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Background: Mild traumatic brain injury (mTBI) generally resolves within weeks. However, 15-30% of patients present persistent pathological and neurobehavioral sequelae that negatively affect their quality of life. Hyperhomocysteinemia (HHCY) is a neurotoxic condition derived from homocysteine accumulation above 15 μM. HHCY can occur in diverse stressful situations, including those sustained by U.S. active-duty service members on the battlefield or during routine combat practice. Mild-TBI accounts for more than 80% of all TBI cases, and HHCY exists in 5-7% of the general population. We recently reported that moderate HHCY exacerbates mTBI-induced cortical injury pathophysiology, including increased oxidative stress. Several studies have demonstrated hippocampus vulnerability to oxidative stress and its downstream effects on inflammation and cell death., Objective: This study aimed to assess the deleterious impact of HHCY on mTBI-associated hippocampal pathological changes. We tested the hypothesis that moderate HHCY aggravates mTBI-induced hippocampal pathological changes., Methods: HHCY was induced in adult male Sprague-Dawley rats with a high methionine dose. Rats were then subjected to mTBI by controlled cortical impact under sustained HHCY. Blood plasma was assessed for homocysteine levels and brain tissue for markers of oxidative stress, blood-brain barrier integrity, and cell death. Endothelial cell ultrastructure was assessed by Electron Microscopy and working memory performance using the Y maze test., Results: HHCY increased the hippocampal expression of nitrotyrosine in astroglial cells and decreased tight junction protein occludin levels associated with the enlargement of the endothelial cell nucleus. Furthermore, HHCY altered the expression of apoptosis-regulating proteins α-ii spectrin hydrolysis, ERK1/2, and AKT phosphorylation, mirrored by exacerbated mTBI-related hippocampal neuronal loss and working memory deficits., Conclusion: Our findings indicate that HHCY is an epigenetic factor that modulates mTBI pathological progression in the hippocampus and represents a putative therapeutic target for mitigating such physiological stressors that increase severity., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Aeromedical evacuation-relevant hypobaria after traumatic brain injury (TBI) leads to increased neurological injury and death in rats relative to those maintained under normobaria. Applicability of rodent brain injury research to humans may be limited, however, by differences in neuroanatomy. Therefore, we developed a model in which ferrets are exposed to polytrauma consisting of controlled cortical impact TBI and hemorrhagic shock subjected 24 h later to 6 h of hypobaria or normobaria. Our objective was to determine whether the deleterious effects of hypobaria observed in rats, with lissencephalic brains, are also present in a species with a human-like gyrencephalic brain. While no deaths were observed, magnetic resonance spectroscopy (MRS) results obtained two days post-injury indicated reduced cortical creatine, N -acetylaspartate, gamma-aminobutyric acid, myo-inositol, and glutamate that were not affected by hypobaria. T 2 -weighted magnetic resonance imaging quantification revealed increased hyperintensity volume representing cortical edema at the site of impact after polytrauma. Hypobaria did not exacerbate this focal edema but did lead to overall reductions in total cortical volume. Both normobaric and hypobaric ferrets exhibited impaired spatial memory six days post-injury on the Object Location Test, but no differences were noted between groups. Finally, cortical lesion volume was not exacerbated by hypobaria exposure on day 7 post-injury. Results suggest that air travel 24 h after polytrauma is associated with structural changes in the ferret brain. Future studies should investigate secondary injury from hypobaria after polytrauma in greater detail including alternative outcome measures, time points, and exposure to multiple flights.

Traumatic brain injury (TBI) is leading cause of morbidity in young children. Acute dysregulation of oxidative glucose metabolism within the first hours after injury is a hallmark of TBI. The developing brain relies on ketones as well as glucose for energy. Thus, the aim of this study was to determine the metabolism of ketones early after TBI injury in the developing brain. Following the controlled cortical impact injury model of TBI, 21-22-day-old rats were infused with [2,4- 13 C]β-hydroxybutyrate during the acute (4 h) period after injury. Using ex vivo 13 C-NMR spectroscopy, we determined that 13 C-β-hydroxybutyrate ( 13 C-BHB) metabolism was increased in both the ipsilateral and contralateral sides of the brain after TBI. Incorporation of the label was significantly higher in glutamate than glutamine, indicating that 13 C-BHB metabolism was higher in neurons than astrocytes in both sham and injured brains. Our results show that (i) ketone metabolism was significantly higher in both the ipsilateral and contralateral sides of the injured brain after TBI; (ii) ketones were extensively metabolized by both astrocytes and neurons, albeit higher in neurons; (iii) the pyruvate recycling pathway determined by incorporation of the label from the metabolism of 13 C-BHB into lactate was upregulated in the immature brain after TBI.

Abstract: Rats exposed to hypobaria equivalent to what occurs during aeromedical evacuation within a few days after isolated traumatic brain injury exhibit greater neurologic injury than those remaining at sea level. Moreover, administration of excessive supplemental O2 during hypobaria further exacerbates brain injury. This study tested the hypothesis that exposure of rats to hypobaria following controlled cortical impact (CCI)-induced brain injury plus mild hemorrhagic shock worsens multiple organ inflammation and associated mortality. In this study, at 24 h after CCI plus hemorrhagic shock, rats were exposed to either normobaria (sea level) or hypobaria (=8,000 ft altitude) for 6 h under normoxic or hyperoxic conditions. Injured rats exhibited mortality ranging from 30% for those maintained under normobaria and normoxia to 60% for those exposed to 6 h under hypobaric and hyperoxia. Lung histopathology and neutrophil infiltration at 2 days postinjury were exacerbated by hypobaria and hyperoxia. Gut and kidney inflammation at 30 days postinjury were also worsened by hypobaric hyperoxia. In conclusion, exposure of rats after brain injury and hemorrhagic shock to hypobaria or hyperoxia results in increased mortality. Based on gut, lung, and kidney histopathology at 2 to 30 days postinjury, increased mortality is consistent with multi-organ inflammation. These findings support epidemiological studies indicating that increasing aircraft cabin pressures to 4,000 ft altitude (compared with standard 8,000 ft) and limiting excessive oxygen administration will decrease critical complications during and following aeromedical transport., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by the Shock Society.)

Background: Aeromedical evacuation can expose traumatically injured patients to low pressure (hypobaria) and hypoxia. Here, we sought to assess the impact of hypobaria on inflammation, organ injury, and mortality in a mouse model of polytrauma., Methods: Eight to 12-week-old male C57BL/6J mice were subjected to sham or polytrauma consisting of bowel ischemia by superior mesenteric artery occlusion, hindlimb muscle crush, and tibia fracture. Two hours after injury, animals were randomized to undergo either 6 h of hypobaria or sea-level, room air conditions. At 8 or 24 h after injury, transthoracic echocardiography was performed. Acute kidney injury (AKI) biomarkers were measured by qRT-PCR. Plasma cytokine and endothelial injury markers were determined by enzyme-linked immunosorbent assay., Results: Eight hours after traumatic injury, mice exhibited a marked increase in plasma IL-6 (57 pg/mL vs. 1,216 pg/mL), AKI with increased Ngal and Kim-1, and endothelial injury as evidenced by significantly increased plasma hyaluronic acid (96 ng/mL vs.199 ng/mL), thrombomodulin (23.2 ng/mL vs. 58.9 ng/mL), syndecan-1 (0.99 ng/mL vs. 4.34 ng/mL), and E-selectin (38.6 ng/mL vs. 62.7 ng/mL). The trauma mice also developed cardiac dysfunction with decreased cardiac output and stroke volume at 8 h postinjury. Hypobaric exposure after polytrauma led to decreased ejection fraction (81.0% vs. 74.2%, P < 0.01) and increased plasma hyaluronic acid (199 ng/mL vs. 260 ng/mL, P < 0.05), thrombomodulin (58.9 ng/mL vs. 75.4 ng/mL, P < 0.05), and syndecan-1 (4.34 ng/mL vs. 8.33 ng/mL, P < 0.001) at 8 h postinjury., Conclusions: Hypobaria exposure appeared to worsen cardiac dysfunction and endothelial injury following polytrauma and thus may represent a physiological "second hit" following traumatic injury., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by the Shock Society.)

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1β, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h ( P  < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

Hyperpolarized magnetic resonance spectroscopic imaging (MRSI) of [1- 13 C]pyruvate metabolism has previously been used to assess the effects of traumatic brain injury (TBI) in rats. Here, we show that MRSI can be used in conjunction with dichloroacetate to measure the phosphorylation state of pyruvate dehydrogenase (PDH) following mild-to-moderate TBI, and that measurements can be repeated in a longitudinal study to monitor the course of injury progression and recovery. We found that the level of 13 C-bicarbonate and the bicarbonate-to-lactate ratio decreased on the injured side of the brain four hours after injury and continued to decrease through day 7. Levels recovered to normal by day 28. Measurements following dichloroacetate administration showed that PDH was inhibited equally by PDH kinase (PDK) on both sides of the brain. Therefore, the decrease in aerobic metabolism is not due to inhibition by PDK.

Background: United States service members injured in combat theatre are often aeromedically evacuated within a few days to regional military hospitals. Animal and epidemiological research indicates that early exposure to flight hypobaria may worsen brain and other injuries. The mechanisms by which secondary exposure to hypobaria worsen trauma outcomes are not well elucidated. This study tested the hypothesis that hypobaria-induced oxidative stress and associated changes in homocysteine levels play a role in traumatic brain injury (TBI) pathological progression caused by hypobaria., Methods: Male Sprague Dawley rats were exposed to a 6 h hypobaria 24 h after mild TBI by the controlled cortical impact. Plasma and brain tissues were assessed for homocysteine levels, oxidative stress markers or glutathione metabolism, and behavioral deficits post-injury in the absence and presence of hypobaria exposure., Results: We found that hypobaria after TBI increased oxidative stress markers, altered homocysteine metabolism, and promoted glutathione oxidation. Increased glutathione metabolism was driven by differential upregulation of glutathione metabolizing genes. These changes correlated with increased anxiety-like behavior., Conclusion: These data provide evidence that hypobaria exposure after TBI increases oxidative stress and alters homocysteine elimination likely through enhanced glutathione metabolism. This pathway may represent a compensatory mechanism to attenuate free radical formation. Thus, hypobaria-induced enhancement of glutathione metabolism represents a potential therapeutic target for TBI management., Competing Interests: Declaration of conflicting interests:The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Many victims of blast traumatic brain injury (TBI) are occupants of vehicles targeted by land mines. A rat model of under-vehicle blast TBI was used to test the hypothesis that the ensuing neuropathology and altered behavior are mitigated by vehicle frame designs that dramatically reduce blast-induced acceleration (G force). Male rats were restrained on an aluminum platform that was accelerated vertically at up to 2850g, in response to detonation of an explosive positioned under a second platform in contact with the top via different structures. The presence of elastomeric, polyurea-coated aluminum cylinders between the platforms reduced acceleration by 80% to 550g compared with 2350g with uncoated cylinders. Moreover, 67% of rats exposed to 2850g, and 20% of those exposed to 2350g died immediately after blast, whereas all rats subjected to 550g blast survived. Assays for working memory (Y maze) and anxiety (Plus maze) were conducted for up to 28 days. Rats were euthanized at 24 h or 29 days, and their brains were used for histopathology and neurochemical measurements. Rats exposed to 2350g blasts exhibited increased cleaved caspase-3 immunoreactive neurons in the hippocampus. There was also increased vascular immunoglobulin (Ig)G effusion and F4/80 immunopositive macrophages/microglia. Blast exposure reduced hippocampal levels of synaptic proteins Bassoon and Homer-1, which were associated with impaired performance in the Y maze and the Plus maze tests. These changes observed after 2350g blasts were reduced or eliminated with the use of polyurea-coated cylinders. Such advances in vehicle designs should aid in the development of the next generation of blast-resistant vehicles.

Cellular oxygen consumption and lactate production rates have been measured in both placental and myometrial cells to study obstetrics-related disease states such as preeclampsia. Platelet metabolic alterations indicate systemic bioenergetic changes that can be useful as disease biomarkers. We tested the hypothesis that platelet mitochondria display functional alterations in preeclampsia. Platelets were harvested from women in the third trimester of either a healthy, non-preeclamptic or preeclamptic pregnancy, and from healthy, non-pregnant women. Using Seahorse respirometry, we analyzed platelets for oxygen consumption (OCR) and extracellular acidification (ECAR) rates, indicators of mitochondrial electron transport and glucose metabolism, respectively. There was a 37% decrease in the maximal respiratory capacity measured in platelets from healthy, non-preeclamptic compared with preeclamptic pregnancy ( P <0.01); this relationship held true for other measurements of OCR, including basal respiration; ATP-linked respiration; respiratory control ratio (RCR); and spare respiratory capacity. RCR, a measure of mitochondrial efficiency, was significantly lower in healthy pregnant compared with non-pregnant women. In contrast with increased OCR, basal ECAR was significantly reduced in platelets from preeclamptic pregnancies compared with either normal pregnancies (-25%; P <0.05) or non-pregnant women (-22%; P <0.01). Secondary analysis of OCR revealed reduced basal and maximal platelet respiration in normal pregnancy prior to 34 weeks' estimated gestational age (EGA) compared with the non-pregnant state; these differences disappeared after 34 weeks. Taken together, findings suggest that in preeclampsia, there exists either a loss or early (before the third trimester) reversal of a normal biologic mechanism of platelet mitochondrial respiratory reduction associated with normal pregnancy., (© 2018 The Author(s).)

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

Objectives: There is a critical need to develop effective treatments for diabetic neuropathy. This study determined if a selective mGluR2/3 receptor agonist prevented or treated experimental diabetic peripheral neuropathy (DPN) through glutamate recycling and improved mitochondrial function., Methods: Adult male streptozotocin treated Sprague-Dawley rats with features of type 1 diabetes mellitus (T1DM) or Low Capacity Running (LCR) rats with insulin resistance or glucose intolerance were treated with 3 or 10 mg/kg/day LY379268. Neuropathy end points included mechanical allodynia, nerve conduction velocities (NCV), and intraepidermal nerve fiber density (IENFD). Markers of oxidative stress, antioxidant response, glutamate recycling pathways, and mitochondrial oxidative phosphorylation (OXPHOS) associated proteins were measured in dorsal root ganglia (DRG)., Results: In diabetic rats, NCV and IENFD were decreased. Diabetic rats treated with an mGluR2/3 agonist did not develop neuropathy despite remaining diabetic. Diabetic DRG showed increased levels of oxidized proteins, decreased levels of glutathione, decreased levels of mitochondrial DNA (mtDNA) and OXPHOS proteins. In addition, there was a 20-fold increase in levels of glial fibrillary acidic protein (GFAP) and the levels of glutamine synthetase and glutamate transporter proteins were decreased. When treated with a specific mGluR2/3 agonist, levels of glutathione, GFAP and oxidized proteins were normalized and levels of superoxide dismutase 2 (SOD2), SIRT1, PGC-1 α , TFAM, glutamate transporter proteins, and glutamine synthetase were increased in DRG neurons., Interpretation: Activation of glutamate recycling pathways protects diabetic DRG and this is associated with activation of the SIRT1-PGC-1 α -TFAM axis and preservation of mitochondrial OXPHOS function.

Blast-related traumatic brain injury (bTBI) resulting from improvised explosive devices is the hallmark injury of recent wars, and affects many returning veterans who experienced either direct or indirect exposure. Many of these veterans have long-term neurocognitive symptoms. However, there is very little evidence to show whether blast-induced acceleration alone, in the absence of secondary impacts, can cause mild TBI. In this study, we examine the effect of under-vehicle blast-induced hyperacceleration (uBIH) of ∼1700 g on the biochemical and microstrucutral changes in the brain using diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS). Two groups of adult male Sprague-Dawley (SD) rats were subjected to a sham procedure and uBIH, respectively. Axonal and neurochemical alterations were assessed using in vivo DTI and MRS at 2 h, 24 h, and 7 days after uBIH. Significant reduction in mean diffusivity, axial diffusivity, and radial diffusivity were observed in the hippocampus, thalamus, internal capsule, and corpus callosum as early as 2 h, and sustained up to 7 days post-uBIH. Total creatine (Cr) and glutamine (Gln) were reduced in the internal capsule at 24 h post-uBIH. The reductions in DTI parameters, Cr and Gln in vivo suggest potential activation of astrocytes and diffuse axonal injury following a single underbody blast, confirming previous histology reports.

Aeromedical evacuation, an important component in the care of many patients with traumatic brain injury (TBI), particularly in war zones, exposes them to prolonged periods of hypobaria. The effects of such exposure on pathophysiological changes and outcome after TBI are largely unexplored. The objective of this study was to investigate whether prolonged hypobaria in rats subjected to TBI alters behavioral and histological outcomes. Adult male Sprague-Dawley rats underwent fluid percussion induced injury at 1.5-1.9 atmospheres of pressure. The effects of hypobaric exposure (6 h duration; equivalent to 0.75 atmospheres) at 6, 24, and 72 h, or 7 days after TBI were evaluated with regard to sensorimotor, cognitive, and histological changes. Additional groups were evaluated to determine the effects of two hypobaric exposures after TBI, representing primary simulated aeromedical evacuation (6 h duration at 24 h after injury) and secondary evacuation (10 h duration at 72 h after injury), as well as the effects of 100% inspired oxygen concentrations during simulated evacuation. Hypobaric exposure up to 7 days after injury significantly worsened cognitive deficits, hippocampal neuronal loss, and microglial/astrocyte activation in comparison with injured controls not exposed to hypobaria. Hyperoxia during hypobaric exposure or two exposures to prolonged hypobaric conditions further exacerbated spatial memory deficits. These findings indicate that exposure to prolonged hypobaria up to 7 days after TBI, even while maintaining physiological oxygen concentration, worsens long-term cognitive function and neuroinflammation. Multiple exposures or use of 100% oxygen further exacerbates these pathophysiological effects.

Altered cerebral metabolism and mitochondrial function have been identified in experimental and clinical studies of pediatric traumatic brain injury (TBI). Metabolic changes detected using (1)H (proton) magnetic resonance spectroscopy correlate with long-term outcomes in children after severe TBI. We previously identified early (4-h) and sustained (24-h and 7-day) abnormalities in brain metabolites after controlled cortical impact (CCI) in immature rats. The current study aimed to identify specific alterations of cerebral glucose metabolism at 24 h after TBI in immature rats. Rats (postnatal days 16-18) underwent CCI to the left parietal cortex. Sham rats underwent craniotomy only. Twenty-four hours after CCI, rats were injected (intraperitoneally) with [1,6-(13)C]glucose. Brains were removed, separated into hemispheres, and frozen. Metabolites were extracted with perchloric acid and analyzed using (1)H and (13)C-nuclear magnetic resonance spectroscopy. TBI resulted in decreases in N-acetylaspartate in both hemispheres, compared to sham contralateral. At 24 h after TBI, there was significant decrease in the incorporation of (13)C label into [3-(13)C]glutamate and [2-(13)C]glutamate in the injured brain. There were no differences in percent enrichment of [3-(13)C]glutamate, [4-(13)C]glutamate, [3-(13)C]glutamine, or [4-(13)C]glutamine. There was significantly lower percent enrichment of [2-(13)C]glutamate in both TBI sides and the sham craniotomy side, compared to sham contralateral. No differences were detected in enrichment of (13)C glucose label in [2-(13)C]glutamine, [2-(13)C]GABA (gamma-aminobutyric acid), [3-(13)C]GABA, or [4-(13)C]GABA, [3-(13)C]lactate, or [3-(13)C]alanine between groups. Results suggest that overall oxidative glucose metabolism in the immature brain recovers at 24 h after TBI. Specific reductions in [2-(13)C]glutamate could be the result of impairments in either neuronal or astrocytic metabolism. Future studies should aim to identify pathways leading to decreased metabolism and develop cell-selective "metabolic rescue."

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO4, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg(2+) (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO4 improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO4 or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO4 protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO4 protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.

Mitochondrial dysfunction may be central to the pathophysiology of traumatic brain injury (TBI) and often can be recognized cytologically by changes in mitochondrial ultrastructure. This study is the first to broadly characterize and quantify mitochondrial morphologic alterations in surgically resected human TBI tissues from three contiguous cortical injury zones. These zones were designated as injury center (Near), periphery (Far), and Penumbra. Tissues from 22 patients with TBI with varying degrees of damage and time intervals from TBI to surgical tissue collection within the first week post-injury were rapidly fixed in the surgical suite and processed for electron microscopy. A large number of mitochondrial structural patterns were identified and divided into four survival categories: normal, normal reactive, reactive degenerating, and end-stage degenerating profiles. A tissue sample acquired at 38 hours post-injury was selected for detailed mitochondrial quantification, because it best exhibited the wide variation in cellular and mitochondrial changes consistently noted in all the other cases. The distribution of mitochondrial morphologic phenotypes varied significantly between the three injury zones and when compared with control cortical tissue obtained from an epilepsy lobectomy. This study is unique in its comparative quantification of the mitochondrial ultrastructural alterations at progressive distances from the center of injury in surviving TBI patients and in relation to control human cortex. These quantitative observations may be useful in guiding the translation of mitochondrial-based neuroprotective interventions to clinical implementation.

Mitochondrial dysfunction contributes to the pathophysiology of acute neurologic disorders and neurodegenerative diseases. Bioenergetic failure is the primary cause of acute neuronal necrosis, and involves excitotoxicity-associated mitochondrial Ca(2+) overload, resulting in opening of the inner membrane permeability transition pore and inhibition of oxidative phosphorylation. Mitochondrial energy metabolism is also very sensitive to inhibition by reactive O(2) and nitrogen species, which modify many mitochondrial proteins, lipids, and DNA/RNA, thus impairing energy transduction and exacerbating free radical production. Oxidative stress and Ca(2+)-activated calpain protease activities also promote apoptosis and other forms of programmed cell death, primarily through modification of proteins and lipids present at the outer membrane, causing release of proapoptotic mitochondrial proteins, which initiate caspase-dependent and caspase-independent forms of cell death. This review focuses on three classifications of mitochondrial targets for neuroprotection. The first is mitochondrial quality control, maintained by the dynamic processes of mitochondrial fission and fusion and autophagy of abnormal mitochondria. The second includes targets amenable to ischemic preconditioning, e.g., electron transport chain components, ion channels, uncoupling proteins, and mitochondrial biogenesis. The third includes mitochondrial proteins and other molecules that defend against oxidative stress. Each class of targets exhibits excellent potential for translation to clinical neuroprotection.

Understanding tissue alterations at an early stage following traumatic brain injury (TBI) is critical for injury management and limiting severe consequences from secondary injury. We investigated the early microstructural and metabolic profiles using in vivo diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy ((1)H MRS) at 2 and 4 h following a controlled cortical impact injury in the rat brain using a 7.0 Tesla animal MRI system and compared profiles to baseline. Significant decrease in mean diffusivity (MD) and increased fractional anisotropy (FA) was found near the impact site (hippocampus and bilateral thalamus; p<0.05) immediately following TBI, suggesting cytotoxic edema. Although the DTI parameters largely normalized on the contralateral side by 4 h, a large inter-individual variation was observed with a trend towards recovery of MD and FA in the ipsilateral hippocampus and a sustained elevation of FA in the ipsilateral thalamus (p<0.05). Significant reduction in metabolite to total creatine ratios of N-acetylaspartate (NAA, p=0.0002), glutamate (p=0.0006), myo-inositol (Ins, p=0.04), phosphocholine and glycerophosphocholine (PCh+GPC, p=0.03), and taurine (Tau, p=0.009) were observed ipsilateral to the injury as early as 2 h, while glutamine concentration increased marginally (p=0.07). These metabolic alterations remained sustained over 4 h after TBI. Significant reductions of Ins (p=0.024) and Tau (p=0.013) and marginal reduction of NAA (p=0.06) were also observed on the contralateral side at 4 h after TBI. Overall our findings suggest significant microstructural and metabolic alterations as early as 2 h following injury. The tendency towards normalization at 4 h from the DTI data and no further metabolic changes at 4 h from MRS suggest an optimal temporal window of about 3 h for interventions that might limit secondary damage to the brain. Results indicate that early assessment of TBI patients using DTI and MRS may provide valuable information on the available treatment window to limit secondary brain damage.

In this study we tested the hypothesis that long-term neuropathological outcome is worsened by hyperoxic compared to normoxic reperfusion in a rat global cerebral ischemia model. Adult male rats were anesthetized and subjected to bilateral carotid arterial occlusion plus bleeding hypotension for 10 min. The rats were randomized to one of four protocols: ischemia/normoxia (21% oxygen for 1 h), ischemia/hyperoxia (100% oxygen for 1 h), sham/normoxia, and sham/hyperoxia. Hippocampal CA1 neuronal survival and activation of microglia and astrocytes were measured in the hippocampi of the animals at 7 and 30 days post-ischemia. Morris water maze testing of memory was performed on days 23-30. Compared to normoxic reperfusion, hyperoxic ventilation resulted in a significant decrease in normal-appearing neurons at 7 and 30 days, and increased activation of microglia and astrocytes at 7, but not at 30, days of reperfusion. Behavioral deficits were also observed following hyperoxic, but not normoxic, reperfusion. We conclude that early post-ischemic hyperoxic reperfusion is followed by greater hippocampal neuronal death and cellular inflammatory reactions compared to normoxic reperfusion. The results of these long-term outcome studies, taken together with previously published results from short-term experiments performed with large animals, support the hypothesis that neurological outcome can be improved by avoiding hyperoxic resuscitation after global cerebral ischemia such as that which accompanies cardiac arrest.

Although studies have shown alterations in cerebral metabolism after traumatic brain injury (TBI), clinical data in the developing brain is limited. We hypothesized that post-traumatic metabolic changes occur early (<24 h) and persist for up to 1 week. Immature rats underwent TBI to the left parietal cortex. Brains were removed at 4 h, 24 h, and 7 days after injury, and separated into ipsilateral (injured) and contralateral (control) hemispheres. Proton nuclear magnetic resonance (NMR) spectra were obtained, and spectra were analyzed for N-acetyl-aspartate (NAA), lactate (Lac), creatine (Cr), choline, and alanine, with metabolite ratios determined (NAA/Cr, Lac/Cr). There were no metabolic differences at any time in sham controls between cerebral hemispheres. At 4 and 24 h, there was an increase in Lac/Cr, reflecting increased glycolysis and/or decreased oxidative metabolism. At 24 h and 7 days, there was a decrease in NAA/Cr, indicating loss of neuronal integrity. The NAA/Lac ratio was decreased ( approximately 15-20%) at all times (4 h, 24 h, 7 days) in the injured hemisphere of TBI rats. In conclusion, metabolic derangements begin early (<24 h) after TBI in the immature rat and are sustained for up to 7 days. Evaluation of early metabolic alterations after TBI could identify novel targets for neuroprotection in the developing brain.

Background and Purpose: Previous reports indicate that compared with normoxia, 100% ventilatory O(2) during early reperfusion after global cerebral ischemia decreases hippocampal pyruvate dehydrogenase activity and increases neuronal death. However, current standards of care after cardiac arrest encourage the use of 100% O(2) during resuscitation and for an undefined period thereafter. Using a clinically relevant canine cardiac arrest model, in this study we tested the hypothesis that hyperoxic reperfusion decreases hippocampal glucose metabolism and glutamate synthesis., Methods: After 10 minutes of cardiac arrest, animals were resuscitated and ventilated for 1 hour with 100% O(2) (hyperoxic) or 21% to 30% O(2) (normoxic). At 30 minutes reperfusion, [1-(13)C]glucose was infused, and at 2 hours, brains were rapidly removed and frozen. Extracted metabolites were analyzed by (13)C nuclear magnetic resonance spectroscopy., Results: Compared with nonischemic controls, the hippocampi from hyperoxic animals had elevated levels of unmetabolized (13)C-glucose and decreased incorporation of (13)C into all isotope isomers of glutamate. These findings indicate impaired neuronal metabolism via the pyruvate dehydrogenase pathway for carbon entry into the tricarboxylic acid cycle and impaired glucose metabolism via the astrocytic pyruvate carboxylase pathway. No differences were observed in the cortex, indicating that the hippocampus is more vulnerable to metabolic changes induced by hyperoxic reperfusion., Conclusions: These results represent the first direct evidence that hyperoxia after cardiac arrest impairs hippocampal oxidative energy metabolism in the brain and challenge the rationale for using excessively high resuscitative ventilatory O(2).

We tested the hypothesis that the response of mitochondrial uptake of calcium and content of Bcl proteins to reversible hemorrhagic shock increases the vulnerability for hepatocellular death. Pentobarbital-anesthetized rats were bled to a mean arterial pressure of 30 to 40 mmHg for 1 h. A subset was then resuscitated (isotonic sodium chloride solution, three times shed volume). Liver mitochondria were isolated at the end of hemorrhage and 1.5 h after the onset of resuscitation. Resuscitation accelerated mitochondrial respiration in the presence of adenosine diphosphate (state 3) above control (P<0.01). The respiratory control ratio ([RCR] state 3/state 4) was calculated using the respiratory rate in the presence of carboxyatractyloside (state 4). The RCR was depressed at the end of hemorrhage and recovered completely in response to resuscitation (P<0.05). The mitochondrial capacity for calcium uptake increased at the end of hemorrhage and remained greater than control (P<0.01) after resuscitation when plasma ornithine carbamoyltransferase (an index of hepatocellular injury) was greater than control (P<0.05). At this time, the capacity for calcium uptake was correlated with plasma ornithine carbamoyltransferase (r=0.819, P<0.01). Mitochondrial content of Bcl-xL, an antiapoptotic protein, was increased at the end of hemorrhage (P<0.03) with no further change after resuscitation and no change in mitochondrial Bak, a proapoptotic protein. Thus, mitochondrial mechanisms are triggered early during reversible hypovolemia that may limit the intensity of intracellular calcium signaling and its potential to cause cellular injury and death.

Mitochondrial dysfunction contributes to the pathophysiology of both acute and chronic neurodegenerative disorders. Quantification of mitochondrial bioenergetic properties generally requires the use of isolated brain mitochondria. However, the involvement of neuronal mitochondrial dysfunction in these disorders is limited by the lack of markers, and therefore isolation procedures, that distinguish neuronal compared with astrocyte mitochondria. To address this and other issues concerning neuronal mitochondria in the CNS, transgenic mice were generated that express a fluorescent protein targeted specifically to neurons. A neuron-specific promoter, CaMKIIalpha (calcium/calmodulin-dependent kinase IIalpha) driven tTA (tetracycline transactivator) mice were crossed with TRE (tetracycline responsive element) driven mitochondrial targeted enhanced yellow fluorescent protein (eYFP) mice. Expression of eYFP in the bigenic mouse brain was observed only in neuronal mitochondria of striatum, forebrain, and hippocampus and was enhanced by the removal of the tetracycline analog doxycycline (Dox) in the diet. The respiratory control ratio of synaptic and nonsynaptic mitochondria isolated from eYFP-expressing mice was the same as control mice, suggesting that neuronal mitochondria expressing eYFP maintain normal bioenergetic functions. More importantly, the development of Dox-inducible, neuron targeted mito/eYFP transgenic mice offer a unique in vivo model for delineating the participation of neuronal mitochondria in neuronal survival and death.

Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.

Background and Purpose: Studies suggest that hyperbaric oxygen (HBO) is neuroprotective after experimental cerebral ischemia, but the mechanism is unknown. This study tested the hypotheses that postischemic HBO affords clinical and histopathological neuroprotection after experimental cardiac arrest and resuscitation (A/R) and that this neuroprotection results from improved cerebral oxygen metabolism after A/R., Methods: Anesthetized adult female beagles underwent A/R and randomization to HBO (2.7-atm absolute [ATA] for 60 minutes, 1 hour after A/R) or control (Po2=80 to 100 mm Hg; 1 ATA). Animals underwent neurological deficit scoring (NDS) 23 hours after A/R. After euthanasia at 24 hours, neuronal death (necrotic and apoptotic) in representative animals was determined stereologically in hippocampus and cerebral neocortex. In experiment 2, arterial and sagittal sinus oxygenation and cerebral blood flow (CBF) were measured. Cerebral oxygen extraction ratio (ERc), oxygen delivery (Do2c), and metabolic rate for oxygen (CMRo2) were calculated (baseline and 2, 30, 60, 120, 180, 240, 300, and 360 minutes after restoration of spontaneous circulation)., Results: NDS improved after A/R in HBO animals (HBO, 35+/-14; controls, 54+/-15; P=0.028). Histopathological examination revealed significantly fewer dying neurons in HBO animals; the magnitude of neuronal injury correlated well with NDS. HBO corrected elevations in ERc (peak, 60+/-14% for controls, 26+/-4% for HBO) but did not increase Do2c or CMRo2, which decreased approximately 50% after A/R in both groups., Conclusions: HBO inhibits neuronal death and improves neurological outcome after A/R; the mechanism of HBO neuroprotection is not due to stimulation of oxidative cerebral energy metabolism.