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Minocycline attenuates brain tissue levels of TNF-? produced by neurons after prolonged hypothermic cardiac arrest in rats.

ABSTRACT: Neuro-cognitive disabilities are a well-recognized complication of hypothermic circulatory arrest. We and others have reported that prolonged cardiac arrest (CA) produces neuronal death and microglial proliferation and activation that are only partially mitigated by hypothermia. Microglia, and possibly other cells, are suggested to elaborate tumor necrosis factor alpha (TNF-?), which can trigger neuronal death cascades and exacerbate edema after CNS insults. Minocycline is neuroprotective in some brain ischemia models in part by blunting the microglial response. We tested the hypothesis that minocycline would attenuate neuroinflammation as reflected by brain tissue levels of TNF-? after hypothermic CA in rats. Rats were subjected to rapid exsanguination, followed by a 6 min normothermic CA. Hypothermia (30 °C) was then induced by an aortic saline flush. After a total of 20 min CA, resuscitation was achieved via cardiopulmonary bypass (CPB). After 5 min reperfusion, minocycline (90 mg kg?1; n = 6) or vehicle (PBS; n = 6) was given. Hypothermia (34 °C) was maintained for 6 h. Rats were sacrificed at 6 or 24 h. TNF-? was quantified (ELISA) in four brain regions (cerebellum, CEREB; cortex, CTX; hippocampus, HIP; striatum, STRI). Naïve rats (n = 6) and rats subjected to the same anesthesia and CPB but no CA served as controls (n = 6). Immunocytochemistry was used to localize TNF-?. Naïve rats and CPB controls had no detectable TNF-? in any brain region. CA markedly increased brain TNF-?. Regional differences were seen, with the highest TNF-? levels in striatum in CA groups (10-fold higher, P < 0.05 vs. all other brain regions). TNF-? was undetectable at 24 h. Minocycline attenuated TNF-? levels in CTX, HIP and STRI (P < 0.05). TNF-? showed unique co-localization with neurons. In conclusion, we report region-dependent early increases in brain TNF-? levels after prolonged hypothermic CA, with maximal increases in striatum. Surprisingly, TNF-? co-localized in neurons and not microglia. Minocycline attenuated TNF-? by approximately 50% but did not totally ablate its production. That minocycline decreased brain TNF-? levels suggests that it may represent a therapeutic adjunct to hypothermia in CA neuroprotection. University of Pittsburgh IACUC 0809278B-3.

SUBMITTER: Drabek T

PROVIDER: S-EPMC3952024 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Minocycline attenuates brain tissue levels of TNF-α produced by neurons after prolonged hypothermic cardiac arrest in rats.

Drabek Tomas T Janata Andreas A Wilson Caleb D CD Stezoski Jason J Janesko-Feldman Keri K Tisherman Samuel A SA Foley Lesley M LM Verrier Jonathan D JD Kochanek Patrick M PM

Resuscitation 20140201 2

Neuro-cognitive disabilities are a well-recognized complication of hypothermic circulatory arrest. We and others have reported that prolonged cardiac arrest (CA) produces neuronal death and microglial proliferation and activation that are only partially mitigated by hypothermia. Microglia, and possibly other cells, are suggested to elaborate tumor necrosis factor alpha (TNF-α), which can trigger neuronal death cascades and exacerbate edema after CNS insults. Minocycline is neuroprotective in som ...[more]

PMID: 24513126

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Project description:BACKGROUND:Hypoxic-ischemic encephalopathy (HIE) has a high morbidity rate and involves severe neurologic deficits, including cerebral palsy. Therapeutic hypothermia (TH) has been shown to decrease the mortality rate and provide neuroprotection in infants with HIE. However, death and disability rates in HIE infants treated with TH remain high. Although the cellular mechanism of the neuroprotective effect of TH remains unclear, astrocytic erythropoietin (EPO) is known to be a key mediator of neuroprotection under hypoxic conditions. In the present study, we investigated the hypothermia effect on EPO expression in astrocytes and determined whether hypothermia attenuates neuronal damage via EPO signaling. METHODS:Astrocytes derived from rat cerebral cortex were cultured under oxygen/glucose deprivation (OGD). The expression of EPO and hypoxia-inducible factor (HIF), a transcription factor of EPO, was assessed. After OGD, astrocytes were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions, and then EPO and HIF expression was assessed. After OGD, rat cortical neurons were cultured in astrocyte-conditioned medium (ACM) derived from the hypothermic group, and neuronal apoptosis was evaluated. RESULTS:OGD induced EPO mRNA and protein expression, although at lower levels than hypoxia alone. HIF-1? and HIF-2? protein expression increased under hypoxia alone and OGD, although OGD increased HIF-2? protein expression less than hypoxia alone. EPO gene and protein expression after OGD was significantly higher under hypothermia. Moreover, expression of HIF-1? and HIF-2? protein was enhanced under hypothermia. In the presence of ACM derived from hypothermic astrocytes following OGD, the number of cleaved caspase 3 and TdT-mediated dUTP nick-end labeling-positive apoptotic neurons was lower than in the presence of ACM from normothermic astrocytes following OGD. Blockade of EPO signaling using anti-EPO neutralization antibody attenuated the anti-apoptotic effect of ACM derived from hypothermic astrocytes following OGD. CONCLUSIONS:Hypothermia after OGD stabilized HIF-EPO signaling in astrocytes, and upregulated EPO expression could suppress neuronal apoptosis. Investigating the neuroprotective effect of EPO from astrocytes under hypothermic conditions may contribute to the development of novel neuroprotection-based therapies for HIE.

Project description:BackgroundDespite the benefits of extracorporeal cardiopulmonary resuscitation (ECPR) in cohorts of selected patients with cardiac arrest (CA), extracorporeal membrane oxygenation (ECMO) includes an artificial oxygenation membrane and circuits that contact the circulating blood and induce excessive oxidative stress and inflammatory responses, resulting in coagulopathy and endothelial cell damage. There is currently no pharmacological treatment that has been proven to improve outcomes after CA/ECPR. We aimed to test the hypothesis that administration of hydrogen gas (H2) combined with ECPR could improve outcomes after CA/ECPR in rats.MethodsRats were subjected to 20 min of asphyxial CA and were resuscitated by ECPR. Mechanical ventilation (MV) was initiated at the beginning of ECPR. Animals were randomly assigned to the placebo or H2 gas treatment groups. The supplement gas was administered with O2 through the ECMO membrane and MV. Survival time, electroencephalography (EEG), brain functional status, and brain tissue oxygenation were measured. Changes in the plasma levels of syndecan-1 (a marker of endothelial damage), multiple cytokines, chemokines, and metabolites were also evaluated.ResultsThe survival rate at 4 h was 77.8% (7 out of 9) in the H2 group and 22.2% (2 out of 9) in the placebo group. The Kaplan-Meier analysis showed that H2 significantly improved the 4 h-survival endpoint (log-rank P = 0.025 vs. placebo). All animals treated with H2 regained EEG activity, whereas no recovery was observed in animals treated with placebo. H2 therapy markedly improved intra-resuscitation brain tissue oxygenation and prevented an increase in central venous pressure after ECPR. H2 attenuated an increase in syndecan-1 levels and enhanced an increase in interleukin-10, vascular endothelial growth factor, and leptin levels after ECPR. Metabolomics analysis identified significant changes at 2 h after CA/ECPR between the two groups, particularly in D-glutamine and D-glutamate metabolism.ConclusionsH2 therapy improved mortality in highly lethal CA rats rescued by ECPR and helped recover brain electrical activity. The underlying mechanism might be linked to protective effects against endothelial damage. Further studies are warranted to elucidate the mechanisms responsible for the beneficial effects of H2 on ischemia-reperfusion injury in critically ill patients who require ECMO support.

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Minocycline attenuates brain tissue levels of TNF-? produced by neurons after prolonged hypothermic cardiac arrest in rats.

Publications

Minocycline attenuates brain tissue levels of TNF-α produced by neurons after prolonged hypothermic cardiac arrest in rats.

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