Pharmacological elevation of glutathione inhibits status epilepticus-induced neuroinflammation and oxidative injury.

Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.

Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.

Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.

Prevalence of celiac disease in China: Meta-analysis and serological survey in high-risk populations.

OBJECTIVE: To summarize data from a serological survey of high-risk populations in Guangdong Province, China, and to perform a meta-analysis to investigate the prevalence and seroprevalence of celiac disease (CD) in the Chinese general and high-risk populations. METHODS: We collected data from the serological survey of high-risk population of CD in Guangdong Province, China (N = 1390) by testing their serum tissue transglutaminase immunoglobulin A (tTG-IgA), deamidated gliadin peptides immunoglobulin A (DGP-IgA) and deamidated gliadin peptides immunoglobulin G (DGP-IgG). Additionally, a literature search was performed on PubMed, EMBASE, Cochrane Library and three Chinese databases for articles published up to 20 December 2020 to estimate the pooled prevalence and seroprevalence of CD in China. RESULTS: In the serological survey, 0.94% (13/1390) of individuals were positive for CD antibodies. In a meta-analysis of 18 studies, the seroprevalence of CD in the general Chinese population was 0.27% (95% confidence interval [CI] 0.02%-0.71%). While that in the high-risk population was 8.34% (95% CI 4.90%-12.54%) (odds ratio 7.27, 95% CI 4.06-13.04). The prevalence of biopsy-confirmed CD in high-risk Chinese populations was 4.44% (95% CI 1.53%-8.58%). The seroprevalence of CD varied with patients' geographical origin, being higher in northern China than in southern China. CONCLUSIONS: Early diagnosis of CD by serological screening in high-risk population and generous serological testing in those with vague symptoms, especially in northern China, are recommended.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

Echo signal envelope fitting based signal processing methods for ultrasonic gas flow-meter.

The echo signal of the ultrasonic gas flow-meter is difficult to locate, and the computation of signal processing methods proposed by others is extensive which affects the meter's real-time performance. Aiming at solving these problems the echo signal envelopes under different flow rates are analyzed. And three kinds of signal processing methods based on echo signal envelope fitting are proposed. The shape of the echo signal remains the "approximate spindle shape" as the flow rate increases, and the envelope gradient of the middle parts of the upper envelope's rising section and the lower envelope's falling section remains the same at different flow rates, so the mathematical models of the two sections are established to obtain the envelope gradient curves of the two sections. With the envelope gradient curves, the ranges of envelope and peak points that linearly distributed are obtained. The least squares fitting is performed on those peak points to obtain two feature straight lines for respectively representing the upper envelope's rising section and the lower envelope's falling section. According to the spatial characteristics of the feature straight lines, the points on the feature straight lines are selected as the feature points for quickly locating the echo signal. According to the offline verification and comparison, the best one of three kinds of signal processing methods is selected, and realized on the hardware system of the two-channel ultrasonic gas flow-meter. The transmitter of two-channel ultrasonic gas flow-meter is developed based on FPGA & DSP dual core structure. It utilizes the parallel processing capacity and logic control ability of FPGA to realize the controlling of the high-speed ADC & DAC and the storage of the data. At the same time, it adopts the high-speed computing capacity of DSP to implement the digital signal processing method. The gas flow calibration experiments were carried out in a national accredited testing agency to verify the effectiveness of the signal processing methods and system. The experimental results show that the improved signal processing method based on echo signal upper and lower envelope fitting can quickly and accurately locate the echo signal. And the measurable range of the ultrasonic gas flow meter based on this signal processing method is broadened to 10 m3/h to 1,300 m3/h, and the turndown ratio is broadened to 1:130.

Aneuploidy of chromosome 8 and mutation of circulating tumor cells predict pathologic complete response in the treatment of locally advanced rectal cancer.

Identifying patients who may or may not achieve pathologic complete response (pathCR) allows for treatment with alternative approaches in the preoperative setting. The aim of the current study was to investigate whether aneuploidy of chromosome 8 and mutations of circulating tumor cells (CTCs) could predict the response of patients with rectal cancer to preoperative chemoradiotherapy. A total of 33 patients with locally advanced rectal cancer (cT3-T4 and/or cN+) treated with neoadjuvant chemoradiotherapy between September 2014 and March 2015 were recruited. Blood samples were collected from 33 patients with pre-chemoradiotherapy rectal cancer. It was demonstrated that ≥5 copies of chromosome 8 was associated with pathCR (univariate logistic regression, P=0.042). Of the 6 patients whose CTCs had <5 copies of chromosome 8, 3 achieved pathCR (3/6, 50%), and of the 27 patients whose CTCs had ≥5 copies of chromosome 8 obtained 3 pathCR (3/27, 11.1%; Chi-square test, P=0.0255). Of the 33 patients with mutations assessed, 8 significant nonsynonymous mutations in CTCs were identified as associated with pathCR (Chi-square test, P-values range, 0.0004-0.0298; mutations in ARID1A, HDAC1, APC, ERBB3, TP53, AMER1 and AR). These results suggest that ≥5 copies of chromosome 8 and 8 nonsynonymous mutations in ARID1A, HDAC1, APC, ERBB3, TP53, AMER1 AR in CTCs were associated with pathCR. This conclusion should be validated further in larger prospective studies and the long-term follow-up survival data of this study will also be reported in the future.

Survival outcomes and patterns of failure after D2 dissection and adjuvant chemoradiotherapy for locally advanced gastric cancer: a retrospective study.

OBJECTIVE:   The goal of the study was to analyze the incidence and patterns of failure in patients with gastric cancer who received D2 dissection and adjuvant chemoradiotherapy (CRT). METHODS:   From January 2004 to October 2015, 324 patients with gastric cancer who underwent radical D2 resection followed by postoperative CRT were enrolled. Clinicopathological characteristics and patterns of failure were retrospectively reviewed to identify factors associated with survival and recurrence. RESULTS:   After a median follow-up of 30 months, the 3-year overall survival and 3-year disease-free survival rates of these patients were 60.3 and 51.1%, respectively. 117 patients had recurrence or metastasis, with peritoneal recurrence as the most frequent (20.7%), followed by distant metastasis (14.2%). The most commonly involved distant organs were the liver (5.9%) and bone (4.9%). Locoregional failure occurred in 39 patients (12.0%), with isolated regional failure occurring in only 23 (7.1%). Further multivariate Cox regression analysis revealed N stage to be an independent risk factor for distant failure-free survival (p = 0.012). Independent risk factors for peritoneal metastasis were tumor differentiation (p = 0.022), T stage (p =0.035) and vascular invasion (p = 0.016). CONCLUSION: Postoperative CRT has a potential effect on optimizing locoregional control, resulting in only 12.0% of locoregional failure. In patients after D2 resection and adjuvant CRT, peritoneal metastasis was the leading pattern of failure, followed by distant metastasis. Advances in knowledge: Peritoneal recurrence was the most common pattern of failure after D2 dissection and adjuvant CRT, followed by distant metastasis, whereas locoregional relapse was relatively rare. Selection of patients based on the predicted risk of each recurrence pattern may be a reasonable approach to the optimization of treatment strategies.

Regulation of kynurenine metabolism by a ketogenic diet.

Ketogenic diets (KDs) are increasingly utilized as treatments for epilepsy, other neurological diseases, and cancer. Despite their long history in suppressing seizures, the distinct molecular mechanisms of action of KDs are still largely unknown. The goal of this study was to identify key metabolites and pathways altered in the hippocampus and plasma of rats fed a KD versus control diet (CD) either ad libitum or calorically restricted to 90% of the recommended intake. This was accomplished using a combination of targeted methods and untargeted MS-based metabolomics analyses. Various metabolites of and related to the tryptophan (TRP) degradation pathway, such as kynurenine (KYN), kynurenic acid as well as enzyme cofactors, showed significant changes between groups fed different diets and/or calorie amounts in plasma and/or the hippocampus. KYN was significantly downregulated in both matrices in animals of the CD-calorically restricted, KD-ad libitum, and KD-calorically restricted groups compared with the CD-ad libitum group. Our data suggest that the TRP degradation pathway is a key target of the KD.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of disulfide high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented disulfide HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

Plasma cysteine/cystine redox couple disruption in animal models of temporal lobe epilepsy.

Currently the field of epilepsy lacks peripheral blood-based biomarkers that could predict the onset or progression of chronic seizures following an epileptogenic injury. Thiol/disulfide ratios have been shown to provide a sensitive means of assessing the systemic redox potential in tissue and plasma. In this study, we utilized a rapid, simple and reliable method for simultaneous determination of several thiol-containing amino acids in plasma using HPLC with electrochemical detection in kainic acid (KA) and pilocarpine rat models of epilepsy. In contrast to GSH and GSSG levels, the levels of cysteine (Cys) were decreased by 42% and 62% and cystine (Cyss) were increased by 46% and 23% in the plasma of KA- and pilocarpine-injected rats, respectively after 48h. In chronically epileptic rats, plasma cysteine was decreased by 40.4% and 37.7%, and plasma GSSG increased by 33.8% and 35.0% following KA and pilocarpine, respectively. Treatment of rats with a catalytic antioxidant, 60min after KA or pilocarpine significant attenuated the decrease of plasma Cys/Cyss ratios at the 48h time point in both models. These observations suggest that the decreased cysteine and ratio of Cys/Cyss in plasma could potentially serve as redox biomarkers in temporal lobe epilepsy.

Variations in CT determination of target volume with active breath co-ordinate in radiotherapy for post-operative gastric cancer.

OBJECTIVE: To investigate interobserver and inter-CT variations in using the active breath co-ordinate technique in the determination of clinical tumour volume (CTV) and normal organs in post-operative gastric cancer radiotherapy. METHODS: Ten gastric cancer patients were enrolled in our study, and four radiation oncologists independently determined the CTVs and organs at risk based on the CT simulation data. To determine interobserver and inter-CT variation, we evaluated the maximum dimensions, derived volume and distance between the centres of mass (CMs) of the CTVs. We assessed the reliability in CTV determination among the observers by conformity index (CI). RESULTS: The average volumes ± standard deviation (cm(3)) of the CTV, liver, left kidney and right kidney were 674 ± 138 (range, 332-969), 1000 ± 138 (range, 714-1320), 149 ± 13 (range, 104-183) and 141 ± 21 (range, 110-186) cm(3), respectively. The average inter-CT distances between the CMs of the CTV, liver, left kidney and right kidney were 0.40, 0.56, 0.65 and 0.6 cm, respectively; the interobserver values were 0.98, 0.53, 0.16 and 0.15 cm, respectively. CONCLUSIONS: In the volume size of CTV for post-operative gastric cancer, there were significant variations among multiple observers, whereas there was no variation between different CTs. The slices in which variations more likely occur were the slices of the lower verge of the hilum of the spleen and porta hepatis, then the paraoesophageal lymph nodes region and abdominal aorta, and the inferior vena cava, and the variation in the craniocaudal orientation from the interobserver was more predominant than that from inter-CT. ADVANCES IN KNOWLEDGE: First, this is the first study to evaluate the interobserver and inter-CT variations in the determination of the CTV and normal organs in gastric cancer with the use of the active breath co-ordinate technique. Second, we analysed the region where variations most likely occur. Third, we investigated the influence of interobserver variation on the dose distribution.

Optomizing Transfection Efficiency of Cervical Cancer Cells Transfected by Cationic Liposomes LipofectamineTM2000.

BACKGROUND: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. MATERIALS AND METHODS: Lipofectamine(TM)2000 as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA , miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. RESULTS: The seeding density of Hela cell line and Siha are 1.5 x 10(4) (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01).MTT assay showed that according to the above conditions which has the lowest cytotoxicity. CONCLUSIONS: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

Immunological subtypes analysis of Uygur diffuse large B-cell lymphoma in Xinjiang and their prognostic significance.

OBJECTIVE: This study aims to explore the application of Choi's typing method in the immunological typing of diffuse large B-cell lymphoma (DLBCL) in Xinjiang Autonomous Region and its prognostic significance. MATERIALS AND METHODS: Seventy-eight cases of DLBCL tumor tissues from Xinjiang were collected to detect the expression of germinal center B (GCB) cell-expressed transcript-1, FOXP1, CD10, bcl-6, and MUM1 using an immunohistochemical method. Then, immunological typing was carried out using Choi's typing method, and the survival analysis was performed using Kaplan-Meier method. Cox proportional hazard model was used to analyze the prognostic factors. RESULTS: GCB-cell-like-DLBCL and non-GCB-DLBCL accounting for 29.5% (23/78) and 70.5% (55/78), respectively. The 3-year overall survival of GCB-DLBCL was 58%, significantly higher than that of non-GCB-DLBCL (39%, P < 0.05). Multivariate analysis showed that International Prognostic Index and immunological typing were two independent prognostic factors for Uygur patients with DLBCL. CONCLUSION: Non-GCB-DLBCL is the main type of DLBCL in Xinjiang and Choi's typing method can be a helpful indicator to determine the prognosis of the Uygur DLBCL in Xinjiang.

Temporal and spatial increase of reactive nitrogen species in the kainate model of temporal lobe epilepsy.

Steady-state levels of reactive oxygen species (ROS) and oxidative damage to cellular macromolecules are increased in the rodent hippocampus during epileptogenesis. However, the role of reactive nitrogen species (RNS) in epileptogenesis remains to be explored. The goal of this study was to determine the spatial and temporal occurrence of RNS i.e. nitric oxide levels in a rat model of temporal lobe epilepsy (TLE). Rats were injected with a single high dose of kainate and monitored by video for behavioral seizures for 6weeks to determine the onset and severity of chronic seizures. RNS and tissue/mitochondrial redox status (glutathione redox couple and coenzyme A:glutathione redox couple) were measured in the hippocampus at 8h, 24h, 48h, 1wk, 3wk and 6wk following kainate to assess the level of reactive species in subcellular compartments. We observed a biphasic increase in RNS levels with a return to control values at the 48h time point. However, both tissue and mitochondrial redox status showed permanent and significant decreases during the entire time course of epilepsy development. 3 nitrotyrosine (3NT) protein adducts were found to gradually increase throughout epileptogenesis, conceivably as a result of the local environment under oxidative and nitrosative stress. Colocalization of 3NT immunostaining with neuron- or astrocyte-specific markers revealed neuron-specific localization of 3NT in hippocampal principal neurons. Persistent and concurrent glutathione oxidation and nitrosative stress occur during epileptogenesis suggesting a favorable environment for posttranslational modifications.

Glutathione deficiency in Gclm null mice results in complex I inhibition and dopamine depletion following paraquat administration.

Depletion of glutathione has been shown to occur in autopsied brains of patients with Parkinson's disease (PD) and in animal models of PD. The goal of this study was to determine whether chronic glutathione (GSH) deficiency per se resulted in complex I inhibition and/or dopamine depletion and whether these indices were further potentiated by aging or administration of paraquat, a redox-cycling herbicide that produces a PD-like neurodegeneration model in rodents (Brooks, A. I., Chadwick, C. A., Gelbard, H. A., Cory-Slechta, D. A., and Federoff, H. J. [1999]. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res. 823, 1-10; McCormack, A. L., Thiruchelvam, M., Manning-Bog, A. B., Thiffault, C., Langston, J. W., Cory-Slechta, D. A., and Di Monte, D. A. [2002]. Environmental risk factors and Parkinson's disease: Selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol. Dis. 10, 119-127.) Deletion of the rate-limiting GSH synthesis gene, glutamate-cysteine ligase modifier subunit (Gclm), leads to significantly lower GSH concentrations in all tissues including brain. Gclm null (Gclm (-/-)) mice provide a model of prolonged GSH depletion to explore the relationship between GSH, complex I inhibition, and dopamine loss in vivo. Despite ~60% depletion of brain GSH in Gclm (-/-) mice of ages 3-5 or 14-16 months, striatal complex I activity, dopamine levels, 3-nitrotyroine/tyrosine ratios, aconitase activity, and CoASH remained unchanged. Administration of paraquat (10mg/kg, twice/week, 3 weeks) to 3- to 5-month-old Gclm (-/-) mice resulted in significantly decreased aconitase activity, complex I activity, and dopamine levels but not in 3- to 5-month-old Gclm (+/+) mice. Furthermore, paraquat-induced inhibition of complex I and aconitase activities in Gclm (-/-) mice was observed in the striatum but not in the cortex. The results suggest that chronic deficiency of GSH in Gclm (-/-) mice was not sufficient to result in complex I inhibition or dopamine depletion perhaps due to homeostatic mechanisms but required an additional oxidative stress insult as shown with paraquat exposure.

Mitochondrial oxidative stress and epilepsy in SOD2 deficient mice: attenuation by a lipophilic metalloporphyrin.

Epileptic seizures are a common feature associated with inherited mitochondrial diseases. This study investigated the role of mitochondrial oxidative stress in epilepsy resulting from mitochondrial dysfunction using cross-bred mutant mice lacking mitochondrial manganese superoxide dismutase (MnSOD or SOD2) and a lipophilic metalloporphyrin catalytic antioxidant. Video-EEG monitoring revealed that in the second to third week of postnatal life (P14-P21) B6D2F2 Sod2(-/-) mice exhibited frequent spontaneous motor seizures providing evidence that oxidative stress-induced mitochondrial dysfunction may contribute to epileptic seizures. To confirm the role of mitochondrial oxidative stress in epilepsy a newly developed lipophilic metalloporphyrin, AEOL 11207, with high potency for catalytic removal of endogenously generated reactive oxygen species was utilized. AEOL 11207-treated Sod2(-/-) mice showed a significant decrease in both the frequency and duration of spontaneous seizures but no effect on seizure severity. A significant increase in the average lifespan of AEOL 11207-treated Sod2(-/-) mice compared to vehicle-treated Sod2(-/-) mice was also observed. Indices of mitochondrial oxidative stress and damage (aconitase inactivation, 3-nitrotyrosine formation, and depletion of reduced coenzyme A) and ATP levels affecting neuronal excitability were significantly attenuated in the brains of AEOL 11207-treated Sod2(-/-) mice compared to vehicle-treated Sod2(-/-) mice. The occurrence of epileptic seizures in Sod2(-/-) mice and the ability of catalytic antioxidant therapy to attenuate seizure activity, mitochondrial dysfunction, and ATP levels suggest that ongoing mitochondrial oxidative stress can contribute to epilepsy associated with mitochondrial dysfunction and disease.

Chelation of mitochondrial iron prevents seizure-induced mitochondrial dysfunction and neuronal injury.

Chelatable iron is an important catalyst for the initiation and propagation of free radical reactions and implicated in the pathogenesis of diverse neuronal disorders. Studies in our laboratory have shown that mitochondria are the principal source of reactive oxygen species production after status epilepticus (SE). We asked whether SE modulates mitochondrial iron levels by two independent methods and whether consequent mitochondrial dysfunction and neuronal injury could be ameliorated with a cell-permeable iron chelator. Kainate-induced SE resulted in a time-dependent increase in chelatable iron in mitochondrial but not cytosolic fractions of the rat hippocampus. Systemically administered N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED), a synthetic iron chelator, ameliorated SE-induced changes in chelatable iron, mitochondrial oxidative stress (8-hydroxy-2' deoxyguanosine and glutathione depletion), mitochondrial DNA integrity and hippocampal cell loss. Measurement of brain HBED levels after systemic administration confirmed its penetration in hippocampal mitochondria. These results suggest a role for mitochondrial iron in the pathogenesis of SE-induced brain damage and subcellular iron chelation as a novel therapeutic approach for its management.

The ketogenic diet increases mitochondrial glutathione levels.

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague-Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of beta-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.

An orally active catalytic metalloporphyrin protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity in vivo.

Parkinson's disease (PD) is an age-related neurodegenerative disease in which the role of reactive oxygen species (ROS) is strongly implicated. The presence of oxidative stress has been detected in human and experimental PD using both direct and indirect indices. Scavenging ROS is, therefore, an important therapeutic avenue for the treatment of PD. Manganic porphyrins are catalytic antioxidants that scavenge a wide range of ROS. In this study, we tested the therapeutic effects of a compound [5,15-bis(methoxycarbonyl)-10,20-bis-trifluoromethyl-porphyrinato manganese (III) chloride (AEOL11207)] belonging to a new generation of lipophilic manganic porphyrins for neuroprotection and oral bioavailability in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. Groups of adult C57BL/6 mice were administered MPTP with varying subcutaneous or oral dosing regimens of AEOL11207. Neurotoxicity was assessed by measurement of striatal dopamine levels and quantification of tyrosine hydroxylase-positive neurons in the substantial nigra pars compacta one week after the first dose of MPTP. Glutathione depletion, lipid peroxidation, and 3-nitrotyrosine (3-NT) formation were measured as indicators of oxidative stress in the ventral midbrain in vivo. AEOL11207 administered either by subcutaneous or oral routes protected against MPTP-induced dopamine depletion in the striatum as well as dopaminergic neuronal loss, glutathione depletion, lipid peroxidation, and 3-NT formation in the ventral midbrain. Neuroprotection correlated with brain metalloporphyrin concentrations. This is the first demonstration of neuroprotection by an orally active catalytic antioxidant in the MPTP mouse model and suggests its potential clinical utility for the treatment of chronic neurodegenerative diseases such as PD.

1-methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons.

Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP(+)). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate-cysteine ligase by 20-25% were observed at early time points and partly account for changes in GSH levels after MPP(+) exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP(+) treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP(+).