Hydrogen-rich saline reverses oxidative stress, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and puncture

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journal homepage: www.JournalofSurgicalResearch.com

Hydrogen-rich saline reverses oxidative stress, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and puncture Jun Zhou, MD,a Ye Chen, MD,b Guo-Qing Huang, MD,c Jun Li, MD,b Gang-Ming Wu, MD,a Li Liu, MD,a Yi-Ping Bai, MD,a and Jian Wang, MDd,* a

Department of Anesthesiology, Affiliated Hospital of Luzhou Medical College, Luzhou, P.R. China Department of Traditional Chinese Medicine, Affiliated Hospital of Luzhou Medical College, Luzhou, P.R. China c Department of Emergency Medicine, Xiangya Hospital of Central South University, Changsha, P.R. China d Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, P.R. China b

article info

abstract

Article history:

Background: Sepsis is associated with high morbidity and mortality, and survivors can

Received 31 August 2011

present with cognitive dysfunction. The present study was performed to investigate the

Received in revised form

effects of hydrogen-rich saline (HRS) on oxidative stress in the brain, cognitive dysfunction,

12 January 2012

and mortality in a rat model of sepsis.

Accepted 25 January 2012

Methods: A rat model of sepsis was induced by cecal ligation and puncture. Physiologic saline

Available online 1 April 2012

or HRS was administered intraperitoneally (2.5 mL/kg or 10 mL/kg) 10 min before the operation. The survival rate was recorded, and cognitive function was tested using the Morris

Keywords:

water maze. The reactive oxygen species and malondialdehyde levels and superoxide dis-

Sepsis

mutase activity in the hippocampus were observed to evaluate the oxidative stress levels.

Hydrogen-rich saline

The caspase 3 levels were measured to detect apoptosis. The histopathologic changes in the

Cognitive function

hippocampus were evaluated by hematoxylin-eosin staining and the terminal deoxy-

Oxidative stress

nucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Results: Cecal ligation and puncture resulted in a poor survival rate, evidence of brain injury, and cognitive dysfunction. The hippocampal reactive oxygen species and malondialdehyde levels increased significantly, and superoxide dismutase activity decreased significantly. HRS reversed these changes in a dose-dependent manner. Conclusions: These findings indicate that HRS could attenuate the consequences of sepsis induced by cecal ligation and puncture in rats, at least in part, by the inhibition of oxidative stress. ª 2012 Elsevier Inc. All rights reserved.

1.

Introduction

Sepsis, a systemic inflammatory disease, is a common, but grave, condition that occurs after infectious insults and

presents with very high mortality and morbidity in intensive care units [1e3]. During sepsis, the brain is one of the first organs to be affected. Septic encephalopathy has been reported to be the most common form of encephalopathy and

* Corresponding author. Department of Anesthesiology, West China Hospital, Sichuan University, no. 37 Wai Nan Guo Xue Xiang, Chengdu, Sichuan, China. Tel.: þ11 86 189 8060 1543; fax: þ86 28 8542 3591. E-mail address: [email protected] (J. Wang). 0022-4804/$ e see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.01.041

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occurs in 8%e70% of septic patients; it is strongly associated with greater mortality rates [4e6]. Septic encephalopathy can be caused by systemic inflammation without direct brain infection. The clinical characteristics include the slowing of mental processes, delirium, disorientation, coma, and, more recently identified, cognitive dysfunction [6,7]. Previous studies have demonstrated that survivors of the cecal ligation and puncture (CLP) animal model had several deficits in their cognitive skills [8]. In addition, clinical studies have shown that septic survivors are frequently left with cognitive impairment [3]. The underlying mechanisms of sepsis are related to the overt generation of cytokines, reactive oxygen species (ROS) and eicosanoids [9]. Although the underlying mechanisms are still not well understood, septic encephalopathy possibly arises from the action of oxidative stress, inflammation, and neuronal apoptosis in the brain [10e12]. ROS, as important mediators of cellular injury, are believed to contribute to the pathology of sepsis and SE. The pro-inflammatory characters of ROS are involved in endothelial cell injuries, recruitment of neutrophils, lipid peroxidation, and oxidation [13]. The oxidative stress induced by ROS plays a significant role in brain injury. Therefore, the inhibition of oxidative stress using antioxidants in the CLP model of sepsis can prevent neutrophil infiltration, improve survival [14], and result in substantial improvements in cognition levels [12,15]. Previous studies have demonstrated that hydrogen gas can provide beneficial effects in different animal models associated with oxidative stress, including neurologic disease [16], inflammation [17], and ischemia-reperfusion injury [18]. Recently, increasing data have shown that hydrogen is an effective and safe antioxidant in animal models of brain injuries [18,19]. Hydrogen can prevent oxidative stress of cells and tissues through selective reduction of the levels of hydroxyl radical and peroxynitrate [20]. However, hydrogen inhalation is dangerous for clinical applications because of its flammable and explosive nature. During its use, special hydrogen gas tanks are needed. Recent studies have shown that hydrogen-rich saline (hydrogen gas-saturated physiologic saline [HRS]) can be easily and safely applied [17,18,21]. Although brain injury can be caused by sepsis clinically, the neuroprotective effects of HRS in sepsis-induced brain damage have not yet been reported. Because of these results, we hypothesized that HRS can reverse brain injury, oxidative damage, cognitive impairment, and mortality in rats submitted to CLP to induce sepsis.

2.

Materials and methods

2.1.

Experimental procedures

2.1.1.

Rats

Adult male Wistar rats, weighing 250e300 g, were used. They were housed five to a cage at a temperature of 22 e24 C and 12-h light/dark cycles and acclimated for 7 d before the manipulations. All rats were fasted for 8 h but were allowed water ad libitum before the experiments. The present study was approved by the Animal Care Committee of Luzhou Medical College (Luzhou, PR China). The animal care and

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handling were performed in accordance with the National Institutes of Health guidelines.

2.1.2.

HRS preparation

HRS was prepared as previously described [22]. In brief, hydrogen gas generated by a hydrogen generator was dissolved in physiologic saline (500 mL) by gassing for 6 h. It was freshly prepared weekly to ensure a constant concentration >0.6 mmol/L. Gas chromatography was used to confirm the level of hydrogen in the saline [20].

2.2.

Experimental protocol

The rats were allocated into one of four groups: (1) a sham group plus normal saline (10 mL/kg); (2) cecal ligation and puncture (CLP) plus normal saline (10 mL/kg); (3) CLP plus HRS (2.5 mL/kg [CLPþH21]); and (4) CLP plus HRS (10 mL/kg [CLPþH22]). Normal saline (in the sham and CLP groups) or HRS (in the CLPþH21 and CLPþH22 groups) was administered intraperitoneally 10 min before the operation. The rats were anesthetized with pentobarbital (30 mg/kg, intraperitoneally). CLP was established as described previously [8]. In brief, under sterile surgical conditions, a 2-cm abdominal incision was made along the ventral surface of the abdomen to expose the cecum, which was then ligated below the ileocecal junction with no bowel obstruction. The cecum was punctured once with an 18-gauge needle, and the fecal contents were allowed to leak into the peritoneum by gently squeezing the cecum. The bowel was then returned to the abdomen and the abdominal cavity closed. The shamoperated rats were submitted to laparotomy, and the cecum was manipulated but neither ligated nor punctured. All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3 mL/100 g body weight) every 6 h for resuscitation. Furthermore, 5% cefoperazone (50 mg/kg) was injected intraperitoneally to avoid infection. In the first experiment, the hydrogen concentration in the blood was determined in the rats from each group (n ¼ 16, 4 rats per measurement point). In the second experiment, the survival rate was monitored for 10 d (n ¼ 20 from each group) after CLP and HRS treatment. In the third experiment, all the rats underwent training for 3 d before CLP and 4 d after CLP for 10 d in the Morris water maze (MWM) to test their cognitive function (n ¼ 8 per group). In the fourth experiment, the rats were allocated into four groups as described and subjected to identical treatment but were excluded from the MWM testing. Instead, they were killed and their brains were removed 48 h after CLP (n ¼ 16 per group). One half of the brain tissues (n ¼ 8) were examined morphologically by hematoxylin-eosin (H&E) staining. Apoptosis was detected by caspase 3 immunohistochemical staining and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. ROS, malondialdehyde (MDA), superoxide dismutase (SOD), and caspase 3 levels were detected in the remaining brain tissues.

2.3.

Specimen preparation

The rats were killed 48 h after CLP, and their aortas were perfused through with 200 mL heparinized saline, followed by

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400 mL freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4, 4 C). The whole brain was carefully removed and post-fixed for 24 h. After dehydration, the brains were frozen and sliced in the coronal plane at 30mm intervals using a cryostat. The sections were then prepared for H&E staining, immunohistochemical staining (cleaved caspase 3), and the TUNEL assay. For Western blot and oxidative stress analyses, the animal brains from another set of experiments were collected at 48 h. All animals were anesthetized and perfused transcardially with 200 mL heparinized 0.9% saline. The brains were removed immediately, and the hippocampal CA1 regions were rapidly microdissected from both sides of the hippocampal fissure by sharp dissection on ice [23]. They were then snap-frozen in liquid nitrogen and stored at 80 C for subsequent testing.

2.4.

Detection of hydrogen concentration in blood

The hydrogen levels were detected in blood using gas chromatography according to a previously described technique [24]. In brief, 5 mL of heparinized arterial blood was collected at 5, 10, 30, or 40 min after injection (n ¼ 16; 4 rats per measurement point). The blood samples were injected immediately into sealed aluminum bags that contained 20 mL of air. The hydrogen was released from the blood into air at room temperature when the aluminum bags were oscillated. For the measurement of the hydrogen content, 100 mL of the rebalanced gas per bag was loaded into a gas chromatograph (Varian CP-3800 GC, Varian Medical Systems, Palo Alto, CA).

2.5.

Survival rate

All rats in the four groups were allowed food and water ad libitum. The survival rate was evaluated daily for the 10-day study period (n ¼ 20 per group).

2.6.

H&E staining

For morphologic examination, the sections were stained with 3% H&E. The stained sections were subsequently examined and photographed with a BX-60 light microscope (Olympus, Southall, U.K.) and an Axiocam digital camera (Zeiss, Gottingen, Germany). Quantitative analysis of the cell numbers in the CA1 area of the hippocampus was performed using Imaging-Pro-Plus (MediaCybernetics, Bethesda, MD). The normal cell numbers in six brain sections of each rat were counted and averaged (three different random high-power fields per sector, magnification 400). The pyramidal neurons of the hippocampal CA1 region with relatively large cell bodies and round and pale stained nuclei were counted as normal pyramid cells; however, neurons with abnormal condensed, pyknotic, and shrunken nuclei were counted as damaged cells [25].

2.7.

Cleaved caspase 3 immunohistochemistry

The sections were stained with cleaved caspase 3, a wellestablished marker of neuronal apoptosis. In brief, the sections were treated with 70% methanol and 0.3% hydrogen

peroxide for 30 min to block endogenous peroxidase and then incubated in 3% blocking phosphate-buffered saline solution containing 0.3% Triton-X and 1% goat serum for 1 h at room temperature. Cleaved caspase 3 was visualized immunohistochemically using rabbit anti-cleaved caspase 3 antibody (1:500; Cell Signaling Technology, Inc., Danvers, MA). After overnight incubation at 4 C with the primary antibodies, the sections were incubated with biotinylated secondary antibodies (goat anti-rabbit IgG,1:200,Chemicon International, Temecula, CA) for 60 min followed by AvidinBiotin-peroxidase complex (Vector Laboratories Inc.) for 60 min at room temperature. The positive cells were visualized with 0.02% 3,3-diaminobenzidine tetrahydrochloride (Sigma) solution containing 0.003% hydrogen peroxide. The sections were then counterstained with hematoxylin. Finally, they were then dehydrated through a gradient of ethanol solutions (70%e100%), cleared with xylene, and covered with a cover slip. The positive stained cells of hippocampal CA1 region were observed under light microscopy (400).

2.8.

Western blot analysis

The homogenized tissue sample was centrifuged at 4 C, 18,000 rpm for 10 min to remove debris. The protein concentration in the supernatants was determined using Coomassie blue dye binding assay (Nanjing Jiancheng Bioengineering Institute, Nanjing City, PR China). Aliquots (60 mg) of proteins from each sample were electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel for 4 h at 100 V. The protein samples were transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). The membrane was blocked with 5% skim milk in Tris-buffered saline and Tween 20 for 2 h and incubated with primary antibodies for cleaved caspase 3 (1:1,000) overnight at 4 C. Subsequently, after three washes with Phosphate Buffer Solution Tween-20, the membranes were incubated for 2 h with relevant species-derived (goat anti-rabbit) horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) at room temperature. Next, the membranes were stripped and reprobed with b-actin antibody (Sigma-Aldrich, Poole, U.K.) to confirm that each lane contained similar amounts of proteins. The protein bands were visualized with enhanced chemiluminescence (New England Biolabs, Ipswich, MA) and then developed on film. After the film was scanned with a GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, CA), densitometric analyses of the bands were completed using Multi-Analyst software (Bio-Rad Laboratories).

2.9.

In situ detection of apoptotic cells

The apoptosis of the hippocampal CA1 region was detected using the TUNEL assay. Apoptotic cells were assessed using an assay kit (Roche Diagnostics, Indianapolis, IN). The sections were counterstained with hematoxylin. The cells with clear nuclear labeling were defined as TUNEL-positive cells. The apoptosis index was calculated as the percentage of stained cells: apoptosis index ¼ number of apoptotic cells  100/total number of nucleated cells.

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2.10.

ROS determination

The ROS levels were detected in hippocampal homogenates using the dye 20 ,70 -dichlorofluorescein diacetate, as previously described [26,27]. In brief, the hippocampi were homogenized and diluted to a 1:10 solution in ice-cold Locke’s buffer to obtain a concentration of 5 mg tissue/mL. The homogenates were then pipetted into 96-well plates (10 mL/well) and allowed to warm to room temperature (25 C) for 5 min. At that point, 5 mL of dichlorofluorescein diacetate (1 mM final concentration) was added, and the mixture was incubated at 37 C in the dark for 2 h. To quantify the free radicals, the fluorescence intensity was measured with a fluorescence spectrophotometer (Varian Cary Eclipse, Darmstadt, Germany) using an excitation and emission wavelength of 488 and 525 nm, respectively. The results are expressed as arbitrary fluorescence units per milligram protein.

2.11.

Statistical analysis

Statistical analysis was performed using SPSS for Windows, version 13.0 (SPSS, Chicago, IL), software. The data are expressed as the mean  standard error of the mean. The chi-square test was used to determine the significance of the differences in survival rates. A two-way repeated-measures analysis of variance (ANOVA) test was used to analyze the training behavioral parameters. A separate two-way ANOVA examined the effects of the operation and time on working memory performance during the hidden platform trial and probe trial test. When ANOVA showed significance, the post hoc Student t-test was used. For all other data, a one-way ANOVA with the least squares difference post hoc test was used to examine the differences. P < 0.05 was considered statistically significant.

3.

Results

3.1.

Changes in hydrogen concentration

MDA and SOD measurements

The MDA levels and SOD activity were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute), and 10% tissue homogenates were prepared according to the manufacturer’s instructions. The MDA levels were assayed for products of lipid peroxidation observed by measurement of thiobarbituric acid-reactive substance levels. The results were calculated as nmol/mg protein. The SOD activity was evaluated by measuring the rate of nitroblue tetrazolium-diformazan formation, which is converted from nitroblue tetrazolium in the presence of O2, which is generated by the xanthine oxidase system and reduced in the presence of SOD. The data are expressed as U/mg protein.

2.12.

2.13.

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MWM for cognitive function testing

To study the cognitive function of rodents, the MWM test is widely used [28]. In brief, the rats were trained for three trials per day for 3 consecutive days during the acquisition phase. The platform was kept in the same position throughout the test. The rats were placed on the platform for 30 s before each trial and gently released into the water facing the wall of the tank from one of four preset entry locations. The trials were automatically ended once the rats reached the platform or 120 s had elapsed. If a rat failed to find the platform within 120 s, it was gently guided to the platform and allowed to remain there for 30 s before starting a new trial. The rats were subjected to CLP on day 4. From the first to the third day after CLP, they were allowed to rest because of high mortality to avoid any confounding motor deficits. Then, they were given the hidden platform trial and probe trial test from days 4 to 10 after CLP. The platform was removed during the probe trial test (120 s). Three measurements were used: (1) the number of crossings of the former platform position, (2) the time needed, and (3) the percentage of time spent in the quadrant where the platform had been previously located. A computer-operated video tracking system (SMART, Barcelona, Spain) was applied to record and analyze latency to the platform, swimming distance, and speed.

The blood hydrogen levels were undetectable in both the sham and CLP groups at all measurement points. After intraperitoneal injection, the blood hydrogen levels in the CLPþH21 and CLPþH22 groups had peaked at approximately 5 min (0.085  0.013 and 0.229  0.034 mmol/L); however, 30 or 40 min later, the hydrogen levels became undetectable in arterial blood (Fig. 1).

3.2.

Survival rates

The survival rate was significantly reduced in the rats that underwent CLP (35%, 7 of 20 rats) compared with the sham controls after 10 d (100%, 20 of 20 rats; P < 0.05). However, the survival rate was significantly greater among rats that had received 10 mL/kg HRS (85%, 17 of 20 rats) after 10 d compared with those that had received no treatment (35%) or had received 2.5 mL/kg HRS (55%, 10 of 20 rats; P < 0.05). No

Fig. 1 e Changes in blood hydrogen concentrations among four experimental groups (n [ 16; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21).

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Fig. 2 e Survival rates among four experimental groups £10 d. Data expressed as percentages (n [ 20; *P < 0.05 versus sham; #P < 0.05 versus CLP; yP < 0.05 versus CLPDH21).

differences were found between the sham and CLPþH22 groups (Fig. 2).

3.3. Pathologic examination of hippocampus by H&E staining The nerve cell bodies in the hippocampal CA1 region were arranged tightly with clear structures and abundant cytoplasm in cells from the sham group, as shown by H&E staining (Fig. 3A). However, most neurons in the CLP group were

shrunken and stained dark, with an enlarged intracellular space (Fig. 3B, arrows). The cells with eumorphism were significantly preserved in the CLPþH21 group (Fig. 3C) and the CLPþH22 group (Fig. 3D). Morphometric analysis showed that the total normal cell count of the hippocampal CA1 region was significantly decreased in the CLP group (206.87  9.87) compared with that in the sham group (295.50  12.91), CLPþH21 group (233.41  7.83), and CLPþH22 group (254.63  11.36; P < 0.05 or P < 0.01). Compared with the CLP group, the number of normal cells significantly increased after HRS treatment (P < 0.05 or P < 0.01), although it was still lower than that in the sham group (P < 0.05 or P < 0.01). Significant differences were seen between the CLPþH21 and CLPþH22 groups (Fig. 3E).

3.4.

Immunohistochemical staining of cleaved caspase 3

Representative micrographs of cleaved caspase 3 immunostaining, which was used to quantify apoptosis, can be found in Fig. 4AeD. The arrows indicate positive cells. The number of cleaved caspase 3-positive cells was significantly greater in the CLP (223.62  25.71), CLPþH21 (142.26  9.89), and CLPþH22 (84.13  12.48) groups than in the sham group (33.06  8.23) 48 h after the CLP event (P < 0.05 or P < 0.01; Fig. 3E). The number of positive cells also differed significantly between the CLP and CLPþH2 groups, with fewer positive cells detectable in the CLPþH22 group than in the CLP group (P < 0.01). Significant differences were found between the CLPþH21 and CLPþH22 groups (P < 0.05).

Fig. 3 e H&E staining in hippocampal CA1 region. Representative sections from hippocampal CA1 region observed 48 h after CLP (magnification 3400). Arrows indicate injured neurons. (A) Regular morphology of hippocampal CA1 region were seen in sham group. (B) Many injured neurons, characterized by shrunken nuclei and stained dark, were observed in CLP group. (C) Normal neurons were significantly increased in CLPDH21 group. (D) Neurons with eumorphism were significantly preserved in CLPDH22 group. (E) Normal neuronal cell counting of hippocampal CA1 field among different groups. Data expressed as mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; y P < 0.05 versus CLPDH21). (Color version of figure is available online.)

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Fig. 4 e Immunostaining for cleaved caspase 3 in hippocampal CA1 region. Cleaved caspase 3 immunopositive cells were darkly stained (arrows) in representative micrographs 48 h after CLP. Magnification 3400. (A) Sham, (B) CLP, (C) CLPDH21, and (D) CLPDH22 groups. (E) Quantitative analysis of positive cells among four groups. Bars represent mean ± standard error mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21). (Color version of figure is available online.)

3.5.

Cleaved caspase 3 expression in hippocampus

The Western blots also supported the results of the immunohistochemistry. They showed that very weak positive signals were found in the brain tissues of the sham group. Significant increases of the intensity of cleaved caspase 3 protein expression were seen in the CLP group. With HRS therapy, the expression of caspase 3 was dramatically reduced after the CLP event. Significant differences were found among all four groups (P < 0.05 or P < 0.01; Fig. 5).

3.6.

TUNEL assay

As presented in Fig. 6, the TUNEL-positive cells, indicating apoptosis, were stained dark brown in the nuclei (arrows). No TUNEL-positive cells were detected in the sham group (Fig. 6A). However, TUNEL-positive cells constituted 66.87% of the total cell population in the CLP group (Fig. 6B), 52.31% in the CLPþH21 group (Fig. 6C) and 37.63% in the CLPþH22 group (Fig. 6D) 48 h after CLP. The number of positive cells differed significantly among all four groups (P < 0.05 or P < 0.01; Fig. 6E).

3.7.

ROS levels in hippocampus

The ROS levels assessed in brain homogenates by dichlorofluorescein diacetate were significantly greater in hippocampus of the CLP group at 48 h than in the sham controls (P < 0.01). The ROS levels of the rats that received HRS were significantly lower than those observed in the CLP group (P < 0.01) but were greater than in the sham controls (P < 0.05;

Fig. 5 e Change in cleaved caspase 3 protein expression in hippocampus after CLP. Western blot analyses demonstrated coordinated increase in cleaved caspase 3 protein within CA1 region of hippocampus at 48 h. Quantitation of Western blots confirmed significant increases in immunoreactive band densities. Data are presented as mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21).

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Fig. 6 e TUNEL staining in hippocampal CA1 region at end of 48 h of CLP. TUNEL-positive cells (arrows) and contain nuclei stained dark brown. Representative micrographs were taken 48 h after CLP event (magnification 3400). (A) Sham, (B) CLP, (C) CLPDH21, and (D) CLPDH22 groups. (E) Quantitative analysis of TUNEL-positive cells of different groups. Bars represent mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21). (Color version of figure is available online.)

Fig. 7). The ROS levels also differed significantly between the CLPþH21 and CLPþH22 group (P < 0.05).

3.8.

Detection of MDA and SOD

The MDA level, an end-product of lipid peroxidation, was elevated in the hippocampi after CLP compared with the level in the sham controls at 48 h (P < 0.01; Fig. 8A). In contrast, the MDA levels were significantly reduced in the rats that received HRS before the CLP event compared with those that did not (P < 0.05 or P < 0.01) but were greater than the levels observed

Fig. 7 e ROS generation in hippocampus by 48 h after CLP. ROS levels were assessed using dichlorofluorescein (DCF) assay. Bars represent mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21).

among the sham controls (P < 0.05 or P < 0.01). The SOD activity in the hippocampi was the inverse of the MDA levels, with the greatest activity observed in the sham group, which differed significantly from the lower activity observed in the CLP (P < 0.01) and CLPþH2 (P < 0.05 or P < 0.01; Fig. 8B) groups. Also, significant differences were found between the CLP and CLPþH2 groups (P < 0.05).

3.9.

Cognitive function measurement

The effect of CLP on spatial learning and memory was evaluated using the MWM method. The swim test data revealed significant effects of day (two-way repeated-measures ANOVA, P < 0.01) on both latency and distance during the acquisition phase (Fig. 9A and B) but not on speed (Fig. 9C). These results demonstrated that all groups rats showed improvements in spatial learning and memory during the acquisition phase over time. No significant differences were found in the latency, swimming distance, and swimming speed among the four groups at the same measurement point before CLP. During the hidden platform trial, longer latency and swimming distances were observed among the CLP group during the hidden platform trials 4e8 d after the CLP event compared with the sham and CLPþH2 groups (P < 0.05 or P < 0.01). Furthermore, the rats in the CLPþH22 and sham groups did not differ significantly for any measured parameter at any measurement point. During the probe trial test, the number of

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Fig. 8 e MDA level and SOD activity in hippocampi at 48 h after CLP. (A) MDA level. (B) SOD activity. Bars represent mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21).

platform crossings (Fig. 10A), retention times (Fig. 10B), and percentage of time spent in the quadrant of the former platform position (Fig. 10C) were significantly decreased (P < 0.05 or P < 0.01) in the CLP group compared with the sham and CLPþH2 groups on days 4 and 7 but not by day 10. However, the sham and CLPþH22 groups did not differ from each other (P > 0.05).

4.

Discussion

In the present study, we demonstrated that the survival rates of rats significantly decreased after CLP but were hardly decreased by 3 d after the insult. This finding indicated that the period of 3 d after injury is a key interval, and close attention needs to be paid to septic patients during this stage. HRS treatment significantly improved the survival rate in the model. We also observed that the rats that underwent CLP showed significant cerebral injury, characterized by histopathologic changes and oxidative damage in the hippocampi, that was significantly attenuated by HRS treatment. To our knowledge, ours is the first study to report the protective potential of HRS on brain injury and the cognitive effects induced by CLP, suggesting it could be of great therapeutic value. Survivors of sepsis are frequently left with cognitive impairment [3,8], and the MWM test is a well-established method to assess cognitive function in the rat model [28]. The hippocampus is involved in spatial navigation and spatial

memory [29]. In the present study, the potential effects of CLP on learning and memory were examined using the MWM method, according to the results of a previous study [30]. The results of the trials before the CLP event demonstrated improvement in spatial learning and memory in a timedependent manner among all experimental groups. However, the rats subjected to CLP had a longer escape latency and swimming distance to reach the hidden platform compared with the sham controls from days 4 to 8. However, the swimming speed did not differ among the experimental groups and was thus not affected by CLP, which suggests the observed differences were cognitive and not due to diminished motor ability or systemic effects induced by the CLP event. Furthermore, the results of the probe trial, which included decreases in the percentage of time spent in the target quadrant, retention time, and the number of crossings of the former platform location, support the hypothesis that CLP results in cognitive impairment in rats. In contrast, rats subjected to CLP that also received HRS performed better on the cognitive assessments than rats that did not receive HRS, because the CLPþH22 group had shorter latency and distance, spent more time in the target quadrant, and crossed the former platform position more often than did the CLP group. The MWM test results for the CLPþH22 and sham groups did not differ significantly from one another. Collectively, the data showed that HRS prevented the detrimental spatial memory impairments that follow CLP. In the CLP group, cognitive dysfunction recovered 10 d after the CLP event, with similar test results observed among the three

Fig. 9 e Cognitive impairment assessed by hidden platform trial. (A) Latency to platform, (B) swimming distance to platform, and (C) swimming speed throughout experiments. Data presented as mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; yP < 0.05 versus CLPDH21 at corresponding measurement points).

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Fig. 10 e Probe trial test. Cognitive impairment occurred at days 4 and 7 after CLP and HRS improved cognitive impairment. (A) Number of crossings in the former platform region. (B) Percentage of target quadrant. (C) Time spent in quadrant of former platform position. Bars represent mean ± standard error of mean (n [ 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ## P < 0.01 versus CLP; yP < 0.05 versus CLPDH21 at corresponding measurement points).

experimental groups. It has been shown that forced exercise can influence learning and memory [31]. We conjecture that the repetitive MWM exercises might be capable of improving the learning and memory of the rats. The mechanisms of organ injury induced by CLP are complex and poorly understood. However, the related brain injury is likely to be a result of oxidative stress [32,33], which includes intermediates associated with several pathologic features, including inflammation, carcinogenesis, and disorder of the central nervous system. The central nervous system is particularly vulnerable to overproduced ROS [34]. Excessive free radicals can cause lipid peroxidation and induce damage of the membranes of the cell and mitochondria, which eventually lead to cell apoptosis and necrosis [35]. MDA, a direct product of lipid peroxidation, can reflect the extent of lipid peroxidation in tissues. SOD activity can reflect the functional status of scavenging oxygen free radicals. The changes in ROS and MDA levels and SOD activity after CLP suggest oxidative stress might be one of the mechanisms involved in the observed cognitive dysfunction and hippocampal CA1 injury occurring after CLP. In the present study, HRS treatment alleviated the increases in ROS and MDA content and decreases in SOD activity in the hippocampus. Oxidative stress could be responsible for the apoptosis of neuronal cells by activating caspase and triggering the apoptotic cascade [36]. Caspase 3, which is located in the final common pathway, is the key protease in the apoptotic cascade. We detected changes in cleaved caspase 3- and TUNEL-positive cells in the hippocampal CA1 region. The expression of apoptotic cells was significantly greater in the rats subjected to CLP compared with sham controls. The results were consistent with those of a previous study [37]. These changes were also consistent with the elevated ROS and MDA levels and reduced SOD activity. Morphologically, we also found that brain injury occurred after CLP, just as demonstrated by a previous report [38]. It has been reported that damaged neurons are observed as early as 6 h after CLP and as long as 48 h later, in particular at 48 h. The oxidative damage in our study also persisted for 48 h. HRS also inhibited the increases in apoptotic cells after CLP, especially to 10 mL/ kg, although the counts were still significantly greater than

those in the sham controls. We hypothesize that HRS alleviates apoptosis in the CA1 hippocampal region by preventing oxidative damage. In a previous study, hydrogen inhalation after the CLP or sham operation significantly improved the survival rate of septic mice in a concentration-dependent manner [39]. Treatment with 1% hydrogen did not significantly increase the 14-day survival rate of moderate CLP. However, 2% and 4% hydrogen treatment increased the 14-day survival rate of moderate CLP to 80% and 90%, respectively. Our results also showed that HRS could significantly attenuate the consequences of septic rats with CLP in a dose-dependent manner. Although the dose of HRS (2.5 mL/kg) alleviated pathologic changes to some extent, it could not improve the survival rate. The present study had several limitations. First, we did not administer HRS to the sham rats. A previous study demonstrated that no significant biochemical and histopathologic differences were present between the sham and sham plus HRS groups [40]; thus, we did not include a sham plus HRS group [18,19]. Second, HRS was not administered after CLP in our animal experiment. Ischemic preconditioning is a potent protective strategy against ischemic organs. Many studies have shown that various forms of preconditioning are beneficial in cases of brain injury, including ischemic, anesthetic, immunologic, and pharmacologic preconditioning techniques. Increasing knowledge of the molecular signaling pathways mediating protection by ischemic preconditioning has provided rational targets for pharmacologic intervention. Various drugs can mimic ischemic preconditioning, including adenosine, adenosine uptake inhibitors, opioids, and anesthetics. The main advantage of pharmacologic preconditioning over ischemic preconditioning-like interventions is its added clinical feasibility [41,42]. After intraperitoneal injection, the blood hydrogen levels in the CLPþH21 and CLPþH22 groups peaked at approximately 5 min; however, 30 or 40 min later, the hydrogen levels became undetectable. These results were extremely similar to those of a previous study [24]. It was interesting that the blood hydrogen levels increased and were maintained for a brief time; however, it provided significant protection against cerebral injury. We speculate that transient hydrogen preconditioning is sufficient to mimic ischemic

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preconditioning to prevent the hazardous insults of sepsis. Moreover, the effective protection of HRS when it is intraperitoneally administered before reperfusion has been previously demonstrated [43]. As stated elsewhere, most patients do not receive medical care until several hours after the insult; thus, the clinical application of neuroprotective drugs in humans is limited. Therefore, drugs that reduce cerebral injury when administered after detrimental insults are attractive targets. The main aim of our report was to explore the effect of HRS on CLP-induced brain damage in an animal model. We will design additional experiments to address postinjury treatment in the future. Third, we did not detect hydrogen levels in the central nervous system, which would have strengthened our present conclusions. Hydrogen can diffuse extremely rapidly into tissues to reach important target subcellular compartments, including the nucleus and mitochondria, owing to its low molecular weight. This is extremely important because mitochondria, as the primary sites of ROS generation, are notoriously difficult to target. Moreover, hydrogen passes through the bloodebrain barrier by gaseous diffusion, although most antioxidant drugs cannot. This is a particular advantage of hydrogen [44]. Previous studies have shown that HRS can provide neuroprotection against brain injury [18,19,21]. Therefore, we conjecture that hydrogen molecules can also cross the bloodebrain barrier to prevent cerebral damage after CLP without the formal detection of hydrogen levels in the central nervous system.

5.

Conclusions

CLP causes cerebral oxidative damage, cognitive dysfunction, and decreased survival in rats. HRS treatment largely prevented the effects associated with CLP, implying that it has great potential as a neuroprotective therapy by the prevention of oxidative stress. The results of the present study support that HRS might be an effective therapeutic strategy for patients with sepsis.

Acknowledgments This study was supported by the Talent Fund Project of Affiliated Hospital of Luzhou Medical College (grant 2011-43-43). The authors thank Drs. Ann Power Smith and Kelly Bonner Engel for their critical reading and language revision of our report.

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