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PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC(50) of 5.95 μM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 hours showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Low-level blast exposure can result in neurological impairment for military personnel. Currently, there is a lack of experimental data using sex as a biological variable in neurovascular outcomes following blast exposure. To model mild blast traumatic brain injury (mbTBI), male and female rats were exposed to a single 11 psi static peak overpressure blast wave using the McMillan blast device and cohorts were then euthanized at 6 h, 24 h, 7 d and 14 d post-blast followed by isolation of the amygdala. After mbTBI, animals experience immediate bradycardia, although no changes in oxygen saturation levels or weight loss are observed. Male mbTBI animals displayed significantly higher levels of anxiety-like behavior (open field and elevated plus maze) compared to male sham groups; however, there was no anxiety phenotype in female mbTBI animals. Blast-induced neurovascular damage was explored by measuring expression of tight junction (TJ) proteins (zonula occludens-1 (ZO-1), occludin and claudin-5), glial fibrillary acidic protein (GFAP) and astrocyte end-feet coverage around the blood–brain barrier (BBB). Western blot analysis demonstrates that TJ protein levels were significantly decreased at 6 h and 24 h post-mbTBI in male rats, but not in female rats, compared to sham. Female animals have decreased GFAP at 6 h post-mbTBI while male animals display decreased GFAP expression at 24 h post-mbTBI. By 7 d post-mbTBI, there were no significant differences in TJ or GFAP levels between groups in either sex. At 24 h post-mbTBI, vascular integrity and astrocytic end-feet coverage around the BBB was significantly decreased in males following mbTBI. These results demonstrate that loss of GFAP expression may be due to astrocytic damage at the BBB. Our findings also demonstrate sex differences in acute vascular and behavioral outcomes after single mbTBI. Female animals display a lack of BBB pathology after mbTBI corresponding to improved acute neuropsychological outcomes as compared to male animals.

Advanced age at the time of spinal cord injury (SCI) exacerbates damage from reactive oxygen species (ROS). Mechanisms underlying this age-dependent response are not well understood and may arise from decreased antioxidant defense. We investigated how spinal cord levels of the antioxidant glutathione (GSH), and its regulation, change with age and SCI. GSH is used by GSH peroxidase to sequester ROS and is recycled by GSH reductase. Male and female, 4- and 14-month-old (MO) mice received a 60 kDyn contusion SCI, and the levels of GSH and its regulatory enzymes were evaluated at one and three days post-injury (dpi). The mice with SCI were treated with N-acetylcysteine-amide (NACA; 150 mg/kg), a cysteine supplement that increases GSH, to determine effects on functional and histological outcomes. GSH was decreased with older age in sham mice, and an SCI-dependent depletion was observed in 4-MO mice by three dpi. Neither age nor injury affected the abundance of proteins regulating GSH synthesis or recycling. GSH peroxidase activity, however, increased after SCI only in 4-MO mice. In contrast, GSH peroxidase activity was increased in 14-MO sham mice, indicating that spinal cords of older mice have an elevated oxidative state. Indeed, 14-MO sham mice had more oxidized protein (3-nitrotyrosine [3-NT]) within their spinal cords compared with 4-MO sham mice. Only 4-MO mice had significant injury-induced increases in 3-NT at three dpi. NACA treatment restored GSH and improved the redox environment in injured 4- and 14-MO mice at one dpi; however, three days of NACA delivery did not improve motor, sensory, or anatomical deficits at 28 dpi in 4-MO mice and trended toward toxicity in all outcomes in 14-MO mice. Our observation suggests that GSH levels at acute stages of SCI play a minimal role in age-dependent outcomes reported after SCI in mice. Collective results implicate elements of injury occurring after three dpi, such as inflammation, as key regulators of age-dependent effects.

Late-life dementia typically develops over a period of many years beginning in midlife. Prevalence of metabolic disturbance also accelerates in middle age and is a prominent risk factor for dementia. Preliminary studies indicate that blueberry supplementation can improve cognitive performance and influence metabolism and brain function and therefore may have a role in early intervention to prevent neurodegeneration. In a randomized controlled trial, we investigated the effects of daily blueberry supplementation in a middle-aged sample of insulin-resistant participants with elevated risk for future dementia. We enrolled overweight men and women, aged 50 to 65 years, with subjective cognitive decline (SCD) and performed pre- and post-intervention assessments of cognition and metabolism and exploratory measures of peripheral mitochondrial function. We observed improved performances for the blueberry group on measures of lexical access, p = 0.003, and memory interference, p = 0.04, and blueberry-treated participants reported reduced memory encoding difficulty in daily life activities, p = 0.03. The blueberry-treated group also exhibited correction of peripheral hyperinsulinemia, p = 0.04, and a modest trend for increased mitochondrial uncoupling, p = 0.11. The cognitive findings indicated improved executive ability in this middle-aged sample. In addition, the changes in metabolic and bioenergetic measures imply potential mechanistic factors associated with anthocyanin and proanthocyanidin actions. The demonstration of these benefits in middle-aged individuals with insulin resistance and SCD suggests that ongoing blueberry supplementation may contribute to protection against cognitive decline when implemented early in at-risk individuals.

BACKGROUNDBeige adipose tissue is associated with improved glucose homeostasis in mice. Adipose tissue contains β3-adrenergic receptors (β3-ARs), and this study was intended to determine whether the treatment of obese, insulin-resistant humans with the β3-AR agonist mirabegron, which stimulates beige adipose formation in subcutaneous white adipose tissue (SC WAT), would induce other beneficial changes in fat and muscle and improve metabolic homeostasis.METHODSBefore and after β3-AR agonist treatment, oral glucose tolerance tests and euglycemic clamps were performed, and histochemical analysis and gene expression profiling were performed on fat and muscle biopsies. PET-CT scans quantified brown adipose tissue volume and activity, and we conducted in vitro studies with primary cultures of differentiated human adipocytes and muscle.RESULTSThe clinical effects of mirabegron treatment included improved oral glucose tolerance (P < 0.01), reduced hemoglobin A1c levels (P = 0.01), and improved insulin sensitivity (P = 0.03) and β cell function (P = 0.01). In SC WAT, mirabegron treatment stimulated lipolysis, reduced fibrotic gene expression, and increased alternatively activated macrophages. Subjects with the most SC WAT beiging showed the greatest improvement in β cell function. In skeletal muscle, mirabegron reduced triglycerides, increased the expression of PPARγ coactivator 1 α (PGC1A) (P < 0.05), and increased type I fibers (P < 0.01). Conditioned media from adipocytes treated with mirabegron stimulated muscle fiber PGC1A expression in vitro (P < 0.001).CONCLUSIONMirabegron treatment substantially improved multiple measures of glucose homeostasis in obese, insulin-resistant humans. Since β cells and skeletal muscle do not express β3-ARs, these data suggest that the beiging of SC WAT by mirabegron reduces adipose tissue dysfunction, which enhances muscle oxidative capacity and improves β cell function.TRIAL REGISTRATIONClinicaltrials.gov NCT02919176.FUNDINGNIH: DK112282, P30GM127211, DK 71349, and Clinical and Translational science Awards (CTSA) grant UL1TR001998.

Background: Alzheimer’s disease (AD) is a progressive age-dependent disorder whose risk is affected by genetic factors. Better models for investigating early effects of risk factors such as apolipoprotein E (APOE) genotype are needed. Objective: To determine whether APOE genotype produces neuropathologies in an AD-susceptible neural system, we compared effects of human APOE ɛ3 (E3) and APOE ɛ4 (E4) alleles on the mouse olfactory epithelium. Methods: RNA-Seq using the STAR aligner and DESeq2, immunohistochemistry for activated caspase-3 and phosphorylated histone H3, glucose uptake after oral gavage of 2-[1,2-3H (N)]-deoxy-D-glucose, and Seahorse Mito Stress tests on dissociated olfactory mucosal cells. Results: E3 and E4 olfactory mucosae show 121 differentially abundant mRNAs at age 6 months. These do not indicate differences in cell type proportions, but effects on 17 odorant receptor mRNAs suggest small differences in tissue development. Ten oxidoreductases mRNAs important for cellular metabolism and mitochondria are less abundant in E4 olfactory mucosae but this does not translate into differences in cellular respiration. E4 olfactory mucosae show lower glucose uptake, characteristic of AD susceptibility and consistent with greater expression of the glucose-sensitive gene, Asns. Olfactory sensory neuron apoptosis is unaffected at age 6 months but is greater in E4 mice at 10 months. Conclusion: Effects of human APOE alleles on mouse olfactory epithelium phenotype are apparent in early adulthood, and neuronal loss begins to increase by middle age (10 months). The olfactory epithelium is an appropriate model for the ability of human APOE alleles to modulate age-dependent effects associated with the progression of AD.

Mechanisms that enhance energy expenditure are attractive therapeutic targets for obesity. Previously we have demonstrated that mice lacking cd47 are leaner, exhibit increased energy expenditure, and are protected against diet-induced obesity. In this study, we further defined the physiological role of cd47 deficiency in regulating mitochondrial function and energy expenditure in both white and brown adipose tissue. We observed that cd47 deficient mice (under normal chow diet) had comparable amount of white fat mass but reduced white adipocyte size as compared to wild-type mice. Subsequent ex vivo and in vitro studies suggest enhanced lipolysis, and not impaired lipogenesis or energy utilization, contributes to this phenotype. In contrast to white adipose tissue, there were no obvious morphological differences in brown adipose tissue between wild-type and knockout mice. However, mitochondria isolated from brown fat of cd47 deficient mice had significantly higher rates of free fatty acid-mediated uncoupling. This suggests that enhanced fuel availability via white adipose tissue lipolysis may perpetuate elevated brown adipose tissue energy expenditure and contributes to the lean phenotype observed in cd47 deficient mice., Summary: Under physiological conditions, CD47 differentially regulates white fat and brown fat function. Through a coordinated manner, altered fuel availability via white adipose tissue lipolysis may influence brown adipose tissue energy expenditure and then body weight management.

BACKGROUND Beige and brown adipose tissue (BAT) are associated with improved metabolic homeostasis. We recently reported that the β3-adrenergic receptor agonist mirabegron induced beige adipose tissue in obese insulin-resistant subjects, and this was accompanied by improved glucose metabolism. Here we evaluated pioglitazone treatment with a combination pioglitazone and mirabegron treatment and compared these with previously published data evaluating mirabegron treatment alone. Both drugs were used at FDA-approved dosages. METHODS We measured BAT by PET CT scans, measured beige adipose tissue by immunohistochemistry, and comprehensively characterized glucose and lipid homeostasis and insulin sensitivity by euglycemic clamp and oral glucose tolerance tests. Subcutaneous white adipose tissue, muscle fiber type composition and capillary density, lipotoxicity, and systemic inflammation were evaluated by immunohistochemistry, gene expression profiling, mass spectroscopy, and ELISAs. RESULTS Treatment with pioglitazone or the combination of pioglitazone and mirabegron increased beige adipose tissue protein marker expression and improved insulin sensitivity and glucose homeostasis, but neither treatment induced BAT in these obese subjects. When the magnitude of the responses to the treatments was evaluated, mirabegron was found to be the most effective at inducing beige adipose tissue. Although monotherapy with either mirabegron or pioglitazone induced adipose beiging, combination treatment resulted in less beiging than either alone. The 3 treatments also had different effects on muscle fiber type switching and capillary density. CONCLUSION The addition of pioglitazone to mirabegron treatment does not enhance beiging or increase BAT in obese insulin-resistant research participants. TRIAL REGISTRATION ClinicalTrials.gov NCT02919176. FUNDING NIH DK112282 and P20GM103527 and Clinical and Translational Science Awards grant UL1TR001998.

PurposeTo test the hypothesis that imaging biomarkers are useful for evaluating in vivo rod photoreceptor cell responses to a mitochondrial protonophore.MethodsIntraperitoneal injections of either the mitochondrial uncoupler 2,4 dinitrophenol (DNP) or saline were given to mice with either higher [129S6/eVTac (S6)] or lower [C57BL/6J (B6)] mitochondrial reserve capacities and were studied in dark or light. We measured: (i) the external limiting membrane-retinal pigment epithelium region thickness (ELM-RPE; OCT), which decreases substantially with upregulation of a pH-sensitive water removal co-transporter on the apical portion of the RPE, and (ii) the outer retina R1 (= 1/(spin lattice relaxation time (T1), an MRI parameter proportional to oxygen / free radical content.ResultsIn darkness, baseline rod energy production and consumption are relatively high compared to that in light, and additional metabolic stimulation with DNP provoked thinning of the ELM-RPE region compared to saline injection in S6 mice; ELM-RPE thickness was unresponsive to DNP in B6 mice. Also, dark-adapted S6 mice given DNP showed a decrease in outer retina R1 values compared to saline injection in the inferior retina. In dark-adapted B6 mice, transretinal R1 values were unresponsive to DNP in superior and inferior regions. In light, with its relatively lower basal rod energy production and consumption, DNP caused ELM-RPE thinning in both S6 and B6 mice.ConclusionsThe present results raise the possibility of non-invasively evaluating the mouse rod mitochondrial energy ecosystem using new DNP-assisted OCT and MRI in vivo assays.

Traumatic brain injury (TBI) results in cognitive impairment, which can be long-lasting after moderate to severe TBI. Currently, there are no FDA-approved therapeutics to treat the devastating consequences of TBI and improve recovery. This study utilizes a prodrug of 2,4-dinitrophenol (DNP), MP201, a mitochondrial uncoupler with extended elimination time, that was administered after TBI to target mitochondrial dysfunction, a hallmark of TBI. Using a model of cortical impact in male C57/BL6 mice, MP201 (80 mg/kg) was provided via oral gavage 2-hours post-injury and daily afterwards. At 25-hours post-injury, mice were euthanized and the acute rescue of mitochondrial bioenergetics was assessed demonstrating a significant improvement in both the ipsilateral cortex and ipsilateral hippocampus after treatment with MP201. Additionally, oxidative markers, 4- hydroxyneneal and protein carbonyls, were reduced compared to vehicle animals after MP201 administration. At 2-weeks post-injury, mice treated with MP201 post-injury (80mg/kg; q.d.) displayed significantly increased cortical sparing (p = 0.0059; 38% lesion spared) and improved cognitive outcome (p = 0.0133) compared to vehicle-treated mice. Additionally, vehicle-treated mice had significantly lower (p = 0.0019) CA3 neuron count compared to sham while MP201-treated mice were not significantly different from sham levels. These results suggest that acute mitochondrial dysfunction can be targeted to impart neuroprotection from reactive oxygen species, but chronic administration may have an added benefit in recovery. This study highlights the potential for safe, effective therapy by MP201 to alleviate negative outcomes of TBI.

Purpose The aims of this work are to establish a time point to determine the most beneficial time to administer GCEE post incident to reduce oxidative damage and secondly, by using redox proteomics, to determine if GCEE can readily suppress 3–NT modification in TBI animals. Experimental design By using a moderate traumatic brain injury model with Wistar rats, it is hypothesized that the role of 3 – NT formation as an intermediate will predict the involvement of protein nitration/nitrosation and oxidative damage in the brain. Results In this experiment, the levels of protein carbonyls, 4-hydroxynonenal, and 3–nitrotyrosine were significantly elevated in TBI injured, saline treated rats compared to those who sustained an injury and were treated with 150 mg/kg of the glutathione mimetic, GCEE. Conclusion and clinical relevance Determining the existence of elevated 3–NT levels provides insight into the relationship between protein nitration/nitrosation and oxidative damage which can determine the pathogenesis and progression of specific neurological diseases. This article is protected by copyright. All rights reserved

Traumatic brain injury (TBI) leads to acute necrosis at the site of injury followed by a sequence of secondary events lasting from hours to weeks and often years. Targeting mitochondrial impairment following TBI has shown improvements in brain mitochondrial bioenergetics and neuronal function. Recently formoterol, a highly selective β

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

Mitochondrial dysfunction is a pivotal event in many neurodegenerative disease states including traumatic brain injury (TBI) and spinal cord injury (SCI). One possible mechanism driving mitochondrial dysfunction is glutamate excitotoxicity leading to Ca(2+)-overload in neuronal or glial mitochondria. Therapies that reduce calcium overload and enhance bioenergetics have been shown to improve neurological outcomes. Pioglitazone, an FDA approved compound, has shown neuroprotective properties following TBI and SCI, but the underlying mechanism(s) are unknown. We hypothesized that the interaction between Pioglitazone and a novel mitochondrial protein called mitoNEET was the basis for neuroprotection following CNS injury. We discovered that mitoNEET is an important mediator of Ca(2+)-mediated mitochondrial dysfunction and show that binding mitoNEET with Pioglitazone can prevent Ca(2+)-induced dysfunction. By utilizing wild-type (WT) and mitoNEET null mice, we show that Pioglitazone mitigates mitochondrial dysfunction and provides neuroprotection in WT mice, though produces no restorative effects in mitoNEET null mice. We also show that NL-1, a novel mitoNEET ligand, is neuroprotective following TBI in both mice and rats. These results support the crucial role of mitoNEET for mitochondrial bioenergetics, its importance in the neuropathological sequelae of TBI and the necessity of mitoNEET for Pioglitazone-mediated neuroprotection. Since mitochondrial dysfunction is a pathobiological complication seen in other diseases such as diabetes, motor neuron disease and cancer, targeting mitoNEET may provide a novel mitoceutical target and therapeutic intervention for diseases that expand beyond TBI.

BACKGROUND. The induction of beige adipocytes in s.c. white adipose tissue (WAT) depots of humans is postulated to improve glucose and lipid metabolism in obesity. The ability of obese, insulin-resistant humans to induce beige adipose tissue is unknown. METHODS. We exposed lean and obese research participants to cold (30-minute ice pack application each day for 10 days of the upper thigh) or treated them with the β3 agonist mirabegron. We determined beige adipose marker expression by IHC and quantitative PCR, and we analyzed mitochondrial bioenergetics and UCP activity with an Oxytherm system. RESULTS. Cold significantly induced UCP1 and TMEM26 protein in both lean and obese subjects, and this response was not associated with age. Interestingly, these proteins increased to the same extent in s.c. WAT of the noniced contralateral leg, indicating a crossover effect. We further analyzed the bioenergetics of purified mitochondria from the abdominal s.c. WAT of cold-treated subjects and determined that repeat ice application significantly increased uncoupled respiration, consistent with the UCP1 protein induction and subsequent activation. Cold also increased State 3 and maximal respiration, and this effect on mitochondrial bioenergetics was stronger in summer than winter. Chronic treatment (10 weeks; 50 mg/day) with the β3 receptor agonist mirabegron induces UCP1, TMEM26, CIDEA, and phosphorylation of HSL on serine660 in obese subjects. CONCLUSION. Cold or β3 agonists cause the induction of beige adipose tissue in human s.c. WAT; this phenomenon may be exploited to increase beige adipose in older, insulin-resistant, obese individuals. TRIAL REGISTRATION. Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02596776","term_id":"NCT02596776"}}NCT02596776, {"type":"clinical-trial","attrs":{"text":"NCT02919176","term_id":"NCT02919176"}}NCT02919176. FUNDING. NIH ({"type":"entrez-nucleotide","attrs":{"text":"DK107646","term_id":"187677713","term_text":"DK107646"}}DK107646, {"type":"entrez-nucleotide","attrs":{"text":"DK112282","term_id":"187405426","term_text":"DK112282"}}DK112282, P20GM103527, and by CTSA grant UL1TR001998).

Summary Mechanisms that regulate metabolites and downstream energy generation are key determinants of T cell cytokine production, but the processes underlying the Th17 profile that predicts the metabolic status of people with obesity are untested. Th17 function requires fatty acid uptake, and our new data show that blockade of CPT1A inhibits Th17-associated cytokine production by cells from people with type 2 diabetes (T2D). A low CACT:CPT1A ratio in immune cells from T2D subjects indicates altered mitochondrial function and coincides with the preference of these cells to generate ATP through glycolysis rather than fatty acid oxidation. However, glycolysis was not critical for Th17 cytokines. Instead, β oxidation blockade or CACT knockdown in T cells from lean subjects to mimic characteristics of T2D causes cells to utilize 16C-fatty acylcarnitine to support Th17 cytokines. These data show long-chain acylcarnitine combines with compromised β oxidation to promote disease-predictive inflammation in human T2D.

The study aim was to determine how peripheral trigeminal nerve injury affects mitochondrial respiration and to test efficacy of combined treatment with 2 Federal Drug Administration approved drugs with potential for improving mitochondrial bioenergetics, pain and anxiety-related behaviors in a chronic orofacial neuropathic pain mouse model.Efficacy of (R)-(+)-4-amino-3-isoxazolidinone (D-cycloserine, DCS), an N-Methyl-D-aspartate antagonist/agonist, and Pioglitazone (PIO), a selective agonist of nuclear receptor peroxisome proliferator-activated receptor gamma was investigate in the trigeminal inflammatory compression (TIC) neuropathic nerve injury mouse model. Combined low doses of these drugs (80 mg/kg DCS and 100 mg/kg PIO) were given as a single bolus or daily for 7 days post-TIC to test ability to attenuate neuropathic nociceptive and associated cognitive dependent anxiety behaviors. In addition, beneficial effects of the DCS/PIO drug combination were explored ex vivo in isolated cortex/brainstem mitochondria at 28 weeks post-TIC.The DCS/PIO combination not only attenuated orofacial neuropathic pain and anxiety-related behaviors associated with trigeminal nerve injury, but it also improved mitochondrial bioenergetics.The DCS/PIO combination uncoupled mitochondrial respiration in the TIC model to improve cortical mitochondrial dysfunction, as well as reduced nociceptive and anxiety behaviors present in mice with centralized chronic neuropathic nerve injury. Combining these drugs could be a beneficial treatment for patients with depression, anxiety, or other psychological conditions due to their chronic pain status.

Purpose Hepatocellular carcinomas (HCCs) bridging two or more Couinaud–Bismuth segments of the liver ("watershed tumors") can recruit multiple segmental arteries. The primary hypothesis of this study was that fewer watershed tumors show complete response (CR) after chemoembolization, with shorter time to local recurrence. Secondary analysis on the impact on transplantation eligibility in the presence of progressive disease was also performed. Materials and Methods A total of 155 transplantation-eligible patients whose HCC met Milan criteria (watershed, n=83; nonwatershed, n=72) and was treated with chemoembolization were included. Cone-beam computed tomography (CT) was used for guidance and for confirmation of circumferential uptake. Local response to chemoembolization per modified Response Evaluation Criteria In Solid Tumors and local disease-free survival (DFS) for the index tumor were calculated. Differences were assessed by univariate and multivariate analyses. Results CR after a single of chemoembolization was observed in 55.4% of watershed tumors and in 72.2% of nonwatershed tumors ( P = .045). Estimated DFS intervals were 151 days (95% confidence interval [CI], 93–245 d) and 336 days (95% CI, 231–747 d; P = .040) in the watershed and nonwatershed groups, respectively. Worse DFS was observed with a Model for End-Stage Liver Disease score > 20 ( P = .0001), higher Child–Pugh–Turcotte score ( P = .049), and watershed location ( P = .040). Waiting list drop-off rates were statistically similar between groups. Conclusions Hepatocellular carcinomas located in the watershed region of the liver have a poorer response to chemoembolization than those located elsewhere. These tumors are associated with worse DFS and require additional treatments to maintain transplantation eligibility per Milan criteria. Cone-beam CT can identify crossover supply and confirm complete geographic drug uptake, possibly reducing (but not eliminating) the risk of incomplete response.

Mitochondrial dysfunction is thought to play a significant role in neurodegeneration observed in Parkinson's disease (PD), yet the mechanisms underlying this pathology remain unclear. Here, we demonstrate that loss of mitoNEET (CISD1), an iron-sulfur containing protein that regulates mitochondrial bioenergetics, results in mitochondrial dysfunction and loss of striatal dopamine and tyrosine hydroxylase. Mitochondria isolated from mice lacking mitoNEET were dysfunctional as revealed by elevated reactive oxygen species (ROS) and reduced capacity to produce ATP. Gait analysis revealed a shortened stride length and decreased rotarod performance in knockout mice, consistent with the loss of striatal dopamine. Together, these data suggest that mitoNEET KO mice exhibit many of the characteristics of early neurodegeneration in PD and may provide a novel drug discovery platform to evaluate compounds for enhancing mitochondrial function in neurodegenerative disorders.

Author(s): Kim, Do Young; Zhang, Fang-Xiong; Nakanishi, Stan T; Mettler, Timothy; Cho, Ik-Hyun; Ahn, Younghee; Hiess, Florian; Chen, Lina; Sullivan, Patrick G; Chen, SR Wayne; Zamponi, Gerald W; Rho, Jong M | Abstract: ObjectivesCarisbamate (CRS) is a novel monocarbamate compound that possesses antiseizure and neuroprotective properties. However, the mechanisms underlying these actions remain unclear. Here, we tested both direct and indirect effects of CRS on several cellular systems that regulate intracellular calcium concentration [Ca2+ ]i .MethodsWe used a combination of cellular electrophysiologic techniques, as well as cell viability, Store Overload-Induced Calcium Release (SOICR), and mitochondrial functional assays to determine whether CRS might affect [Ca2+ ]i levels through actions on the endoplasmic reticulum (ER), mitochondria, and/or T-type voltage-gated Ca2+ channels.ResultsIn CA3 pyramidal neurons, kainic acid induced significant elevations in [Ca2+ ]i and long-lasting neuronal hyperexcitability, both of which were reversed in a dose-dependent manner by CRS. Similarly, CRS suppressed spontaneous rhythmic epileptiform activity in hippocampal slices exposed to zero-Mg2+ or 4-aminopyridine. Treatment with CRS also protected murine hippocampal HT-22 cells against excitotoxic injury with glutamate, and this was accompanied by a reduction in [Ca2+ ]i . Neither kainic acid nor CRS alone altered the mitochondrial membrane potential (ΔΨ) in intact, acutely isolated mitochondria. In addition, CRS did not affect mitochondrial respiratory chain activity, Ca2+ -induced mitochondrial permeability transition, and Ca2+ release from the ER. However, CRS significantly decreased Ca2+ flux in human embryonic kidney tsA-201 cells transfected with Cav 3.1 (voltage-dependent T-type Ca2+ ) channels.SignificanceOur data indicate that the neuroprotective and antiseizure activity of CRS likely results in part from decreased [Ca2+ ]i accumulation through blockade of T-type Ca2+ channels.

Mitochondrial dysfunction is one of the key posttraumatic neuropathological events observed in various experimental models of traumatic brain injury (TBI). The extent of mitochondrial dysfunction has been associated with the severity and time course of secondary injury following brain trauma. Critically, several mitochondrial targeting preclinical drugs used in experimental TBI models have shown improved mitochondrial bioenergetics, together with cortical tissue sparing and cognitive behavioral outcome. Mitochondria, being a central regulator of cellular metabolic pathways and energy producer of cells, are of a great interest for researchers aiming to adopt cutting-edge methodology for mitochondrial bioenergetics assessment. The traditional way of mitochondrial bioenergetics analysis utilizing a Clark-type oxygen electrode (aka. oxytherm) is time-consuming and labor-intensive. In the present chapter, we describe an advanced and high-throughput method for mitochondrial bioenergetics assessments utilizing the Seahorse Biosciences XF(e)24 Flux Analyzer. This allows for simultaneous measurement of multiple samples with higher efficiency than the oxytherm procedure. This chapter provides helpful guidelines for conducting mitochondrial isolation and studying mitochondrial bioenergetics in brain tissue homogenates following experimental TBI.

Mitochondria play a pivotal role in the development of pathology associated with Parkinson's disease (PD), traumatic brain injury (TBI), and following exposure to the environmental toxin trichloroethylene (TCE). Evidence from humans indicates that both TBI and TCE can play a role in the development of PD and that each of these insults result in significant mitochondrial dysfunction. In the current studies we hypothesized that exposure to both TCE and TBI would result in increased pathology associated with PD. To test this hypothesis, 16 week old male Fischer 344 rats were administered TCE for either one or two weeks by oral gavage. Following exposure to TCE, rats were subjected to either a sham, mild (1.0mm), or moderate (2.0mm) controlled cortical impact TBI. Given the strong connection between mitochondrial function and PD, TBI, and TCE, tissue from the striatum and substantia nigra were analyzed 6h after the TBI. Neither TCE exposure, TBI, nor the combination of the two insults resulted in mitochondrial deficits at 6h post-TBI in the substantia nigra. Unlike the substantia nigra, the striatum exhibited significant mitochondrial dysfunction. Exposure to TCE alone for two weeks resulted in approximately a 75% reduction in mitochondrial function (p

The analysis of mitochondrial bioenergetic function typically has required 50–100 μg of protein per sample and at least 15 min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 μg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p < 0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10 pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12 min after administration (p < 0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions.

Mitochondrial dysfunction plays a pivotal role in secondary cell death mechanisms following traumatic brain injury (TBI). Several reports have demonstrated that inhibition of the mitochondrial permeability transition pore with the immunosuppressant drug cyclosporine A (CsA) is efficacious. Accordingly, CsA is being moved forward into late-stage clinical trials for the treatment of moderate and severe TBI. However, several unknowns exist concerning the optimal therapeutic window for administering CsA at the proposed dosages to be used in human studies. The present study utilized a moderate (1.75 mm) unilateral controlled cortical impact model of TBI to determine the most efficacious therapeutic window for initiating CsA therapy. Rats were administered an IP dose of CsA (20 mg/kg) or vehicle at 1, 3, 4, 5, 6, and 8 h post-injury. Immediately following the initial IP dose, osmotic mini-pumps were implanted at these time points to deliver 10 mg/kg/d of CsA or vehicle. Seventy-two hours following the initiation of treatment the pumps were removed to stop CsA administration. Quantitative analysis of cortical tissue sparing 7 days post-injury revealed that CsA treatment initiated at any of the post-injury initiation times out to 8 h resulted in significantly less cortical damage compared to animals receiving vehicle treatment. However, earlier treatment begun in the first 3 h was significantly more protective than that begun at 4 and 8 h. Treatment initiated at 1 h post-injury (∼68% decrease) was not significantly different than that seen at 3 h (∼46% decrease), but resulted in significantly greater cortical tissue sparing compared to CsA treatment initiated at least 4 h post-injury (28% decrease). Together these results illustrate the importance of initiating therapeutic interventions such as CsA as soon as possible following TBI, preferably within 4 h post-injury, to achieve the best possible neuroprotective effect. However, the drug appears to retain some protective efficacy even when initiated as late as 8 h post-injury.

Trichloroethylene, a chlorinated solvent widely used as a degreasing agent, is a common environmental contaminant. Emerging evidence suggests that chronic exposure to trichloroethylene may contribute to the development of Parkinson's disease. The purpose of this study was to determine if selective loss of nigrostriatal dopaminergic neurons could be reproduced by systemic exposure of adult Fisher 344 rats to trichloroethylene. In our experiments, oral administration of trichloroethylene induced a significant loss of dopaminergic neurons in the substantia nigra pars compacta in a dose-dependent manner, whereas the number of both cholinergic and GABAergic neurons were not decreased in the striatum. There was a robust decline in striatal levels of 3, 4-dihydroxyphenylacetic acid without a significant depletion of striatal dopamine. Rats treated with trichloroethylene showed defects in rotarod behavior test. We also found a significantly reduced mitochondrial complex I activity with elevated oxidative stress markers and activated microglia in the nigral area. In addition, we observed intracellular alpha-synuclein accumulation in the dorsal motor nucleus of the vagus nerve, with some in nigral neurons, but little in neurons of cerebral cortex. Overall, our animal model exhibits some important features of Parkinsonism, and further supports that trichloroethylene may be an environmental risk factors for Parkinson's disease.

Experimental traumatic brain injury (TBI) leads to a rapid and extensive necrosis at the primary site of injury that appears to be driven in part by significant mitochondrial dysfunction. The present study is based on the hypothesis that TBI-induced, aberrant glutamate release increases mitochondrial Ca(2+) cycling/overload ultimately leading to mitochondrial damage. Previous work from our laboratory demonstrates that mitochondrial uncoupling during the acute phases of TBI-induced excitotoxicity can reduce mitochondrial Ca(2+) uptake (cycling), ROS production and mitochondrial damage resulting in neuroprotection and improved behavioral outcome. The current study was designed to determine the optimal dosage and therapeutic window of opportunity for the potent mitochondrial uncoupler FCCP following moderate TBI. For this study, we used young adult male Sprague-Dawley rats (300-350 g); either sham-operated or moderately (1.5 mm) injured using the controlled cortical impactor (CCI) model of TBI. In the first set of studies animals were injected with either vehicle (100% DMSO) or different concentrations of FCCP (0.5, 1, 2.5 and 5 mg/kg in 100% DMSO) intraperitoneally at 5 min post-injury; tested behaviorally at 10 days and cortical sparing assessed at 18 days post-injury. The results demonstrate that of all the dosages tested, 2.5 mg/kg rendered the maximum improvement in behavioral outcomes and tissue spared. Using this optimal dose (2.5 mg/kg) and time point for intervention (5 min post-injury), we assessed mitochondrial bioenergetics and mitochondrial structural integrity 24 h post-injury. Furthermore, using this dosage we assessed mitochondrial bioenergetics and Ca(2+) loading at 3 and 6 h post-injury to further verify our target mechanism and establish these assessments as a valid endpoint to use as a means to determine the therapeutic window of FCCP. To begin to address the window of opportunity for maintaining mitochondrial homeostasis, the optimal dose of FCCP was then administered at 5 min, 3, 6, or 24 h post-injury and several parameters of mitochondrial function were used as outcome measures. The results demonstrate that a prolonged window of opportunity exists for targeting mitochondrial dysfunction using uncouplers following TBI and give insight into the cellular pathology associated with TBI.

Traumatic brain injury (TBI) occurs suddenly and has damaging effects to the brain that are dependent on the severity of insult. Symptoms can be mild, moderate, or severe. Oxidative damage is associated with traumatic brain injury through reactive oxygen/nitrogen species production. One such species, peroxynitrite, is elevated in TBI brain tissue (Orihara et al. [ 2001] Forensic Sci. Int. 123:142–149; Deng et al. [ 2007] Exp. Neurol. 205:154–165). Peroxynitrite can react with carbon dioxide and decompose to produce NO2 and carbonate radicals, which in turn can lead to 3-nitrotyrosine, an index of protein nitration. Gamma-glutamylcysteine ethyl ester (GCEE) is an ethyl ester moiety of gamma-glutamylcysteine, an agent that up-regulates glutathione (GSH) production in brain (Drake et al. [ 2002] J. Neurosci. Res. 68:776–784). Many preclinical studies of TBI have employed pretreatment of animals with proposed beneficial agents prior to the injury itself. However, in the real world of TBI, treatment begins postinjury. Hence, insights into agents that improve outcome following injury are desperately needed. This study is one of the first to investigate a potential GSH-based therapy for TBI postinjury. Protein carbonyls, an index of protein oxidation, were significantly elevated in brain of animals subjected to TBI. However, if, after TBI, GCEE was administered i.p., protein carbonyl levels were significantly reduced. Similarly, 3-nitrotyrosine levels were elevated in brain following TBI but significantly decreased following TBI if GCEE was administered i.p. Redox proteomics analysis showed that several brain proteins were nitrated after TBI. However, if GCEE was given i.p. following TBI, many of these proteins were protected from nitration. The results are encouraging and are discussed with reference to potential therapeutic strategies for TBI involving elevated GSH. © 2008 Wiley-Liss, Inc.

Region specific declines in the cerebral glucose metabolism are an early and progressive feature of Alzheimer's disease (AD). Such declines occur pre-symptomatically and offer a potential point of intervention in developing AD therapeutics. Medium chain triglycerides (MCTs), which are rapidly converted to ketone bodies, were tested for their ability to provide an alternate energy source to neurons suffering from compromised glucose metabolism. The present study determined the short-term effects of ketosis in aged dogs, a natural model of amyloidosis. The animals were administered a 2 g/kg/day dose of MCTs for 2 months. Mitochondrial function and oxidative damage assays were then conducted on the frontal and parietal lobes. Amyloid-beta (Abeta), amyloid precursor protein (APP) processing and beta-site APP cleaving enzyme (BACE1) assays were conducted on the frontal, parietal and occipital lobes. Aged dogs receiving MCTs, as compared to age-matched controls, showed dramatically improved mitochondrial function, as evidenced by increased active respiration rates. This effect was most prominent in the parietal lobe. The improved mitochondrial function may have been due to a decrease in oxidative damage, which was limited to the mitochondrial fraction. Steady-state APP levels were also decreased in the parietal lobe after short-term MCT administration. Finally, there was a trend towards a decrease in total Abeta levels in the parietal lobe. BACE1 levels remained unchanged. Combined, these findings suggest that short-term MCT administration improves energy metabolism and decreases APP levels in the aged dog brain.

This report documents an analysis of post-traumatic neurodegeneration during the first 7 days after controlled cortical impact (CCI) traumatic brain injury (TBI) in mice and rats using the de Olmos aminocupric silver staining method, which selectively stains degenerating axons and nerve terminals, compared to the fluorojade method, which stains degenerating neuronal cell bodies. A progressive increase in cortical, hippocampal, and thalamic degeneration was observed over the first 48 h after injury in both species. Approximately 50% of the ipsilateral cortical volume was stained at 48 h. Similarly, the dorsal hippocampus showed widespread degeneration in all of the subfields. This included CA1, CA3, CA4, and dentate cell bodies revealed by fluorojade together with a high degree of axonal degeneration in areas carrying afferent and efferent hippocampal projections that is identified by silver staining. These results show that previous CCI studies which have relied on conventional histological methods that show cell body staining alone have underestimated the degree of axonal damage associated with the CCI-TBI model. In order to capture the full extent of the injury to both axons and cell bodies, the combination of silver staining and fluorojade staining is needed, respectively. Future studies of potential neuroprotective agents should probably not rely on the measure of cortical lesion volume or volume of spared cortical tissue using conventional histological stains alone, since these fail to identify the complete extent of the posttraumatic neuropathology that some agents which reduce cortical lesion volume may not be able to effect.

Objective To analyze a cluster of 30 industrial coworkers with Parkinson's disease and parkinsonism subjected to long-term (8–33 years) chronic exposure to trichloroethylene. Methods Neurological evaluations were conducted on the 30 coworkers, including a general physical and neurological examination and the Unified Parkinson's Disease Rating Scale. In addition, fine motor speed was quantified and an occupational history survey was administered. Next, animal studies were conducted to determine whether trichloroethylene exposure is neurotoxic to the nigrostriatal dopamine system that degenerates in Parkinson's disease. The experiments specifically analyzed complex 1 mitochondrial neurotoxicity because this is a mechanism of action of other known environmental dopaminergic neurotoxins. Results The three workers with workstations adjacent to the trichloroethylene source and subjected to chronic inhalation and dermal exposure from handling trichloroethylene-soaked metal parts had Parkinson's disease. Coworkers more distant from the trichloroethylene source, receiving chronic respiratory exposure, displayed many features of parkinsonism, including significant motor slowing. Neurotoxic actions of trichloroethylene were demonstrated in accompanying animal studies showing that oral administration of trichloroethylene for 6 weeks instigated selective complex 1 mitochondrial impairment in the midbrain with concomitant striatonigral fiber degeneration and loss of dopamine neurons. Interpretation Trichloroethylene, used extensively in industry and the military and a common environmental contaminant, joins other mitochondrial neurotoxins, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and some pesticides, as a risk factor for parkinsonism. Ann Neurol 2007

To determine the neuroprotective effect of fasting after traumatic brain injury (TBI) and to elucidate the potential underlying mechanisms, we used a controlled cortical impact (CCI) injury model to induce either a moderate or a severe injury to adult male Sprague Dawley rats. Tissue-sparing assessments were used to determine the level of neuroprotection of fasting, hypoglycemia (insulin 10 U), or ketone (1.66 mmoles/kg/day or 0.83 mmoles/kg/day; D-beta-hydroxtbutyrate) administration. Mitochondrial isolation and respiratory studies were utilized to determine the functionality of mitochondria at the site of injury. We also investigated biomarkers of oxidative stress, such as lipid/protein oxidation, reactive oxygen species (ROS) production, and intramitochondrial calcium load, as a secondary measure of mitochondrial homeostasis. We found that fasting animals for 24 hr, but not 48 hr, after a moderate (1.5 mm), but not severe (2.0 mm), CCI resulted in a significant increase in tissue sparing. This 24-hr fast also decreased biomarkers of oxidative stress and calcium loading and increased mitochondrial oxidative phosphorylation in mitochondria isolated from the site of injury. Insulin administration, designed to mimic the hypoglycemic effect seen during fasting did not result in significant tissue sparing after moderate CCI injury and in fact induced increased mortality at some injection time points. However, the administration of ketones resulted in increased tissue sparing after moderate injury. Fasting for 24 hr confers neuroprotection, maintains cognitive function, and improves mitochondrial function after moderate (1.5 mm) TBI. The underlying mechanism appears to involve ketosis rather than hypoglycemia.

Following traumatic brain injury (TBI), mitochondrial function becomes compromised. Mitochondrial dysfunction is characterized by intra-mitochondrial Ca(2+) accumulation, induction of oxidative damage, and mitochondrial permeability transition (mPT). Experimental studies show that cyclosporin A (CsA) inhibits mPT. However, CsA also inhibits calcineurin. In the present study, we conducted a dose-response analysis of NIM811, a non-calcineurin inhibitory CsA analog, on mitochondrial dysfunction following TBI in mice, and compared the effects of the optimal dose of NIM811 (10 mg/kg i.p.) against an optimized dose of CsA (20 mg/kg i.p.). Male CF-1 mice were subjected to severe TBI utilizing the controlled cortical impact model. Mitochondrial respiration was assessed from animals treated with either NIM811, CsA, or vehicle 15 min post-injury. The respiratory control ratio (RCR) of mitochondria from vehicle-treated animals was significantly (p

Mitochondria isolated from synaptosomes are more sensitive to Ca2+overload and the resultant opening of the mitochondrial permeability transition pore (mPTP) than nonsynaptic mitochondria. To identify the mechanisms underlying these differences in Ca2+dynamics, we examined relative levels of mPTP components in synaptic versus nonsynaptic mitochondria. Synaptic mitochondria had higher levels of cyclophilin D when compared with nonsynaptic mitochondria, whereas levels of the voltage-dependent anion channel and the adenine nucleotide translocase were similar in the two mitochondrial fractions. These differences in Ca2+handling between synaptic and nonsynaptic mitochondria were greatly reduced in cyclophilin D null [Ppif−/−(peptidylprolyl isomerase F)] mice. Higher concentrations of cyclosporine A, which interacts with cyclophilin D to delay mPTP opening, were necessary to increase the Ca2+uptake capacity of synaptic versus nonsynaptic mitochondria. To determine whether the differences in Ca2+handling might reflect the relative abundance of neuronal and glial mitochondria in the two mitochondrial fractions, we compared cyclophilin D levels in primary cortical neurons and astrocytes. Primary rat cortical neurons possess higher cyclophilin D levels than do primary astrocytes. In the adult rat brain, cyclophilin D immunoreactivity was abundant in neurons but sparse in astrocytes. Together, these results demonstrate that the Ca2+handling differences observed in synaptic versus nonsynaptic mitochondria are primarily the result of the high levels of cyclophilin D in synaptic mitochondria, reflecting the greater proportion of neuronal mitochondria in this fraction. The high levels of cyclophilin D in neuronal mitochondria result in their greater vulnerability to mPT and in higher levels of cyclosporine A being required to inhibit mPTP opening.

Peroxynitrite-mediated oxidative damage has been implicated in brain mitochondrial respiratory dysfunction after traumatic brain injury (TBI), which precedes the onset of neuronal loss. The aim of this study was to investigate the detrimental effects of the peroxynitrite donor SIN-1 (3-morpholinosydnonimine) on isolated brain mitochondria and to screen penicillamine, a stoichiometric (1:1) peroxynitrite-scavenging agent, and tempol, a catalytic scavenger of peroxynitrite-derived radicals, as antioxidant mitochondrial protectants. Exposure of the isolated mitochondria to SIN-1 caused a significant dose-dependent decrease in the respiratory control ratio and was accompanied by a significant increase in state II respiration, followed by significant decreases (P < 0.05) in states III and V. These functional alterations occurred together with significant increases in mitochondrial protein carbonyl (PC), lipid peroxidation-related 4-hydroxynonenal (4-HNE), and 3-nitrotyrosine (3-NT) content. Penicillamine hydrochloride (10 microM) partially but significantly (P < 0.05) protected against SIN-1-induced decreases in states III and V. However, a 2.5 microM concentration of tempol was able to significantly antagonize a 4-fold molar excess (10 microM) concentration of SIN-1 as effectively as were higher tempol concentrations, consistent with the likelihood that tempol works by a catalytic mechanism. The protection of mitochondrial respiration by penicillamine and tempol occurred in parallel with attenuation of PC, 4-HNE, and 3-NT. These results indicate that SIN-1 causes mitochondrial oxidative damage and complex I dysfunction and that antioxidant compounds that target either peroxynitrite or its radicals may be effective mitochondrial protectants in the treatment of neural injury.

Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI, however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI, however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12 hour following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI.

International genetic bull evaluations of somatic cell counts (SCC) from 8 different Holstein populations and clinical mastitis from 3 of these populations were inferred simultaneously using a multiple-trait–multiple-country evaluation (MT-MACE) model. This model considered effective independent weighting factors and multivariately deregressed national genetic evaluations for countries with multiple-trait national models. Predictions of genetic merit from MT-MACE and their reliabilities were compared with the corresponding results from 2 separate single-trait–multiple-country evaluations (ST-MACE) for different groups of bulls. The assumed heritabilities for clinical mastitis (h 2 = 0.02 to 0.05) were substantially lower than the heritabilities for SCC (h 2 = 0.08 to 0.27). The predictive ability of MT-MACE was essentially equal to or better than the predictive ability of ST-MACE for all country-trait combinations, but both methods yielded effectively unbiased and consistent consecutive predictions (correlation>0.93). Both sets of predictions also agreed well with future national genetic evaluations for bulls receiving additional daughter information (correlation>0.96), except for evaluations for which within-country correlations were utilized internationally, but not nationally (correlation = 0.86 to 0.97). The reliabilities for MT-MACE were essentially equal to or higher than reliabilities for ST-MACE, depending on the trait and group of bulls in question. Reliabilities increased most for young bulls, and for clinical mastitis in countries that did not use the within-country correlations with SCC in the national evaluation (up to a 23% increase in average reliability).

An age-related Ca(2+) dysregulation and increased production of reactive oxygen species (ROS) may contribute to late-onset neurodegenerative disorders. These alterations are often attributed to impaired mitochondrial function yet few studies have directly examined mitochondria isolated from various regions of the aged brain. The purpose of this study was to examine Ca(2+)-buffering and ROS production in mitochondria isolated from Fischer 344 rats ranging in age from 4 to 25 months. Mitchondria isolated from the cortex of the 25 month rat brain exhibited greater rates of ROS production and mitochondrial swelling in response to increasing Ca(2+) loads as compared to mitochondria isolated from younger (4, 13 month) animals. The increased swelling is indicative of opening of the mitochondrial permeability transition pore indicating impaired Ca(2+) buffering/cycling in aged animals. These age-related differences were not observed in mitochondria isolated from cerebellum. Together, these results demonstrate region specific, age-related, alterations in mitochondrial responses to Ca(2+).

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage resulting in significant neurological dysfunction. The identification of cell death pathways that mediate this secondary traumatic injury have not been elucidated, however recent studies have implicated a role for apoptosis in the neuropathology of traumatic brain injury. The present study utilized a controlled cortical impact model of brain injury to assess the involvement of apoptotic pathways: release of cytochrome c from mitochondria and the activation of caspase-1- and caspase-3-like proteases in the injured cortex at 6, 12 and 24 h post-injury. Collectively, these results demonstrate cytochrome c release from mitochondria and its redistribution into the cytosol occurs in a time-dependent manner following TBI. The release of cytochrome c is accompanied by a time-dependent increase in caspase-3-like protease activity with no apparent increase in caspase-1-like activity. However, pretreatment with a general caspase inhibitor had no significant effect on the amount of cortical damage observed at 7 days post-injury. Our data suggest that several pro-apoptotic events occur following TBI, however the translocation of cytochrome c itself and/or other events upstream of caspase activation/inhibition may be sufficient to induce neuronal cell death.

Glucocorticoid activation of two types of adrenocorticosteroid receptors (ACRs), the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), influences the vulnerability of hippocampal neurons to insult. To examine the potential impact of ACR activation following traumatic brain injury (TBI), the current study assesses regulation of MR and GR expression and glucocorticoid levels following controlled cortical impact (CCI). Male Sprague-Dawley rats were pretreated for 48 h with vehicle, the MR antagonist spironolactone, or the GR antagonist mifepristone (RU486). On day three, subjects were sham-operated or injured by unilateral CCI. In situ hybridization analysis determined that pretreatment with either MR antagonist spironolactone or GR antagonist RU486 increased 24-h hippocampal GR mRNA levels in sham-operated animals only, suggesting that MR and GR regulation of GR mRNA is suppressed following TBI. Injury decreased GR mRNA levels in the ipsilateral dentate gyrus of all pretreatment groups and bilaterally increased MR mRNA levels in CA3 of antagonist-pretreated animals. One day post-injury, plasma corticosterone levels were comparable in sham and injured animals pretreated with vehicle. A separate group of animals that did not receive pretreatment injections prior to sham operation or injury were included for a 24-h time course analysis of plasma ACTH and corticosterone levels. Injury increased hypothalamic-pituitary-adrenal (HPA) activation for approximately 6 h following injury. These results indicate that hippocampal ACR mRNA levels and HPA activation are altered by TBI. Regulation of MR and GR expression following TBI may influence hippocampal neuron viability by modulating glucocorticoid signaling after injury.

The present study addresses mineralocorticoid receptor and glucocorticoid receptor effects on hippocampal neuron viability after experimental traumatic brain injury. Rats were pretreated for 48 h with vehicle, the mineralocorticoid receptor antagonist spironolactone, or the glucocorticoid receptor antagonist mifepristone (RU486) and subsequently subjected to sham operation or unilateral controlled cortical impact injury. To determine the effects of receptor antagonist pretreatments on cell survival, neurons in regions CA1, CA3, and dentate gyrus of the hippocampal formation were counted 24 h post-injury using the optical fractionator method. Injury decreased the number of viable neurons in CA1 and CA3 of vehicle-pretreated animals. Notably, this cell loss was prevented in CA1 by RU486 pretreatment. Neuronal loss was also observed in dentate gyrus. The effects of receptor blockade and injury on the expression of viability-related genes were also assessed by comparing relative bcl-2, bax, and p53 messenger RNA levels using in situ hybridization analysis. Spironolactone and RU486 decreased basal bcl-2 messenger RNA levels in CA1 and dentate gyrus but did not affect basal bax or p53 levels. Injury decreased bcl-2 messenger RNA levels in dentate gyrus but did not affect bax or p53 levels in vehicle-pretreated animals. These data demonstrate that RU486 pretreatment prevents the loss of CA1 pyramidal neurons 24 h after traumatic brain injury. RU486 modulation of bcl-2, bax, or p53 messenger RNA expression does not predict neuronal viability at this time point, suggesting that RU486-mediated preservation of CA1 neurons does not involve transcriptional regulation of these cell death-related genes.

Traumatic brain injury (TBI) has become a growing epidemic but no approved pharmacological treatment has been identified. Our previous work indicates that mitochondrial oxidative stress/damage and loss of bioenergetics play a pivotal role in neuronal cell death and behavioral outcome following experimental TBI. One tactic that has had some experimental success is to target glutathione using its precursor N-acetylcysteine (NAC). However, this approach has been hindered by the low CNS bioavailability of NAC. The current study evaluated a novel, cell permeant amide form of N-acetylcysteine (NACA), which has high permeability through cellular and mitochondrial membranes resulting in increased CNS bioavailability. Cortical tissue sparing, cognitive function and oxidative stress markers were assessed in rats treated with NACA, NAC, or vehicle following a TBI. At 15 days post-injury, animals treated with NACA demonstrated significant improvements in cognitive function and cortical tissue sparing compared to NAC or vehicle treated animals. NACA treatment also was shown to reduce oxidative damage (HNE levels) at 7 days post-injury. Mechanistically, post-injury NACA administration was demonstrated to maintain levels of mitochondrial glutathione and mitochondrial bioenergetics comparable to sham animals. Collectively these data provide a basic platform to consider NACA as a novel therapeutic agent for treatment of TBI.

Acute neuropathology following experimental traumatic brain injury results in the rapid necrosis of cortical tissue at the site of injury. This primary injury is exacerbated in the ensuing hours and days via the progression of secondary injury mechanism(s) leading to significant neurological dysfunction. Recent evidence from our laboratory demonstrates that the immunosuppressant cyclosporin A significantly ameliorates cortical damage following traumatic brain injury. The present study extends the previous findings utilizing a unilateral controlled cortical impact model of traumatic brain injury in order to establish a dose-response curve and optimal dosing regimen of cyclosporin A. Following injury to adult rats, cyclosporin A was administrated at various dosages and the therapy was initiated at different times post-injury. In addition to examining the effect of cyclosporin A on the acute disruption of the blood-brain barrier following controlled cortical impact, we also assessed the efficacy of cyclosporin A to reduce tissue damage utilizing the fluid percussion model of traumatic brain injury. The findings demonstrate that the neuroprotection afforded by cyclosporin A is dose-dependent and that a therapeutic window exists up to 24h post-injury. Furthermore, the optimal cyclosporin dosage and regimen markedly reduces disruption of the blood-brain barrier acutely following a cortical contusion injury, and similarly affords significant neuroprotection following fluid percussion injury. These findings clearly suggest that the mechanisms responsible for tissue necrosis following traumatic brain injury are amenable to pharmacological intervention.

Changes in the expression of central nervous system (CNS) neurotransmitter receptors may contribute to behavioral and physiological deficits that occur following traumatic brain injury (TBI). Studies investigating the neurochemical basis for the protracted cognitive dysfunction that follows TBI have focused in part on cholinergic mechanisms. The present study compared the effects of mild and moderate cortical contusion injury (CCI) on the density of cholinergic receptor subtypes, NMDA-type glutamate receptors, and calcium channel expression. Quantitative autoradiography was used to determine the effects of CCI on receptor expression, 48 h following injury. The most robust and consistent change in receptor binding was in the density of alpha7 nicotinic receptors as determined by alpha-[125I]-bungarotoxin (BTX) binding. Bilateral deficits in BTX binding were present following both mild and moderate levels of injury. In contrast, changes in the density of alpha3/alpha4 nAChr's, muscarinic AChr's, NMDA-type glutamate receptors, and L-type calcium channel expression were more regionally restricted and lower in magnitude, as compared to changes in BTX binding. The high calcium permeability of the alpha7 nAChr may be related to the extensive decrease in BTX binding that occurs following TBI.

Traumatic brain injury (TBI) results in the rapid necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the original damage resulting in significant neurological dysfunction. Recent reports from our lab demonstrate that a bolus injection of the immunosuppressant cyclosporin A (CsA) is neuroprotective following TBI. CsA transiently inhibits the opening of the mitochondrial permeability transition pore and maintains calcium homeostasis in isolated mitochondria. The present study utilized a unilateral controlled cortical impact model of TBI to assess whether the neuroprotective effects of CsA could be extended by chronic infusion. Adult rats were subjected to a moderate (2 mm) cortical deformation and the extent of cortical damage was assessed using modern stereological techniques. Animals were administrated a 20 mg/kg intraperitoneal bolus of CsA or vehicle 15 min postinjury and osmotic minipumps were implanted subcutaneously to deliver CsA (4.5 or 10 mg/kg/day) or vehicle. All animals receiving CsA demonstrated a significant reduction in lesion volume, with the highest dose offering the most neuroprotection (74% reduction in lesion volume). These results extend our previous findings and demonstrate that chronic infusion of CsA is neuroprotective following TBI. These findings also suggest that the mechanisms responsible for tissue necrosis following TBI are amenable to manipulation.

Mitochondrial dysfunction is known to play a pivotal role in cell death mechanisms following traumatic brain injury (TBI). N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclosporin A (CsA) analog, inhibits the mitochondrial permeability transition pore (mPTP) and has been shown to be neuroprotective following TBI in mice. However, the translation of the neuroprotective effects of mPTP inhibitors, including CsA and NIM811, into improved cognitive end points has yet to be fully investigated. Therefore, to build upon these results, a severe unilateral controlled cortical impact model of TBI was used in the present study to establish a dose–response curve for NIM811 in rats. The findings demonstrate that the neuroprotection afforded by NIM811 is dose dependent, with the 10 mg/kg dose being the most effective dose. Once the dose response was established, we evaluated the effect of the optimal dose of NIM811 on behavior, mitochondrial bioenergetics, and mitochondrial oxidative damage following TBI. For behavioral studies, rats were administered NIM811 at 15 min and 24 h post-injury, with cognitive testing beginning 10 days post-injury. Mitochondrial studies involved a single injection of NIM811 at 15 min post-injury followed by mitochondrial isolation at 6 h post-injury. The results revealed that the optimal dose of NIM811 improves cognition, improves mitochondrial functioning, and reduces oxidative damage following TBI.

Traumatic brain injury (TBI) as a consequence of exposure to blast is increasingly prevalent in military populations, with the underlying pathophysiological mechanisms mostly unknown. In the present study, we utilized an air-driven shock tube to investigate the effects of blast exposure (120 kPa) on rat brains. Immediately following exposure to blast, neurological function was reduced. BBB permeability was measured using IgG antibody and evaluating its immunoreactivity in the brain. At 3 and 24 hr postexposure, there was a transient significant increase in IgG staining in the cortex. At 3 days postexposure, IgG immunoreactivity returned to control levels. Quantitative immunostaining was employed to determine the temporal course of brain oxidative stress following exposure to blast. Levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) were significantly increased at 3 hr postexposure and returned to control levels at 24 hr postexposure. The response of microglia to blast exposure was determined by autoradiographic localization of (3) H-PK11195 binding. At 5 days postexposure, increased binding was observed in the contralateral and ipsilateral dentate gyrus. These regions also displayed increased binding at 10 days postexposure; in addition to these regions there was increased binding in the contralateral ventral hippocampus and substantia nigra at this time point. By using antibodies against CD11b/c, microglia morphology characteristic of activated microglia was observed in the hippocampus and substantia nigra of animals exposed to blast. These results indicate that BBB breakdown, oxidative stress, and microglia activation likely play a role in the neuropathology associated with TBI as a result of blast exposure.

Following traumatic brain injury, mitochondria sustain structural and functional impairment, which contributes to secondary damage that can continue for days after the initial injury. The present study investigated mitochondrial bioenergetic changes in the rat neocortex at 1 and 3 h after mild, moderate, and severe injuries. Brains from young adult Sprague-Dawley rats were harvested from the injured and contralateral cortex to assess possible changes in mitochondrial respiration abilities following a unilateral cortical contusion injury. Differential centrifugation was used to isolate synaptic and extrasynaptic mitochondria from cortical tissue. Bioenergetics was assessed using a Clark-type electrode and results were graphed as a function of injury severity and time post-injury. Respiration was significantly affected by all injury severity levels compared to uninjured tissue. Complex 1- and complex 2-driven respirations were affected proportionally to the severity of the injury, indicating that damage to mitochondria may occur on a gradient. Total oxygen utilization, respiratory control ratio, ATP production, and maximal respiration capabilities were all significantly decreased in the injured cortex at both 1 and 3 h post-trauma. Although mitochondria displayed bioenergetic deficits at 1 h following injury, damage was not exacerbated by 3 h. This study stresses the importance of early therapeutic intervention and suggests a window of approximately 1–3 h before greater dysfunction occurs.

Dogs develop cognitive decline and a progressive accumulation of oxidative damage. In a previous longitudinal study, we demonstrated that aged dogs treated with either an antioxidant diet or with behavioral enrichment show cognitive improvement. The antioxidant diet included cellular antioxidants (vitamins E and C, fruits and vegetables) and mitochondrial cofactors (lipoic acid and carnitine). Behavioral enrichment consisted of physical exercise, social enrichment, and cognitive training. We hypothesized that the antioxidant treatment improved neuronal function through increased mitochondrial function. Thus, we measured reactive oxygen species (ROS) production and bioenergetics in mitochondria isolated from young, aged, and treated aged animals. Aged canine brain mitochondria show significant increases in ROS production and a reduction in NADH-linked respiration. Mitochondrial function (ROS and NADH-linked respiration) was improved selectively in aged dogs treated with an antioxidant diet. In contrast, behavioral enrichment had no effect on any mitochondrial parameters. These results suggest that an antioxidant diet improves cognition by maintaining mitochondrial homeostasis, which may be an independent molecular pathway not engaged by behavioral enrichment.

Background: Sporadic Parkinson’s disease (PD) is a progressive neurodegenerative disorder with unknown cause, but it has been suggested that neuroinflammation may play a role in pathogenesis of the disease. Neuroinflammatory component in process of PD neurodegeneration was proposed by postmortem, epidemiological and animal model studies. However, it remains unclear how neuroinflammatory factors contribute to dopaminergic neuronal death in PD. Findings: In this study, we analyzed the relationship among inducible nitric oxide synthase (iNOS)-derived NO, mitochondrial dysfunction and dopaminergic neurodegeneration to examine the possibility that microglial neuroinflammation may induce dopaminergic neuronal loss in the substantia nigra. Unilateral injection of lipopolysaccharide (LPS) into the striatum of rat was followed by immunocytochemical, histological, neurochemical and biochemical analyses. In addition, behavioral assessments including cylinder test and amphetamine-induced rotational behavior test were employed to validate ipsilateral damage to the dopamine nigrostriatal pathway. LPS injection caused progressive degeneration of the dopamine nigrostriatal system, which was accompanied by motor impairments including asymmetric usage of forelimbs and amphetamine-induced turning behavior in animals. Interestingly, some of the remaining nigral dopaminergic neurons had intracytoplasmic accumulation of a-synuclein and ubiquitin. Furthermore, defect in the mitochondrial respiratory chain, and extensive S-nitrosylation/nitration of mitochondrial complex I were detected prior to the dopaminergic neuronal loss. The mitochondrial injury was prevented by treatment with L-N 6 -(l-iminoethyl)-lysine, an iNOS inhibitor, suggesting that iNOS-derived NO is associated with the mitochondrial impairment. Conclusions: These results implicate neuroinflammation-induced S-nitrosylation/nitration of mitochondrial complex I in mitochondrial malfunction and subsequent degeneration of the nigral dopamine neurons.

Prediction of genetic merit for missing traits is possible by combining available indicator traits. Indicator traits were combined using genetic correlations obtained from multiple regression equations of estimated genetic correlations among available indicator traits on variables explaining production circumstances and trait definitions. This prediction of missing traits was closer to actual breeding values than breeding values for any of the indicator traits. This was verified by evaluating clinical mastitis in each of the Nordic countries as a missing trait. The derived methodology was used to predict breeding values for clinical mastitis in the United States for local and international bulls with an average reliability of 43%.