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 Ca2+ 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 O2 and nitrogen species, which modify many mitochondrial proteins, lipids, and DNA/RNA, thus impairing energy transduction and exacerbating free radical production. Oxidative stress and Ca2+-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.
biogenesis; mitophagy; Nrf2; oxidative stress; peroxisome proliferator-activated receptor-gamma coactivator 1a; protein kinase C
Diffusion Kurtosis Imaging (DKI) provides quantifiable information on the non-Gaussian behavior of water diffusion in biological tissue. Changes in water diffusion tensor imaging (DTI) parameters and DKI parameters in several white and grey matter regions were investigated in a mild controlled cortical impact (CCI) injury rat model at both the acute (2 hours) and the sub-acute (7 days) stages following injury. Mixed model ANOVA analysis revealed significant changes in temporal patterns of both DTI and DKI parameters in the cortex, hippocampus, external capsule and corpus callosum. Post-hoc tests indicated acute changes in mean diffusivity (MD) in the bilateral cortex and hippocampus (p < 0.0005) and fractional anisotropy (FA) in ipsilateral cortex (p < 0.0005), hippocampus (p = 0.014), corpus callosum (p = 0.031) and contralateral external capsule (p = 0.011). These changes returned to baseline by the sub-acute stage. However, mean kurtosis (MK) was significantly elevated at the sub-acute stages in all ipsilateral regions and scaled inversely with the distance from the impacted site (cortex and corpus callosum: p < 0.0005; external capsule: p = 0.003; hippocampus: p = 0.011). Further, at the sub-acute stage increased MK was also observed in the contralateral regions compared to baseline (cortex: p = 0.032; hippocampus: p = 0.039) while no change was observed with MD and FA. An increase in mean kurtosis was associated with increased reactive astrogliosis from immunohistochemistry analysis. Our results suggest that DKI is sensitive to microstructural changes associated with reactive astrogliosis which may be missed by standard DTI parameters alone. Monitoring changes in MK allows the investigation of molecular and morphological changes in vivo due to reactive astrogliosis and may complement information available from standard DTI parameters. To date the use of diffusion tensor imaging has been limited to study changes in white matter integrity following traumatic insults. Given the sensitivity of DKI to detect microstructural changes even in the gray matter in vivo, allows the extension of the technique to understand patho-morphological changes in the whole brain following a traumatic insult.
Magnetic Resonance Imaging; diffusion tensor imaging; diffusion kurtosis imaging; traumatic brain injury; astrogliosis; rat brain
The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 hr following 3 hr OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24 hr time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.
zinc; oxidative stress; mitochondria; ischemia; stroke; fluorescence biosensor; ratiometric; carbonic anhydrase II
Exposure of mitochondria to oxidative stress and elevated Ca2+ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 hr later. Respiring mitochondria actively accumulated added Ca2+, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or that directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca2+ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies.
This study tested the hypothesis that sensitivity to the Ca2+-induced loss of mitochondrial membrane potential (ΔΨm) and the sensitivity of the loss of ΔΨ to mitochondrial permeability transition pore (PTP) inhibitors are different for neurons and astrocytes. Primary cultures of rat cortical neurons and astrocytes were exposed to the Ca2+ ionophore 4-Br-A23187 and ΔΨm was monitored with the fluorescent probe tetramethylrhodamine methyl ester (TMRM). Ca2+ ionophore caused a decline in ΔΨm in both cell types that was partially inhibited by cyclosporin A (CsA) in astrocytes but not in neurons. Another PTP inhibitor, 2-aminoethoxy-diphenylborate, was ineffective at protecting against mitochondrial depolarization but depolarization was inhibited by FK506, an immunosuppressant drug similar to CsA that does not inhibit the PTP. CsA and FK506 both significantly reduced the ionophore-induced rise in [Ca2+]i in both neurons and astrocytes. We conclude that the protective effects of CsA and FK506 against Ca2+ ionophore-induced mitochondrial membrane depolarization in intact astrocytes is not due to PTP inhibition but is possibly a consequence of inhibiting the rise in [Ca2+]i.
Permeability transition pore; immunophilin; TMRM; calcium homeostasis; mitochondria; 4-Br-A23187
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 (1H 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.
controlled cortical impact; diffusion tensor imaging; early changes; MR spectroscopy; traumatic brain injury
Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in children and is characterized by reduced aerobic cerebral energy metabolism early after injury, possibly due to impaired activity of the pyruvate dehydrogenase complex. Exogenous acetyl-l-carnitine (ALCAR) is metabolized in the brain to acetyl coenzyme A and subsequently enters the tricarboxylic acid cycle. ALCAR administration is neuroprotective in animal models of cerebral ischemia and spinal cord injury, but has not been tested for TBI. This study tested the hypothesis that treatment with ALCAR during the first 24 h following TBI in immature rats improves neurologic outcome and reduces cortical lesion volume. Postnatal day 21–22 male rats were isoflurane anesthetized and used in a controlled cortical impact model of TBI to the left parietal cortex. At 1, 4, 12 and 23 h after injury, rats received ALCAR (100 mg/kg, intraperitoneally) or drug vehicle (normal saline). On days 3–7 after surgery, behavior was assessed using beam walking and novel object recognition tests. On day 7, rats were transcardially perfused and brains were harvested for histological assessment of cortical lesion volume, using stereology. Injured animals displayed a significant increase in foot slips compared to sham-operated rats (6 ± 1 SEM vs. 2 ± 0.2 on day 3 after trauma; n = 7; p < 0.05). The ALCAR-treated rats were not different from shams and had fewer foot slips compared to vehicle-treated animals (2 ± 0.4; n = 7; p< 0.05). The frequency of investigating a novel object for saline-treated TBI animals was reduced compared to shams (45 ± 5% vs. 65 ± 10%; n = 7; p < 0.05), whereas the frequency of investigation for TBI rats treated with ALCAR was not significantly different from that of shams but significantly higher than that of saline-treated TBI rats (68 ± 7; p < 0.05). The left parietal cortical lesion volume, expressed as a percentage of the volume of tissue in the right hemisphere, was significantly smaller in ALCAR-treated than in vehicle-treated TBI rats (14 ± 5% vs. 28 ± 6%; p < 0.05). We conclude that treatment with ALCAR during the first 24 h after TBI improves behavioral outcomes and reduces brain lesion volume in immature rats within the first 7 days after injury.
Controlled cortical impact; Sensorimotor function; Memory; Hippocampus; Acetyl-l-carnitine; Neuroprotection; Novel object recognition
Oxidative stress promotes Ca2+-dependent opening of the mitochondrial inner membrane permeability transition pore (PTP), causing bioenergetic failure and subsequent cell death in many paradigms, including those related to acute brain injury. One approach to preconditioning against oxidative stress is pharmacologic activation of the Nrf2/ARE pathway of antioxidant gene expression by agents such as sulforaphane (SFP). This study tested the hypothesis that administration of SFP to normal rats increases resistance of isolated brain mitochondria to redox-sensitive PTP opening. SFP or DMSO vehicle was administered intraperitoneally to adult male rats at 10 mg/kg 40 h prior to isolation of non-synaptic brain mitochondria. Mitochondria were suspended in medium containing a respiratory substrate and were exposed to an addition of Ca2+ below the threshold for PTP opening. Subsequent addition of tert-butyl hydroperoxide (tBOOH) resulted in a cyclosporin A-inhibitable release of accumulated Ca2+ into the medium, as monitored by an increase in fluorescence of Calcium Green 5N within the medium, and was preceded by a decrease in the autofluorescence of mitochondrial NAD(P)H. SFP treatment significantly reduced the rate of tBOOH-induced Ca2+ release but did not affect NAD(P)H oxidation or inhibit PTP opening induced by the addition of phenylarsine oxide, a direct sulfhydryl oxidizing agent. SFP treatment had no effect on respiration by brain mitochondria and had no effect on PTP opening or respiration when added directly to isolated mitochondria. We conclude that SFP confers resistance of brain mitochondria to redox-regulated PTP opening, which could contribute to neuroprotection observed with SFP.
Nrf2; Calcium; Peroxide; Pyridine nucleotide; Oxidation/reduction
Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexanoic acid (DHA) and eicosapentanoic acid (EPA) exerts cardioprotective effects, and suppresses Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca2+-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca2+-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca2+ retention capacity and decreased Ca2+-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca2+-induced MPTP opening.
cardiac; eicosapentaenoic acid; docosahexaenoic acid; fish oil; heart; mitochondrial permeability transition pore
There are several forms of acute pediatric brain injury, including neonatal asphyxia, pediatric cardiac arrest with global ischemia, and head trauma, that result in devastating, lifelong neurologic impairment. The only clinical intervention that appears neuroprotective is hypothermia initiated soon after the initial injury. Evidence indicates that oxidative stress, mitochondrial dysfunction, and impaired cerebral energy metabolism contribute to the brain cell death that is responsible for much of the poor neurologic outcome from these events. Recent results obtained from both in vitro and animal models of neuronal death in the immature brain point toward several molecular mechanisms that are either induced or promoted by oxidative modification of macromolecules, including consumption of cytosolic and mitochondrial NAD+ by poly-ADP ribose polymerase, opening of the mitochondrial inner membrane permeability transition pore, and inactivation of key, rate-limiting metabolic enzymes, e.g., the pyruvate dehydrogenase complex. In addition, the relative abundance of pro-apoptotic proteins in immature brains and neurons, and particularly within their mitochondria, predisposes these cells to the intrinsic, mitochondrial pathway of apoptosis, mediated by Bax- or Bak-triggered release of proteins into the cytosol through the mitochondrial outer membrane. Based on these pathways of cell dysfunction and death, several approaches toward neuroprotection are being investigated that show promise toward clinical translation. These strategies include minimizing oxidative stress by avoiding unnecessary hyperoxia, promoting aerobic energy metabolism by repletion of NAD+ and by providing alternative oxidative fuels, e.g., ketone bodies, directly interfering with apoptotic pathways at the mitochondrial level, and pharmacologic induction of antioxidant and anti-inflammatory gene expression.
Metabolism; Calcium; Permeability transition pore; poly(ADP-ribose) polymerase; Reactive oxygen species; Pyruvate dehydrogenase; Cytochrome c; Nicotinamide adenine dinucleotide
Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity which can be an important pathogenic factor in acute brain injury. Induced expression of Phase II detoxification enzymes through activation of the anti-oxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress-induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O2 and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective at protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death and both pre- and co-treatment with SFP were remarkably effective at blocking cytotoxicity. RT-PCR analysis indicated that several Nrf2-dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1) and glutamate-cysteine ligase modifier subunit (GCLM) that is involved in glutathione biosynthesis, were upregulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.
Nrf2; oxidative stress; ischemia/reperfusion; hemin; hippocampus
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.
astrocyte; cardiac arrest; hippocampus; microglia; Morris water maze; resuscitation
A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD+ that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD+ glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and at 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration.
Nicotinamide adenine dinucleotides; Nitrotetrazolium blue chloride; Formazan; Histo-enzymatic; Cerebral ischemia; Cryostat brain sections
Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury and neurodegenerative diseases. Our animal models of brain injury caused by cardiac arrest illustrate this relationship and demonstrate that both oxidative molecular modifications and mitochondrial metabolic impairment are exacerbated by reoxygenation of the brain using 100% ventilatory O2 compared to lower levels that maintain normoxemia. Numerous molecular mechanisms may be responsible for mitochondrial dysfunction caused by oxidative stress, including oxidation and inactivation of mitochondrial proteins, promotion of the mitochondrial membrane permeability transition, and consumption of metabolic cofactors and intermediates, e.g., NAD(H). Moreover, the relative contribution of these mechanisms to cell injury and death is likely different among different types of brain cells, e.g., neurons and astrocytes. In order to better understand these oxidative stress mechanisms and their relevance to neurologic disorders, we have undertaken studies with primary cultures of astrocytes and neurons exposed to O2 and glucose deprivation and reoxygenation and compared the results of these studies to those using a rat model of neonatal asphyxic brain injury. These results support the hypothesis that release and or consumption of mitochondrial NAD(H) is at least partially responsible for respiratory inhibition, particularly in neurons.
pyruvate dehydrogenase; respiration; nicotinamide adenine dinucleotide
Consumption of ω-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA+DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA+DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospholipid composition, respiration, and sensitivity to mitochondrial permeability transition pore (MPTP) opening in normal and infarcted myocardium. Rats were subjected to sham surgery or myocardial infarction by coronary artery ligation (n=10–14), and fed a standard diet, or supplemented with EPA+DHA (2.3% of energy intake) for 12 weeks. EPA+DHA altered fatty acid composition of total mitochondrial phospholipids and cardiolipin by reducing arachidonic acid content and increasing DHA incorporation. EPA+DHA significantly increased calcium uptake capacity in both subsarcolemmal and intrafibrillar mitochondria from sham rats. This treatment effect persisted with the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration or coupling, or cyclophilin D protein expression. Myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction. Infarcted LV myocardium had decreased mitochondrial protein yield and activity of mitochondrial marker enzymes, however respiratory function of isolated mitochondria was normal. EPA+DHA had no effect on LV function, mitochondrial respiration, or MPTP opening in rats with heart failure. In conclusion, dietary supplementation with EPA+DHA altered mitochondrial membrane phospholipid fatty acid composition in normal and infarcted hearts, but delayed MPTP opening only in normal hearts.
eicosapentaenoic acid; docosahexaenoic acid; myocardial infarction; mitochondrial permeability transition pore
Traumatic brain injury (TBI) results in a cerebral metabolic crisis that contributes to poor neurologic outcome. The aim of this study was to characterized changes in oxidative glucose metabolism in early periods after injury in the brains of immature animals. At 5 hours after controlled cortical impact TBI or sham surgery to the left cortex, 21-22 day old rats were injected intraperitoneally with [1,6-13C]glucose and brains removed 15 min, 30 min and 60 min later and studied by ex vivo 13C-NMR spectroscopy. Oxidative metabolism, determined by incorporation of 13C into glutamate, glutamine and GABA over 15 to 60 min, was significantly delayed in both hemispheres of brain from TBI rats. The most striking delay was in labeling of the C4 position of glutamate from neuronal metabolism of glucose in injured, ipsilateral hemisphere which peaked at 60 min, compared to the contralateral and sham-operated brains, where metabolism peaked at 30 and 15 min, respectively. Our findings indicate that 1) neuronal specific oxidative metabolism of glucose at 5-6h after TBI is delayed in both injured and contralateral sides compared to sham brain; 2) labeling from metabolism of glucose via the pyruvate carboxylase pathway in astrocytes was also initially delayed in both sides of TBI brain compared to sham brain; 3) despite this delayed incorporation, at 6 hours after TBI, both sides of brain showed apparent increased neuronal and glial metabolism, reflecting accumulation of labeled metabolites, suggesting impaired malate aspartate shuttle activity. The presence of delayed metabolism, followed by accumulation of labeled compounds is evidence of severe alterations in homeostasis that could impair mitochondrial metabolism in both ipsilateral and contralateral sides of brain after TBI. However, ongoing oxidative metabolism in mitochondria in injured brain suggests that there is a window of opportunity for therapeutic intervention up to at least 6 hrs after injury.
traumatic brain injury; immature brain; glucose; metabolism; 13C NMR spectroscopy; PDHC; PC; glutamate; GABA
Oxidative stress is an important molecular mechanism of astrocyte injury and death following ischemia reperfusion and may be an effective target of intervention. One therapeutic strategy for detoxifying the many different reactive oxygen and nitrogen species that are produced under these conditions is induction of the Phase II gene response by the use of chemicals or conditions that promote the translocation of the transcriptional activating factor NRF2 from the cytosol to the nucleus, where it binds to genomic antioxidant response elements. This study tested the hypothesis that pre- or post-treatment of cultured cortical astrocytes with sulforaphane, an alkylating agent known to activate the NRF2 pathway of gene expression protects against death of astrocytes caused by transient exposure to O2 and glucose deprivation (OGD). Rat cortical astrocytes were exposed to 5 μM sulforaphane either 48 hr prior to, or for 48 hr after a 4 hr period of OGD. Both pre- and post-treatments significantly reduced cell death at 48 hr after OGD. Immunostaining for 8-hydroxy-2-deoxyguanosine, a marker of DNA/RNA oxidation, was reduced at 4 hr reoxygenation with sulforaphane pretreatment. Sulforaphane exposure was followed by an increase in cellular and nuclear NRF2 immunoreactivity. Moreover, sulforaphane also increased the mRNA, protein level, and enzyme activity of NADPH/Quinone Oxidoreductase 1, a known target of NRF2 transcriptional activation. We conclude that sulforaphane stimulates the NRF2 pathway of antioxidant gene expression in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion.
Ischemia/Reperfusion; NRF2; NQO1; Oxidative stress; 8-hydroxy-2-deoxyguanosine
Brain mitochondria are relatively resistant to calcium-induced mitochondrial permeability transition (MPT) with heterogenic response to the insult. Since the cause for this heterogeneity is not clear we studied the distribution of a key regulator of the MPT, cyclophilin D (cypD) within the rat brain by using immunohistology and western blotting. Motor and parietal cortex, hippocampus, striatum, substantia nigra, ventral tegmental area, septum, and mammillary nucleus displayed a strong immunoreactivity to cypD within specific subpopulation of neurons. The staining was punctate and intense particularly in perinuclear regions of cells. Apart from neurons, a subpopulation of astrocytes and NG2 positive cells showed higher cypD immunoreactivity. The double staining of cypD with cytochrome oxidase confirmed the mitochondrial specificity of cypD immunoreactivity. The neurons with high levels of cypD also expressed glutamate decarboxylase (GABA) and the calcium binding protein parvalbumin or calbinding D-28k, identifying these cells as interneurons. Western blots confirmed our immunohistochemical findings showing significantly higher levels of cypD in crude mitochondria of substantia nigra when compared to cortex or striatum. Furthermore, nonsynaptic mitochondria representing mainly mitochondria from cell bodies of neurons and glia have about 16 % higher levels of cypD when compared to synaptic mitochondria that are localized in pre-synaptic buttons. These data suggest that the underlying factor of heterogenic response of isolated brain mitochondria to MPT-inducing insults can be the different expression levels of cypD with mitochondria originated from interneurons as the most sensitive.
cyclophilin D; calcium-binding proteins; interneurons; mitochondria; brain
Pesticide exposure has been suggested as an increased risk factor in developing Parkinson’s disease (PD). While the molecular mechanism underlying this association is not clear, several studies have demonstrated a role for mitochondrial dysfunction and oxidative damage in PD. Although data on specific pesticides associated with PD are often lacking, several lines of evidence point to the potential involvement of the organochlorine class of pesticides. Previously, we have found that the organochlorine pesticide methoxychlor (mxc) causes mitochondrial dysfunction and oxidative stress in isolated mitochondria. Here, we sought to determine whether mxc-induced mitochondrial dysfunction results in oxidative damage and dysfunction of the dopamine system. Adult female CD1 mice were dosed with either vehicle (sesame oil) or mxc (16, 32, or 64 mg/kg/day) for 20 consecutive days. Following treatment, we observed a dose-related increase in protein carbonyl levels in non-synaptic mitochondria, indicating oxidative modification of mitochondrial proteins which may lead to mitochondrial dysfunction. Mxc exposure also caused a dose-related decrease in striatal levels of dopamine (16–31%), which were accompanied by decreased levels of the dopamine transporter (DAT; 35–48%) and the vesicular monoamine transporter 2 (VMAT2; 21–44%). Because mitochondrial dysfunction, oxidative damage, and decreased levels of DAT and VMAT2 are found in PD patients, our data suggests that mxc should be investigated as a possible candidate involved in the association of pesticides with increased risk for PD, particularly in highly-exposed populations.
Methoxychlor; Parkinson’s disease; dopamine; complex I; mitochondria; neurodegeneration; dopamine transporter; vesicular monoamine transporter 2; oxidative stress
Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, due to a reversed transmembrane chloride gradient. GABAA receptor activation in immature neurons is sufficient to open L-type voltage gated calcium channels. As propofol is a GABAA agonist, we hypothesized that it and more specific GABAA modulators would increase intracellular free calcium ([Ca2+]i), resulting in the death of neonatal rat hippocampal neurons. Neuronal [Ca2+]i was monitored using Fura2-AM fluorescence imaging. Cell death was assessed by double-staining with propidium iodide and Hoechst 33258 at 1 h (acute) and 48 h (delayed) after 5 h exposure of neurons to propofol or the GABAA receptor agonist, muscimol, in the presence and absence of the GABA receptor antagonist, bicuculline, or the L-type Ca2+ channel blocker, nifedipine. Fluorescent measurements of caspase-3,-7 activities were performed at 1 h after exposure. Both muscimol and propofol induced a rapid increase in [Ca2+]i in day in vitro (DIV) 4, but not in DIV 8 neurons, that was inhibited by nifedipine and bicuculline. Caspase-3,-7 activities and cell death increased significantly in DIV 4 but not DIV 8 hippocampal neuronal cultures 1 h after a 5 h exposure to propofol, but not muscimol, and were inhibited by the presence of bicuculline or nifedipine. We conclude that an increase in [Ca2+]i, due to activation of GABAA receptors and opening of L-type calcium channels, is necessary for propofol-induced death of immature rat hippocampal neurons but that additional mechanisms not elicited by GABAA activation alone also contribute to cell death.
calcium; apoptosis; anesthetic; caspase
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 (∼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.
N-Acetyl aspartate; development; lactate; mitochondria; NMR spectroscopy
Background and Purpose
Previous reports indicate that compared with normoxia, 100% ventilatory O2 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% O2 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.
After 10 minutes of cardiac arrest, animals were resuscitated and ventilated for 1 hour with 100% O2 (hyperoxic) or 21% to 30% O2 (normoxic). At 30 minutes reperfusion, [1-13C]glucose was infused, and at 2 hours, brains were rapidly removed and frozen. Extracted metabolites were analyzed by 13C nuclear magnetic resonance spectroscopy.
Compared with nonischemic controls, the hippocampi from hyperoxic animals had elevated levels of unmetabolized 13C-glucose and decreased incorporation of 13C 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.
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 O2
13C-glucose; pyruvate dehydrogenase; mitochondria; cardiac arrest; glutamate; energy metabolism
Mitochondria contribute to cytosolic Ca2+ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca2+ release from Ca2+-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na+/Ca2+ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca2+ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca2+ and relatively slowreuptake, a secondary progressive release of Ca2+ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca2+ efflux is abolished by La3+ (1mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca2+ efflux. Ca2+-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGsinduced Ca2+ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brainmitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La3+. The presence of a second messenger-sensitive Ca2+ release mechanism in mitochondria could have an important impact on intracellular Ca2+ homeostasis.
Mitochondria; calcium; diacylglycerol; mitoplast; cation channel; permeability transition pore; protein kinase C; transient receptor potential; OAG
Background and Purpose
Current guidelines suggest that cardiac arrest (CA) survivors should be ventilated with 100% O2 after resuscitation. Breathing 100% O2 may worsen neurological outcome after experimental CA. This study tested the hypothesis that graded reoxygenation, with oximetry guidance, can safely reduce FiO2 after resuscitation, avoiding hypoxia while promoting neurological recovery.
Mature dogs underwent 10 minutes of CA and restoration of spontaneous circulation with100% O2. Animals were randomized to 1-hour additional ventilation on 100% FiO2 or to rapid lowering of arterial O2 saturation to <96% but >94% with pulse oximeter guidance. Animals were awakened at hour 23, and the neurological deficit score (0=normal; 100=brain-dead) was measured. Reanesthetized animals were perfusion-fixed and the brains removed for histopathology.
The neurological deficit score was significantly better in oximetry (O) dogs. O dogs appeared aware of their surroundings, whereas most hyperoxic (H) animals were stuporous (neurological deficit score=43.0±5.9 [O] versus 61.0±4.2 [H]; n=8, P<0.05). Stereological analysis revealed fewer injured CA1 neurons in O animals (cresyl violet: 35.5±4.3% [O] versus 60.5±3.3% [H]; P<0.05). There were also fewer fluoro-Jade B-stained degenerating CA1 neurons in O animals (3320±267 [O] versus 6633±356 [H] per 0.1 mm3; P<0.001).
A clinically applicable protocol designed to reduce postresuscitative hyperoxia after CA results in significant neuroprotection. Clinical trials of controlled normoxia after CA/restoration of spontaneous circulation should strongly be considered.
canine model; cerebral ischemia; neurological deficit; oxygen utilization
The induction of GRIM-19 has been shown to be essential for interferon-β (IFN-β)-induced and retinoic acid (RA)-induced tumor cell death. We have studied the localization and levels of GRIM-19 in IFN/RA-induced cell death in neural cells and in focal cerebral ischemia. Exposure to IFN/RA caused a ~15-fold increase in GRIM-19 protein levels and induced >50% cell death in human neuroblastoma SH-SY5Y cells. In rats subjected to permanent focal cerebral ischemia, increased oxidative stress, as well as increased GRIM mRNA levels (32-fold) and increased GRIM-19 (>50%) protein levels were noted in the ipsilateral (affected) hemisphere compared with the contralateral (unaffected) hemisphere. These results suggest that GRIM-19 may play a role in ischemia-induced neuronal cell death.