RESUMO
Mechanical impact-induced primary injury after traumatic brain injury (TBI) leads to acute microglial pro-inflammatory activation and consequently mediates neurodegeneration, which is a major secondary brain injury mechanism. However, the detailed pathologic cascades have not been fully elucidated, partially because of the pathologic complexity in animal TBI models. Although there are several in vitro TBI models, none of them closely mimic post-TBI microglial activation. In the present study, we aimed to establish an in vitro TBI model, specifically reconstituting the pro-inflammatory activation and associated neurodegeneration following TBI. We proposed three sets of experiments. First, we established a needle scratch injured neuron-induced microglial activation and neurodegeneration in vitro model of TBI. Second, we compared microglial pro-inflammatory cytokines profiles between the in vitro TBI model and TBI in male mice. Additionally, we validated the role of injured neurons-derived damage-associated molecular patterns in amplifying microglial pro-inflammatory pathways using the in vitro TBI model. Third, we applied the in vitro model for the first time to characterize the cellular metabolic profile of needle scratch injured-neuron-activated microglia, and define the role of metabolic reprogramming in mediating pro-inflammatory microglial activation and mediated neurodegeneration. Our results showed that we successfully established a novel in vitro TBI model, which closely mimics primary neuronal injury-triggered microglial pro-inflammatory activation and mediated neurodegeneration after TBI. This in vitro model provides an advanced and highly translational platform for dissecting interactions in the pathologic processes of neuronal injury-microglial activation-neuronal degeneration cascade, and elucidating the detailed underlying cellular and molecular insights after TBI.SIGNIFICANCE STATEMENT Microglial activation is a key component of acute neuroinflammation that leads to neurodegeneration and long-term neurologic outcome deficits after TBI. However, it is not feasible to truly dissect primary neuronal injury-induced microglia activation, and consequently mediated neurodegeneration in vivo Furthermore, there is currently lacking of in vitro TBI models closely mimicking the TBI primary injury-mediated microglial activation. In this study, we successfully established and validated a novel in vitro TBI model of microglial activation, and for the first time, characterized the cellular metabolic profile of microglia in this model. This novel microglial activation in vitro TBI model will help in elucidating microglial inflammatory activation and consequently associated neurodegeneration after TBI.
Assuntos
Lesões Encefálicas Traumáticas , Microglia , Camundongos , Masculino , Animais , Microglia/metabolismo , Lesões Encefálicas Traumáticas/patologia , Macrófagos/metabolismo , Neurônios/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Risk factors contributing to dementia are multifactorial. Accumulating evidence suggests a role for pathogens as risk factors, but data is largely correlative with few causal relationships. Here, we demonstrate that intermittent murine cytomegalovirus (MCMV) infection of mice, alters blood brain barrier (BBB) permeability and metabolic pathways. Increased basal mitochondrial function is observed in brain microvessels cells (BMV) exposed to intermittent MCMV infection and is accompanied by elevated levels of superoxide. Further, mice score lower in cognitive assays compared to age-matched controls who were never administered MCMV. Our data show that repeated systemic infection with MCMV, increases markers of neuroinflammation, alters mitochondrial function, increases markers of oxidative stress and impacts cognition. Together, this suggests that viral burden may be a risk factor for dementia. These observations provide possible mechanistic insights through which pathogens may contribute to the progression or exacerbation of dementia.
Assuntos
Transtornos Cognitivos , Disfunção Cognitiva , Infecções por Citomegalovirus , Demência , Animais , Camundongos , Infecções por Citomegalovirus/complicações , CogniçãoRESUMO
Plasma soluble prorenin receptor (sPRR) displays sexual dimorphism and is higher in women with type 2 diabetes mellitus (T2DM). However, the contribution of plasma sPRR to the development of vascular complications in T2DM remains unclear. We investigated if plasma sPRR contributes to sex differences in the activation of the systemic renin-angiotensin-aldosterone system (RAAS) and vascular damage in a model of high-fat diet (HFD)-induced T2DM. Male and female C57BL/6J mice were fed either a normal fat diet (NFD) or an HFD for 28 wk to assess changes in blood pressure, cardiometabolic phenotype, plasma prorenin/renin, sPRR, and ANG II. After completing dietary protocols, tissues were collected from males to assess vascular reactivity and aortic reactive oxygen species (ROS). A cohort of male mice was used to determine the direct contribution of increased systemic sPRR by infusion. To investigate the role of ovarian hormones, ovariectomy (OVX) was performed at 32 wk in females fed either an NFD or HFD. Significant sex differences were found after 28 wk of HFD, where only males developed T2DM and increased plasma prorenin/renin, sPRR, and ANG II. T2DM in males was accompanied by nondipping hypertension, carotid artery stiffening, and aortic ROS. sPRR infusion in males induced vascular thickening instead of material stiffening caused by HFD-induced T2DM. While intact females were less prone to T2DM, OVX increased plasma prorenin/renin, sPRR, and systolic blood pressure. These data suggest that sPRR is a novel indicator of systemic RAAS activation and reflects the onset of vascular complications during T2DM regulated by sex.NEW & NOTEWORTHY High-fat diet (HFD) for 28 wk leads to type 2 diabetes mellitus (T2DM) phenotype, concomitant with increased plasma soluble prorenin receptor (sPRR), nondipping blood pressure, and vascular stiffness in male mice. HFD-fed female mice exhibiting a preserved cardiometabolic phenotype until ovariectomy revealed increased plasma sPRR and blood pressure. Plasma sPRR may indicate the status of systemic renin-angiotensin-aldosterone system (RAAS) activation and the onset of vascular complications during T2DM in a sex-dependent manner.
Assuntos
Diabetes Mellitus Tipo 2 , Hipertensão , ATPases Vacuolares Próton-Translocadoras , Feminino , Masculino , Camundongos , Animais , Renina , Receptor de Pró-Renina , Dieta Hiperlipídica/efeitos adversos , Espécies Reativas de Oxigênio , Camundongos Endogâmicos C57BL , Sistema Renina-Angiotensina/genética , Receptores de Superfície Celular/genética , Pressão SanguíneaRESUMO
Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency.NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.
Assuntos
Encéfalo/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxidos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Catálise , Respiração Celular , Potencial da Membrana Mitocondrial , Metaloporfirinas/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Ácido Peroxinitroso/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.
Assuntos
Inibidores Enzimáticos/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Consumo de Oxigênio/efeitos dos fármacos , Amidinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ornitina/análogos & derivados , Ornitina/farmacologiaRESUMO
Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito-Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito-Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice.NEW & NOTEWORTHY We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.
Assuntos
Circulação Cerebrovascular/fisiologia , Endotélio Vascular/metabolismo , Mitocôndrias/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação MultifotônicaRESUMO
One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROS-mediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance (P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS.NEW & NOTEWORTHY We have characterized for the first time the dynamics of mitochondrial function during the progression of type 2 diabetes mellitus with regard to mitochondrial respiration, protein expression, and reactive oxygen species production. In addition, this is the first measurement of rattin levels in the cerebral vasculature, which could potentially lead to novel treatment options.
Assuntos
Artérias Cerebrais/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Animais , Glicemia/metabolismo , Respiração Celular , Artérias Cerebrais/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Masculino , Mitocôndrias/patologia , Proteínas/metabolismo , Ratos Zucker , Superóxidos/metabolismo , Fatores de TempoAssuntos
Disfunção Cognitiva , Diabetes Mellitus Experimental , Ferroptose , Acidente Vascular Cerebral , Ratos , Feminino , Animais , Terapia por Quelação , Desferroxamina , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/prevenção & controle , Ferro , Hemorragia , Acidente Vascular Cerebral/prevenção & controleRESUMO
The actions of hydrogen sulfide (H2S) on the heart and vasculature have been extensively reported. However, the mechanisms underlying the effects of H2S are unclear in the anesthetized rat. The objective of the present study was to investigate the effect of H2S on the electrocardiogram and examine the relationship between H2S-induced changes in heart rate (HR), mean arterial pressure (MAP), and respiratory function. Intravenous administration of the H2S donor Na2S in the anesthetized Sprague-Dawley rat decreased MAP and HR and produced changes in respiratory function. The administration of Na2S significantly increased the RR interval at some doses but had no effect on PR or corrected QT(n)-B intervals. In experiments where respiration was maintained with a mechanical ventilator, we observed that Na2S-induced decreases in MAP and HR were independent of respiration. In experiments where respiration was maintained by mechanical ventilation and HR was maintained by cardiac pacing, Na2S-induced changes in MAP were not significantly altered, whereas changes in HR were abolished. Coadministration of glybenclamide significantly increased MAP and HR responses at some doses, but methylene blue, diltiazem, and ivabradine had no significant effect compared with control. The decreases in MAP and HR in response to Na2S could be dissociated and were independent of changes in respiratory function, ATP-sensitive K+ channels, methylene blue-sensitive mechanism involving L-type voltage-sensitive Ca2+ channels, or hyperpolarization-activated cyclic nucleotide-gated channels. Cardiovascular responses observed in spontaneously hypertensive rats were more robust than those in Sprague-Dawley rats.NEW & NOTEWORTHY H2S is a gasotransmitter capable of producing a decrease in mean arterial pressure and heart rate. The hypotensive and bradycardic effects of H2S can be dissociated, as shown with cardiac pacing experiments. Responses were not blocked by diltiazem, ivabradine, methylene blue, or glybenclamide.
Assuntos
Pressão Arterial/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Sulfetos/farmacologia , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Estimulação Cardíaca Artificial , Eletrocardiografia/efeitos dos fármacos , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Canais KATP/efeitos dos fármacos , Masculino , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Respiração ArtificialRESUMO
Mitochondrial dysfunction has been suggested as a potential underlying cause of pathological conditions associated with type 2 diabetes (T2DM). We have previously shown that mitochondrial respiration and mitochondrial protein levels were similar in the large cerebral arteries of insulin-resistant Zucker obese rats and their lean controls. In this study, we extend our investigations into the mitochondrial dynamics of the cerebral vasculature of 14-week-old Zucker diabetic fatty obese (ZDFO) rats with early T2DM. Body weight and blood glucose levels were significantly higher in the ZDFO group, and basal mitochondrial respiration and proton leak were significantly decreased in the large cerebral arteries of the ZDFO rats compared with the lean controls (ZDFL). The expression of the mitochondrial proteins total manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC) were significantly lower in the cerebral microvessels, and acetylated MnSOD levels were significantly reduced in the large arteries of the ZDFO group. Additionally, superoxide production was significantly increased in the microvessels of the ZDFO group. Despite evidence of increased oxidative stress in ZDFO, exogenous SOD was not able to restore mitochondrial respiration in the ZDFO rats. Our results show, for the first time, that mitochondrial respiration and protein levels are compromised during the early stages of T2DM.
Assuntos
Artérias Cerebrais/metabolismo , Transtornos Cerebrovasculares/etiologia , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/etiologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Acetilação , Animais , Glicemia/metabolismo , Peso Corporal , Respiração Celular , Artérias Cerebrais/efeitos dos fármacos , Transtornos Cerebrovasculares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Angiopatias Diabéticas/metabolismo , Modelos Animais de Doenças , Sequestradores de Radicais Livres/farmacologia , Masculino , Microvasos/metabolismo , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo , Ratos Zucker , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Little is known about mitochondrial functioning in the cerebral vasculature during insulin resistance (IR). We examined mitochondrial respiration in isolated cerebral arteries of male Zucker obese (ZO) rats and phenotypically normal Zucker lean (ZL) rats using the Seahorse XFe24 analyzer. We investigated mitochondrial morphology in cerebral blood vessels as well as mitochondrial and nonmitochondrial protein expression levels in cerebral arteries and microvessels. We also measured reactive oxygen species (ROS) levels in cerebral microvessels. Under basal conditions, the mitochondrial respiration components (nonmitochondrial respiration, basal respiration, ATP production, proton leak, and spare respiratory capacity) showed similar levels among the ZL and ZO groups with the exception of maximal respiration, which was higher in the ZO group. We examined the role of nitric oxide by measuring mitochondrial respiration following inhibition of nitric oxide synthase with N(ω)-nitro-l-arginine methyl ester (l-NAME) and mitochondrial activation after administration of diazoxide (DZ). Both ZL and ZO groups showed similar responses to these stimuli with minor variations.l-NAME significantly increased the proton leak, and DZ decreased nonmitochondrial respiration in the ZL group. Other components were not affected. Mitochondrial morphology and distribution within vascular smooth muscle and endothelium as well as mitochondrial protein levels were similar in the arteries and microvessels of both groups. Endothelial nitric oxide synthase (eNOS) and ROS levels were increased in cerebral microvessels of the ZO. Our study suggests that mitochondrial function is not significantly altered in the cerebral vasculature of young ZO rats, but increased ROS production might be due to increased eNOS in the cerebral microcirculation during IR.
Assuntos
Artérias Cerebrais/metabolismo , Resistência à Insulina , Microvasos/metabolismo , Mitocôndrias/metabolismo , Obesidade/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular , Endotélio Vascular/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismoRESUMO
The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.
Assuntos
Artérias Cerebrais/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/enzimologia , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Comunicação Parácrina , Vasodilatação , Animais , Benzopiranos/farmacologia , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/inervação , Diazóxido/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Indazóis/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios Nitrérgicos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Comunicação Parácrina/efeitos dos fármacos , Fosforilação , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Cultura Primária de Células , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Serina , Transdução de Sinais , Vasodilatação/efeitos dos fármacosRESUMO
We examined the role of the mechanistic target of rapamycin (mTOR) pathway in delayed diazoxide (DZ)-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for 3 days with 500 µM DZ or feeding medium and then exposed to 3 h of continuous normoxia in Dulbecco's modified eagle medium with glucose or with 3 h of oxygen-glucose deprivation (OGD) followed by normoxia and feeding medium. The OGD decreased viability by 50%, depolarized mitochondria, and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Neuroprotection by DZ was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). The mTOR inhibitors rapamycin and Torin-1, as well as S6K-targeted siRNA abolished the protective effects of DZ. The effects of DZ on mitochondrial membrane potential and reactive oxygen species production were not affected by rapamycin. Preconditioning with DZ also changed mitochondrial and non-mitochondrial oxygen consumption rates. We conclude that in addition to reducing reactive oxygen species (ROS) production and mitochondrial membrane depolarization, DZ protects against OGD by activation of the Akt-mTOR-S6K pathway and by changes in mitochondrial respiration. Ischemic strokes have limited therapeutic options. Diazoxide (DZ) preconditioning can reduce neuronal damage. Using oxygen-glucose deprivation (OGD), we studied Akt/mTOR/S6K signaling and mitochondrial respiration in neuronal preconditioning. We found DZ protects neurons against OGD via the Akt/mTOR/S6K pathway and alters the mitochondrial and non-mitochondrial oxygen consumption rate. This suggests that the Akt/mTOR/S6k pathway and mitochondria are novel stroke targets.
Assuntos
Diazóxido/farmacologia , Precondicionamento Isquêmico , Proteínas do Tecido Nervoso/fisiologia , Neurônios/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Meios de Cultura/farmacologia , Ativação Enzimática/efeitos dos fármacos , Técnicas In Vitro , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/metabolismo , Oxigênio/farmacologia , Consumo de Oxigênio , Fosforilação , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
The objective of the present study was to determine whether mitochondrial function in the cerebral vasculature is maintained after transient middle cerebral artery (MCA) occlusion (tMCAO) in rats. Sprague-Dawley rats were exposed to 90 min of tMCAO followed by 4 or 48 h of reperfusion. MCAs from ischemic (ipsilateral) and nonischemic (contralateral) sides were compared with control MCAs from sham-operated rats. We determined 1) vasoreactivity to diazoxide (DZ; a mitochondrial ATP-activated K(+) channel opener), ACh, bradykinin (BK), serotonin, and sodium nitroprusside; 2) levels of mitochondrial and nonmitochondrial proteins and mitochondrial DNA; and 3) vascular levels of tetramethylrhodamine ethyl ester (an indicator of mitochondrial membrane potential). All dilator responses, including those with DZ, were intact 4 h post-tMCAO. Dilator responses to ACh, BK, and sodium nitroprusside were reduced in ipsilateral MCAs at 48 h compared with contralateral MCAs, but DZ responses were comparable with control MCAs. Surprisingly, contralateral responses to ACh, BK, and serotonin were reduced compared with control MCAs at 48 h. Ipsilateral vasodilation to DZ at 48 h was eliminated by endothelial denudation and endothelial nitric oxide synthase (eNOS) inhibition but was only reduced in control MCAs. Mitochondrial proteins, phosphorylated eNOS, mitochondrial DNA, and mitochondrial membrane potential were higher in ipsilateral compared with contralateral MCAs. In conclusion, contrary to conventional wisdom, mitochondria remain functional for at least 48 h after severe ischemic stress in MCAs, and DZ-induced dilation is preserved due to maintained mitochondrial mass, probably in the endothelium, and eNOS signaling. Our findings support the concept that functioning vascular mitochondria are an unexpected target for novel stroke therapies.
Assuntos
Artérias Cerebrais/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Mitocôndrias/metabolismo , Acetilcolina/farmacologia , Animais , Bradicinina/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/fisiologia , Diazóxido/farmacologia , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/farmacologia , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologia , VasoconstriçãoRESUMO
Mitochondrial depolarization following ATP-sensitive potassium (mitoKATP) channel activation has been shown to induce cerebral vasodilation by generation of mitochondrial reactive oxygen species (ROS), which sequentially promotes frequency of calcium sparks and activation of large conductance calcium-activated potassium channels (BKCa) in vascular smooth muscle (VSM). We previously demonstrated that cerebrovascular insulin resistance accompanies aging and obesity. It is unclear whether mitochondrial depolarization without the ROS generation enhances calcium sparks and vasodilation in phenotypically normal [Sprague Dawley (SD); Zucker lean (ZL)] and insulin-resistant [Zucker obese (ZO)] rats. We compared the mechanisms underlying the vasodilation to ROS-dependent (diazoxide) and ROS-independent [BMS-191095 (BMS)] mitoKATP channel activators in normal and ZO rats. Arterial diameter studies from SD, ZL, and ZO rats showed that BMS as well as diazoxide induced vasodilation in endothelium-denuded cerebral arteries. In normal rats, BMS-induced vasodilation was mediated by mitochondrial depolarization and calcium sparks generation in VSM and was reduced by inhibition of BKCa channels. However, unlike diazoxide-induced vasodilation, scavenging of ROS had no effect on BMS-induced vasodilation. Electron spin resonance spectroscopy confirmed that diazoxide but not BMS promoted vascular ROS generation. BMS- as well as diazoxide-induced vasodilation, mitochondrial depolarization, and calcium spark generation were diminished in cerebral arteries from ZO rats. Thus pharmacological depolarization of VSM mitochondria by BMS promotes ROS-independent vasodilation via generation of calcium sparks and activation of BKCa channels. Diminished generation of calcium sparks and reduced vasodilation in ZO arteries in response to BMS and diazoxide provide new insights into mechanisms of cerebrovascular dysfunction in insulin resistance.
Assuntos
Artérias Cerebrais/metabolismo , Resistência à Insulina , Mitocôndrias Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Vasodilatação , Animais , Benzopiranos/farmacologia , Sinalização do Cálcio , Artérias Cerebrais/fisiologia , Diazóxido/farmacologia , Imidazóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Potencial da Membrana Mitocondrial , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Vasodilatadores/farmacologiaRESUMO
OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.
Assuntos
Artérias Cerebrais/metabolismo , Circulação Cerebrovascular , Células Endoteliais/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Canais de Potássio/metabolismo , Vasodilatação , Animais , Benzopiranos/farmacologia , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.
Assuntos
Hipocampo , Transtornos de Estresse Pós-Traumáticos , Ratos , Animais , Espécies Reativas de Oxigênio/farmacologia , Hipocampo/metabolismo , Região CA3 Hipocampal/metabolismo , Aprendizagem da Esquiva/fisiologia , Giro Denteado/metabolismoRESUMO
ANG II-stimulated production of reactive oxygen species (ROS) through NADPH oxidase is suggested to activate MAPK pathways, which are implicated in neurally mediated pressor effects of ANG II. Emerging evidence suggests that ANG-(1-7) up regulates MAPK phosphatases to reduce MAPK signaling and attenuate actions of ANG II. Whether angiotensin peptides participate in long-term regulation of these systems in the brain is not known. Therefore, we determined tissue and mitochondrial ROS, as well as expression and activity of MAPK phosphatase-1 (MKP-1) in brain dorsal medullary tissue of hypertensive transgenic (mRen2)27 rats exhibiting higher ANG II/ANG-(1-7) tone or hypotensive transgenic rats with targeted decreased glial expression of angiotensinogen, ASrAOGEN (AS) exhibiting lower ANG II/ANG-(1-7) tone compared with normotensive Sprague-Dawley (SD) rats that serve as the control strain. Transgenic (mRen2)27 rats showed higher medullary tissue NADPH oxidase activity and dihydroethidium fluorescence in isolated mitochondria vs. SD or AS rats. Mitochondrial uncoupling protein 2 was lower in AS and unchanged in (mRen2)27 compared with SD rats. MKP-1 mRNA and protein expression were higher in AS and unchanged in (mRen2)27 compared with SD rats. AS rats also had lower phosphorylated ERK1/2 and JNK consistent with higher MKP-1 activity. Thus, an altered brain renin-angiotensin system influences oxidative stress status and regulates MKP-1 expression. However, there is a dissociation between these effects and the hemodynamic profiles. Higher ROS was associated with hypertension in (mRen2)27 and normal MKP-1, whereas the higher MKP-1 was associated with hypotension in AS, where ROS was normal relative to SD rats.
Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , Hipertensão/enzimologia , Bulbo/enzimologia , Estresse Oxidativo , Sistema Renina-Angiotensina , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Angiotensinogênio/genética , Angiotensinogênio/metabolismo , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Fosfatase 1 de Especificidade Dupla/genética , Regulação da Expressão Gênica , Hipertensão/genética , Hipertensão/fisiopatologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NADPH Oxidases/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Renina/genética , Renina/metabolismo , Sistema Renina-Angiotensina/genética , Transdução de Sinais , Proteína Desacopladora 2RESUMO
Differentially expressed (DE) proteins in the cortical microvessels (MVs) of young, middle-aged, and old male and female mice were evaluated using discovery-based proteomics analysis (> 4,200 quantified proteins/group). Most DE proteins (> 90%) showed no significant differences between the sexes; however, some significant DE proteins showing sexual differences in MVs decreased from young (8.3%), to middle-aged (3.7%), to old (0.5%) mice. Therefore, we combined male and female data for age-dependent comparisons but noted sex differences for examination. Key proteins involved in the oxidative stress response, mRNA or protein stability, basement membrane (BM) composition, aerobic glycolysis, and mitochondrial function were significantly altered with aging. Relative abundance of superoxide dismutase-1/-2, catalase and thioredoxin were reduced with aging. Proteins participating in either mRNA degradation or pre-mRNA splicing were significantly increased in old mice MVs, whereas protein stabilizing proteins decreased. Glycolytic proteins were not affected in middle age, but the relative abundance of these proteins decreased in MVs of old mice. Although most of the 41 examined proteins composing mitochondrial complexes I-V were reduced in old mice, six of these proteins showed a significant reduction in middle-aged mice, but the relative abundance increased in fourteen proteins. Nidogen, collagen, and laminin family members as well as perlecan showed differing patterns during aging, indicating BM reorganization starting in middle age. We suggest that increased oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability, BM integrity, and ATP synthesis capacity.
Assuntos
Mitocôndrias , Proteômica , Animais , Membrana Basal , Encéfalo/metabolismo , Feminino , Glicólise/genética , Masculino , Camundongos , Microvasos/metabolismo , Mitocôndrias/metabolismo , Estabilidade Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
In the pathophysiology of hemorrhagic stroke, the perturbation of the neurovascular unit (NVU), a functional group of the microvascular and brain intrinsic cellular components, is implicated in the progression of secondary injury and partially informs the ultimate patient outcome. Given the broad NVU functions in maintaining healthy brain homeostasis through its maintenance of nutrients and energy substrates, partitioning central and peripheral immune components, and expulsion of protein and metabolic waste, intracerebral hemorrhage (ICH)-induced dysregulation of the NVU directly contributes to numerous destructive processes in the post-stroke sequelae. In ICH, the damaged NVU precipitates the emergence and evolution of perihematomal edema as well as the breakdown of the blood-brain barrier structural coherence and function, which are critical facets during secondary ICH injury. As a gateway to the central nervous system, the NVU is among the first components to interact with the peripheral immune cells mobilized toward the injured brain. The release of signaling molecules and direct cellular contact between NVU cells and infiltrating leukocytes is a factor in the dysregulation of NVU functions and further adds to the acute neuroinflammatory environment of the ICH brain. Thus, the interactions between the NVU and immune cells, and their reverberating consequences, are an area of increasing research interest for understanding the complex pathophysiology of post-stroke injury. This review focuses on the interactions of T-lymphocytes, a major cell of the adaptive immunity with expansive effector function, with the NVU in the context of ICH. In cataloging the relevant clinical and experimental studies highlighting the synergistic actions of T-lymphocytes and the NVU in ICH injury, this review aimed to feature emergent knowledge of T cells in the hemorrhagic brain and their diverse involvement with the neurovascular unit in this disease.