Elsevier

Brain Research

Volume 1256, 23 February 2009, Pages 129-137
Brain Research

Research Report
Neuroprotective effects of hydrogen saline in neonatal hypoxia–ischemia rat model

https://doi.org/10.1016/j.brainres.2008.11.048Get rights and content

Abstract

Cerebral hypoxia–ischemia (HI) represents a major cause of brain damage in the term newborn. This study aimed to examine the short and long-term neuroprotective effect of hydrogen saline (H2 saline) using an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 °C). H2 saturated saline was administered by peritoneal injection (5 ml/kg) immediately and again at 8 h after HI insult. At 24 h after HI, the pups were decapitated and brain morphological injury was assessed by 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, and TUNEL staining. Acute cell death, inflammation and oxidative stress were evaluated at 24 h by studying caspase-3 activity, MDA measurement as well as Iba-1 immunochemistry in the brain. At 5 weeks after HI, spontaneous activity test and Morris water maze test were conducted. We observed that H2 saline treatment reduced the caspase activity, MDA, Iba-1 levels, the infarct ratio, and improved the long-term neurological and neurobehavioral functions. H2 saline has potentials in the clinical treatment of HI and other ischemia-related cerebral diseases.

Introduction

Cerebral hypoxia–ischemia (HI) represents a major cause of brain damage in the term newborn. Although the mechanisms involved in HI were not completely understood, neuronal cell death either necrosis or apoptosis may play a critical role (Martin et al., 2000, Northington et al., 2001). Specially, apoptosis represents a treatable target which may occur in penumbra areas for days after the initial insult (Pulera et al., 1998). However, there is no specific treatment which is available to HI patients (Perlman, 2006).

Inflammation and oxidative stress are the two major causes of apoptosis identified after ischemic brain injury including neonatal HI (Kriz, 2006). Microglia was involved in the inflammatory process induced by the HI (Stoll et al., 1998). Allograft inflammatory factor-1 (AIF-1) in microglia is an index for the activation of microglia (Postler et al., 2000). Oxidative stress after HI damages DNA, membrane and proteins and contributes to apoptotic changes. The malondialdehyde (MDA) is the product of lipid membrane oxidation and a marker of the oxidative damage.

Hydrogen gas was found to be protective in the brain, heart and liver after ischemia–reperfusion damage (Ohsawa et al., 2007, Fukuda et al., 2007, Hayashida et al., 2008). Hydrogen gas neutralizes free radicals and reduces oxidative stress. However, application of hydrogen gas presents a clinical issue for safety and convenience. In this study, we produced and tested short-term and long-term neuroprotective effect of intraperitoneal application of saturated hydrogen saline (H2 saline) in an established neonatal HI model.

Section snippets

Nissl staining

We tested three different doses of hydrogen saline in the treatments to identify the proper dose in the Nissl staining. Fig. 1 shows representative samples of Nissl staining from the cerebral cortex and hippocampus of pups 24 h after HI insult. Extensive neuronal changes in the cortex and CA1 sector of the hippocampus were noticed with features of considerable dark, pyknotic neurons in HI group (B1–4). More Nissl stained cells (D1–4) were observed in H2W group than in HI group. We used 5 ml/Kg

Discussion

We investigated the neuroprotective effect of peritoneal administration of saturated H2 saline in neonatal HI rats. The short-term results indicated H2 saline treatment significantly reduced the infarct ratio, increased the number of survival neurons, reduced the number of apoptotic cells, suppressed caspase-3 activity, prevented activation of microglia, and decreased the level of oxidative stress (MDA). These short-term effects were translated into long-term neurological functional

Experimental groups

7-day-old Sprague–Dawley rat pups were randomly assigned to the following three groups: 1) control group (no carotid ligation and hypoxia) (n =  40), 2) HI group (carotid ligation and hypoxia) (n =  80), 3) HI + H2W group (carotid ligation, hypoxia and H2 saturated saline treatment) (n =  80). Pups in each group were obtained from different litters to obtain parity within the groups. The Animal and Ethics Review Committee at the Second Military Medical University evaluated and approved the protocol used

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Jianmei Cai and Zhimin Kang contributed equally to this work.

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