Ethyl pyruvate protects against hypoxic-ischemic brain injury via anti-cell death and anti-inflammatory mechanisms

https://doi.org/10.1016/j.nbd.2009.12.010Get rights and content

Abstract

Ethyl pyruvate (EP) is protective in experimental models of many illnesses. This study investigates whether EP can protect against neonatal hypoxic-ischemic (H-I) brain injury. Pre-treatment with EP significantly reduced brain damage at 7 days post-H-I, with 50 mg/kg EP achieving over 50% recovery in tissue loss compared to vehicle-treated animals. Delayed treatment with EP until 30 min after H-I was still neuroprotective. EP-afforded brain protection, together with neurological function improvement, was observed up to 2 months after H-I. We further demonstrated an inhibitory effect of EP on cell death, both in an in vivo model of H-I and in in vitro neuronal cultures subjected to OGD, by reducing calpain activation and calcium dysregulation. Moreover, EP exerted an anti-inflammatory effect in microglia by inhibiting NF-κB activation and subsequent release of inflammatory mediators. Taken together, our results suggest that EP confers potent neuroprotection against neonatal H-I brain injury via its anti-cell death and anti-inflammatory actions. EP is a potential novel therapeutic agent for neonatal H-I brain injury.

Introduction

Perinatal hypoxic-ischemic (H-I) brain injury is a common cause of neurological deficits in children (Chang and Huang, 2006). The mechanisms of H-I-induced brain damage are heterogeneous. A complex series of pathological events including apoptosis, inflammation, oxidative stress and excitotoxicity after H-I injury are known to precipitate neuronal demise and subsequent neurological dysfunction (Benjelloun, 1999, Blomgren and Hagberg, 2006, Johnston, 2005, Northington, 2001). H-I lesion to the brain has been shown to result in earlier acute necrotic neuronal death in the core of the infarction, followed by a delayed but prolonged apoptotic-like neuronal death in the surrounding zone (Nakajima, 2000, Northington, 2001). A rapidly expanding body of evidence also indicates that cerebral inflammation, characterized by activation of microglia and macrophages, leukocyte infiltration, release of pro-inflammatory cytokines, and increased expression of endothelial adhesion molecules (Barone and Feuerstein, 1999, del Zoppo, 2000), also contributes substantially to the pathogenesis of perinatal H-I brain injury (Benjelloun et al., 1999). The synergistic actions of these pathological processes exacerbate brain injury, leading to deterioration of neurological functions (Barone and Feuerstein, 1999). Therefore, therapeutic strategies that can target multiple mechanisms could be extremely useful in limiting post-H-I neuronal damage.

Ethyl pyruvate (EP) is a stable lipophilic ester derivative of pyruvate. EP was initially reported to ameliorate structural and functional damage associated with mesenteric ischemia and reperfusion in rats (Sims et al., 2001). Subsequent in vivo studies documented therapeutic benefits of EP in experimental models of many illnesses, such as coronary ischemia and reperfusion injury (Woo et al., 2004), hemorrhagic shock (Tawadrous, 2002, Yang, 2002), severe sepsis (Ulloa, 2002, Zhang, 2009), and acute pancreatitis (Yang et al., 2004b, 2008). The neuroprotective effect of EP was also observed in post-ischemic brain using an adult rat model of middle cerebral artery occlusion (MCAO) (Kim, 2005, Yu, 2005). The effect of EP in immature brain after H-I, however, is unknown. Multiple mechanisms such as metabolic augmentation, inflammatory response suppression and radical scavenging have been suggested to be involved in the protective effect of EP (Kim, 2008, Tokumaru, 2009). However, the specific molecular target of EP that produces neuroprotection remains to be elucidated.

In the current study, we explored the protective effect of EP in neonatal brain after H-I injury and investigated the molecular mechanisms underlying the action of EP. Using a rat model of neonatal H-I brain injury that induces considerable unilateral damage in cortical, hippocampal and striatal regions, we found that intraperitoneal injections of EP resulted in marked and prolonged neuroprotection. We further demonstrated that EP protects against H-I via dual mechanisms. On one hand, EP directly protects neurons against H-I by inhibiting calpain activation and subsequent pro-death pathways. On the other hand, EP offers indirect neuroprotection via suppression of microglial-mediated inflammatory response.

Section snippets

Rat model of H-I injury and EP administration

All animal protocols used in this study were approved by the Institutional Animal Care and Use Committee. Animals were treated humanely and with regard for alleviating suffering. Housing and breeding of animals were done in accordance with National Institutes of Health guidelines. The procedures for the modeling of neonatal H-I injury were based on the modification of the Levine method using Sprague–Dawley rat litters at postnatal day 7 (P7) (Levine, 1960, Rice, 1981). Pups were anesthetized

EP protects against brain tissue loss after neonatal H-I

To evaluate the neuroprotective effect of EP against neonatal H-I-induced brain injury, EP was administered intraperitoneally at different doses (10, 30, 50, 100, 250 and 500 mg/kg) 1 h after the left common carotid artery occlusion (CCAO) and 30 min before hypoxia (Fig. 1A). The extent of brain damage was determined 7 days after H-I by calculating the degree of tissue loss in each section. As shown in Fig. 1B, EP at concentrations ranging from 30 to 500 mg/kg significantly attenuated post-H-I

Discussion

Neonatal H-I brain injury is an important clinical problem associated with high morbidity and mortality. Current available therapies for this disease are rather limited. In this study, we demonstrated, for the first time, that EP significantly reduced brain damage and improved neurological outcomes evaluated up to 2 months after neonatal H-I injury. Brain tissue loss was ameliorated even when EP administration was delayed for up to 30 min following the H-I insult. Further mechanistic studies

Acknowledgments

H.S. and X.H. contributed equally to this manuscript. This project was supported by NIH/NINDS grants NS36736, NS43802, and NS45048 (to J.C.). Y.G. was supported by the Chinese Natural Science Foundation (grants 30470592, 30670642 and J0730860). C.L. was supported by the Chinese Natural Science Foundation (grant J0730860). We thank Carol Culver for excellent editorial assistance.

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      The effects of EP on ischemia–reperfusion injury have also been demonstrated. Specifically, Hu et al. have shown that EP reduces myocardial ischemia–reperfusion injury by inhibiting HMGB1 protein expression in rats [35], while Shen et al. have shown that EP improves hypoxic-ischemic brain injury through anti-inflammatory and anti-cell death mechanisms [36]. Tsung et al. have reported that EP protects against hepatic ischemia/reperfusion (I/R) injury by reducing hepatic necrosis and apoptosis [37].

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