Combined Injury Model Research Articles

191 Treatment with GM-CSF Reverses TBI-Induced Immune Suppression in a Combined Injury Model

INTRODUCTION: Traumatic brain injury (TBI) is a potentially devastating injury for which there are no pharmacotherapies. Frequently, TBI is accompanied by injuries outside the CNS, and we have previously shown that TBI with concurrent extra-cranial injury reliably leads to post-injury immunosuppression, which puts patients at risk for infection and ultimately a poor outcome. To reverse post-injury immune suppression, we have proposed the use of the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF). METHODS: To induce post-injury immunosuppression, we used a previously described model. 28 day old rats were injured via controlled cortical impact and then had 25% blood volume removed. Following injury, animals were treated with vehicle, 5 μg/kg, or 50 μg/kg GM-CSF daily for 7 days. Blood was obtained pre-injury, and on 1, 3, and 7 days post-injury. Brain and spleen were harvested at 7 days post-injury. Adaptive and innate immune function (measured by ex vivo cytokine production in response to an immunostimulant) were assessed in blood and spleen. Flow cytometry was performed on blood and spleen samples. Cytokine levels at the lesion and penumbra were measured by ELISA. RESULTS: As shown previously, injury induced reduction of both innate and adaptive immune function (approximately 50% decrease), which was reversed with GM-CSF. Treatment with GM-CSF significantly expanded the percentage of monocytes. In spleen, injury was associated with an increase in percentage of CD4 cells expressing programmed death-1 (PD-1), and this was also reversed with GM-CSF. ELISA for cytokines in brain samples from lesion and penumbra from GM-CSF treated animals were not significantly different from vehicle. CONCLUSIONS: These data add to a body of literature suggesting that severe trauma leads to immune suppression. Our findings suggest that treatment with GM-CSF may normalize post-injury immune response.

Recombinant human erythropoietin induces neuroprotection, activates MAPK/CREB pathway, and rescues fear memory after traumatic brain injury with delayed hypoxemia in mice

Therapeutic interventions targeting secondary insults, such as delayed hypoxemia, provide a unique opportunity for treatment in severe traumatic brain injury (TBI). Erythropoietin (EPO) is a hypoxia-responsive cytokine with important roles in neurodevelopment, neuroprotection and neuromodulation. We hypothesized that recombinant human erythropoietin (rhEPO) administration would mitigate injury in a combined injury model of TBI and delayed hypoxemia. Utilizing a clinically relevant murine model of TBI and delayed hypoxemia, we characterized how ongoing rhEPO administration influenced neurogenesis, neuroprotection, synaptic density and, behavioral outcomes early after TBI, and the impact on long-lasting outcomes 6 months after injury. We employed novel object recognition (NOR) and fear conditioning to assess long-term memory. At 1-month post-injury, we observed a significant increase in cued-fear memory response in the rhEPO-injured mice compared with vehicle-injured mice. This was associated with neuroprotection and neurogenesis in the hippocampus and mitogen-activated protein kinase (MAPK)/cAMP response element-binding protein (CREB) signaling activation and increased of excitatory synaptic density in the amygdala. Early rhEPO treatment after injury reduced neurodegeneration and increased excitatory synaptic density in the hippocampus and amygdala at 6 months post-injury. However at 6 months post-injury (4 months after discontinuation of rhEPO), we did not observe changes in behavioral assessments nor MAPK/CREB pathway activation. In summary, these data demonstrate that ongoing rhEPO treatment initiated at a clinically feasible time point improves neurological, cognitive, and histological outcomes after TBI in the setting of secondary hypoxemic insults.

Characterization of the Basal and mTOR-Dependent Acute Pulmonary and Systemic Immune Response in a Murine Model of Combined Burn and Inhalation Injury.

Severe burn injury leads to a cascade of local and systemic immune responses that trigger an extreme state of immune dysfunction, leaving the patient highly susceptible to acute and chronic infection. When combined with inhalation injury, burn patients have higher mortality and a greater chance of developing secondary respiratory complications including infection. No animal model of combined burn and inhalation injury (B+I) exists that accurately mirrors the human clinical picture, nor are there any effective immunotherapies or predictive models of the risk of immune dysfunction. Our earlier work showed that the mechanistic/mammalian target of rapamycin (mTOR) pathway is activated early after burn injury, and its chemical blockade at injury reduced subsequent chronic bacterial susceptibility. It is unclear if mTOR plays a role in the exacerbated immune dysfunction seen after B+I injury. We aimed to: (1) characterize a novel murine model of B+I injury, and (2) investigate the role of mTOR in the immune response after B+I injury. Pulmonary and systemic immune responses to B+I were characterized in the absence or presence of mTOR inhibition at the time of injury. Data describe a murine model of B+I with inhalation-specific immune phenotypes and implicate mTOR in the acute immune dysfunction observed.

A Novel Therapeutic Approach With Sodium Pyruvate on Vital Signs, Acid-Base, and Metabolic Disturbances in Rats With a Combined Blast and Hemorrhagic Shock.

BackgroundBlast injuries from improvised explosive devices (IEDs) are known to cause blast traumatic brain injuries (bTBIs), hemorrhagic shock (HS), organ damage, mitochondrial dysfunction, and subsequent free radical production. A pre-citric acid cycle reagent, pyruvate, is suggested to improve mitochondrial ATP production through the activation of the mitochondrial gatekeeper enzyme “pyruvate dehydrogenase complex (PDH).” Our study aimed to investigate the role of physiologic, metabolic, and mitochondrial effects of hypertonic sodium pyruvate resuscitation in rats with a combined blast and HS injury.MethodsA pre-clinical rat model of combined injury with repetitive 20 PSI blast exposure accompanied with HS and fluid resuscitation (sodium pyruvate as metabolic adjuvant or hypertonic saline as control), followed by transfusion of shed blood was used in this study. Control sham animals (instrumental and time-matched) received anesthesia and cannulation, but neither received any injury nor treatment. The mean arterial pressure and heart rate were recorded throughout the experiment by a computerized program. Blood collected at T0 (baseline), T60 (after HS), and T180 (end) was analyzed for blood chemistry and mitochondrial PDH enzyme activity.ResultsSodium pyruvate resuscitation significantly improved the mean arterial pressure (MAP), heart rate (HR), pulse pressure (PP), hemodynamic stability (Shock index), and autonomic response (Kerdo index) after the HS and/or blast injury. Compared with the baseline values, plasma lactate and lactate/pyruvate ratios were significantly increased. In contrast, base excess BE/() was low and the pH was also acidotic <7.3, indicating the sign of metabolic acidosis after blast and HS in all animal groups. Sodium pyruvate infusion significantly corrected these parameters at the end of the experiment. The PDH activity also improved after the sodium pyruvate infusion.ConclusionIn our rat model of a combined blast and HS injury, hypertonic sodium pyruvate resuscitation was significantly effective in hemodynamic stabilization by correcting the acid–base status and mitochondrial mechanisms via its pyruvate dehydrogenase enzyme.

46 Bardoxolone-methyl Microparticles Ameliorate Immune Dysfunction Following Burn and Inhalation Injury Through the NRF2/KEAP1 Axis

IntroductionSevere burn injury leads to many systemic stresses that can seriously impact multiple systems throughout the body. These stresses are further exacerbated if there is a combined burn and inhalation injury, which leads to increased morbidity and mortality for many patients. Combined burn and inhalation injury causes an intense systemic inflammatory response and activation of the innate immune system which can lead to inflammatory complications, such as systemic inflammatory response syndrome and multiple organ failure. Nuclear Factor-Erythroid-2-Related Factor (NRF2) is a transcription factor that acts to downregulate overt damaging pro-inflammatory and oxidative responses and maintain immune homeostasis. This transcription factor remains bound to Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm. Under oxidative stress, NRF2 dissociates from KEAP1 and translocates to the nucleus where it facilitates the transcription of anti-inflammatory and antioxidant genes. We hypothesized that NRF2 is a key regulator after burn and inhalation injury, and activation of NRF2 can limit the severity of burn and inhalation injury.MethodsTo test this, we have developed a mouse model of combined cutaneous burn and woodsmoke inhalation injury. After burn and inhalation injury, we found that NRF2-/- knockout mice have higher mortality compared to wild-type (WT) mice and suffer from increased vascular permeability and lung edema, suggesting that this transcription factor is important for controlling morbidity and mortality. In WT mice, NRF2 is activated following burn and inhalation injury, however, based on immunohistochemical staining, it is not sufficiently induced following this insult since it remains in the cytoplasm and is unable to transcribe anti-inflammatory genes. Therefore, we treated WT mice with an intraperitoneal injection of bardoxolone-methyl microparticles, a NRF2 activator that separates KEAP1 from NRF2 in the cytoplasm, immediately after burn and inhalation injury in WT miceResultsWe observed significant decreases in mortality as well as reduced concentrations of certain pro-inflammatory cytokines in the blood and bronchoalveolar-lavage fluid of these mice. In the lungs of these mice, there was an upregulation in numerous pathways involved in the management of inflammation and immune response compared to mice that did not receive bardoxolone-methyl microparticles.ConclusionsIn conclusion, treatment with bardoxolone-methyl microparticles immediately after burn and inhalation injury might be effective for reducing the severity of the inflammatory response and limiting inflammatory complications.

17\u03b1-Ethinyl estradiol-3-sulfate increases survival and hemodynamic functioning in a large animal model of combined traumatic brain injury and hemorrhagic shock: a randomized control trial

BackgroundTraumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) represent leading causes of trauma-induced mortality, especially when co-occurring in pre-hospital settings where standard therapies are not readily available. The primary objective of this study was to determine if 17α-ethinyl estradiol-3-sulfate (EE-3-SO4) increases survival, promotes more rapid cardiovascular recovery, or confers neuroprotection relative to Placebo following TBI + HS.MethodsAll methods were approved by required regulatory agencies prior to study initiation. In this fully randomized, blinded preclinical study, eighty (50% females) sexually mature (190.64 ± 21.04 days old; 28.18 ± 2.72 kg) Yucatan swine were used. Sixty-eight animals received a closed-head, accelerative TBI followed by removal of approximately 40% of circulating blood volume. Animals were then intravenously administered EE-3-SO4 formulated in the vehicle at 5.0 mg/mL (dosed at 0.2 mL/kg) or Placebo (0.45% sodium chloride solution) via a continuous pump (0.2 mL/kg over 5 min). Twelve swine were included as uninjured Shams to further characterize model pathology and replicate previous findings. All animals were monitored for up to 5 h in the absence of any other life-saving measures (e.g., mechanical ventilation, fluid resuscitation).ResultsA comparison of Placebo-treated relative to Sham animals indicated evidence of acidosis, decreased arterial pressure, increased heart rate, diffuse axonal injury and blood–brain barrier breach. The percentage of animals surviving to 295 min post-injury was significantly higher for the EE-3-SO4 (28/31; 90.3%) relative to Placebo (24/33; 72.7%) cohort. EE-3-SO4 also restored pulse pressure more rapidly post-drug administration, but did not confer any benefits in terms of shock index. Primary blood-based measurements of neuroinflammation and blood brain breach were also null, whereas secondary measurements of diffuse axonal injury suggested a more rapid return to baseline for the EE-3-SO4 group. Survival status was associated with biological sex (female > male), as well as evidence of increased acidosis and neurotrauma independent of EE-3-SO4 or Placebo administration.ConclusionsEE-3-SO4 is efficacious in promoting survival and more rapidly restoring cardiovascular homeostasis following polytraumatic injuries in pre-hospital environments (rural and military) in the absence of standard therapies. Poly-therapeutic approaches targeting additional mechanisms (increased hemostasis, oxygen-carrying capacity, etc.) should be considered in future studies.

Feasibility of nitric oxide synthesis inhibitor for the treatment of combined radiation injuries

The model of combined radiation-thermal injury (CRTI) in mice was experimentally and histologically verified: exposure to 60Co-radiation at a dose of 7 Gy (0.39 Gy/min) on the Agat device and the subsequent thermal effect on the GL6 device on the back, leading to the development of a 3B degree burn on 10% of the body surface. This model was used to confirm the positive influence of the NOS inhibitor T1023 on the course of CRTI. It was found that a single i.p. administration of T1023 72 h after CRTI application caused a nearly statistically significant (p = 0.051) increase in the 30-day survival rate of animals. The data obtained indicate the need for further detailed study of NOS inhibitor effects on the course of acute radiation syndrome (ARS) and CRTI.

CORM-3 ameliorates neurodegeneration in the amygdala and improves depression- and anxiety-like behavior in a rat model of combined traumatic brain injury and hemorrhagic shock

ObjectiveEmotional disturbances characterized by depression and anxiety among survivors of traumatic brain injury (TBI) impact the quality of life severely. Currently, there is a lack of effective drug treatment for neurodegeneration induced by TBI, mainly due to failed efficacy of compounds such as corticosteroids, calcium channel blockers, and excitatory amino acid inhibitors. Thus, we sought to continue with our investigation on CORM-3, a water-soluble exogenous carbon monoxide-releasing molecule with excellent anti-inflammatory actions employed in a previous study using a rat model of combined TBI with hemorrhage shock and resuscitation (HSR). MethodsRats were administrated with CORM-3 after induction of TBI and HSR and examined depressive and anxiety-like behaviors, along with cerebral function employing functional magnetic resonance imaging (MRI) 30-days post-trauma. Also, the following variables were measured: 1) neuronal pyroptosis and apoptosis 24 h post-trauma, 2) the roles of PKG-ERK1/2 signaling pathways with the use of the protein kinase G (PKG) specific inhibitor, KT5823. ResultsCORM-3-treated rats displayed significant ameliorated depression- and anxiety-like behaviors, improved cerebral blood flow, and fractional anisotropy (FA), showed less neuronal pyroptosis and apoptosis in the amygdala, and upregulated the phosphorylation of Vasodilator-stimulated phosphoprotein (VASP) and ERK1/2. However, CORM-3 neuroprotective effects against trauma were only partially reversed by KT5823. ConclusionCORM-3 ameliorated the emotional deficits and neuronal death induced in the amygdala post-TBI and HSR rat model, and PKG-ERK1/2 signaling might be implicated in the underlying mechanism.

A Combination of Kidney Ischemia and Injection of Isolated Mitochondria Leads to Activation of Inflammation and Increase in Mortality Rate in Rats.

We studied the development of acute kidney injury and animal death in the model of combined injury caused by kidney ischemia/reperfusion with simultaneous systemic administration of mitochondria. It was found that intraperitoneal injection of isolated mitochondria led to the appearance of mitochondrial DNA in the peripheral blood that could activate innate immunity. After administration of mitochondria, as well as after renal ischemia/reperfusion, proinflammatory changes were observed, primarily leukocytosis and granulocytosis. The combination of ischemia/reperfusion with injection of mitochondria caused a sharp increase in animal death, which may indicate a direct link between activation of TLR-signaling and high mortality of patients with combined injuries and multiple-organ failure in intensive care units. Treatment with mitochondria-targeted antioxidant increased animal survival, which indicated the participation of mitochondrial ROS in the development of systemic inflammatory response and death caused by acute renal failure.

Innate immune suppression after traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma

Traumatic injury in children is known to cause immune suppression. Polytrauma involving a traumatic brain injury (TBI) may increase this degree of immune suppression, which increases the risk of developing nosocomial infections, potentially causing secondary brain injury and worsening patient outcomes. Despite the high prevalence of polytrauma with TBI in children, mechanisms of immune suppression following such injuries remain poorly understood. Here, we used a combined animal injury model of TBI and hemorrhage to assess immune function after polytrauma. Pre-pubescent rats were injured using a prefrontal controlled cortical impact method and a controlled hemorrhage by femoral arteriotomy. Immune function was measured by whole blood ex-vivo tumor necrosis factor alpha production capacity following incubation with lipopolysaccharide, measuring the percentage of monocytes by flow cytometry, and by examining concentrations of plasma cytokines. The degree of brain injury was sufficient to produce deficits in spatial memory testing (Barnes maze). Both hemorrhage and TBI with hemorrhage (combined injury) reduced several of the measured plasma cytokines, as compared with TBI alone. The combined injury correlated with reduced concentration of monocytes and reduced tumor necrosis factor alpha production capacity at post-injury day 1. These results demonstrate that this animal model can be used to study post-injury immune suppression.

EFFECTS OF PARTIALLY HYDROGENATED PYRIDINES (CYANOTHIOACETAMIDE DERIVATIVES) ON THE BLOOD OF RATS WITH COMBINED PARACETAMOL-ALCOHOLIC LIVER INJURY

Aim. In this research, we studied the parameters of blood biochemical analysis in rats with combined paracetamol-alcoholic liver injury after its pharmacological correction by newly synthesized partially hydrogenated pyridines (cyanothioacetamide derivatives).Materials and methods. 50 samples of new derivatives of partially hydrogenated pyridines synthesized on the basis of the “Chemex” Research Laboratory of the Vladimir Dal’ Lugansk National University underwent virtual bioscreening, which allowed the selection of 4 compounds, the biological activity of which may be associated with the effect on the protein synthesis and detoxifi cation function of the liver. The experiment was conducted on 48 white outbred male rats. For 14 days, the rats of the control and experimental groups were intragastrically injected with 1 ml of 40% ethanol and paracetamol at a dose of 500 mg/kg once a day. Pharmacocorrection with a thiotriazoline reference drug (50 mg/kg) and pyridine derivatives at a dose of 5 mg/kg was performed from the fourth day. On the 15th day, blood was sampled to determine the level of total bilirubin, alanine aminotransferase, aspartate aminotransferase and thymol turbidity test.Results. The value of total bilirubin in the blood of the rats treated with CV046 compounds was 34.88% lower than that in the control group. The animals of the test groups receiving CV047 also showed signifi cant differences with the control group in terms of bilirubin level (a decrease of 31.78%). The AST values in the blood of rats in the test groups had no signifi cant differences as compared with intact animals. Following 10 days of pharmacological correction of combined paracetamol-alcoholic liver injury with CV146, the total bilirubin level in the blood decreased by 26.36% in comparison with the control group, with the activity of ALT and AST demonstrating the levels of intact values.Conclusions. The conducted screening studies of four partially hydrogenated pyridines — cyanothioacetamide derivatives — using the model of combined paracetamol-alcoholic liver injury when used at a dose of 5 mg/kg during ten days have shown a high hepatoprotective and detoxifying activity of the following three compounds: СV046 (2-[(9-tert-butyl-1,5-dicyano4-oxo-3-azaspiro[5,5]undec-1-en-2-yl)thio]-N-(2-ethylphenyl)acetamide — IUPAC), СV047 (2-[(9-tert-butyl-1,5-dicyano-4-oxo-3-azaspiro[5,5]undec-1-en-2-yl)thio]-N-(4-fl uorophenyl) acetamide) and СV146 (benzyl 4-(4-chlorophenyl)-5-cyano-6-({2-[(3,4-dimethylphenyl)amino]-2-oxoethyl}thio)-2-methyl-1,4-dihydropyridine-3-carboxylate).

Variable saline resuscitation in a murine model of combined traumatic brain injury and haemorrhage

ABSTRACTBackground: Resuscitation strategies for combined traumatic brain injury (TBI) with haemorrhage in austere environments are not fully established. Our aim was to establish the effects of various saline concentrations in a murine model of combined TBI and haemorrhage, and identify an effective resuscitative strategy for the far-forward environment.Methods: Male C57BL/6 mice underwent closed head injury and subjected to controlled haemorrhage to a systolic blood pressure of 25 mmHg via femoral artery cannulation for 60 min. Mice were resuscitated with a fixed volume bolus or variable volumes of fluid to achieve a systolic blood pressure goal of 80 mmHg with 0.9% saline, 3% saline, 0.1-mL bolus of 23.4% saline, or a 0.1-mL bolus of 23.4% saline followed by 0.9% saline (23.4+).Results: 23.4% saline and 23.4+ resulted in higher mortality at 6 h compared to 0.9% saline. Use of 3% saline required less volume to achieve targeted resuscitation, did not affect survival, and did not exacerbate post-traumatic inflammation. While 23.4+ resuscitation utilized lower volume, it resulted in hypernatremia, azotemia, and elevated systemic pro-inflammatory cytokines. All groups except 3% saline demonstrated progression of neuron damage, with cerebral oedema highest with 0.9% saline.Conclusions: 3% saline demonstrated favourable balance of survival, blood pressure restoration, minimization of inflammation, and prevention of ongoing neurologic injury without contributing to significant physiologic derangements. 23.4% saline administration may not be appropriate in the setting of concomitant hypotension.

РОЗВИТОК СИНДРОМУ ЕНДОГЕННОЇ ІНТОКСИКАЦІЇ ЗА УМОВ ПОЄДНАНОЇ ТРАВМИ ОРГАНІВ ЧЕРЕВНОЇ ПОРОЖНИНИ НА ФОНІ ГІПОВОЛЕМІЧНОГО ШОКУ ТА РЕПЕРФУЗІЙНОГО СИНДРОМУ КІНЦІВКИ

Endogenous intoxication is a complicated multicomponent process caused by pathological biological activity of endogenous products or dysfunction of natural detoxification systems. The article presents the results of the experiment on ischemic-reperfusion syndrome as an aggravating factor in the development of endogenous intoxication in the model of combined injury of the abdominal cavity in the background of hypovolemic shock.The aim of the study – to learn the effect of ischemic-reperfusion syndrome on the changes of indicators of endogenous intoxication in the model of combined injury of the abdominal cavity in the background of hypovolemic shock.Materials and Methods. The experiment was conducted on 80 nonlinear male rats weighing 190–220 grams. In experimental groups ischemic-reperfusion syndrome was simulated in combination with trauma of the abdominal cavity in the background of hypovolemic shock and contents of endogenous intoxication markers (molecules of average mass fractions determined at wavelengths of 238, 254, 260 and 280) and the erythrocytes index of intoxication were analyzed. The probability of differences between the control group and the study group was evaluated using non-parametric Mann-Whitney U-test.Results and Discussion. The applications of haemostatic tourniquets on two lower limbs caused a significant reliable increase of EII indicator in all study groups. The maximum increase of EII indicator was in the group where the applications of the tourniquets were combined with the trauma of the abdominal cavity and hypovolemic shock. These data proved the potentiating effect of ischemicreperfusion syndrome on the development of systemic changes in combined trauma. It has also been shown that the manifestation of endogenous intoxication within the first 24 hours after the applications of haemostatic tourniquets is similar to that arising from the combined injury of the abdominal cavity in the background of massive external bleeding.Conclusions. The applications of haemostatic tourniquets to the two lower limbs for two hours enhance the development of endogenous intoxication in the model of combined injury of the abdominal cavity in the background of hypovolemic shock, that was confirmed by significant increase of EII indicator and the median mass molecules seven days after reperfusion.

Glibenclamide Produces Region-Dependent Effects on Cerebral Edema in a Combined Injury Model of Traumatic Brain Injury and Hemorrhagic Shock in Mice.

Cerebral edema is critical to morbidity/mortality in traumatic brain injury (TBI) and is worsened by hypotension. Glibenclamide may reduce cerebral edema by inhibiting sulfonylurea receptor-1 (Sur1); its effect on diffuse cerebral edema exacerbated by hypotension/resuscitation is unknown. We aimed to determine if glibenclamide improves pericontusional and/or diffuse edema in controlled cortical impact (CCI) (5m/sec, 1 mm depth) plus hemorrhagic shock (HS) (35 min), and compare its effects in CCI alone. C57BL/6 mice were divided into five groups (n = 10/group): naïve, CCI+vehicle, CCI+glibenclamide, CCI+HS+vehicle, and CCI+HS+glibenclamide. Intravenous glibenclamide (10 min post-injury) was followed by a subcutaneous infusion for 24 h. Brain edema in injured and contralateral hemispheres was subsequently quantified (wet-dry weight). This protocol brain water (BW) = 80.4% vehicle vs. 78.3% naïve, p < 0.01) but was not reduced by glibenclamide (I%BW = 80.4%). Ipsilateral edema also developed in CCI alone (I%BW = 80.2% vehicle vs. 78.3% naïve, p < 0.01); again unaffected by glibenclamide (I%BW = 80.5%). Contralateral (C) %BW in CCI+HS was increased in vehicle (78.6%) versus naive (78.3%, p = 0.02) but unchanged in CCI (78.3%). At 24 h, glibenclamide treatment in CCI+HS eliminated contralateral cerebral edema (C%BW = 78.3%) with no difference versus naïve. By 72 h, contralateral cerebral edema had resolved (C%BW = 78.5 ± 0.09% vehicle vs. 78.3 ± 0.05% naïve). Glibenclamide decreased 24 h contralateral cerebral edema in CCI+HS. This beneficial effect merits additional exploration in the important setting of TBI with polytrauma, shock, and resuscitation. Contralateral edema did not develop in CCI alone. Surprisingly, 24 h of glibenclamide treatment failed to decrease ipsilateral edema in either model. Interspecies dosing differences versus prior studies may play an important role in these findings. Mechanisms underlying brain edema may differ regionally, with pericontusional/osmolar swelling refractory to glibenclamide but diffuse edema (via Sur1) from combined injury and/or resuscitation responsive to this therapy. TBI phenotype may mandate precision medicine approaches to treat brain edema.

Treatment of combined traumatic brain injury and hemorrhagic shock with fractionated blood products versus fresh whole blood in a rat model.

Treatment of combined traumatic brain injury and hemorrhagic shock, poses a particular challenge due to the possible conflicting consequences. While restoring diminished volume is the treatment goal for hypovolemia, maintaining adequate cerebral perfusion pressure and avoidance of secondary damage remains a treatment goal for the injured brain. Various treatment modalities have been proposed, but the optimal resuscitation fluid and goals have not yet been clearly defined. A growing body of evidence suggests that in hypovolemic shock, resuscitation with fresh whole blood (FWB) may be superior to component therapy without platelets (which are likely to be unavailable in the pre-hospital setting). Nevertheless, the effects of this approach have not been studied in the combined injury. Previously, in a rat model of combined injury we have found that mild resuscitation to MABP of 80mmHg with FWB is superior to fluid resuscitation or aggressive resuscitation with FWB. In this study, we investigate the physiological and neurological outcomes in a rat model of combined traumatic brain injury (TBI) and hypovolemic shock, submitted to treatment with varying amounts of FWB, compared to similar resuscitation goals with fractionated blood products-red blood cells (RBCs) and plasma in a 1:1 ratio regimen. 40 male Lewis rats were divided into control and treatment groups. TBI was inflicted by a free-falling rod on the exposed cranium. Hypovolemia was induced by controlled hemorrhage of 30% blood volume. Treatment groups were treated either with fresh whole blood or with RBC + plasma in a 1:1 ratio, achieving a resuscitation goal of a mean arterial blood pressure (MAP) of 80mmHg at 15min. MAP was assessed at 60min, and neurological outcomes and mortality in the subsequent 24h. At 60min, hemodynamic parameters were improved compared to controls, but not significantly different between treatment groups. Survival rates at 48h were 100% for both of the mildly resuscitated groups (MABP 80mmHg) with FWB and RBC + plasma. The best neurological outcomes were found in the group mildly resuscitated with FWB and were better when compared to resuscitation with RBC + plasma to the same MABP goal (FWB: Neurological Severity Score (NSS) 6 ± 2, RBC + plasma: NSS 10 ± 2, p = 0.02). In this study, we find that mild resuscitation with goals of restoring MAP to 80mmHg (which is lower than baseline) with FWB, provided better hemodynamic stability and survival. However, the best neurological outcomes were found in the group resuscitated with FWB. Thus, we suggest that resuscitation with FWB is a feasible modality in the combined TBI + hypovolemic shock scenario, and may result in improved outcomes compared to platelet-free component blood products.

A Murine Combined Injury Model of Total Body Irradiation and Skin Wound

In the radiation terrorism scenario there will be combined injuries; we have developed a mouse model of combined injury (total body irradiation (TBI) plus defined skin wound). We measured the effect of TBI doses from 7.0 to 9.25 Gy irradiation on survival and healing of the skin wound. C57BL/6NTac female mice (8 weeks of age) were irradiated to doses of 0 Gy, 7.0 Gy, 8.0 Gy, 8.5 Gy, 9.0 Gy and 9.25 Gy TBI. Twenty-four hours before irradiation, the lower backs were shaved followed by depilation with NAIR. One hour after TBI, mice were anesthetized and a 25 mm length, 5 mm deep scalpel incision/wound was made in the skin. The wound was closed using staples. Control mice received no irradiation and no skin wound. Numbers of surviving mice were recorded daily. At 14, or 20 days after irradiation, groups of control and 0 Gy + wounded mice were sacrificed and wound healing was determined by measuring the tensile strength of each of ten 2 mm strips of skin per mouse across the wound using a 45N capacity tensile tester. On day 28 following irradiation, all remaining mice were tested. The LD50/30 for C57BL/6NTac mice was 9.25 Gy. In the combined injury model the LD50/30 was 8.5 Gy with significantly increased lethality with wounding at 8.5 Gy and 9.0 Gy. Mice irradiated to 9.0 Gy + wound and 8.5 Gy + wound had a statistically significant decrease in survival compared to those receiving 9.0 Gy or 8.5 Gy alone (p = 0.0073 and 0.0115, respectively). The number of deaths in the 8.5 Gy, 9.0 Gy and 9.25 Gy + wound groups was high. We tested the tensile strength in wounds from mice receiving 0 Gy + wound, 7.0 Gy + wound and 8.0 Gy + wound. Compared to the control (non-irradiated, non-wounded) mice tensile strength, the wounds in the combined injury mice were less completely healed at all times. At days 14 and 20, the healing in the 0 Gy + wound was so minimal that we did not measure the other groups until day 28. At 28 days, the mice in the 8.0 Gy + wound group still showed a significantly decreased tensile strength (1.1 ± 0.1 newtons) compared to 1.4 ± 0.1 newtons for the 0 Gy + wound (p = 0.0013). This murine combined injury model of TBI irradiation + wound revealed a clear decreased survival compared to mice receiving TBI only. While longer observation times are needed to monitor the rates of wound healing, mice receiving 8.0 Gy + wound had decreased wound healing compared to 0 Gy + wound.

Hemorrhage enhances cytokine, complement component 3, and caspase-3, and regulates microRNAs associated with intestinal damage after whole-body gamma-irradiation in combined injury.

Hemorrhage following whole-body γ-irradiation in a combined injury (CI) model increases mortality compared to whole-body γ-irradiation alone (RI). The decreased survival in CI is accompanied by increased bone marrow injury, decreased hematocrit, and alterations of miRNA in the kidney. In this study, our aim was to examine cytokine homeostasis, susceptibility to systemic bacterial infection, and intestinal injury. More specifically, we evaluated the interleukin-6 (IL-6)-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), Flt-3 ligand, and corticosterone. CD2F1 male mice received 8.75 Gy 60Co gamma photons (0.6 Gy/min, bilateral) which was followed by a hemorrhage of 20% of the blood volume. In serum, RI caused an increase of IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17A, IL-18, G-CSF, CM-CSF, eotaxin, IFN-γ, MCP-1, MIP, RANTES, and TNF-α, which were all increased by hemorrhage alone, except IL-9, IL-17A, and MCP-1. Nevertheless, CI further elevated RI-induced increases of these cytokines except for G-CSF, IFN- γ and RANTES in serum. In the ileum, hemorrhage in the CI model significantly enhanced RI-induced IL-1β, IL-3, IL-6, IL-10, IL-12p70, IL-13, IL-18, and TNF-α concentrations. In addition, Proteus mirabilis Gram(-) was found in only 1 of 6 surviving RI mice on Day 15, whereas Streptococcus sanguinis Gram(+) and Sphingomonas paucimobilis Gram(-) were detected in 2 of 3 surviving CI mice (with 3 CI mice diseased due to inflammation and infection before day 15) at the same time point. Hemorrhage in the CI model enhanced the RI-induced increases in C3 and decreases in CRP concentrations. However, hemorrhage alone did not alter the basal levels, but hemorrhage in the CI model displayed similar increases in Flt-3 ligand levels as RI did. Hemorrhage alone altered the basal levels of corticosterone early after injury, which then returned to the baseline, but in RI mice and CI mice the increased corticosterone concentration remained elevated throughout the 15 day study. CI increased 8 miRNAs and decreased 10 miRNAs in serum, and increased 16 miRNA and decreased 6 miRNAs in ileum tissue. Among the altered miRNAs, CI increased miR-34 in the serum and ileum which targeted an increased phosphorylation of ERK, p38, and increased NF-κB, thereby leading to increased iNOS expression and activation of caspase-3 in the ileum. Further, let-7g/miR-98 targeted the increased phosphorylation of STAT3 in the ileum, which is known to bind to the iNOS gene. These changes may correlate with cell death in the ileum of CI mice. The histopathology displayed blunted villi and villus edema in RI and CI mice. Based on the in silico analysis, miR-15, miR-99, and miR-100 were predicted to regulate IL-6 and TNF. These results suggest that CI-induced alterations of cytokines/chemokines, CRP, and C3 cause a homeostatic imbalance and may contribute to the pathophysiology of the gastrointestinal injury. Inhibitory intervention in these responses may prove therapeutic for CI and improve recovery of the ileal morphologic damage.

Impact of tranexamic acid on coagulation and inflammation in murine models of traumatic brain injury and hemorrhage

BackgroundPosttraumatic coagulopathy and inflammation can exacerbate secondary cerebral damage after traumatic brain injury (TBI). Tranexamic acid (TXA) has been shown clinically to reduce mortality in hemorrhaging and head-injured trauma patients and has the potential to mitigate secondary brain injury with its reported antifibrinolytic and antiinflammatory properties. We hypothesized that TXA would improve posttraumatic coagulation and inflammation in a murine model of TBI alone and in a combined injury model of TBI and hemorrhage (TBI/H). MethodsAn established murine weight drop model was used to induce a moderate TBI. Mice were administered intraperitoneal injections of 10 mg/kg TXA or equivalent volume of saline 10 min after injury. An additional group of mice was subjected to TBI followed by hemorrhagic shock using a pressure-controlled model. TBI/H mice were given intraperitoneal injections of TXA or saline during resuscitation. Blood was collected at intervals after injury to assess coagulation by rotational thromboelastometry (ROTEM) and inflammation by Multiplex cytokine analysis. Soluble P-selectin, a biomarker of platelet activation, and serum neuron-specific enolase, a biomarker of cerebral injury, were measured at intervals. Brain homogenates were analyzed for inflammatory changes by Multiplex enzyme-linked immunosorbent assay, and splenic tissue was collected for splenic cell population assessment by flow cytometry. ResultsThere were no coagulation, serum or cerebral cytokine, P-selectin, or neuron-specific enolase differences between mice treated with TXA or saline after TBI. After the addition of hemorrhagic shock and resuscitation to TBI, TXA administration still did not affect coagulation parameters, systemic or cerebral inflammation, or platelet activation, as compared with saline alone. At 24 hours after TBI, mice given TXA demonstrated lower splenic total cell counts central memory CD8, effector CD8, B cell, and increased naive CD4 cell populations. By contrast, TXA did not affect splenic leukocyte populations after combined TBI/H. ConclusionsDespite clinical data suggesting a mortality benefit, TXA did not modulate coagulation, inflammation, or biomarker generation in either the TBI or TBI/H murine models. Administration of TXA after TBI altered splenic leukocyte populations, which may contribute to a change in posttraumatic immune status. Future studies should be done to investigate the role of TXA in the development of posttraumatic immunosuppression and risk of nosocomial infections.

Characterization of erythropoietin and hepcidin in the regulation of persistent injury-associated anemia.

The cause of persistent injury-associated anemia is multifactorial and includes acute blood loss, an altered erythropoietin (EPO) response, dysregulation of iron homeostasis, and impaired erythropoiesis in the setting of chronic inflammation/stress. Hepcidin plays a key role in iron homeostasis and is regulated by anemia and inflammation. Erythropoietin is a main regulator of erythropoiesis induced by hypoxia. A unique rodent model of combined lung injury (LC)/hemorrhagic shock (HS) (LCHS)/chronic restraint stress (CS) was used to produce persistent injury-associated anemia to further investigate the roles of EPO, hepcidin, iron, ferritin, and the expression of EPO receptors (EPOr). Male Sprague-Dawley rats were randomly assigned into one of the four groups of rodent models: naive, CS alone, combined LCHS, or LCHS/CS. Plasma was used to evaluate levels of EPO, hepcidin, iron, and ferritin. RNA was isolated from bone marrow and lung tissue to evaluate expression of EPOr. Comparisons between models were performed by t tests followed by one-way analysis of variance. After 7 days, only LCHS/CS was associated with persistent anemia despite significant elevation of plasma EPO. Combined LCHS and LCHS/CS led to a persistent decrease in EPOr expression in bone marrow on Day 7. The LCHS/CS significantly decreased plasma hepcidin levels by 75% on Day 1 and 84% on Day 7 compared to LCHS alone. Hepcidin plasma levels are inversely proportional to EPO plasma levels (Pearson R = -0.362, p < 0.05). Tissue injury, hemorrhagic shock, and stress stimulate and maintain high levels of plasma EPO while hepcidin levels are decreased. In addition, bone marrow EPOr and plasma iron availability are significantly reduced following LCHS/CS. The combined deficit of reduced iron availability and reduced bone marrow EPOr expression may play a key role in the ineffective EPO response associated with persistent injury-associated anemia.

Combined Neurotrauma Models: Experimental Models Combining Traumatic Brain Injury and Secondary Insults.

Patients with severe traumatic brain injury (TBI) frequently present with concomitant injuries that may cause secondary brain injury and impact outcomes. Animal models have been developed that combine contemporary models of TBI with a secondary neurologic insult such as hypoxia, shock, long bone fracture, and radiation exposure. Combined injury models may be particularly useful when modeling treatment strategies and in efforts to map basic research to a heterogeneous patient population. Here, we review these models and their collective contribution to the literature on TBI. In addition, we provide protocols and notes for two well-characterized models of TBI plus hemorrhagic shock.