Early local delivery of vancomycin suppresses ectopic bone formation in a rat model of trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is a debilitating sequela of high-energy injuries. It frequently requires surgical excision once symptomatic and there is no practical prophylaxis for combat-injured patients. In this study, we examined the effect of local vancomycin powder on HO formation in a small animal model of blast-related, post-traumatic HO. Male Sprague-Dawley rats were subjected to a polytraumatic extremity injury and amputation with or without methicillin-resistant Staphylococcus aureus infection. Animals were randomized to receive a single local application of vancomycin (20 mg/kg) at the time of injury (POD-0, n = 34) or on postoperative day-3 (POD-3, n = 11). Quantitative volumetric measurement of ectopic bone was calculated at 12-weeks post-injury by micro-CT. Bone marrow and muscle tissues were also collected to determine the bacterial burden. Blood for serum cytokine analysis was collected at baseline and post-injury. Vancomycin treatment on POD-0 suppressed HO formation by 86% and prevented bone marrow and soft tissue infections. We concurrently observed a marked reduction histologically in nonviable tissue, chronic inflammatory cell infiltrates, bone infection, fibrous tissue, and areas of bone necrosis within this same cohort. Delayed treatment was significantly less efficacious. Neither treatment had a marked effect on the production of pro-inflammatory cytokines. Our study demonstrates that local vancomycin treatment at the time of injury significantly reduces HO formation in both the presence and absence of infection, with decreased efficacy if not given early. These findings further support the concept that the therapeutic window for prophylaxis is narrow, highlighting the need to develop early treatment strategies for clinical management. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2397-2406, 2017.

Double-blinded, placebo-controlled study of early tranexamic acid treatment in swine uncontrolled hemorrhage model.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that was shown to increase survival in trauma patients, but the mechanisms remain unclear. The purpose of this double-blinded, randomized placebo-controlled study was to determine if TXA with hypotensive resuscitation with Hextend (HEX) or fresh frozen plasma (FFP) reduced blood loss (BL) and improved survival in a model of uncontrolled hemorrhage. METHODS: Instrumented, anesthetized pigs (n = 11 per group) were subjected to 24-mL/kg controlled hemorrhage, followed by transection of the spleen. After 15 minutes of bleeding, TXA (1.43 mg/kg/min) or normal saline (NS) was given over 10 minutes, and then 15-mL/kg HEX or FFP was administered. At 90 minutes, a second infusion of TXA or NS was given. BL, coagulation status, and 5-hour survival were determined. Tissue plasminogen activator (tPA) was added to blood samples collected before and after TXA administration to confirm that the TXA inhibited fibrinolysis. In addition, a comparison of a dose response to tPA-induced fibrinolysis was made between swine and human plasma in vitro. RESULTS: TXA prevented the rise in d-dimers that occurred after spleen injury. However, there was no significant effect of TXA on survival or BL compared with NS with HEX (HEX + NS, 17 ± 2 mL/kg vs. HEX + TXA, 17 ± 2 mL/kg) or FFP (FFP + NS, 7 ± 2 mL/kg vs. FFP + TXA, 12 ± 3 mL/kg), while FFP significantly reduced BL and increased survival compared with HEX in the NS-treated animals. The tPA-induced fibrinolysis was inhibited in the blood from TXA-treated animals, yet in fibrinolysis sensitivity studies, human plasma was 30 times more sensitive to tPA-induced fibrinolysis than swine plasma. CONCLUSION: TXA did not reduce BL, even though TXA was antifibrinolytic in the pigs. The possibility remains that the pig is highly resistant to fibrinolysis and not a good model to study the effects of antifibrinolytics or that fibrinolysis is not a major factor in bleeding from splenic injury.

Fat Intravasation from Intraosseous Flush and Infusion Procedures.

STUDY HYPOTHESIS: The primary study objective was to delineate the procedural aspects of intraosseous (IO) infusions responsible for fat intravasation by testing the hypothesis that the fat content of effluent blood increases during IO infusions. METHODS: IO cannulas were inserted into the proximal tibiae of 35 anesthetized swine (Sus scrofa, 50.1 ± 3.5 kg) and intravasated fat was assessed using a lipophilic fluoroprobe (Nile red) and by vascular ultrasound imaging. Effluent blood bone marrow fat was assessed at baseline, during flush, and with regimens of controlled infusion pressures (73-300 mmHg) and infusion flow rates (0.3-3.0 mL per second). Fat intravasation was also assessed with IO infusions at different tibial cannulation sites and in the distal femur. In 7 animals, the lipid uptake of alveolar macrophages and lung tissue assessed for fat embolic burden using oil red O stain 24 hours post infusion. Additionally, bone marrow shear-strain was assessed radiographically with IO infusions. RESULTS: Fat intravasation was observed during all IO infusion regimens, with subclinical pulmonary fat emboli persisting 24 hours post infusion. It was noted that initial flush was a significant factor in fat intravasation, low levels of intravasation occurred with infusions ≤300 mmHg, fat intravasation and bone marrow shear-strain increased with IO infusion rates, and intravasation was influenced by cannula insertion site. Ultrasound findings suggest that echogenic particles consistent with fat emboli are carried in fast and slow venous blood flow fields. Echo reflective densities were observed to rise to the nondependent endovascular margins and coalesce in accordance with Stoke's law. In addition, ultrasound findings suggested that intravasated bone marrow fat was thrombogenic. CONCLUSION: Results suggest that in swine the intravasation of bone marrow fat is a common consequence of IO infusion procedures and that its magnitude is influenced by the site of cannulation and infusion forces. Although the efficacy and benefits of IO infusions for emergent care are well established, emergency care providers also should be cognizant that infusion procedures affect bone marrow fat intravasation.

Toxoplasmosis-associated abortion in an alpaca (Vicugna pacos) fetus.

A near-full-term alpaca (Vicugna pacos) was stillborn 2 days before the expected date of delivery; necropsy examination was conducted within 6 hr of delivery. Gross lesions were enlarged liver and hydrocephalus. On histologic examination, mild inflammatory lesions were identified in the placenta, liver, and lungs, although no etiology was recognized. Within the brain, there was a mild nonsuppurative meningoencephalitis, hydrocephalus, and hydromyelia. Both kidneys had inflammatory foci in cortex and medulla associated with protozoal tissue cysts. Protozoa in brain and kidneys were identified as Toxoplasma gondii based on immunoreactivity to T. gondii polyclonal antibodies that stain both tachyzoites and tissue cysts and BAG1 T. gondii antibodies that are bradyzoite specific. The tissue cysts exhibited intense positivity to T. gondii and mild immunoreactivity to Neospora caninum antibodies. The dam had a high antibody titer (1 : 12,800) to T. gondii and low titer (1 : 100) to N. caninum using their respective agglutination tests. This is the first report of toxoplasmosis-associated abortion in alpaca.

In vivo assessment of the Combat Ready Clamp to control junctional hemorrhage in swine.

BACKGROUND: Junctional wounds and associated hemorrhage have become more common and more lethal in the current war. The Combat Ready Clamp (CRoC) has been developed and deployed for treating junctional hemorrhage on the battlefield. This study examined the efficacy of CRoC and its acute effects in an animal model. METHODS: Anesthetized pigs (n = 6) were subjected to laparotomy, splenectomy, and abdominal closure. Next, coagulopathy was induced in animals by hemodilution and hypothermia. The left femoral artery was isolated, punctured (6-mm hole), and allowed to bleed for 15 seconds. The groin wound was packed with gauze, and a CRoC applied and tightened until hemorrhage stopped. It was kept in place for 1 hour (treatment period) and then released for another hour or less (control-period) if animal exsanguinated. Fluid resuscitation was administered, and vascular blood flow was examined by Doppler and CT scans. After death, local tissues were collected for histology. RESULTS: CRoC generated 800 to 900 mm Hg pressure on the wounds, which stopped the hemorrhage and prevented rebleeding during the first hour in all animals. Blood loss was minimal (≤137 mL), and mean arterial pressure remained at or higher than the target level (65 mm Hg) during this period. Removal of the clamp promptly led to rebleeding and exsanguination of five of six pigs during the second hour despite fluid resuscitation. Blood loss, survival, shock indices, and other measures were significantly (p < 0.01) different between the two periods. Doppler tests and CT scans showed no blood flow in the proximal, distal, and collateral arteries of the clamped leg. Minor inflammation was seen on blood vessels (endothelium) and nerves. CONCLUSION: CRoC functioned as an effective hemostatic adjunct for compression and control of groin hemorrhage. Although no acute histological damages were seen in compressed tissues, the short- and long-term effects of CRoC application (e.g., total ischemia) on limb function remain unknown and warrant investigation.

Negative-pressure wound therapy: a hemostatic adjunct for control of coagulopathic hemorrhage in large soft tissue wounds.

BACKGROUND: Negative-pressure wound therapy has been commonly used for treating chronic wounds and recently applied for treatment of traumatic wounds. We investigated the potential hemostatic benefit of negative-pressure wound therapy for control of refractory hemorrhage in a soft tissue wound model in swine. METHODS: Coagulopathy was induced in pigs (n = 38, 36 kg) by hemodilution and hypothermia. Next, a large soft tissue wound (diameter, approximately 20 cm) was created by slicing the gluteus maximus muscle. Free bleeding was allowed for 1 minute, and wounds were then randomly dressed with either laparotomy gauze (G) alone or TraumaPad (TP, a kaolin-coated dressing) alone or in combination with negative pressure (NP, approximately -500 mm Hg). All wounds were sealed with adhesive drapes. Fluid resuscitation was administered and targeted to mean arterial pressure of 60 mm Hg. Pigs were observed for 150 minutes or until death after which tissues were sampled for histologic examination. RESULTS: Induced coagulopathy as measured by increases in prothrombin time (12%) and activated partial thromboplastin time (22%) and decreases in fibrinogen (48%) were similar in all groups. There were no differences in initial bleeding rates (4.5 mL/kg/min). Dressing the wounds with G or TP produced hemostasis only in one pig (1 of 18 pigs). Addition of NP to these dressings secured hemostasis in 70% (G) and 90% (TP) of animals with average hemostasis time of 34 minutes and 25 minutes, respectively. Blood losses and fluid resuscitation requirements were significantly less, and survival times were significantly longer in NP adjunct groups than in the other groups. Survival rates were 80% (G+NP) and 90% (TP+NP) versus 0% (G) and 10% (TP) in the respective groups. Histologic examination showed similar superficial myofibril damages in all groups. CONCLUSION: To our knowledge, the present data provide the first evidence that NP serves as an effective hemostatic adjunct and when combined with standard hemostatic dressing it is able to stop lethal coagulopathic bleeding in large soft tissue wounds.