Wound Infections Department, Bacterial Diseases Branch, Center for Infectious Diseases Research, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Oak Ridge Institute for Science and Education, Oak Ridge, TN 37830, USA.
Mil Med. 2024 Aug 19;189(Suppl 3):694-701. doi: 10.1093/milmed/usae233.
Extensive trauma, commonly seen in wounded military Service Members, often leads to a severe sterile inflammation termed systemic inflammatory response syndrome (SIRS), which can progress to multiple organ dysfunction syndrome (MODS) and death. MODS is a serious threat to wounded Service Members, historically causing 10% of all deaths in trauma admissions at a forward deployed combat hospital. The importance of this problem will be exacerbated in large-scale combat operations, in which evacuation will be delayed and care of complex injuries at lower echelons of care may be prolonged. The main goal of this study was to optimize an existing mouse model of lethal SIRS/MODS as a therapeutic screening platform for the evaluation of immunomodulatory drugs.
Male C57BL/6 mice were euthanized, and the bones and muscles were collected and blended into a paste termed tissue-bone matrix (TBX). The TBX at 12.5%-20% relative to body weight of each recipient mouse was implanted into subcutaneous pouches created on the dorsum of anesthetized animals. Mice were observed for clinical scores for up to 48 hours postimplantation and euthanized at the preset point of moribundity. To test effects of anesthetics on TBX-induced mortality, animals received isoflurane or ketamine/xylazine (K/X). In a separate set of studies, mice received TBX followed by intraperitoneal injection with 20 mg/kg or 40 mg/kg Eritoran or a placebo carrier. All Eritoran studies were performed in a blinded fashion.
We observed that K/X anesthesia significantly increased the lethality of the implanted TBX in comparison to inhaled anesthetics. Although all the mice anesthetized with isoflurane and implanted with 12.5% TBX survived for 24 hours, 60% of mice anesthetized with K/X were moribund by 24 hours postimplantation. To mimic more closely the timing of lethal SIRS/MODS following polytrauma in human patients, we extended observation to 48 hours. We performed TBX dose-response studies and found that as low as 15%, 17.5%, and 20% TBX caused moribundity/mortality in 50%, 80%, and 100% mice, respectively, over a 48-hour time period. With 17.5% TBX, we tested if moribundity/mortality could be rescued by anti-inflammatory drug Eritoran, a toll-like receptor 4 antagonist. Neither 20 mg/kg nor 40 mg/kg doses of Eritoran were found to be effective in this model.
We optimized a TBX mouse model of SIRS/MODS for the purpose of evaluating novel therapeutic interventions to prevent trauma-related pathophysiologies in wounded Service Members. Negative effects of K/X on lethality of TBX should be further evaluated, particularly in the light of widespread use of ketamine in treatment of pain. By mimicking muscle crush, bone fracture, and necrosis, the TBX model has pleiotropic effects on physiology and immunology that make it uniquely valuable as a screening tool for the evaluation of novel therapeutics against trauma-induced SIRS/MODS.
在受伤的军人中，广泛的创伤通常会导致严重的无菌性炎症，称为全身炎症反应综合征（SIRS），它可能进展为多器官功能障碍综合征（MODS）和死亡。MODS 是受伤军人的严重威胁，在前沿部署的战斗医院，它曾导致创伤住院患者中 10%的死亡。在大规模作战行动中，这一问题的重要性将更加突出，因为在这些行动中，伤员的后送会延迟，在较低的医疗层级对复杂损伤的治疗可能会延长。本研究的主要目标是优化现有的致命性 SIRS/MODS 小鼠模型，作为免疫调节药物治疗筛选平台。
处死雄性 C57BL/6 小鼠，收集骨骼和肌肉并混合成一种称为组织-骨基质（TBX）的糊状物。将相当于每个接受者小鼠体重 12.5%-20%的 TBX 植入到麻醉动物背部的皮下囊中。在植入后长达 48 小时的时间内观察小鼠的临床评分，并在预定的濒死点处死。为了测试麻醉剂对 TBX 诱导的死亡率的影响，动物接受异氟烷或氯胺酮/二甲苯胺（K/X）麻醉。在一组单独的研究中，小鼠接受 TBX 后，腹腔内注射 20mg/kg 或 40mg/kg 的艾立布林或安慰剂载体。所有的艾立布林研究均以盲法进行。
我们观察到，与吸入麻醉剂相比，K/X 麻醉显著增加了植入 TBX 的致死率。尽管所有接受异氟烷麻醉并植入 12.5%TBX 的小鼠在 24 小时内均存活，但接受 K/X 麻醉的小鼠中有 60%在植入后 24 小时内处于濒死状态。为了更紧密地模拟人类患者在遭受多发伤后发生致命性 SIRS/MODS 的时间，我们将观察时间延长至 48 小时。我们进行了 TBX 剂量反应研究，发现低至 15%、17.5%和 20%的 TBX 在 48 小时的时间内分别导致 50%、80%和 100%的小鼠出现濒死/死亡。在使用 17.5%TBX 的情况下，我们测试了抗炎症药物艾立布林（一种 Toll 样受体 4 拮抗剂）是否能挽救濒死/死亡率。在这种模型中，20mg/kg 和 40mg/kg 剂量的艾立布林均未被发现有效。
我们优化了一种基于 TBX 的 SIRS/MODS 小鼠模型，用于评估预防创伤相关病理生理学的新治疗干预措施，以用于受伤军人。应进一步评估 K/X 对 TBX 致死率的负面影响，特别是在广泛使用氯胺酮治疗疼痛的情况下。通过模拟肌肉挤压、骨折和坏死，TBX 模型对生理学和免疫学具有多种影响，使其作为评估创伤性 SIRS/MODS 新型治疗药物的筛选工具具有独特的价值。
