(+)-Naltrexone is neuroprotective and promotes alternative activation in the mouse hippocampus after cardiac arrest/cardiopulmonary resuscitation

doi:10.1016/j.bbi.2015.03.005

Brain, Behavior, and Immunity

Volume 48, August 2015, Pages 115-122

https://doi.org/10.1016/j.bbi.2015.03.005 Get rights and content

Highlights

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TLR4 blockade by (+)-naltrexone is neuroprotective after CA/CPR.
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(+)-Naltrexone decreased expression of CD11b and pro-inflammatory cytokines.
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(+)-Naltrexone elevated Arginase-1 expression, an alternative activation marker.
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(+)-Naltrexone may clinically alleviate memory impairment after CA/CPR.

Abstract

Despite dramatic improvement in cardiopulmonary resuscitation (CPR) and other techniques for cardiac arrest (CA), the majority of survivors continue to show signs of decreased memory or executive cognitive function. Such memory impairment may be due to hippocampal CA1 neuronal death, which is delayed by several days after CA/CPR. Classical microgliosis in the CA1 region may contribute to neuronal death, yet the role of a key activation receptor Toll Like Receptor 4 (TLR4) has not been previously investigated for such neuronal death after CA/CPR. We show that (+)-naltrexone was neuroprotective after CA/CPR. TLR4 blockade was associated with decreased expression of markers for microglial/macrophage activation and T cell and B cell infiltration, as well as decreased pro-inflammatory cytokine levels. Notably, IL-10 expression was elevated in response to CA/CPR, but was not attenuated by (+)-naltrexone, suggesting that the local monocyte/microglial phenotype had shifted towards alternative activation. This was confirmed by elevated expression of Arginase-1, and decreased expression of NFκB p65 subunit. Thus, (+)-naltrexone and other TLR4 antagonists may represent a novel therapeutic strategy to alleviate the substantial burden of memory or executive cognitive function impairment after CA/CPR.

Introduction

Cardiac arrest (CA) is a leading cause of mortality in developed nations (Go et al., 2014). Despite dramatic improvement in cardiopulmonary resuscitation (CPR) techniques and the widespread adoption of hypothermia as a treatment option, the majority of survivors continue to show signs of decreased memory or executive cognitive function, which is a cause of significant and sustained disability (Allen and Buckberg, 2012, Cronberg et al., 2009, Mateen et al., 2011). Such memory impairment is believed to be caused by death of hippocampal CA1 neurons, which are especially sensitive to transient global ischemia (Allen and Buckberg, 2012, Garcia, 1988, Garcia and Anderson, 1989). Since neuronal death is delayed by several days after CA/CPR (Bottiger et al., 1998), there is a unique window of opportunity for therapeutic intervention. Despite the fact that trained medical personnel are present during this window, there are no routine interventions to prevent delayed neuronal death after CA/CPR.

Microglia are the tissue specific macrophages of the central nervous system that rapidly respond to insult, sometimes creating a neurotoxic environment (Kettenmann et al., 2011). Microgliosis may occur in response to a broad array of challenges, including ischemia (Kettenmann et al., 2011), and markers of microgliosis are increased in the CA1 region of the hippocampus after CA/CPR (Norman et al., 2011, Wang et al., 2013a). Attenuation of microgliosis and the neurotoxic products of microglia is generally associated with neuroprotection after global ischemia (Neigh et al., 2009, Takeuchi et al., 2008, Tang et al., 2010, Wang et al., 2013a, Webster et al., 2013, Zhang et al., 2012). Moreover, induction of interleukin 10 (IL-10) producing microglia (alternatively activated microglia) (Locati et al., 2013) is also neuroprotective for CA1 hippocampal neurons after CA/CPR (Wang et al., 2013a). Therefore, classical microgliosis may contribute to neuronal death after CA/CPR. One classical activation pathway is the activation of Toll-Like Receptor 4 (TLR4), which is a key receptor by which microglia and macrophages sense danger in their local environment. Upon cell stress or sterile injury, such as that occurring under ischemic conditions, cells release Danger Associated Molecular Patterns (DAMPs), such as extracellular matrix proteins and heat shock proteins that are agonists at TLR4 (Kawai and Akira, 2010). TLR4 signaling is considered to induce classical activation in peripheral macrophages (Locati et al., 2013), and results in the production of a wide range of neurotoxic mediators by microglia (e.g., tumor necrosis factor (TNF) and IL-1β). We have previously shown that (+)-naltrexone (which does not bind the stereoselective mu opioid receptor), blocks TLR4 signaling and attenuates expression of microglial activation markers and pro-inflammatory cytokine release (Hutchinson et al., 2008). However the neuroprotective capacity of (+)-naltrexone in hippocampal neuronal death after CA/CPR has not been previously addressed. Hence, we aimed to test whether TLR4 blockade by (+)-naltrexone would attenuate CA/CPR-induced neuronal death, microgliosis and production of pro-inflammatory mediators in the hippocampus.

Section snippets

Experimental animals

All experimental protocols were approved by the University of Colorado Denver Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guidelines for the care and use of animals in research. Adult male C57Bl/6 mice (8–12 week old; Charles River Laboratory, Hollister, CA) were used for this study. Mice were housed in temperature- (18–21 °C) and light-controlled (12 h light/dark cycle; lights on at 07:00 h) rooms with standard rodent food and water available ad

(+)-Naltrexone is neuroprotective for hippocampal CA1 neurons after CA/CPR

Male C57Bl/6 mice were subjected to 8 min CA/CPR, resulting in neuronal injury in the CA1 region of hippocampus that was analyzed 3 days after resuscitation. Immediate asystole was observed in all mice following injection of KCl and were successfully resuscitated within the CPR time window of 2 min. We tested the ability of two (+)-naltrexone doses to protect neurons against CA/CPR-induced damage. Mice treated with 3 mg/kg intraperitoneal (+)-naltrexone were significantly protected against ischemic

Discussion

In this study, we show for the first time that TLR4 blockade by (+)-naltrexone is neuroprotective for hippocampal CA1 neurons after CA/CPR. This was primarily associated with elevated expression of the microglial activation marker CD11b, as well as several pro-inflammatory cytokines. These were directly decreased by TLR4 blockade. Notably, the only anti-inflammatory cytokine to be modified under any treatment conditions in the hippocampus was IL-10, whose expression level was furthermore

Acknowledgments

Supported by the University of Colorado Boulder Innovative Seed Grant Program. P.M.G. is an NHMRC CJ Martin Fellow (ID: 1054091) and an American Australian Association Sir Keith Murdoch Fellow. The work of the Drug Design and Synthesis Section, CBRB, NIDA, and NIAAA was supported by the NIH Intramural Research Programs of the National Institute on Drug Abuse (NIDA) and the National Institute of Alcohol Abuse and Alcoholism (NIAAA). The authors have no conflict of interest to declare.

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¹: Authors contributed equally.

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