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Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through in vivo delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.
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INTRODUCTION: Hemorrhage is responsible for 91% of preventable prehospital deaths in combat. Bleeding from anatomic junctions such as the groin, neck, and axillae make up 19% of these deaths, and reports estimate that effective control of junctional hemorrhage could have prevented 5% of fatalities in Afghanistan. Hemostatic dressings are effective but are time-consuming to apply and are limited when proper packing and manual pressure are not feasible, such as during care under fire. CounterFlow-Gauze is a hemostatic dressing that is effective without compression and delivers thrombin and tranexamic acid into wounds. Here, an advanced prototype of CounterFlow-Gauze, containing a range of low thrombin doses, was tested in a lethal swine model of junctional hemorrhage. Outcomes were compared with those of Combat Gauze, the current dressing recommended by Tactical Combat Casualty Care. MATERIALS AND METHODS: CounterFlow-Gauze containing thrombin doses of 0, 20, 200, and 500 IU was prepared. Swine received femoral arteriotomies, and CounterFlow-Gauze was packed into wounds without additional manual compression. In a separate study using a similar model of junctional hemorrhage without additional compression, CounterFlow-Gauze containing 500 IU thrombin was tested and compared with Combat Gauze. In both studies, the primary outcomes were survival to 3 h and volume of blood loss. RESULTS: CounterFlow-Gauze with 200 and 500 IU had the highest 3-h survival, achieving 70 and 75% survival, respectively. CounterFlow-Gauze resulted in mean peak plasma tranexamic acid concentrations of 9.6 ± 1.0 µg/mL (mean ± SEM) within 3 h. In a separate study with smaller injury, CounterFlow-Gauze with 500 IU achieved 100% survival to 3 h compared with 92% in Combat Gauze animals. CONCLUSIONS: An advanced preclinical prototype of CounterFlow-Gauze formulated with a minimized thrombin dose is highly effective at managing junctional hemorrhage without compression. These results demonstrate that CounterFlow-Gauze could be developed into a feasible alternative to Combat Gauze for hemorrhage control on the battlefield.
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Hemostáticos , Ácido Tranexámico , Animales , Porcinos , Trombina/uso terapéutico , Ácido Tranexámico/farmacología , Ácido Tranexámico/uso terapéutico , Técnicas Hemostáticas , Modelos Animales de Enfermedad , Hemorragia/tratamiento farmacológico , Hemostáticos/farmacología , Hemostáticos/uso terapéutico , Vendajes , CegueraRESUMEN
BACKGROUND AND AIMS: Hemostatic powders used to manage upper GI bleeding continue to exhibit high recurrent bleeding rates. Previously, self-propelling thrombin powder (SPTP) sprayed endoscopically managed severe Forrest class 1A bleeding. Here, we evaluate SPTP in a 3-day recovery model of diffuse ulcerated bleeding. METHODS: Five anesthetized pigs underwent an endoscopic mucosal snare resection to trigger diffuse ulcer bleeding and were treated with SPTP. The time to hemostasis and the amount of powder delivered were measured. Pigs were recovered and monitored. RESULTS: Five pigs achieved hemostasis in 4.5 ± 1.2 minutes At 3 days after the procedure, the pigs were rescoped and showed no recurrent bleeding. Measured blood parameters were not significantly different from baseline. There were no signs of foreign bodies or thromboembolism during gross necropsy and histopathology of key organs. CONCLUSIONS: SPTP is a promising novel material that stopped diffuse ulcer bleeding in 5 pigs without recurrent bleeding or adverse local or systemic events.
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Hemostasis Endoscópica , Hemostáticos , Trombosis , Porcinos , Animales , Polvos , Trombina/uso terapéutico , Hemostasis Endoscópica/métodos , Úlcera/terapia , Hemostáticos/uso terapéutico , Hemorragia Gastrointestinal/tratamiento farmacológico , HemostasisRESUMEN
BACKGROUND: Noncompressible truncal hemorrhage (NCTH) remains a leading cause of preventable death on the battlefield. Definitively managing severe NCTH requires surgery within the first hour after injury, which is difficult when evacuating casualties from remote and austere environments. During delays to surgery, hemostatic interventions that are performed prehospital can prevent coagulopathy and hemorrhagic shock and increase the likelihood that casualties survive to receive definitive care. We previously reported that a self-propelling thrombin-containing powder (SPTP) can be delivered percutaneously into the abdomen as a minimally invasive intervention and can self-disperse through pooled blood to deliver the hemostatic agents thrombin and tranexamic acid locally to noncompressible intracavitary wounds. We hypothesized that, in swine with massive NCTH, dilutional coagulopathy, and hypothermia, delivering SPTP could extend survival times. METHODS: Ten swine (n = 5 per group) underwent NCTH from a Grade V liver injury following a midline laparotomy. The laparotomy was closed with sutures afterwards, creating a hemoperitoneum, and animals were managed with crystalloid fluid resuscitation, or crystalloid resuscitation and SPTP. Self-propelling thrombin-containing powder was delivered into the closed abdomen using a CO 2 -powered spray device and a catheter placed into the hemoperitoneum, entering through the upper right quadrant using the Seldinger technique. Survival to 1 and 3 hours was recorded. In an additional animal, hemorrhage was created laparoscopically, and SPTP was imaged in situ within the abdomen to visually track dispersion of the particles. RESULTS: Self-propelling thrombin-containing powder dispersed as far as 35 ± 5.0 cm within the abdomen. It increased survival to 1 and 3 hours (Kaplan-Meier p = 0.007 for both). The median survival time was 61 minutes with SPTP and 31 minutes without ( p = 0.016). CONCLUSION: Self-propelling thrombin-containing powder effectively disperses medications throughout a hemoperitoneum and increases survival in a model of NCTH. It is a promising strategy for nonsurgical management of NCTH, warranting further testing of its safety and efficacy.
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Trastornos de la Coagulación Sanguínea , Hemostáticos , Hipotermia , Animales , Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/terapia , Soluciones Cristaloides , Modelos Animales de Enfermedad , Hemoperitoneo , Hemostáticos/uso terapéutico , Polvos , Resucitación , Porcinos , TrombinaRESUMEN
INTRODUCTION: COVID-19 shutdowns in many research facilities across North America impacted preclinical trauma-related research and development. Shutdown limited the speed and resources available for large animal experiments necessary for advancing medical devices and technologies. However, the pandemic led to the rapid adoption and expansion of videoconferencing in social circles, workplaces, and primary care health settings. Here, we describe the use of simple videoconferencing equipment to plan and carry out 3 total weeks of large animal experiments with a large, cross-continent, interdisciplinary team testing a novel technology in swine models of noncompressible intraabdominal hemorrhage and junctional hemorrhage. MATERIALS AND METHODS: Animal experiments using swine were scheduled over 3 weeks in February and March 2021 to take place in Toronto, Canada. All relevant animal protocols and COVID-19 site-specific risk assessments were completed and approved by the responsible institutional committees. Experiments were conducted by connecting 12 total research personnel from 3 sites by a simple video conferencing setup which included low-cost, high-definition webcams and standard smartphones streaming to Zoom. RESULTS: Video conferencing allowed for 3 weeks of trauma experiments to take place at the height of Toronto's third peak of COVID-19 cases. Up to 3 experiments were completed for models requiring 6 hours of monitoring, and up to 5 experiments were completed for models requiring 3 hours of monitoring. The large amount of digital data collected during these experiments was rapidly shared with our network of collaborators, who analyzed results and interpreted findings in real time. CONCLUSIONS: The system described in this paper has the potential to reduce costs of trauma animal model development and allow for rapid testing and implementation of life-saving devices in settings with limited onsite personnel as experienced during the COVID-19 pandemic.
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INTRODUCTION: Non-compressible intra-abdominal hemorrhage (NCIAH) is a major cause of preventable death on the battlefield and in civilian trauma. Currently, it can only be definitively managed with surgery, as there are limited strategies for controlling ongoing NCIAH in the prehospital environment. We hypothesized that a self-propelling thrombin-containing powder (SPTP) could increase survival in a swine model of NCIAH when delivered percutaneously into the closed abdomen using an engineered spray system. MATERIALS AND METHODS: Nineteen swine underwent surgical laparotomy followed by a Grade V liver injury that created massive hemorrhage, before closing the abdomen with sutures. Animals either received treatment with standard of care fluid resuscitation (n=9) or the SPTP spray system (n=10), which consisted of a spray device and a 14 Fr catheter. Using the spray system, SPTP was delivered into a hemoperitoneum identified using a focused assessment with sonography in trauma (FAST) exam. Lactated Ringer's solution was administered to all animals to maintain a mean arterial pressure (MAP) of >50 mmHg. The primary outcome was percentage of animals surviving at three hours following injury. RESULTS: In the swine model of NCIAH, a greater percentage of animals receiving SPTP survived to three hours, although differences were not significant. The SPTP spray system increased the median survival of animals from 1.6 hr in the fluid resuscitation group to 4.3 hr. The SPTP spray system delivered a total mass of 18.5 ± 1.0 g of SPTP. The mean change in intra-abdominal pressure following SPTP delivery was 5.2 ± 1.8 mmHg (mean ± SEM). The intervention time was 6.7 ± 1.7 min. No adverse effects related to the SPTP formulation or the spray system were observed. SPTP was especially beneficial in animals that had either severely elevated lactate concentrations or low mean arterial pressure of <35 mmHg shortly after injury. CONCLUSIONS: This demonstrates proof-of-concept for use of a new minimally invasive procedure for managing NCIAH, which could extend survival time to enable patients to reach definitive surgical care.
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Hemorragia , Hemostáticos , Abdomen , Animales , Modelos Animales de Enfermedad , Fluidoterapia , Hemorragia/terapia , Hemostáticos/farmacología , Humanos , Polvos , Resucitación/métodos , PorcinosRESUMEN
Platelet transfusions are an integral component of balanced hemostatic resuscitation protocols used to manage severe hemorrhage following trauma. Enhancing the hemostatic potential of platelets could lead to further increases in the efficacy of transfusions, particularly for non-compressible torso hemorrhage or severe hemorrhage with coagulopathy, by decreasing blood loss and improving overall patient outcomes. Advances in gene therapies, including RNA therapies, are leading to new strategies to enhance platelets for better control of hemorrhage. This review will highlight three approaches for creating modified platelets using gene therapies: (i) direct transfection of transfusable platelets ex vivo, (ii) in vitro production of engineered platelets from platelet-precursor cells, and (iii) modifying the bone marrow for in vivo production of modified platelets. In summary, modifying platelets to enhance their hemostatic potential is an exciting new frontier in transfusion medicine, but more preclinical development as well as studies testing the safety and efficacy of these agents are needed.