RESUMO
A short ischemic episode preceding sustained ischemia is known to increase tolerance against ischemic cell death. We report early-onset long-lasting neuroprotection against in vitro hypoxia by preceding selective chemical inhibition of oxidative phosphorylation: "chemical preconditioning." The amplitude of CA1 population spikes (psap) in hippocampal slices prepared from control animals (control slices) was 31 +/- 27% (mean +/- SD) upon 45-min recovery from 15-min in vitro hypoxia. In slices prepared from animals treated in vivo with 20 mg/kg 3-nitropropionate (3-np) 1-24 h prior to slice preparation (preconditioned slices), psap improved to 90 +/- 15% (p < 0.01). Posthypoxic oxygen free radicals were reduced to 65 +/- 10% (mean +/- SD) of control in preconditioned slices (p < 0.05). Posthypoxic neuronal density improved from 52 +/- 15% (mean +/- SD) in control slices to 97 +/- 23% in preconditioned slices (p < 0.001). Glibenclamide, an antagonist at KATP-channels, partly reversed increased hypoxic tolerance. We conclude that chemical preconditioning induces early-onset long-lasting tolerance against in vitro hypoxia. Ultimately, this strategy may be applicable as a neuroprotective strategy in humans.
Assuntos
Hipóxia Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Propionatos/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Isquemia Encefálica/tratamento farmacológico , Radicais Livres , Glibureto/farmacologia , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia Encefálica/tratamento farmacológico , Masculino , NAD/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nitrocompostos , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio , Succinato Desidrogenase/antagonistas & inibidoresRESUMO
Antibiotics are used extensively, but in addition to their anti-infectious effects some inhibit cellular energy metabolism. We investigated hypoxic tolerance following in vivo pretreatment with erythromycin and kanamycin, or in vitro pretreatment with ampicillin. Recovery of the CA1 population spike amplitude in hippocampal slices upon 15 min hypoxia improved time-dependently following single i.p. in vivo pretreatment with erythromycin (maximum at 6 h: recovery 90+/-7% (mean s.d.) vs 30% in untreated controls; p<0.01). The hypoxia-induced increase in NADH was smaller in slices that recovered from hypoxia. We conclude that antibiotics increase cellular hypoxic tolerance to a varying extent. Use of antibiotics in experimental studies may, therefore, distort conclusions about hypoxic sensitivity and confounding mechanisms. In contrast, antibiotics may provide an effective strategy to induce chemical preconditioning in humans.
Assuntos
Antibacterianos/farmacologia , Isquemia Encefálica/prevenção & controle , Hipóxia Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ampicilina/farmacologia , Análise de Variância , Animais , Citoproteção , Avaliação Pré-Clínica de Medicamentos , Eritromicina/farmacologia , Fluorometria , Hipocampo/irrigação sanguínea , Técnicas In Vitro , Canamicina/farmacologia , Masculino , NAD/análise , Ratos , Ratos Wistar , Fatores de RiscoRESUMO
Microglial activation is a prominent feature of affected brain areas in multiple system atrophy. Microglia express proinflammatory peptides, which may be a result of activation of nuclear factor-KB. We investigated the nuclear presence of RelA, the 65 kDa subunit of the NF-KB/RelA family in striatum and brain stem of patients with multiple system atrophy. Affected brain areas of patients with multiple system atrophy showed a marked immunoreactivity for nuclear Rel A p65, which was almost exclusively localized in activated microglia. Interestingly nuclear translocation of Rel A was not detected in striatal tissue of controls and Parkinson disease patients. Thus, NF-kappaB/Rel A complexes may play a role in mediating microglial activation in multiple system atrophy.
Assuntos
Microglia/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , NF-kappa B/fisiologia , Idoso , Tronco Encefálico/química , Tronco Encefálico/patologia , Núcleo Celular/química , Corpo Estriado/química , Corpo Estriado/patologia , Citoplasma/química , Humanos , Imuno-Histoquímica , Antígeno de Macrófago 1/análise , Microglia/química , Microglia/patologia , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/patologia , NF-kappa B/análise , Fator de Transcrição RelARESUMO
Glutamate antagonists mitigate hypoxic damage upon acute inhibition of energy metabolism. The goal of this study was to investigate their effect on increased hypoxic tolerance induced by preceding chemical inhibition of energy metabolism. While recovery of population spike amplitude (psap) is 30% of onset in slices prepared from control animals (15 min hypoxia, 45 min recovery), recovery exceeds 90% in slices prepared from animals that underwent mild chemical hypoxia in vivo by treatment with 20 mg/kg 3-nitropropionic acid 1 h prior to slice preparation (p-slices). In p-slices perfused for 5 min with D(-)-2-amino-5-phosphonopentanoic acid (APV) (100 microM) 45 min prior to hypoxia, recovery declines to 42 +/- 13% (mean +/- SEM). In contrast, posthypoxic recovery after similar perfusion with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) is 72 +/- 15% (P < 0.05). We conclude that increased hypoxic tolerance is abolished by N-methyl-D-aspartate (NMDA)-antagonists but not non-NMDA-antagonists.
Assuntos
Hipóxia Encefálica/fisiopatologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Metabolismo Energético/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipóxia Encefálica/induzido quimicamente , Hipóxia Encefálica/metabolismo , Técnicas In Vitro , Masculino , Nitrocompostos , Consumo de Oxigênio/fisiologia , Propionatos/farmacologia , Ratos , Ratos WistarRESUMO
Chemical preconditioning with low dose inhibition of succinic dehydrogenase by 3-nitropropionic acid (3-np) increases tolerance against succeeding hypoxia. Supraphysiological doses of nerve growth factor (NGF) repeatedly were shown to protect against ischemic damage. We investigated whether increased tolerance against hypoxia results from increased or accelerated production of endogenous NGF. Average recovery of population spike amplitude after 15 min of hypoxia and 45 min of reoxygenation was 31 +/- 9% (mean +/- SE) in control hippocampal slices. After pretreatment with 3-np (single i.p. injection of 20 mg/kg body weight 1 h to 3 days prior to slice preparation), recovery exceeded 90% (P < 0.01). However, NGF content did not increase upon slice preparation, hypoxia in vitro, and pretreatment with 3-np in vivo 1 h to 1 day prior to slice preparation with and without additional hypoxia in vitro. We conclude that early-onset tolerance to hypoxia induced by 3-np treatment is not caused by induction of endogenous NGF production.
Assuntos
Inibidores Enzimáticos/farmacologia , Hipocampo/fisiopatologia , Hipóxia Encefálica/fisiopatologia , Precondicionamento Isquêmico , Fatores de Crescimento Neural/fisiologia , Propionatos/farmacologia , Succinato Desidrogenase/antagonistas & inibidores , Animais , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Nitrocompostos , Fosforilação Oxidativa/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Energy metabolism is impaired in the Cu,Zn superoxide dismutase transgenic mouse model of amyotrophic lateral sclerosis. The goal was to investigate tolerance against single and repetitive hypoxia in C57B6SJL-TgN(SOD1-G93A)1GUR mice (G93A mice). Posthypoxic recovery (15 min hypoxia, 45 min recovery) of population spike amplitude in hippocampal region CA1 was 38 +/- 29% (mean +/- SD) in controls and 67 +/- 41% (ns) in G93A mice at day 40. Upon in vivo pretreatment with 20 mg/kg 3-nitropropionate posthypoxic recovery increased to 82 +/- 32% (P < 0.01) in controls and decreased to 35 +/- 33% in G93A mice (P < 0.05 to pretreated controls). Results at day 80 and 110 were similar. We conclude that G93A mice show a long-lasting impairment to sustain repetitive hypoxic episodes whereas tolerance to a single hypoxic episode is comparable to controls.
Assuntos
Hipóxia Celular/genética , Hipocampo/fisiologia , Superóxido Dismutase/genética , Transmissão Sináptica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Masculino , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND AND PURPOSE: Treatment with acetylsalicylic acid (ASA) is established for secondary stroke prevention. Recent studies showed neuroprotection of ASA against glutamatergic excitants. The goal of this study was to investigate the time course of neuroprotection of ASA against indirect excitotoxicity by hypoxic hypoxia and chemical hypoxia. METHODS: Population spike amplitude (PSA) and ATP content were measured in hippocampal slices from untreated control animals (c-slices) and slices prepared from animals pretreated in vivo with a single intraperitoneal injection of 20 mg/kg body wt ASA 1 to 48 hours before slice preparation (p-slices). RESULTS: Posthypoxic recovery of PSA was 30% in c-slices (15 minutes of hypoxia, 45 minutes of recovery). When c-slices were treated in vitro for 15 minutes with 20 mg/L ASA 30 minutes before hypoxia, posthypoxic recovery improved to 82 +/- 4% (mean +/- SE, P < .01). In p-slices, posthypoxic recovery of PSA improved in a time-dependent manner. With a time interval of 1 hour between in vivo pretreatment with ASA and slice preparation, posthypoxic recovery of PSA was 64 +/- 16% (P < .05). With time intervals of 6 hours, 24 hours, and 48 hours, posthypoxic recovery of PSA was 87 +/- 19% (P < .01), 59 +/- 12%, and 40 +/- 9%, respectively. Pretreatment with ASA in vitro or in vivo decreased the decline of ATP content during hypoxic hypoxia and chemical hypoxia (inhibition of succinic dehydrogenase by 3-nitropropionic acid). When extracellular glucose was reduced to 4 mmol/L, no difference was observed between c-slices and p-slices. CONCLUSIONS: We conclude that ASA is neuroprotective against hypoxic hypoxia and chemical hypoxia and delays the decline of intracellular ATP content.
Assuntos
Aspirina/uso terapêutico , Hipóxia/induzido quimicamente , Hipóxia/fisiopatologia , Fármacos Neuroprotetores/uso terapêutico , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Animais , Espaço Extracelular/metabolismo , Glucose/metabolismo , Hipocampo/metabolismo , Masculino , Nitrocompostos , Propionatos , Ratos , Ratos Wistar , Succinato Desidrogenase/antagonistas & inibidores , Fatores de TempoRESUMO
Brain slices are used extensively for biochemical, electrophysiological and molecular investigations. However, only the time frame for electrophysiological and biochemical investigations has as yet been defined. The goal of the present study was to investigate the time course of nuclear structure in live brain slices. Hippocampal slices (300 microm) were prepared from male CD1 mice (25-30 g), stained with Hoechst 33342 (10 microM), calcein-AM (2 microM) and ethidium homodimer (4 microM), and imaged with single- and dual-photon microscopy. The volume of CA1 pyramidal cell nuclei decreased from 759+/-229 microm3 in 40-50 microm depth 25 min after preparation to 453+/-169 microm3 (P<0.001) after 60 min, 315+/-112 microm3 (P<0.001) after 120 min and 128+/-71 microm3 (P<0.001) after 8 h. Similar results were obtained on a prolonged time scale in 70-80 microm depth and with an accelerated time scale in 20-30 microm depth. Live-dead staining showed that cell damage is progressing from the surface to deeper layers of the slices in a time-dependent fashion. We conclude that nuclei of CA1 hippocampal pyramidal cells show a time- and depth-dependent shrinkage converging 8 h after slice preparation to a volume of 90-130 microm; in any depth between 20 and 80 microm. The nucleus in the superficial 80 microm of each side appears dysfunctional even at times suitable for electrophysiological and biochemical experimentation in hippocampal slices. Molecular analysis of cell regulation in brain slices may, therefore, be time-dependently distorted by progressing cell death in at least half of the tissue under investigation.
Assuntos
Morte Celular/fisiologia , Núcleo Celular/patologia , Sobrevivência Celular/fisiologia , Etídio/análogos & derivados , Hipocampo/patologia , Degeneração Neural/patologia , Células Piramidais/patologia , Absorciometria de Fóton , Animais , Benzimidazóis , Tamanho Celular/fisiologia , Fluoresceínas , Corantes Fluorescentes , Hipocampo/fisiopatologia , Substâncias Intercalantes , Masculino , Camundongos , Degeneração Neural/fisiopatologia , Rodamina 123 , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Exogenous application of estrogens or progesterone ameliorates hypoxic/ischemic cell damage. This study investigates whether values of primary and induced hypoxic tolerance vary endogenously during the estrus cycle in female mice. METHODS: Population spike amplitude (PSA) and NADH were measured during hypoxic hypoxia and recovery in hippocampal slices from untreated control animals (C slices) and slices prepared from animals pretreated in vivo with a single intraperitoneal injection of 3-nitropropionate (3NP) (3NP slices) or acetylsalicylate (ASA) (ASA slices). RESULTS: Posthypoxic recovery of PSA was dose dependent in 3NP slices from males, with maximal recovery on pretreatment attained with 20 mg/kg 3NP (82+/-32% [mean+/-SD]; C slices, 38+/-29%; P<0.01). PSA recovered to 17+/-12% in C slices during proestrus, 43+/-23% during estrus, and 63+/-44% during diestrus. In 3NP slices, recovery of PSA increased to 57+/-36% (P<0. 05) during proestrus. Hypoxic tolerance was not increased in other stages of the estrus cycle. Hypoxic NADH increase during proestrus declined from 212+/-76% in C slices to 133+/-11% in 3NP slices (P<0. 05). Recovery of PSA in ASA slices was 75+/-36% (P<0.01 versus control) in males and 48+/-34% during proestrus (P<0.05 versus ASA slices from males). CONCLUSIONS: Primary and induced hypoxic tolerance are endogenously modulated during the estrus cycle. Differences in hypoxic oxidative energy metabolism mediate part of the differential tolerance. Experimental and clinical therapeutic strategies against cerebral ischemia/hypoxia need to consider sex-related dependence.