Intravenous Administration of Anti-CD47 Antibody Augments Hematoma Clearance, Mitigates Acute Neuropathology, and Improves Cognitive Function in a Rat Model of Penetrating Traumatic Brain Injury.

Traumatic brain injury (TBI)-induced intracerebral hematoma is a major driver of secondary injury pathology such as neuroinflammation, cerebral edema, neurotoxicity, and blood-brain barrier dysfunction, which contribute to neuronal loss, motor deficits, and cognitive impairment. Cluster of differentiation 47 (CD47) is an antiphagocytic cell surface protein inhibiting hematoma clearance. This study was designed to evaluate the safety and efficacy of blockade of CD47 via intravenous (i.v.) administration of anti-CD47 antibodies following penetrating ballistic-like brain injury (PBBI) with significant traumatic intracerebral hemorrhage (tICH). The pharmacokinetic (PK) profile of the anti-CD47 antibody elicited that antibody concentration decayed over 7 days post-administration. Blood tests and necropsy analysis indicated no severe adverse events following treatment. Cerebral hemoglobin levels were significantly increased after injury, however, anti-CD47 antibody administration at 0.1 mg/kg resulted in a significant reduction in cerebral hemoglobin levels at 72 h post-administration, indicating augmentation of hematoma clearance. Immunohistochemistry assessment of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) demonstrated a significant reduction of GFAP levels in the lesion core and peri-lesional area. Based on these analyses, the optimal dose was identified as 0.1 mg/kg. Lesion volume showed a reduction following treatment. Rotarod testing revealed significant motor deficits in all injured groups but no significant therapeutic benefits. Spatial learning performance revealed significant deficits in all injured groups, which were significantly improved by the last testing day. Anti-CD47 antibody treated rats showed significantly improved attention deficits, but not retention scores. These results provide preliminary evidence that blockade of CD47 using i.v. administration of anti-CD47 antibodies may serve as a potential therapeutic for TBI with ICH.

EFFECTS OF TRANEXAMIC ACID ON NEUROPATHOLOGY, ELECTROENCEPHALOGRAPHY, AND CEREBRAL FIBRIN DEPOSITION IN A RAT MODEL OF POLYTRAUMA WITH CONCOMITANT PENETRATING TRAUMATIC BRAIN INJURY.

ABSTRACT: Several studies have demonstrated the clinical utility of tranexamic acid (TXA) for use in trauma patients presenting with significant hemorrhage. Tranexamic acid is an antifibrinolytic that inhibits plasminogen activation, and plasmin activity has been shown to mitigate blood loss and reduce all-cause mortality in the absence of adverse vascular occlusive events. Recent clinical developments indicate TXA is safe to use in patients with concomitant traumatic brain injury (TBI); however, the prehospital effects are not well understood. Importantly, TXA has been associated with seizure activity. Therefore, this study sought to evaluate the effects of early administration of TXA on neurological recovery and electroencephalogram (EEG) abnormalities following penetrating TBI with concomitant hypoxemia and hemorrhagic shock. We hypothesized that early administration of TXA will provide hemodynamic stabilization and reduce intracerebral hemorrhage, which will result in improved neurological function. To test this hypothesis, Sprague-Dawley rats received a unilateral, frontal penetrating ballistic-like brain injury by inserting a probe into the frontal cortex of the anesthetized rat. Five minutes following brain injury, animals underwent 30 min of respiratory distress and 30 min of hemorrhage. Upon completion of the hemorrhage phase, animals received the initial dose of drug intravenously over 10 min after which the prehospital phase was initiated. During the prehospital phase, animals received autologous shed whole blood as needed to maintain a MAP of 65 mm Hg. After 90 min, "in-hospital" resuscitation was performed by administering the remaining shed whole blood providing 100% oxygen for 15 min. Upon recovery from surgery, animals were administered their second dose of vehicle or TXA intravenously over 8 h. Tranexamic acid induced an early improvement in neurologic deficit, which was statistically significant compared with vehicle at 24, 48, and 72 h at three doses tested. Analysis of cerebral hemoglobin content and intracerebral lesion progression revealed 100 mg/kg provided the optimal effects for improvement of neuropathology and was continued for determination of adverse treatment effects. We observed no exacerbation of cerebral thrombosis, but TXA treatment caused an increased risk of EEG abnormalities. These results suggest that TXA following polytrauma with concomitant brain injury may provide mild neuroprotective effects by preventing lesion progression, but this may be associated with an increased risk of abnormal EEG patterns. This risk may be associated with TXA inhibition of glycine receptors and may warrant additional considerations during the use of TXA in patients with severe TBI.

Prehospital Whole Blood Resuscitation Reduces Fluid Requirement While Maintaining Critical Physiology in a Model of Penetrating Traumatic Brain Injury and Hemorrhage: Implications on Resource-Limited Combat Casualty Care.

ABSTRACT: Prehospital resuscitation using whole blood (WB) is the standard of care for hemorrhagic shock (HS) but there is no consensus recommendation for resuscitation in the presence of traumatic brain injury (TBI) due to a lack of sufficient evidence. In order to evaluate the optimal resuscitation strategies for TBI+HS, Sprague-Dawley rats were randomized into four groups based on resuscitation fluid and prehospital mean arterial pressure (MAP) threshold (n = 9-10/group): Lactated Ringer's (LR)-60 mm Hg (LR60), LR-70 mm Hg (LR70), WB-60 mm Hg (WB60), WB-70 mm Hg (WB70). All groups received a frontal penetrating ballistic-like brain injury followed by a 35-min period of HS. During the prehospital phase, rats received an initial bolus of resuscitation fluid (WB or LR) followed by LR as needed to maintain MAP above the designated threshold for 90 min. During the in-hospital phase, rats received definitive resuscitation with shed WB. Physiological parameters were recorded continuously and cerebral edema was measured at 3 and 24 h postinjury. The WB60 group demonstrated a significantly lower prehospital fluid requirement compared WB70, LR60, and LR70 (P < 0.05). Compared to the respective LR groups, both the WB60 and WB70 groups also demonstrated improved MAP, cerebral perfusion pressure, brain tissue oxygen tension, and cerebral edema. The edema benefits were observed at 3 h, but not 24 h postinjury, and were localized to the injury site. Together, these results provide evidence that prehospital WB resuscitation and lower MAP resuscitation thresholds can reduce the prehospital fluid requirement while still maintaining critical cerebral physiology in a model of HS and concomitant TBI.

Alterations in Peripheral Organs following Combined Hypoxemia and Hemorrhagic Shock in a Rat Model of Penetrating Ballistic-Like Brain Injury.

Polytrauma, with combined traumatic brain injury (TBI) and systemic damage are common among military and civilians. However, the pathophysiology of peripheral organs following polytrauma is poorly understood. Using a rat model of TBI combined with hypoxemia and hemorrhagic shock, we studied the status of peripheral redox systems, liver glycogen content, creatinine clearance, and systemic inflammation. Male Sprague-Dawley rats were subjected to hypoxemia and hemorrhagic shock insults (HH), penetrating ballistic-like brain injury (PBBI) alone, or PBBI followed by hypoxemia and hemorrhagic shock (PHH). Sham rats received craniotomy only. Biofluids and liver, kidney, and heart tissues were collected at 1 day, 2 days, 7 days, 14 days, and 28 days post-injury (DPI). Creatinine levels were measured in both serum and urine. Glutathione levels, glycogen content, and superoxide dismutase (SOD) and cytochrome C oxidase enzyme activities were quantified in the peripheral organs. Acute inflammation marker serum amyloid A-1 (SAA-1) level was quantified using western blot analysis. Urine to serum creatinine ratio in PHH group was significantly elevated on 7-28 DPI. Polytrauma induced a delayed disruption of the hepatic GSH/GSSG ratio, which resolved within 2 weeks post-injury. A modest decrease in kidney SOD activity was observed at 2 weeks after polytrauma. However, neither PBBI alone nor polytrauma changed the mitochondrial cytochrome C oxidase activity. Hepatic glycogen levels were reduced acutely following polytrauma. Acute inflammation marker SAA-1 showed a significant increase at early time-points following both systemic and brain injury. Overall, our findings demonstrate temporal cytological/tissue level damage to the peripheral organs due to combined PBBI and systemic injury.

The Effects of Balloon Occlusion of the Aorta on Cerebral Blood Flow, Intracranial Pressure, and Brain Tissue Oxygen Tension in a Rodent Model of Penetrating Ballistic-Like Brain Injury.

Trauma is among the leading causes of death in the United States. Technological advancements have led to the development of resuscitative endovascular balloon occlusion of the aorta (REBOA) which offers a pre-hospital option to non-compressible hemorrhage control. Due to the prevalence of concomitant traumatic brain injury (TBI), an understanding of the effects of REBOA on cerebral physiology is critical. To further this understanding, we employed a rat model of penetrating ballistic-like brain injury (PBBI). PBBI produced an injury pattern within the right frontal cortex and striatum that replicates the pathology from a penetrating ballistic round. Aortic occlusion was initiated 30 min post-PBBI and maintained continuously (cAO) or intermittently (iAO) for 30 min. Continuous measurements of mean arterial pressure (MAP), intracranial pressure (ICP), cerebral blood flow (CBF), and brain tissue oxygen tension (PbtO2) were recorded during, and for 60 min following occlusion. PBBI increased ICP and decreased CBF and PbtO2. The arterial balloon catheter effectively occluded the descending aorta which augmented MAP in the carotid artery. Despite this, CBF levels were not changed by aortic occlusion. iAO caused sustained adverse effects to ICP and PbtO2 while cAO demonstrated no adverse effects on either. Temporary increases in PbtO2 were observed during occlusion, along with restoration of sham levels of ICP for the remainder of the recordings. These results suggest that iAO may lead to prolonged cerebral hypertension following PBBI. Following cAO, ICP, and PbtO2 levels were temporarily improved. This information warrants further investigation using TBI-polytrauma model and provides foundational knowledge surrounding the non-hemorrhage applications of REBOA including neurogenic shock and stroke.

Selective Brain Cooling Reduces Motor Deficits Induced by Combined Traumatic Brain Injury, Hypoxemia and Hemorrhagic Shock.

Selective brain cooling (SBC) can potentially maximize the neuroprotective benefits of hypothermia for traumatic brain injury (TBI) patients without the complications of whole body cooling. We have previously developed a method that involved extraluminal cooling of common carotid arteries, and demonstrated the feasibility, safety and efficacy for treating isolated TBI in rats. The present study evaluated the neuroprotective effects of 4-h SBC in a rat model of penetrating ballistic-like brain injury (PBBI) combined with hypoxemic and hypotensive insults (polytrauma). Rats were randomly assigned into two groups: PBBI+polytrauma without SBC (PHH) and PBBI+polytrauma with SBC treatment (PHH+SBC). All animals received unilateral PBBI, followed by 30-min hypoxemia (fraction of inspired oxygen = 0.1) and then 30-min hemorrhagic hypotension (mean arterial pressure = 40 mmHg). Fluid resuscitation was given immediately following hypotension. SBC was initiated 15 min after fluid resuscitation and brain temperature was maintained at 32-33°C (core temperature at ~36.5°C) for 4 h under isoflurane anesthesia. The PHH group received the same procedures minus the cooling. At 7, 10, and 21 days post-injury, motor function was assessed using the rotarod task. Cognitive function was assessed using the Morris water maze at 13-17 days post-injury. At 21 days post-injury, blood samples were collected and the animals were transcardially perfused for subsequent histological analyses. SBC transiently augmented cardiovascular function, as indicated by the increase in mean arterial pressure and heart rate during cooling. Significant improvement in motor functions were detected in SBC-treated polytrauma animals at 7, 10, and 21 days post-injury compared to the control group (p < 0.05). However, no significant beneficial effects were detected on cognitive measures following SBC treatment in the polytrauma animals. In addition, the blood serum and plasma levels of cytokines interleukin-1 and -10 were comparable between the two groups. Histological results also did not reveal any between-group differences in subacute neurodegeneration and astrocyte/ microglial activation. In summary, 4-h SBC delivered through extraluminal cooling of the common carotid arteries effectively ameliorated motor deficits induced by PBBI and polytrauma. Improving cognitive function or mitigating subacute neurodegeneration and neuroinflammation might require a different cooling regimen such as extended cooling, a slow rewarming period and a lower temperature.

Neurochemical changes following combined hypoxemia and hemorrhagic shock in a rat model of penetrating ballistic-like brain injury: A microdialysis study.

BACKGROUND: Energy metabolic dysfunction is a key determinant of cellular damage following traumatic brain injury and may be worsened by additional insults. This study evaluated the acute/subacute effects of combined hypoxemia (HX) and hemorrhagic shock (HS) on cerebral interstitial levels of glucose, lactate, and pyruvate in a rat model of penetrating ballistic-like brain injury (PBBI). METHODS: Rats were randomly assigned into the sham control, PBBI, and combined injury (P + HH) groups. The P + HH group received PBBI followed by 30-minute HX and 30 minute HS. Samples were collected from striatum (perilesional region) using intracerebral microdialysis at 1 to 3 hours after injury and then at 1 to 3, 7, and 14 days after injury. Glucose, lactate, and pyruvate were measured in the dialysate samples. RESULTS: Glucose levels dropped significantly up to 24 hours following injury in both PBBI and P + HH groups (p < 0.05). A reduction in pyruvate was observed in the PBBI group from 24 to 72 hours after injury (vs. sham). In the P + HH group, the pyruvate was significantly reduced from 2 to 24 hours after injury (p < 0.05 vs. PBBI). This prominent reduction persisted for 14 days after injury. In contrast, lactate levels were significantly increased in the PBBI group during the first 24 hours after injury and remained elevated out to 7 days. The P + HH group exhibited a similar trend of lactate increase as did the PBBI group. Critically, P + HH further increased the lactate-to-pyruvate ratio by more than twofold (vs. PBBI) during the first 24 hours. The ratio reached a peak at 2 hours and then gradually decreased, but the level remained significantly higher than that in the sham control from 2 to 14 days after injury (p < 0.05). CONCLUSION: This study identified the temporal profile of energy-related neurochemical dysregulation induced by PBBI and combined injury in the perilesional region. Furthermore, combined HX and HS further reduced the pyruvate level and increased the lactate-to-pyruvate ratio following PBBI, indicating the exacerbation of posttraumatic metabolic perturbation.

Increased latencies to initiate cocaine self-administration following laterodorsal tegmental nucleus lesions.

Cholinergic input to the ventral tegmental area (VTA), origin of the mesocorticolimbic dopamine system that is critical for cocaine reward, is important for both cocaine seeking and cocaine taking. The laterodorsal tegmental nucleus (LDTg) provides one of the two major sources of excitatory cholinergic input to the VTA, but little is known of the role of the LDTg in cocaine reward. LDTg cholinergic cells express urotensin-II receptors and here we used local microinjections of a conjugate of the endogenous ligand for these receptors with diphtheria toxin (Dtx::UII) to lesion the cholinergic cells of the LDTg in rats previously trained to self-administer cocaine (1mg/kg/infusion, i.v.). Lesioned rats showed long latencies to initiate cocaine self-administration after treatment with the toxin, which resulted in a reduction in cocaine intake per session. Priming injections reduced latencies to initiate responding for cocaine in lesioned rats, and once they began to respond the rats regulated their moment-to-moment cocaine intake within normal limits. Thus we conclude that while LDTg cholinergic cell loss does not significantly alter the rewarding effects of cocaine, LDTg lesions can reduce the rat's responsiveness to cocaine-predictive stimuli.

Propofol limits microglial activation after experimental brain trauma through inhibition of nicotinamide adenine dinucleotide phosphate oxidase.

BACKGROUND: Microglial activation is implicated in delayed tissue damage after traumatic brain injury (TBI). Activation of microglia causes up-regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, with the release of reactive oxygen species and cytotoxicity. Propofol appears to have antiinflammatory actions. The authors evaluated the neuroprotective effects of propofol after TBI and examined in vivo and in vitro whether such actions reflected modulation of NADPH oxidase. METHODS: Adult male rats were subjected to moderate lateral fluid percussion TBI. Effect of propofol on brain microglial activation and functional recovery was assessed up to 28 days postinjury. By using primary microglial and BV2 cell cultures, the authors examined propofol modulation of lipopolysaccharide and interferon-γ-induced microglial reactivity and neurotoxicity. RESULTS: Propofol improved cognitive recovery after TBI in novel object recognition test (48 ± 6% for propofol [n = 15] vs. 30 ± 4% for isoflurane [n = 14]; P = 0.005). The functional improvement with propofol was associated with limited microglial activation and decreased cortical lesion volume and neuronal loss. Propofol also attenuated lipopolysaccharide- and interferon-γ-induced microglial activation in vitro, with reduced expression of inducible nitric oxide synthase, nitric oxide, tumor necrosis factor-α, interlukin-1ß, reactive oxygen species, and NADPH oxidase. Microglial-induced neurotoxicity in vitro was also markedly reduced by propofol. The protective effect of propofol was attenuated when the NADPH oxidase subunit p22 was knocked down by small interfering RNA. Moreover, propofol reduced the expression of p22 and gp91, two key components of NADPH oxidase, after TBI. CONCLUSION: The neuroprotective effects of propofol after TBI appear to be mediated, in part, through the inhibition of NADPH oxidase.

Cognitive deficits in a mouse model of pre-manifest Parkinson's disease.

Early cognitive deficits are increasingly recognized in patients with Parkinson's disease (PD), and represent an unmet need for the treatment of PD. These early deficits have been difficult to model in mice, and their mechanisms are poorly understood. α-Synuclein is linked to both familial and sporadic forms of PD, and is believed to accumulate in brains of patients with PD before cell loss. Mice expressing human wild-type α-synuclein under the Thy1 promoter (Thy1-aSyn mice) exhibit broad overexpression of α-synuclein throughout the brain and dynamic alterations in dopamine release several months before striatal dopamine loss. We now show that these mice exhibit deficits in cholinergic systems involved in cognition, and cognitive deficits in domains affected in early PD. Together with an increase in extracellular dopamine and a decrease in cortical acetylcholine at 4-6 months of age, Thy1-aSyn mice made fewer spontaneous alternations in the Y-maze and showed deficits in tests of novel object recognition (NOR), object-place recognition, and operant reversal learning, as compared with age-matched wild-type littermates. These data indicate that cognitive impairments that resemble early PD manifestations are reproduced by α-synuclein overexpression in a murine genetic model of PD. With high power to detect drug effects, these anomalies provide a novel platform for testing improved treatments for these pervasive cognitive deficits.