Shock/Sepsis/Trauma/Critical Care
The Effect of Hydrogen-Rich Saline on the Brain of Rats with Transient Ischemia

https://doi.org/10.1016/j.jss.2011.01.057Get rights and content

Background

Due to its antioxidant and anti-inflammatory properties, hydrogen gas (H2) has protective effects on a variety of organs from damage induced by ischemia/reperfusion (I/R). In this study, we tested the protective effect of hydrogen-rich saline on the brain in a global cerebral I/R model.

Materials and Methods

We used a four-vessel occlusion model of global cerebral ischemia (15 min) and reperfusion with rats. The rats were divided into four groups (n = 96): sham, I/R plus physiologic saline injected intraperitoneally, I/R plus hydrogen-rich saline injected intraperitoneally at the beginning of reperfusion, and I/R plus hydrogen-rich saline injected intraperitoneally 6 h after reperfusion began. One group of rats was sacrificed after 24 h of reperfusion. Malondialdehyde (MDA) was measured to quantify the oxidative stress. Caspase-3 was measured to indicate the status of apoptosis. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nuclear factor-κB (NF-κB) were measured to monitor the inflammation. Another group of rats was sacrificed after 72 h of reperfusion to measure the histologic damages in hippocampus by hematoxylin and eosin staining and Nissl staining.

Results

Compared with rats with I/R only, hydrogen-rich saline treatment significantly improved the amount of surviving cells. NF-κB, TNF-α, IL-6, MDA, and caspase-3 were all increased significantly by I/R injury. Hydrogen-rich saline reduced all these markers.

Conclusions

Our data demonstrate that intraperitoneal injection of hydrogen-rich saline has strong protective effect on the transient global cerebral ischemia-reperfusion rats.

Introduction

Transient global cerebral ischemia-reperfusion (I/R) injury is one of the major complications to occur during the perioperative period of cardiac arrest and resuscitation [1]. For patients undergoing these surgeries, pharmacologic interventions that can protect the brain against cerebral I/R injury would be very beneficial.

During the process of cerebral ischemic reperfusion, a variety of pathologic mechanisms are involved. Oxidative stress is an important one 2, 3. Ischemia disrupts the balance of endogenous oxidants and antioxidants and overproduces toxic free radicals. Reperfusion also comes with massive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that potentiate initial brain damage. Hydroxyl radicals easily react with cellular macromolecules, including DNA, proteins, and lipids, to exert a strong cytotoxic effect [4]. Reactive oxygen species and other free radicals/oxidants, released by inflammatory cells, can severely threaten tissue viability in the brain. Another important pathologic change is the prominent inflammatory response. Proinflammatory cytokines, such as TNF-α, IL-1, and IL-6, are produced to propagate the inflammation cascade [5]. Other mechanisms include excitatory amino acid toxicity [6] and intracellular calcium overload [7].

An agent that has antioxidant and anti-inflammatory properties provides greater hope. Hydrogen (H2), which could react with hydroxyl radical to produce water, emerged as such a potential candidate [8]. H2 reacts with only the strongest oxidants. H2 is often too mild to disturb metabolic oxidation-reduction reactions or to disrupt ROS involved in cell signaling [9]. Recently, it has been demonstrated in numerous studies that H2 has high protective properties, including the protective effect on lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance [10], transplantation induced intestinal graft injury [11], chronic liver inflammation [12], arteriosclerosis [13], myocardial ischemia/reperfusion (I/R) injury [14], neonatal hypoxia acute pancreatitis [15], intestinal I/R injury 16, 17, and acute oxidative stress [9]. Besides, hydrogen-rich saline is easy to use, safe, and economical. All these findings make hydrogen-rich saline a potential good candidate to provide the protective effect on the brain against cerebral I/R injuries. In this study, we tested whether hydrogen-rich saline can protect the brain against global cerebral ischemia-reperfusion injury in rats.

Section snippets

Animals

Adult male Sprague-Dawley rats weighing 280 ± 20 g were purchased from the Experimental Animal Central of Jinling Hospital. All animals were maintained under the 12 h light/dark cycle with constant temperature and free access to standard rodent chow and tap water. The animals were fasted for 12 h before operation. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Jinling Hospital, Nanjing, China.

Hydrogen-Rich Saline Preparation

Hydrogen was dissolved in physiologic saline 6 h

Histologic Examination of the Hippocampus by H and E Staining

In Sham group, cells were big and had abundant cytoplasm, with one or two round and big nuclei. The cell outline was clear and the structure was compact (Fig. 1A-1, A-2). In I/R group, cells were arranged sparsely and the cell outline was fuzzy. There were rare neuronal cells left (Fig. 1B-1, B-2). In I/R+H21 group and I/R+H22 group, there was less neuronal loss and fewer dead cells. There were significantly more cells with eumorphism in the I/R group (Fig. 1C-1, C-2, D-1, D-2).

NISSL Assay

In CA1 region of

Discussion

Plenty of ROS are generated during an acute ischemic stroke and there is considerable evidence that oxidative stress is an important mediator of tissue injury in acute ischemic stroke [21]. Overproduced free radicals, combined with damaged antioxidant system, cause lipid peroxidatic reaction and induce damage of the membranes of the cell, mitochondria, and lysosome. These eventually lead to cell apoptosis and necrosis [22]. MDA is produced when lipid peroxidatic reaction is triggered on cell

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