11065-07</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8</td>
			<td>GraphPad by Dotmatics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating mats</td>
			<td>TEMSEGA</td>
			<td>OPT/THERM_MATELASSTEREORATS</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>PANPHARMA&#160;</td>
			<td>N/A</td>
			<td>5 mL bottle, 5,000 UI/mL</td>
		</tr>
		<tr>
			<td>IOX2 software</td>
			<td>emka TECHNOLOGIES</td>
			<td>IOX_BASE_4c + IOX_FULLCARDIO_4a</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - ToughCut</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Fresenius</td>
			<td>N/A</td>
			<td>10 mL bootle, 8.11 mg, lidocaine hydrochloride</td>
		</tr>
		<tr>
			<td>MiniHub-V3.2</td>
			<td>TEMSEGA</td>
			<td>PF006</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria 201/A Vessel Clamp - Straight</td>
			<td>Fine Science Tools</td>
			<td>18320-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Non sterile compresses</td>
			<td>Raffin</td>
			<td>70189</td>
			<td></td>
		</tr>
		<tr>
			<td>Non sterile drape</td>
			<td>Dutscher&#160;</td>
			<td>30786</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder with Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing PE10</td>
			<td>PHYMEP</td>
			<td>BTPE-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing PE50</td>
			<td>PHYMEP</td>
			<td>BTPE-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Rats</td>
			<td>Charles Rivers</td>
			<td>-</td>
			<td>Male WISTAR HAN (10 weeks)</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>TEMSEGA</td>
			<td>SONDE_TEMP_RATS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer Lactates</td>
			<td>Fresenius Kabi</td>
			<td>964175</td>
			<td></td>
		</tr>
		<tr>
			<td>Scrub Betadine 4%</td>
			<td>Mylan</td>
			<td>N/A</td>
			<td>125 mL bottle</td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Abbott</td>
			<td>N/A</td>
			<td>250 mL bottle, gas 100%</td>
		</tr>
		<tr>
			<td>Sevoflurane Vaporizer</td>
			<td>TEMSEGA</td>
			<td>SEVOTEC3NSELEC</td>
			<td></td>
		</tr>
		<tr>
			<td>StatStrip lactate test strips</td>
			<td>Nova Biomedical</td>
			<td>47486</td>
			<td></td>
		</tr>
		<tr>
			<td>StatStrip Xpress lactate Meter</td>
			<td>Nova Biomedical</td>
			<td>47486</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile compresses</td>
			<td>Laboratoire SYLAMED</td>
			<td>211S05-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile drape</td>
			<td>M&#246;lnlycke</td>
			<td>800330</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriles gloves</td>
			<td>MEDLINE</td>
			<td>MSG7275</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Optilene</td>
			<td>3097141</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture for vessels</td>
			<td>SMI</td>
			<td>8150046</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Vial m&#233;dical</td>
			<td>16010</td>
			<td></td>
		</tr>
		<tr>
			<td>usbAMP</td>
			<td>emka TECHNOLOGIES</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Cooper</td>
			<td>N/A</td>
			<td>10 mL vial</td>
		</tr>
		<tr>
			<td>Vitamin A Dulcis (ALLERGAN)</td>
			<td>Allergan</td>
			<td>N/A</td>
			<td>10 g tube, Retinol</td>
		</tr>
	</tbody>
</table>

developing a clinically relevant hemorrhagic shock model in rats

近几十年来，动物模型的发展使我们能够更好地了解各种病理并确定新的治疗方法。失血性休克，即由于大量血液快速丢失而导致的器官衰竭，与涉及多个通路的高度复杂的病理生理学有关。许多现有的出血性休克动物模型都试图复制人类身上发生的情况，但这些模型在临床相关性、可重复性或标准化方面存在局限性。本研究的目的是改进这些模型以开发一种新的失血性休克模型。简而言之，在雄性 Wistar Han 大鼠 （11-13 周龄） 中，通过受控放血诱导出血性休克，导致平均动脉压下降。下一阶段 75 min 是维持较低的平均动脉血压，在 32 mmHg 和 38 mmHg 之间，以触发失血性休克的病理生理途径。该方案的最后阶段通过静脉输液乳酸林格液来模拟患者护理，以升高血压。方案开始后 16 小时评估乳酸和行为评分，受伤后 24 小时评估血流动力学参数和血浆标志物。出血性休克诱导后 24 小时，出血性休克组的平均动脉压和舒张压降低 （p < 0.05）。心率和收缩压保持不变。所有器官损伤标志物均随着出血性休克而增加 （p < 0.05）。与假手术组相比，乳酸血症和行为评分增加 （p < 0.05）。总之，我们证明此处描述的方案是失血性休克的相关模型，可用于后续研究，特别是用于评估新分子的治疗潜力。

Hemorrhagic shock (HS) is a state of shock characterized by significant blood volume loss, resulting in tissue dysoxia. HS is a complex pathology that associates hemodynamic and metabolic changes along with pro- and anti-inflammatory responses. Approximately 1.9 million deaths worldwide are attributed to the hemorrhage and its consequences each year1. Current guidelines for care primarily involve intravenous fluid administration (supplemented or not with vasoactive molecules) and oxygen therapy. However, these treatments are symptomatic and can be ineffective, which explains why the HS-associated mortality remains high2. This justifies the importance of identifying new molecular and cellular mechanisms and, thus, treatments to reduce mortality.
Animal models allow for deciphering the pathophysiological mechanisms involved in diseases and testing new therapeutic strategies. Numerous animal models of hemorrhagic shock exist in the literature. These models differ not only in the species used but also in the means of inducing HS (e.g., fixed pressure vs. fixed volume) (Table 1, Table 2)3,4,5,6,7,8,9,10,11,12,13. Also, protocols vary within the same type of model (e.g., time of hemorrhage, targeted mean arterial pressure) (Table 3)14,15,16,17,18,19,20. Considering the wide variety of existing hemorrhagic shock models and the complexity of replicating the clinical situation, the preclinical study of this pathology remains limited. The development of a reproducible, standardizable, and easy-to-implement hemorrhagic shock model is in everyone&#39;s interest. This would facilitate comparison between the various studies and thus unravel the complex pathophysiology of the hemorrhagic shock. The aim of this protocol was to develop a new clinically relevant model of hemorrhagic shock in rats using two successive phases of hemorrhage with fixed volume followed by a fixed low blood pressure phase.
Table 1: Species used as a model for hemorrhagic shock3,4,5,6,7,8,9,10,11,12,13. <a href="https://www.jove.com/files/ftp_upload/66523/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: The different types of hemorrhagic shock13. <a href="https://www.jove.com/files/ftp_upload/66523/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Example of the diversity of experimental models of hemorrhagic shock in rats induced by a fixed pressure protocol. Summary of parameters for different experimental models of hemorrhagic shock. Vessels shown in red are arteries, and those shown in blue are veins. For resuscitation, the volume of blood sampled is used as a reference (blood: resuscitation with a volume identical to that of blood sampled during shock; x2: resuscitation with a volume twice that of blood sampled during shock; x4: resuscitation with a volume four times that of blood sampled during shock). MAP: Mean Arterial Pressure; RL: Ringer Lactate14,15,16,17,18,19,20. <a href="https://www.jove.com/files/ftp_upload/66523/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>

作者聚焦：为失血性休克研究开发创新的治疗策略

开发大鼠临床相关的出血性休克模型

Hemorrhagic shock is responsible for two million deaths worldwide every year. We are studying the pathological mechanism of hemorrhagic shock, with the aim of identifying new targets, and thus proposing innovative therapeutic strategies. Hemorrhagic shock is a difficult condition in its own right, combining hemodynamic alteration with metabolic damage and organ dysfunctions.The variety of experimental protocols makes its study even more complex. In this study, we propose to develop a reproductive, standardized, and clinically relevant rat model of hemorrhagic shock. We hope that this model of hemorrhagic shock can become one of the standard models for future study.The use of a standard model would facilitate advances in the field, not least because it would make it possible to compare results between different teams working on hemorrhagic shock.

按照上述方案，我们在诱导失血性休克后 24 小时评估了几个血流动力学参数。假休克组和失血性休克组之间的基础平均动脉压（出血性休克方案开始前）相似（图 2A）。正如预期的那样，出血性休克方案的平均动脉压显着降低，这可以通过舒张压下降来解释（平均动脉压：假：92 mmHg ± 3 mmHg;HS：82 毫米汞柱± 2 毫米汞柱;舒张压：73 mmHg ± 3 mmHg;HS：61 mmHg ± 2 mmHg）（图 2B、C）。失血性休克不会影响收缩压、脉压和心率（图 2D-F）。休克指数 （心率/收缩压比值） 和改良休克指数 （MSI） （心率/平均血压比值） 是重症患者死亡率的两个预测指标14,15。值越高，死亡风险越大。在该模型中，两组之间的休克指数保持不变，而修正的休克指数在出血性休克中趋于增加（MSI：假：4.24 ± 0.11;HS：4.70 ± 0.15）（图 2G，H）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66523/66523fig02.jpg"> 图 2：失血性休克对血流动力学参数的影响。 （A） 假休克动物和出血性休克动物的基础平均动脉压，（B） 平均动脉压，（C） 舒张压，（D） 收缩压，（E） 脉压，（F） 心率，（G） 休克指数和 （H） 改良休克指数。结果表示为均值 ± SEM。通过未配对的 t 检验评估统计显着性。*： p < 0.05;：p < 0.001。n = 6-12。 <a href="https://www.jove.com/files/ftp_upload/66523/66523fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
失血性休克期间的整体代谢损害可通过乳酸血症进行评估。正如预期的那样，失血性休克方案后和后 16 小时（方案结束：假手术：1.13 mmol/L ± 0.14 mmol/L;HS：5.98 mmol/L ± 0.39 mmol/L;H+16： 假手术： 1.95 mmol/L ± 0.23 mmol/L;HS：2.95 mmol/L ± 0.19 mmol/L）（图 3A、B）。体温和呼吸频率是全身炎症反应综合征 （SIRS） 的两个组成部分，SIRS 是休克状态的促炎反应特征。出血性休克诱导后 16 小时，两组之间的温度和呼吸速率均未改变（图 3C，D）。我们评估了失血性休克对一些行为参数的影响，如姿势、活动等（补充文件 1）。方案后 16 小时出血性休克组的行为评分增加 （假动作： 0.33 ± 0.21;HS：2.27 ± 0.69）（图 3E）。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66523/66523fig03.jpg"> 图 3：失血性休克对乳酸血症、体温、呼吸频率和行为评分的影响。 （A） 失血性休克方案结束时的乳酸血症，（B） 乳酸血症，（C） 温度，（D） 呼吸率和 （E） 假动物和失血性休克动物在失血性休克诱导后 16 小时的行为评分。结果表示为均值 ± SEM。通过未配对的 t 检验评估统计显着性。*： p < 0.05;**： p < 0.01;：p < 0.001。n = 6-12。 <a href="https://www.jove.com/files/ftp_upload/66523/66523fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
失血性休克与器官功能障碍有关。为了评估该模型是否具有临床相关性，我们在方案后 24 小时评估了器官损伤的血浆标志物。肌酐血症 （假手术：19.13 μmol/L ± 0.33 μmol/L;HS：28.88 μmol/L ± 2.69 μmol/L）、心肌肌钙蛋白 T （假手术：9.38 ng/L ± 1.87 ng/L;HS：35.62 ng/L ± 2.28 ng/L）以及天冬氨酸和丙氨酸氨基转移酶（ASAT：假：221 UI/L ± 48 UI/L;HS：963 UI/L ± 144 UI/L;ALAT： 假： 36 UI/L ± 4 UI/L;HS：323 UI/L ± 13 UI/L）分别反映了对肾脏、心脏和肝脏的损害，都随着出血性休克而显着增加（图 4）。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66523/66523fig04.jpg"> 图 4：出血性休克模型与器官功能障碍有关。 （A） 假动物和失血性休克动物在失血性休克诱导后 24 小时出现肌酐血症，（B） 心肌肌钙蛋白 T，（C） 天冬氨酸氨基转移酶和 （D） 丙氨酸氨基转移酶水平。结果表示为均值 ± SEM。通过未配对的 t 检验评估统计显着性。*： p < 0.05;**： p < 0.01;：p < 0.001。n = 4 <a href="https://www.jove.com/files/ftp_upload/66523/66523fig04large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
补充文件 1：行为评分详细信息 <a href="https://www.jove.com/files/ftp_upload/66523/Supplementary%20File%201.zip" target="_blank">请单击此处下载此文件。</a>

Read this Scientific Article Protocol about Author Spotlight: Developing Innovative Therapeutic Strategies for Hemorrhagic Shock Research at JoVE.com

全球每年有 190 万人死于失血性休克。小动物经常被用作出血性休克模型，但与标准化、可重复性和临床意义问题有关，因此限制了它们的相关性。本文介绍了在大鼠中开发一种新的临床相关的出血性休克模型。

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic ...

失血性休克每年导致全球 200 万人死亡。我们正在研究失血性休克的病理机制，目的是确定新的靶点，从而提出创新的治疗策略。失血性休克本身就是一种困难的疾病，结合了血流动力学改变、代谢损伤和器官功能障碍。实验方案的多样性使其研究更加复杂。在这项研究中，我们建议开发一种生殖、标准化和临床相关的失血性休克大鼠模型。我们希望这种失血性休克模型能够成为未来研究的标准模型之一。使用标准模型将促进该领域的进步，尤其是因为它可以比较研究失血性休克的不同团队之间的结果。

developing-a-clinically-relevant-hemorrhagic-shock-model-in-rats

Research

JoVE Journal

Medicine

Developing a Clinically Relevant Hemorrhagic Shock Model in Rats

629 次观看.  Nantes Université. 失血性休克每年导致全球 200 万人死亡。我们正在研究失血性休克的病理机制，目的是确定新的靶点，从而提出创新的治疗策略。失血性休克本身就是一种困难的疾病，结合了血流动力学改变、代谢损伤和器官功能障碍。实验方案的多样性使其研究更加复杂。在这项研究中，我们建议开发一种生殖、标准化和临床相关的失血性休克大鼠模型。我们希望这种失血性休克模?...

视频: 作者聚焦：为失血性休克研究开发创新的治疗策略

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p&#160;&#60; 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p &#60; 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p &#60; 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

Hemorrhagic shock kills 1.9 million people worldwide every year. Small animals are frequently used as hemorrhagic shock models but are associated with issues of standardization, reproducibility, and clinical significance, thus limiting their relevance. This article describes developing a new clinically relevant hemorrhagic shock model in rats.

科研

医学

Jugular Vein and Femoral Artery Cannulation with Hemorrhagic Shock Induction and Resuscitation in Rats

To begin, insert a lubricated rectal probe to control the temperature of the anesthetized rat during surgery. After locating the jugular region in the lower right neck region of the anesthetized rat above the clavicle, use DeBakey Atraumatic Forceps to gently grasp the skin and make a precise incision with fine sharp scissors or a scalpel. Gently slash the tissue with standard pattern forceps.Locate and gently liberate the jugular vein from its muscular compartment. Place a 4-0 silk suture thread to securely ligate the distal side of the jugular vein. Use the suture with the needle holders to tension the jugular vein.Place a 4-0 suture on the proximal side of the jugular vein and prepare a surgeon knot without tightening it. Using Vannas micro dissecting scissors, make a small incision in the jugular vein. After carefully grasping the vein wall with small forceps, insert the PE50 catheter in the jugular vein.Slightly advance the catheter, and to confirm its correct placement in the vein, draw a small amount of blood and re-inject it. Tighten the previously prepared knot to secure the catheter. Apply a drop of lidocaine to the incised area.Cover the incision with a moist sterile gauze pad. Use DeBakey Atraumatic Forceps to grasp the skin of the Scarpa triangle on the left leg, and make an incision with fine sharp scissors or a scalpel. Gently dilacerate tissue with standard pattern forceps.Locate the femoral triad and place one forceps under the triad. Then with a second standard pattern forceps, carefully separate the artery from the nerve and femoral vein. Place a 4-0 suture to ligate the femoral artery distally and take the suture with the needle holders to put the femoral artery in tension.Now place a 4-0 suture on the proximal side of the femoral artery and prepare a surgeon knot without closing it. After clamping the artery, use Vannas micro dissecting scissors to make a transverse incision in the femoral artery. Grasp the artery wall with standard pattern forceps and cannulate the femoral artery with the catheter holding it with forceps.Gently unclamp the artery and ensure that the blood is not flowing back into the catheter. If the signal is good, advance the catheter slightly into the artery and tighten the previously prepared surgeon knot. Administer heparin into the rat, and place a moistened sterile gauze pad over the incised area.After 10 minutes of stabilization, record the basal hemodynamic values for five minutes. To induce hemorrhagic shock, use a one milliliter syringe to draw five milliliters of blood from the femoral artery over five minutes. Prepare a mix of 50%lactated Ringer solution and 50%collected blood at room temperature.Place a two milliliter syringe filled with the mix at the end of the jugular vein cannula. For hemorrhagic shock maintenance, when mean arterial pressure exceeds 38 millimeters of mercury, withdraw 200 microliters of blood from the femoral artery. If pressure drops below 32 millimeters of mercury, inject 200 microliters of the 50%blood, 50%lactated Ringer's mixture through the jugular vein.After sterile preparation of the tail with a gauze pad, use the tip of the needle to draw a blood drop from the end of the tail to measure peripheral blood lactate. Finally, resuscitate the rat with a lactated Ringer solution using a 20 milliliter syringe through the jugular catheter with a syringe pump.

大鼠颈静脉和股动脉插管出血性休克诱导和复苏

Post-Surgical Monitoring and Hemodynamic Evaluation Following Hemorrhagic Shock in Rats

After hemorrhagic shock treatment, clamp the jugular vein of the anesthetized rat. Remove the catheter from the veins. Upon ligation, ensure that there is no blood leakage from the vessels.Now use a 5-0 sterile suture to close incised areas with subcutaneous and cutaneous stitches. Disinfect the surgical area with 10%povidone iodine solution. Using a one milliliter syringe fitted with a 26 gauge needle, subcutaneously inject buprenorphine.Upon awakening, place the rat on the heating mat in its cage. After 16 hours of hemorrhagic shock induction, draw blood drop from the end of the tail to measure blood lactate. Place the anesthetized rat on the surgical table and insert the rectal probe.For carotid artery cannulation, use DeBakey atraumatic forceps to grasp the skin and make an incision in the middle of the neck with fine and sharp scissors or a scalpel. With standard pattern forceps, gently dilacerate tissue. Separate the salivary glands and open the tracheal muscle to reveal the tracheal rings.Isolate the left carotid artery from the nerve with standard pattern forceps and ligate it distally with a 4-0 silk thread. Now place the suture thread on the proximal side of the artery and prepare a surgeon's knot without closing it. After clamping, use Vannas micro dissecting scissors to incise the carotid artery.With standard operating forceps, gently grasp the artery wall to enlarge the opening and cannulate the artery with the catheter provided, holding it using forceps. Gently unclamp the artery and ensure that the blood is not flowing back into the catheter. After confirming the correct blood pressure signal, advance the catheter slightly into the artery and tighten the previously prepared surgeon knot.Basal mean arterial pressure was similar between sham and hemorrhagic shock groups 24 hours post-hemorrhagic shock induction. The mean arterial pressure significantly decreased in the hemorrhagic shock group due to a reduction in diastolic blood pressure. No changes were observed in systolic blood pressure, pulse pressure, and heart rate across groups.While the shock index remained unchanged, the modified shock index exhibited a notable increase in the hemorrhagic shock group. Lactatemia, indicative of global metabolic impairment, showed a significant increase following hemorrhagic shock, further elevating after 16 hours of hemorrhagic shock. Temperature and respiration rate, markers of systemic inflammatory response syndrome remained unchanged between groups 16 hours after hemorrhagic shock induction.Behavioral assessments demonstrated an increased behavioral score in the hemorrhagic shock group, suggesting altered postures and activities.