Sang Won Suh

The protective effects of interleukin-18 and interferon-γ on neuronal damages in the rat hippocampus following status epilepticus.

To elucidate whether interleukin-18 (IL-18) or interferon-γ (IFN-γ) participates in neurodegeneartion, we investigated the changes in IL-18 and IFN-γ systems within the rat hippocampus following status epilepticus (SE). In non-SE induced animals, IL-18, IL-18 receptor α (IL-18Rα), IFN-γ and IFN-γ receptor α (IFN-γRα) immunoreactivity was not detected in the hippocampus. Following SE, IL-18 immunoreactivity was increased in CA1-3 pyramidal cells as well as dentate granule cells. IL-18 immunoreactivity was also up-regulated in astrocytes and microglia/macrophages. IL-18Rα immunoreactivity was detected in astrocytes and microglia/macrophages. IFN-γ immunoreactivity was detected only in astrocytes within all regions of the hippocampus. IFN-γRα immunoreactivity was increased in neurons as well as astrocytes. Intracerebroventricular infusions of recombinant rat IL-18 or IFN-γ alleviated SE-induced neuronal damages, while neutralization of IL-18, IFN-γ or their receptors aggravated them, as compared to saline-infused animals. These findings suggest that astroglial-mediated IFN-γ pathway in response to IL-18 induction may play an important role in alleviation of SE-induced neuronal damages.

Copper/zinc chelation by clioquinol reduces spinal cord white matter damage and behavioral deficits in a murine MOG-induced multiple sclerosis model

The present study aimed to evaluate the therapeutic potential of clioquinol (CQ), a metal chelator, on multiple sclerosis pathogenesis. Experimental autoimmune encephalomyelitis was induced by immunization with myelin oligodendrocyte glycoprotein (MOG(35-55)) in female mice. Three weeks after the initial immunization, demyelination and immune cell infiltration in the spinal cord were analyzed. CQ (30mg/kg) was given by gavage once per day for the entire experimental course. CQ profoundly reduced the daily clinical score and incidence rate of EAE mice. The CQ-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination and reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20 and F4/80 positive cells. CQ also remarkably inhibited EAE-associated BBB disruption and MMP-9 activation. Autophagy contributes to clearance of aggregated proteins in astrocytes and neurons. The present study found that EAE increased the induction of autophagy and CQ further increased this expression. Furthermore, the present study found that post-treatment with CQ also reduced the clinical score of EAE and spinal cord demyelination. These results demonstrate that CQ inhibits the clinical features and neuropathological changes associated with EAE. The present study suggests that transition metals may be involved in several steps of multiple sclerosis pathogenesis.

Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death

The present study sought to evaluate the neuroprotective effects of apocynin, an NADPH oxidase assembly inhibitor, on seizure-induced neuronal death. Apocynin, also known as acetovanillone, is a natural organic compound isolated from the root of Canadian hemp (Apocynum cannabium). It has been extensively studied to determine its disease-fighting capabilities and application in several brain insults, such as traumatic brain injury and stroke. Here we tested the hypothesis that post-treatment of apocynin may prevent seizure-induced neuronal death by suppression of NADPH oxidase-mediated superoxide production. Temporal lobe epilepsy (TLE) was induced by intraperitoneal injection of pilocarpine (25mg/kg) in male rats. Apocynin (30mg/kg, i.p.) was injected into the intraperitoneal space two hours after seizure onset. A second injection was performed 24h after seizure. To test whether apocynin inhibits NADPH oxidase activation-induced reactive oxygen species (ROS) production, dihydroethidium (dHEt, 5mg/kg, i.p.) was injected before onset of seizure and ROS production was detected five hours after seizure onset. Neuronal oxidative injury (4HNE), neuronal death (Fluoro Jade-B), blood brain barrier (BBB) disruption (IgG leak), neurotrophil infiltration (MPO) and microglia activation (CD11b) in the hippocampus was evaluated at three days after status epilepticus (SE). Pilocarpine-induced seizure increased p47 immunofluorescence in the plasma membrane of hippocampal neurons at 12h post-insult and apocynin treatment prevented this increase. The present study found that apocynin post-treatment decreased ROS production and lipid peroxidation after seizure and decreased the number of degenerating hippocampal neurons. Apocynin also reduced seizure-induced BBB disruption, neurotrophil infiltration and microglial activation. Taken together, the present results suggest that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce seizure-induced neuronal dysfunction.

Zinc chelation reduces hippocampal neurogenesis after pilocarpine-induced seizure.

Several studies have shown that epileptic seizures increase hippocampal neurogenesis in the adult. However, the mechanism underlying increased neurogenesis after seizures remains largely unknown. Neurogenesis occurs in the subgranular zone (SGZ) of the hippocampus in the adult brain, although an understanding of why it actively occurs in this region has remained elusive. A high level of vesicular zinc is localized in the presynaptic terminals of the SGZ. Previously, we demonstrated that a possible correlation may exist between synaptic zinc localization and high rates of neurogenesis in this area after hypoglycemia. Using a lithium-pilocarpine model, we tested our hypothesis that zinc plays a key role in modulating hippocampal neurogenesis after seizure. Then, we injected the zinc chelator, clioquinol (CQ, 30 mg/kg), into the intraperitoneal space to reduce brain zinc availability. Neuronal death was detected with Fluoro Jade-B and NeuN staining to determine whether CQ has neuroprotective effects after seizure. The total number of degenerating and live neurons was similar in vehicle and in CQ treated rats at 1 week after seizure. Neurogenesis was evaluated using BrdU, Ki67 and doublecortin (DCX) immunostaining 1 week after seizure. The number of BrdU, Ki67 and DCX positive cell was increased after seizure. However, the number of BrdU, Ki67 and DCX positive cells was significantly decreased by CQ treatment. Intracellular zinc chelator, N,N,N0,N-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), also reduced seizure-induced neurogenesis in the hippocampus. The present study shows that zinc chelation does not prevent neurodegeneration but does reduce seizure-induced progenitor cell proliferation and neurogenesis. Therefore, this study suggests that zinc has an essential role for modulating hippocampal neurogenesis after seizure.

Adipose-Derived Mesenchymal Stem Cells Reduce Neuronal Death After Transient Global Cerebral Ischemia Through Prevention of Blood-Brain Barrier Disruption and Endothelial Damage

Global cerebral ischemia (GCI) is the leading cause of a poor prognosis even after successful resuscitation from cardiac arrest. Therapeutic induction of hypothermia (TH) is the only proven therapy—and current standard care—for GCI after cardiac arrest; however, its application has been significantly limited owing to technical difficulties. Mesenchymal stem cells (MSCs) are known to suppress neuronal death after cerebral ischemia. The prevention of blood‐brain barrier (BBB) disruption has not been suggested as a mechanism of MSC treatment but has for TH. We evaluated the therapeutic effect of MSC administration on BBB disruption and neutrophil infiltration after GCI. To evaluate the therapeutic effects of MSC treatment, rats were subjected to 7 minutes of transient GCI and treated with MSCs immediately after reperfusion. Hippocampal neuronal death was evaluated at 7 days after ischemia using Fluoro‐Jade B (FJB). BBB disruption, endothelial damage, and neutrophil infiltration were evaluated at 7 days after ischemia by immunostaining for IgG leakage, Rat endothelial antigen‐1, and myeloperoxidase (MPO). Rats treated with MSCs showed a significantly reduced FJB+ neuron count compared with the control group. They also showed reduced IgG leakage, endothelial damage, and MPO+ cell counts. The present study demonstrated that administration of MSCs after transient GCI provides a dramatic protective effect against hippocampal neuronal death. We hypothesized that the neuroprotective effects of MSC treatment might be associated with the prevention of BBB disruption and endothelial damage and a decrease in neutrophil infiltration.

Zinc chelation reduces traumatic brain injury-induced neurogenesis in the subgranular zone of the hippocampal dentate gyrus.

Numerous studies have demonstrated that traumatic brain injury (TBI) increases hippocampal neurogenesis in the rodent brain. However, the mechanisms underlying increased neurogenesis after TBI remain unknown. Continuous neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in the adult brain. The mechanism that maintains active neurogenesis in the hippocampal area is not known. A high level of vesicular zinc is localized in the presynaptic terminals of the SGZ (mossy fiber). The mossy fiber of dentate granular cells contains high levels of chelatable zinc in their terminal vesicles, which can be released into the extracellular space during neuronal activity. Previously, our lab presented findings indicating that a possible correlation may exist between synaptic zinc localization and high rates of neurogenesis in this area after hypoglycemia or epilepsy. Using a weight drop animal model to mimic human TBI, we tested our hypothesis that zinc plays a key role in modulating hippocampal neurogenesis after TBI. Thus, we injected a zinc chelator, clioquinol (CQ, 30mg/kg), into the intraperitoneal space to reduce brain zinc availability twice per day for 1 week. Neuronal death was evaluated with Fluoro Jade-B and NeuN staining to determine whether CQ has neuroprotective effects after TBI. The number of degenerating neurons (FJB (+)) and live neurons (NeuN (+)) was similar in vehicle and in CQ-treated rats at 1 week after TBI. Neurogenesis was evaluated using BrdU, Ki67 and doublecortin (DCX) immunostaining 1 week after TBI. The number of BrdU, Ki67 and DCX positive cell was increased after TBI. However, the number of BrdU, Ki67 and DCX positive cells was significantly decreased by CQ treatment. The present study shows that zinc chelation did not prevent neurodegeneration but did reduce TBI-induced progenitor cell proliferation and neurogenesis. Therefore, this study suggests that zinc has an essential role for modulating hippocampal neurogenesis after TBI.

Pyruvate Administration Reduces Recurrent/Moderate Hypoglycemia-Induced Cortical Neuron Death in Diabetic Rats

Recurrent/moderate (R/M) hypoglycemia is common in type 1 diabetes patients. Moderate hypoglycemia is not life-threatening, but if experienced recurrently it may present several clinical complications. Activated PARP-1 consumes cytosolic NAD, and because NAD is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Pyruvate, however, can be metabolized in the absence of cytosolic NAD. We therefore hypothesized that pyruvate may be able to improve the outcome in diabetic rats subjected to insulin-induced R/M hypoglycemia by terminating hypoglycemia with glucose plus pyruvate, as compared with delivering just glucose alone. In an effort to mimic juvenile type 1 diabetes the experiments were conducted in one-month-old young rats that were rendered diabetic by streptozotocin (STZ, 50mg/kg, i.p.) injection. One week after STZ injection, rats were subjected to moderate hypoglycemia by insulin injection (10U/kg, i.p.) without anesthesia for five consecutive days. Pyruvate (500mg/kg) was given by intraperitoneal injection after each R/M hypoglycemia. Three hours after last R/M hypoglycemia, zinc accumulation was evaluated. Three days after R/M hypoglycemia, neuronal death, oxidative stress, microglial activation and GSH concentrations in the cerebral cortex were analyzed. Sparse neuronal death was observed in the cortex. Zinc accumulation, oxidative injury, microglial activation and GSH loss in the cortex after R/M hypoglycemia were all reduced by pyruvate injection. These findings suggest that when delivered alongside glucose, pyruvate may significantly improve the outcome after R/M hypoglycemia by circumventing a sustained impairment in neuronal glucose utilization resulting from PARP-1 activation.

Impairment of Autophagic Flux Promotes Glucose Reperfusion-Induced Neuro2A Cell Death after Glucose Deprivation

Hypoglycemia-induced brain injury is a common and serious complication of intensive insulin therapy experienced by Type 1 diabetic patients. We previously reported that hypoglycemic neuronal death is triggered by glucose reperfusion after hypoglycemia rather than as a simple result of glucose deprivation. However, the precise mechanism of neuronal death initiated by glucose reperfusion is still unclear. Autophagy is a self-degradation process that acts through a lysosome-mediated trafficking pathway to degrade and recycle intracellular components, thereby regulating metabolism and energy production. Recent studies suggest that autophagic and lysosomal dysfunction leads to abnormal protein degradation and deposition that may contribute to neuronal death. Here, we focused on the relationship between autophagy and lysosomal dysfunction in hypoglycemia-induced neuronal death. In neuronal cells, glucose reperfusion after glucose deprivation resulted in inhibition of autophagy, which may promote cell death. This cell death was accompanied with activation of caspase3 and the lysosomal proteases cathepsin B and D, which indicated impairment of autophagic flux. Taken together, these results suggest that interplay of autophagy, caspase3 activation and lysosomal proteases serve as a basis for neuronal death after hypoglycemia. Thus, we provide the molecular mechanism of neuronal death by glucose reperfusion and suggest some clues for therapeutic strategies to prevent hypoglycemia-induced neuronal death.

EAAC1 gene deletion alters zinc homeostasis and enhances cortical neuronal injury after transient cerebral ischemia in mice.

The excitatory amino acids glutamate and cysteine are actively transported into neurons from the extracellular space by the high affinity glutamate transporter EAAC1. The astrocyte glutamate transporters, GLT1 and GLAST, are the primary mediators of glutamate clearance. EAAC1 has a limited role in this function. However, uptake of cysteine into neurons via EAAC1 contributes to neuronal antioxidant function by providing cysteine substrate for glutathione synthesis. Mice in which the EAAC1 gene has been deleted were seen to have enhanced susceptibility to neuronal oxidative stress and developed brain atrophy and cognitive function decline with aging. The aim of the current study was to evaluate if EAAC1 confers protection against ischemic events. Young adult CD-1 wild-type or EAAC1(-/-) mice were subjected to 30 min of bilateral common carotid artery occlusion and evaluated for neuronal death and zinc translocation. The intensity of TSQ fluorescence in the cytoplasm of cortical neurons in the EAAC1(-/-) mice was significantly higher than wild-type mice, indicating that the cortical neurons of EAAC1(-/-) mice contain higher cytoplasmic concentrations of labile (or free) zinc. Zinc translocation into cortical neurons was also enhanced in EAAC1(-/-) mice. Three days after ischemia, Fluoro-Jade B staining revealed that EAAC1(-/-) mice had more than twice as many degenerating neurons as wild-type mice. N-acetylcysteine, a membrane-permeant cysteine pro-drug, normalized basal zinc levels, reduced TSQ (+) neurons and reduced ischemic neuronal death in the EAAC1(-/-) mice when delivered in a pre-treatment fashion. Taken together, this study implicates EAAC1-dependent cysteine uptake as an endogenous source of enhancing antioxidant function and zinc homeostasis in neurons in the ischemic brain.

EAAC1 gene deletion increases neuronal death and blood brain barrier disruption after transient cerebral ischemia in female mice.

EAAC1 is important in modulating brain ischemic tolerance. Mice lacking EAAC1 exhibit increased susceptibility to neuronal oxidative stress in mice after transient cerebral ischemia. EAAC1 was first described as a glutamate transporter but later recognized to also function as a cysteine transporter in neurons. EAAC1-mediated transport of cysteine into neurons contributes to neuronal antioxidant function by providing cysteine substrates for glutathione synthesis. Here we evaluated the effects of EAAC1 gene deletion on hippocampal blood vessel disorganization after transient cerebral ischemia. EAAC1−/− female mice subjected to transient cerebral ischemia by common carotid artery occlusion for 30 min exhibited twice as much hippocampal neuronal death compared to wild-type female mice as well as increased reduction of neuronal glutathione, blood–brain barrier (BBB) disruption and vessel disorganization. Pre-treatment of N-acetyl cysteine, a membrane-permeant cysteine prodrug, increased basal glutathione levels in the EAAC1−/− female mice and reduced ischemic neuronal death, BBB disruption and vessel disorganization. These findings suggest that cysteine uptake by EAAC1 is important for neuronal antioxidant function under ischemic conditions.