Next-Generation REBOA (Resuscitative Endovascular Balloon Occlusion of the Aorta) Device Precisely Achieves Targeted Regional Optimization in a Porcine Model of Hemorrhagic Shock.

INTRODUCTION: Limitations such as time-dependent distal ischemia have slowed the adoption of resuscitative endovascular balloon occlusion of the aorta (REBOA) for noncompressible hemorrhage. Next-generation REBOA technologies may allow for controlled partial flow, known as targeted regional optimization, to reduce distal ischemia. We aimed to characterize the efficacy of one such catheter in a porcine model of lethal hemorrhagic shock. METHODS: Noncompressible hemorrhage from an iliac injury was induced in anesthetized swine (Sus scrofa) (70-90 kg), targeting 30% total blood volume. Animals were then randomized to partial aortic occlusion (PO) with targeted distal mean arterial pressure (MAP) of 35-40 mm of mercury (mm Hg) and complete aortic occlusion (CO) (n = 8 per group) for 90 min. All groups were then resuscitated during a two-h critical care (CC) phase, with flow rate and MAP recorded continuously at the distal infrarenal aorta and proximal carotid artery, and analyzed with two-way repeated measures analysis of variance with S-N-K post-hoc test. RESULTS: During aortic occlusion, MAP distal to the balloon was consistently maintained at 35.8 ± 0.3 mm Hg in the PO group compared to 27.1 ± 0.3 mm Hg in the CO group (P < 0.05), which also corresponded to higher flow rates (202.9 ± 4.8 mL/min PO versus 25.9 ± 0.8 mL/min CO; P < 0.05). MAP proximal to the balloon was significantly higher with CO versus PO (109.2 ± 2.3 mm Hg versus 85.2 ± 2.3 mm Hg; P < 0.05). During the CC phase, distal aortic flow and MAP were not significantly different between groups. However, creatinine returned to baseline levels by the end of the study in the PO group, but not the CO group. One animal died in the CO group, whereas none died in the PO group. CONCLUSIONS: This is the first examination of the next-generation pREBOA-PRO in a porcine model of lethal hemorrhagic shock. We show technical feasibility of this technique to precisely achieve targeted regional optimization without device failure or complication. The ability to titrate balloon inflation and thus distal flow/pressure may extend the therapeutic window of REBOA by mitigating distal ischemia.

Targeted Regional Optimization in Action: Dose-dependent End-organ Ischemic Injury with Partial Aortic Occlusion in the Setting of Ongoing Liver Hemorrhage.

INTRODUCTION: Targeted regional optimization (TRO) describes partial resuscitative endovascular balloon occlusion of the aorta strategy that allows for controlled distal perfusion to balance hemostasis and tissue perfusion. This study characterized hemodynamics at specific targeted distal flow rates in a swine model of uncontrolled hemorrhage to determine if precise TRO by volume was possible. METHODS: Anesthetized swine were subjected to liver laceration and randomized into TRO at distal flows of 300 mL/min (n = 8), 500 mL/min (n = 8), or 700 mL/min (n = 8). After 90 min, the animals received damage control packing and were monitored for 6 h. Hemodynamic parameters were measured continuously, and hematology and serologic labs obtained at predetermined intervals. RESULTS: During TRO, the average percent deviation from the targeted flow was lower than 15.9% for all cohorts. Average renal flow rates were significantly different across all cohorts during TRO phase (P < 0.0001; TRO300 = 63.1 ±â€Š1.2; TRO500 = 133.70 ±â€Š1.93; TRO700 = 109.3 ±â€Š2.0), with the TRO700 cohort having less renal flow than TRO500. The TRO500 and TRO700 average renal flow rates inverted during the intensive care unit phase (P < 0.0001; TRO300 = 86.20 ±â€Š0.40; TRO500 = 148.50 ±â€Š1.45; TRO700 = 181.1 ±â€Š0.70). There was higher blood urea nitrogen, creatinine, and potassium in the TRO300 cohort at the end of the experiment, but no difference in lactate or pH between cohorts. CONCLUSION: This study demonstrated technical feasibility of TRO as a strategy to improve outcomes after prolonged periods of aortic occlusion and resuscitation in the setting of ongoing solid organ hemorrhage. A dose-dependent ischemic end-organ injury occurs beginning with partial aortic occlusion that progresses through the critical care phase, with exaggerated effect on renal function.

Blood-Based Brain and Global Biomarker Changes after Combined Hypoxemia and Hemorrhagic Shock in a Rat Model of Penetrating Ballistic-Like Brain Injury.

Penetrating traumatic brain injury (pTBI) often occurs with systemic insults such as hemorrhagic shock (HS) and hypoxemic (HX). This study examines rat models of penetrating ballistic-like brain injury (PBBI) and HX+HS to assess whether the blood levels of brain and systemic response biomarkers phosphorylated neurofilament-heavy protein (pNF-H), neurofilament-light protein (NF-L), αII-spectrin, heat shock protein (HSP70), and high mobility group box 1 protein (HMGB1) can distinguish pTBI from systemic insults and guide in pTBI diagnosis, prognosis, and monitoring. Thirty rats were randomly assigned to sham, PBBI, HS+HX, and PBBI+HS+HX groups. PBBI and sham groups underwent craniotomy with and without probe insertion and balloon expansion, respectively. HX and HS was then simulated by blood withdrawal and fraction of inspired oxygen (FIO2) reduction. Biomarker serum concentrations were determined at one (D1) and two (D2) days post-injury with enzyme-linked immunosorbent assay (ELISA) methods. Axonal injury-linked biomarkers pNF-H and NF-L serum levels in PBBI groups were higher than those in sham and HX+HS groups at D1 and D2 post-injury. The same was true for PBBI+HX+HS compared with sham (D2 only for pNF-H) and HX+HS groups. However, pNF-H and NF-L levels in PBBI+HX+HS groups were not different than their PBBI counterparts. At D1, αII-spectrin levels in the HX+HS and PBBI+HS+HX groups were higher than the sham groups. αII-spectrin levels in the HX+HS group were higher than the PBBI group. This suggests HX+HS as the common insult driving αII-spectrin elevations. In conclusion, pNF-H and NF-L may serve as specific serum biomarkers of pTBI in the presence or absence of systemic insults. αII-spectrin may be a sensitive acute biomarker in detecting systemic insults occurring alone or with pTBI.

Time-Course Evaluation of Brain Regional Mitochondrial Bioenergetics in a Pre-Clinical Model of Severe Penetrating Traumatic Brain Injury.

Mitochondrial dysfunction is a pivotal target for neuroprotection strategies for traumatic brain injury (TBI). However, comprehensive time-course evaluations of mitochondrial dysfunction are lacking in the pre-clinical penetrating TBI (PTBI) model. The current study was designed to characterize temporal responses of mitochondrial dysfunction from 30 min to 2 weeks post-injury after PTBI. Anesthetized adult male rats were subjected to either PTBI or sham craniectomy (n = 6 animals per group × 7 time points). Animals were euthanized at 30 min, 3 h, 6 h, 24 h, 3 days, 7 days, and 14 days post-PTBI, and mitochondria were isolated from the ipsilateral hemisphere of brain regions near the injury core (i.e., frontal cortex [FC] and striatum [ST]) and a more distant region from the injury core (i.e., hippocampus [HIP]). Mitochondrial bioenergetics parameters were measured in real time using the high-throughput procedures of the Seahorse Flux Analyzer (Agilent Technologies, Santa Clara, CA). The post-injury time course of FC + ST showed a biphasic mitochondrial bioenergetics dysfunction response, indicative of reduced adenosine triphosphate synthesis rate and maximal respiratory capacity after PTBI. An initial phase of energy crisis was detected at 30 min (-42%; p < 0.05 vs. sham), which resolved to baseline levels between 3 and 6 h (non-significant vs. sham). This was followed by a second and more robust phase of bioenergetics dysregulation detected at 24 h that remained unresolved out to 14 days post-injury (-55% to -90%; p < 0.05 vs. sham). In contrast, HIP mitochondria showed a delayed onset of mitochondrial dysfunction at 7 days (-74%; p < 0.05 vs. sham) that remained evident out to 14 days (-51%; p < 0.05 vs. sham) post-PTBI. Collectively, PTBI-induced mitochondrial dysfunction responses were time and region specific, evident differentially at the injury core and distant region of PTBI. The current results provide the basis that mitochondrial dysfunction may be targeted differentially based on region specificity post-PTBI. Even more important, these results suggest that therapeutic interventions targeting mitochondrial dysfunction may require extended dosing regimens to achieve clinical efficacy after 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.

Multifaceted Benefit of Whole Blood Versus Lactated Ringer's Resuscitation After Traumatic Brain Injury and Hemorrhagic Shock in Mice.

BACKGROUND: Despite increasing use in hemorrhagic shock (HS), whole blood (WB) resuscitation for polytrauma with traumatic brain injury (TBI) is largely unexplored. Current TBI guidelines recommend crystalloid for prehospital resuscitation. Although WB outperforms lactated Ringer's (LR) in increasing mean arterial pressure (MAP) in TBI + HS models, effects on brain tissue oxygenation (PbtO2), and optimal MAP remain undefined. METHODS: C57BL/6 mice (n = 72) underwent controlled cortical impact followed by HS (MAP = 25-27 mmHg). Ipsilateral hippocampal PbtO2 (n = 40) was measured by microelectrode. Mice were assigned to four groups (n = 18/group) for "prehospital" resuscitation (90 min) with LR or autologous WB, and target MAPs of 60 or 70 mmHg (LR60, WB60, LR70, WB70). Additional LR (10 ml/kg) was bolused every 5 min for MAP below target. RESULTS: LR requirements in WB60 (7.2 ± 5.0 mL/kg) and WB70 (28.3 ± 9.6 mL/kg) were markedly lower than in LR60 (132.8 ± 5.8 mL/kg) or LR70 (152.2 ± 4.8 mL/kg; all p < 0.001). WB70 MAP (72.5 ± 2.9 mmHg) was higher than LR70 (59.8 ± 4.0 mmHg, p < 0.001). WB60 MAP (68.7 ± 4.6 mmHg) was higher than LR60 (53.5 ± 3.2 mmHg, p < 0.001). PbtO2 was higher in WB60 (43.8 ± 11.6 mmHg) vs either LR60 (25.9 ± 13.0 mmHg, p = 0.04) or LR70 (24.1 ± 8.1 mmHg, p = 0.001). PbtO2 in WB70 (40.7 ± 8.8 mmHg) was higher than in LR70 (p = 0.007). Despite higher MAP in WB70 vs WB60 (p = .002), PbtO2 was similar. CONCLUSION: WB resuscitation after TBI + HS results in robust improvements in brain oxygenation while minimizing fluid volume when compared to standard LR resuscitation. WB resuscitation may allow for a lower prehospital MAP without compromising brain oxygenation when compared to LR resuscitation. Further studies evaluating the effects of these physiologic benefits on outcome after TBI with HS are warranted, to eventually inform clinical trials.

The Cardiac Physiology Underpinning Exsanguination Cardiac Arrest: Targets for Endovascular Resuscitation.

ABSTRACT: Exsanguination leading to cardiac arrest is the terminal phase of uncontrolled hemorrhage. Resuscitative interventions have focused on preload and afterload support. Outcomes remain poor due to several factors but poor coronary perfusion undoubtedly plays a role. The aim of this study is to characterize the relationship between arterial pressure and flow during hemorrhage in an effort to better describe the terminal phases of exsanguination.Male swine weighing 60 kg to 80 kg underwent splenectomy and instrumentation followed by a logarithmic exsanguination until asystole. Changes in hemodynamic parameters over time were compared using one-way, repeated measures analysis of variance.Nine animals weighing 69 ±â€Š15 kg were studied. Asystole occurred at 53 ±â€Š13 min when 52 ±â€Š11% of total blood volume has been shed. The greatest fall in mean hemodynamic indices were noted in the first 15 min: SBP (80-42 mm Hg, P = 0.02), left ventricular end-diastolic volume (94-52 mL, P = 0.04), cardiac output (4.8-2.4 L/min, P = 0.03), coronary perfusion pressure (57-30 mm Hg, P = 0.01), and stroke volume (60-25 mL, P = 0.02). This corresponds to the greatest rate of exsanguination. Organized cardiac activity was observed until asystole without arrythmias. Coronary flow was relatively preserved throughout the study, with a precipitous decline once mean arterial pressure was less than 20 mm Hg, leading to asystole.In this model, initial hemodynamic instability was due to preload failure, with asystole occurring relatively late, secondary to failure of coronary perfusion. Future resuscitative therapies need to directly address coronary perfusion failure if effective attempts are to be made to salvage these patients.

A New Pressure-Regulated, Partial Resuscitative Endovascular Balloon Occlusion of the Aorta Device Achieves Targeted Distal Perfusion.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) reduces blood loss and improves hemodynamics. Complete occlusion results in distal ischemia, limiting its use for prolonged care. This study evaluated two next-generation partial REBOA (pREBOA) catheters and their ability to achieve targeted distal aortic flow. MATERIALS AND METHODS: Swine underwent hemorrhagic shock, complete aortic occlusion, controlled continuous balloon deflation, and targeted distal perfusion (TDP; 300-mL/min) phases. They were randomized into three groups (n = 6/group), one managed with the current ER-REBOA (ER), and two with the new pREBOA technologies: a bilobed (BL) device and a semicompliant pREBOA-PRO (PRP). Hemodynamics including flow rates and mean arterial pressures at the carotid artery and infrarenal aorta were recorded. RESULTS: Hemodynamics were comparable between groups during hemorrhage and complete occlusion phases. During the controlled continuous balloon deflation phase, the distal aortic flow rate strongly correlated with percent balloon volume in BL and PRP groups, suggesting a precise control of distal perfusion. The slope of flow-balloon-volume curves was greater in the ER group than BL and PRP groups, indicating the change in distal aortic flow rate was more sensitive to the balloon volume (less titratable) when using ER. During the TDP phase, variation in distal aortic flow and mean arterial pressure with respect to the target flow was lower in ER and PRP groups, than the BL group. CONCLUSIONS: Pressure-regulated occlusion using the next-generation pREBOA catheters is more controlled than the first-generation ER-REBOA catheter and allow for targeted and precise distal perfusion.

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.

Chronic Cognitive Deficits and Associated Histopathology Following Closed-Head Concussive Injury in Rats.

Close-head concussive injury, as one of the most common forms of traumatic brain injury (TBI), has been shown to induce cognitive deficits that are long lasting. A concussive impact model was previously established in our lab that produces clinically relevant signs of concussion and induced acute pathological changes in rats. To evaluate the long-term effects of repeated concussions in this model, we utilized a comprehensive Morris water maze (MWM) paradigm for cognitive assessments at 1 and 6 months following repeated concussive impacts in rats. As such, adult Sprague-Dawley rats received either anesthesia (sham) or repeated concussive impacts (4 consecutive impacts at 1 h interval). At 1 month post-injury, results of the spatial learning task showed that the average latencies to locate the hidden "escape" platform were significantly longer in the injured rats over the last 2 days of the MWM testing compared to sham controls (p < 0.05). In the memory retention task, rats subjected to repeated concussive impacts also spent significantly less time in the platform zone searching for the missing platform during the probe trial (p < 0.05). On the working memory task, the injured rats showed a trend toward worse performance, but this failed to reach statistical significance compared to sham controls (p = 0.07). At 6 months post-injury, no differences were detected between the injured group and sham controls in either the spatial learning or probe trials. However, rats with repeated concussive impacts exhibited significantly worsened working memory performance compared to sham controls (p < 0.05). In addition, histopathological assessments for axonal neurodegeneration using silver stain showed that repeated concussive impacts induced significantly more axonal degeneration in the corpus callosum compared to sham controls (p < 0.05) at 1 month post-injury, whereas such difference was not observed at 6 months post-injury. Overall, the results show that repeated concussive impacts in our model produced significant cognitive deficits in both spatial learning abilities and in working memory abilities in a time-dependent fashion that may be indicative of progressive pathology and warrant further investigation.

Comprehensive Profile of Acute Mitochondrial Dysfunction in a Preclinical Model of Severe Penetrating TBI.

Mitochondria constitute a central role in brain energy metabolism, and play a pivotal role in the development of secondary pathophysiology and subsequent neuronal cell death following traumatic brain injury (TBI). Under normal circumstances, the brain consumes glucose as the preferred energy source for adenosine triphosphate (ATP) production over ketones. To understand the comprehensive picture of substrate-specific mitochondrial bioenergetics responses following TBI, adult male rats were subjected to either 10% unilateral penetrating ballistic-like brain injury (PBBI) or sham craniectomy (n = 5 animals per group). At 24 h post-injury, mitochondria were isolated from pooled brain regions (frontal cortex and striatum) of the ipsilateral hemisphere. Mitochondrial bioenergetics parameters were measured ex vivo in the presence of four sets of metabolic substrates: pyruvate+malate (PM), glutamate+malate (GM), succinate (Succ), and ß-hydroxybutyrate+malate (BHBM). Additionally, mitochondrial matrix dehydrogenase activities [i.e., pyruvate dehydrogenase complex (PDHC), alpha-ketoglutarate dehydrogenase complex (α-KGDHC), and glutamate dehydrogenase (GDH)] and mitochondrial membrane-bound dehydrogenase activities [i.e., electron transport chain (ETC) Complex I, II, and IV] were compared between PBBI and sham groups. Furthermore, mitochondrial coenzyme contents, including NAD(t) and FAD(t), were quantitatively measured in both groups. Collectively, PBBI led to an overall significant decline in the ATP synthesis rates (43-50%; * p < 0.05 vs. sham) when measured using each of the four sets of substrates. The PDHC and GDH activities were significantly reduced in the PBBI group (42-53%; * p < 0.05 vs. sham), whereas no significant differences were noted in α-KGDHC activity between groups. Both Complex I and Complex IV activities were significantly reduced following PBBI (47-81%; * p < 0.05 vs. sham), whereas, Complex II activity was comparable between groups. The NAD(t) and FAD(t) contents were significantly decreased in the PBBI group (27-35%; * p < 0.05 vs. sham). The decreased ATP synthesis rates may be due to the significant reductions in brain mitochondrial dehydrogenase activities and coenzyme contents observed acutely following PBBI. These results provide a basis for the use of "alternative biofuels" for achieving higher ATP production following severe penetrating brain trauma.

Serum-Based Phospho-Neurofilament-Heavy Protein as Theranostic Biomarker in Three Models of Traumatic Brain Injury: An Operation Brain Trauma Therapy Study.

Glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase (UCH-L1), markers of glial and neuronal cell body injury, respectively, have been previously selected by the Operation Brain Trauma Therapy (OBTT) pre-clinical therapy and biomarker screening consortium as drug development tools. However, traumatic axonal injury (TAI) also represents a major consequence and determinant of adverse outcomes after traumatic brain injury (TBI). Thus, biomarkers capable of assessing TAI are much needed. Neurofilaments (NFs) are found exclusively in axons. Here, we evaluated phospho-neurofilament-H (pNF-H) protein as a possible new TAI marker in serum and cerebrospinal fluid (CSF) across three rat TBI models in studies carried out by the OBTT consortium, namely, controlled cortical impact (CCI), parasagittal fluid percussion (FPI), and penetrating ballistics-like brain injury (PBBI). We indeed found that CSF and serum pNF-H levels are robustly elevated by 24 h post-injury in all three models. Further, in previous studies by OBTT, levetiracetam showed the most promising benefits, whereas nicotinamide showed limited benefit only at high dose (500 mg/kg). Thus, serum samples from the same repository collected by OBTT were evaluated. Treatment with 54 mg/kg intravenously of levetiracetam in the CCI model and 170 mg/kg in the PBBI model significantly attenuated pNF-H levels at 24 h post-injury as compared to respective vehicle groups. In contrast, nicotinamide (50 or 500 mg/kg) showed no reduction of pNF-H levels in CCI or PBBI models. Our current study suggests that pNF-H is a useful theranostic blood-based biomarker for TAI across different rodent TBI models. In addition, our data support levetiracetam as the most promising TBI drug candidate screened by OBTT to date.

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.

Early Microglial Activation Following Closed-Head Concussive Injury Is Dominated by Pro-Inflammatory M-1 Type.

Microglial activation is a pathological hallmark of traumatic brain injury (TBI). Following brain injury, activated microglia/macrophages adopt different phenotypes, generally categorized as M-1, or classically activated, and M-2, or alternatively activated. While the M-1, or pro-inflammatory phenotype is detrimental to recovery, M-2, or the anti-inflammatory phenotype, aids in brain repair. Recent findings also suggest the existence of mixed phenotype following brain injury, where activated microglia simultaneously express both M-1 and M-2 markers. The present study sought to determine microglial activation states at early time points (6-72 h) following single or repeated concussive injury in rats. Closed-head concussive injury was modeled in rats using projectile concussive impact injury, with either single or repeated impacts (4 impacts, 1 h apart). Brain samples were examined using immunohistochemical staining, inflammatory gene profiling and real-time polymerase chain reaction analyses to detect concussive injury induced changes in microglial activation and phenotype in cortex and hippocampal regions. Our findings demonstrate robust microglial activation following concussive brain injury. Moreover, we show that multiple concussions induced a unique rod-shaped microglial morphology that was also observed in other diffuse brain injury models. Histological studies revealed a predominance of MHC-II positive M-1 phenotype in the post-concussive microglial milieu following multiple impacts. Although there was simultaneous expression of M-1 and M-2 markers, gene expression results indicate a clear dominance in M-1 pro-inflammatory markers following both single and repeated concussions. While the increase in M-1 markers quickly resolved after a single concussion, they persisted following repeated concussions, indicating a pro-inflammatory environment induced by multiple concussions that may delay recovery and contribute to long-lasting consequences of concussion.

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.

Enduring Neuroprotective Effect of Subacute Neural Stem Cell Transplantation After Penetrating TBI.

Traumatic brain injury (TBI) is the largest cause of death and disability of persons under 45 years old, worldwide. Independent of the distribution, outcomes such as disability are associated with huge societal costs. The heterogeneity of TBI and its complicated biological response have helped clarify the limitations of current pharmacological approaches to TBI management. Five decades of effort have made some strides in reducing TBI mortality but little progress has been made to mitigate TBI-induced disability. Lessons learned from the failure of numerous randomized clinical trials and the inability to scale up results from single center clinical trials with neuroprotective agents led to the formation of organizations such as the Neurological Emergencies Treatment Trials (NETT) Network, and international collaborative comparative effectiveness research (CER) to re-orient TBI clinical research. With initiatives such as TRACK-TBI, generating rich and comprehensive human datasets with demographic, clinical, genomic, proteomic, imaging, and detailed outcome data across multiple time points has become the focus of the field in the United States (US). In addition, government institutions such as the US Department of Defense are investing in groups such as Operation Brain Trauma Therapy (OBTT), a multicenter, pre-clinical drug-screening consortium to address the barriers in translation. The consensus from such efforts including "The Lancet Neurology Commission" and current literature is that unmitigated cell death processes, incomplete debris clearance, aberrant neurotoxic immune, and glia cell response induce progressive tissue loss and spatiotemporal magnification of primary TBI. Our analysis suggests that the focus of neuroprotection research needs to shift from protecting dying and injured neurons at acute time points to modulating the aberrant glial response in sub-acute and chronic time points. One unexpected agent with neuroprotective properties that shows promise is transplantation of neural stem cells. In this review we present (i) a short survey of TBI epidemiology and summary of current care, (ii) findings of past neuroprotective clinical trials and possible reasons for failure based upon insights from human and preclinical TBI pathophysiology studies, including our group's inflammation-centered approach, (iii) the unmet need of TBI and unproven treatments and lastly, (iv) present evidence to support the rationale for sub-acute neural stem cell therapy to mediate enduring neuroprotection.

Alterations in brain-derived neurotrophic factor and insulin-like growth factor-1 protein levels after penetrating ballistic-like brain injury in rats.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) are essential for neuroplasticity and neuronal survival. Despite the importance of these endogenous factors in mediating posttraumatic recovery, little is known about their response after penetrating type traumatic brain injury. The objective of this study was to quantify the expression levels BDNF and IGF-1, two well-known neuroplasticity mediators, after penetrating ballistic-like brain injury (PBBI). METHODS: Rats were randomly assigned to receive unilateral sham or PBBI injuries. Using enzyme-linked immunosorbent assay and immunohistochemistry, we performed a comprehensive evaluation of BDNF and IGF-1 expression at acute (1 hour, 6 hours, 1 day) and subacute (2, 3, 7, and 14 days) timepoints after injury. RESULTS: BDNF and IGF-1 expression was transiently upregulated in both cortex and hippocampus after PBBI. Although BDNF levels increased at acute timepoints, IGF-1 expression peaked at 3 days in cortical homogenates. Although there was loss of staining in cells bordering the cavity, increased BDNF and IGF-1 immunoreactivity was observed in scattered neurons away from the contusion site. Glial upregulation of both growth factors was observed at early timepoints in the hippocampus. CONCLUSION: Our findings demonstrate that PBBI results in a brief upregulation of BDNF and IGF-1 during early posttraumatic period, providing critical information for interventions aiming to enhance neuronal survival and brain plasticity.

Functional and Molecular Correlates after Single and Repeated Rat Closed-Head Concussion: Indices of Vulnerability after Brain Injury.

Closed-head concussive injury is one of the most common causes of traumatic brain injury (TBI). Isolated concussions frequently produce acute neurological impairments, and individuals typically recover spontaneously within a short time frame. In contrast, brain injuries resulting from multiple concussions can result in cumulative damage and elevated risk of developing chronic brain pathologies. Increased attention has focused on identification of diagnostic markers that can prognostically serve as indices of brain health after injury, revealing the temporal profile of vulnerability to a second insult. Such markers may demarcate adequate recovery periods before concussed patients can return to required activities. We developed a noninvasive closed-head impact model that captures the hallmark symptoms of concussion in the absence of gross tissue damage. Animals were subjected to single or repeated concussive impact and examined using a battery of neurological, vestibular, sensorimotor, and molecular metrics. A single concussion induced transient, but marked, acute neurological impairment, gait alterations, neuronal death, and increased glial fibrillary acidic protein (GFAP) expression in brain tissue. As expected, repeated concussions exacerbated sensorimotor dysfunction, prolonged gait abnormalities, induced neuroinflammation, and upregulated GFAP and tau. These animals also exhibited chronic functional neurological impairments with sustained astrogliosis and white matter thinning. Acute changes in molecular signatures correlated with behavioral impairments, whereas increased times to regaining consciousness and balance impairments were associated with higher GFAP and neuroinflammation. Overall, behavioral consequences of either single or repeated concussive impact injuries appeared to resolve more quickly than the underlying molecular, metabolic, and neuropathological abnormalities. This observation, which is supported by similar studies in other mTBI models, underscores the critical need to develop more objective prognostic measures for guiding return-to-play decisions.

Air-puff induced vocalizations: A novel approach to detecting negative affective state following concussion in rats.

BACKGROUND: Negative emotional states resulting from concussion are of increasing concern. In the current study, we developed a model to investigate negative affect following concussion in the projectile concussive impact (PCI) model. High frequency ultrasonic vocalizations (22kHz USVs) are associated with negative affective stimuli in rats. Changes in negative affective state were examined following PCI using a mild air-puff stimulus to elicit 22kHz USVs. NEW METHOD: Forty-eight hours post-injury, animals were placed into a clean acrylic box lined with bedding. A 5min baseline recording was followed by 15 air puffs (55psi) spaced 15s apart aimed at the upper back and neck. RESULTS: Injured animals produced on average 153.5±55.13 more vocalizations than shams, vocalizing on average 4min longer than shams. Additionally, concussed animals vocalized to fewer air-puffs, exhibiting a 1.5 fold lower threshold for the expression of negative affect. COMPARISON WITH EXISTING METHODS: Studies currently used to test negative affective states following concussion in animals, such as the elevated plus maze and forced swim task have, as of yet, been unsuccessful in demonstrating injury effects in the PCI model. While the air-puff test has been applied in other fields, to our knowledge it has not been utilized to study traumatic brain injury. CONCLUSION: The current study demonstrates that the air-puff vocalization test may be a valuable tool in assessing negative mood states following concussion in rat models and may be used to evaluate novel therapies following brain injury for the treatment of mood dysfunction.

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.