Edaravone Attenuates Brain Damage in Rats After Acute CO Poisoning Through Inhibiting Apoptosis and Oxidative Stress Qin Li,1 Ming Jun Bi,1 Wei Kang Bi,2 Hai Kang,1 Le Jing Yan,1 Yun-Liang Guo3 1
Emergency Centre, Yantai Yuhuangding Hospital Affiliated Hospital of Qingdao University Medical College, Yantai Shandong 264000, People’s Republic of China
2
Department of Clinical Medicine, Qingdao University Medical College, Qingdao Shandong 266003, People’s Republic of China
3
Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College, Qingdao Shandong 266003, People’s Republic of China
Received 24 August 2013; accepted 10 September 2014 ABSTRACT: Acute carbon monoxide (CO) poisoning is the most common cause of death from poisoning all over the world and may result in neuropathologic and neurophysiologic changes. Acute brain damage and delayed encephalopathy are the most serious complication, yet their pathogenesis is poorly understood. The present study aimed to evaluate the neuroprotective effects of Edaravone against apoptosis and oxidative stress after acute CO poisoning. The rat model of CO poisoning was established in a hyperbaric oxygen chamber by exposed to CO. Ultrastructure changes were observed by transmission electron microscopy (TEM). TUNEL stain was used to assess apoptosis. Immunohistochemistry and immunofluorescence double stain were used to evaluate the expression levels of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf-2) protein and their relationship. By dynamically monitored the carboxyhemoglobin (HbCO) level in blood, we successfully established rat model of severe CO poisoning. Ultrastructure changes, including chromatin condensation, cytoplasm dissolution, vacuoles formation, nucleus membrane and cell organelles decomposition, could be observed after CO poisoning. Edaravone could improve the ultrastructure damage. CO poisoning could induce apoptosis. Apoptotic cells were widely distributed in cortex, striatum and hippocampus. Edaravone treatment attenuated neuronal apoptosis as compared with the poisoning group (P < 0.01). Basal expressions of HO-1 and Nrf-2 proteins were found in normal brain tissue. CO poisoning could activate HO-1/Nrf-2 pathway, start oxidative stress response. After the administration of Edaravone, the expression of HO-1 and Nrf-2 significantly increased (P < 0.01). These findings suggest that Edaravone may inhibit apoptosis, activate the Keapl-Nrf/ARE pathway, and thus improve the ultrastructure damage and neurophysiologic changes C 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 372–379, 2016. following acute CO poisoning. V Keywords: CO poisoning; edaravone; ultrastructure changes; apoptosis; oxidative stress response; HO-1; Nrf-2
INTRODUCTION Correspondence to: Q. Li; e-mail: [email protected] Published online 28 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22052
Carbon monoxide (CO) inhalation is a relatively frequent cause of death, as indicated by the Centers for Disease Control and Prevention (Centers for Disease Control and
C 2014 Wiley Periodicals, Inc. V
372
EDARAVONE ATTENUATES BRAIN DAMAGE IN RATS
Prevention, 2007; Hampson et al., 2007). CO poisoning may result in neuropathologic and neurophysiologic changes, such as acute brain damage and delayed encephalopathy, which seriously affect the life quality of patients. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly elucidated. Hyperbaric oxygen is considered to be one of the most effective treatment, has been widely used in the patients of acute CO poisoning. However, there has been controversy over the effectiveness of hyperbaric oxygen treatment on CO poisoning (Hampson et al., 2008; Kusuba et al., 2012; Garrabou et al., http://www.ncbi.nlm.nih.gov/pubmed/214249752011). Some people insisted that hyperbaric oxygen could rapidly improve the clinical symptoms, reduce the mortality and the neurologic morbidity in mild and moderate patients. Others stated that hyperbaric oxygen had no positive effect on white matter damage after CO poisoning, and whether or not it could reduce neurological sequelae was still unclear. Hyperbaric oxygen therapy might be harmful, or at least no good for the nervous system complications induced by CO poisoning. It is imperative that novel effective neuroprotective strategy be designed for repairing the injured cerebral structure and improving neurologic function and the life quality of patients with acute severe CO poisoning. Edaravone, as a new free radical scavengers, has been widely used in patients and animal models with acute cerebral infarction. It can effectively eliminate active oxygen and cytotoxic hydroxy radical, inhibit lipid peroxidation, prevent the damage of vascular endothelial cell, suppress delayed neurocyte death (Kikuchi et al., 2012) and thus significantly improve the neurological symptoms of patients with cerebral infarction. Whether it has the same neuro-protective effects on brain damage after CO poisoning or not need to be further explored. The aim of this study was to evaluate the neuro-protection of Edaravone on the injured brain tissue after CO poisoning. We hypothesized that Edaravone might be comparably efficient in the pathologic and physiologic amelioration of brain damage in rat models.
MATERIALS AND METHODS Experimental Model and Group Total of 90 adult healthy male Sprague–Dawley rats, weighing (230 6 20) g and SPF grade, were obtained from Shangdong Academy of Medical Sciences, China. The laboratory animals were treated according to the animal ethics guidelines of the Chinese National Health and Medical Research Council (NHMRC). All rats were kept in an air-conditioned room with a 12-h light/dark cycle, and food and water available ad libitum. After being adapted to the environment for 7 days at the room temperature of (25 6 2) C, rats were randomly selected as a normal group (Group A, n 5 30), and the rest were exposed to CO. Rat model of CO poisoning was established as described previously (Han et al., 2007;
373
Guoping et al., 2009). Briefly, the rats were shut in a hyperbaric oxygen chamber and intoxicated with 1000 ppm CO (Shangdong Gas, China) for 40 min, followed with 3000 ppm CO for another 20 min until loss of consciousness. Then they were removed from the chamber to breathe in fresh air and retrieve consciousness. During the whole experiment, core body temperature of all rats were maintained at 36–37 C using a homeothermic blanket control unit (Shandong Apparatus, China). About 0.3 mL whole blood was drawn from the left femoral artery for carboxyhemoglobin (HbCO) assay after proper anesthesia via 3% pentobarbital intraperitoneal injection. A blood gas analyzer (RapidLab, Bayer HealthCare, Leverkusen, Germany) was used for HbCO detection. Rats with coma and high HbCO concentration (>40%) were regarded as the successful models of acute severe CO poisoning. Three halfway death cases were removed and not included in the experimental statistics. The rats from group A were given intraperitoneally the same dose of phosphate-buffered saline (PBS, pH 7.4) at 1 h after inhaling fresh air. Sixty poisoning rats were randomly subdivided into one of the following two groups: a poisoning control group (Group B, n 5 30), each was injected PBS intraperitoneally 2 h after CO exposure; a treatment group (Group C, n 5 30), each was administered Edaravone (10 mg kg21) intraperitoneally 2 h after CO poisoning, twice a day till decapitated. By dynamically monitored the level of HbCO, we found that the HbCO concentration in group B and group C was up to 58.9% 6 6.1%, and consistent with the standard of severe CO poisoning, whereas it was only 0.5% 6 0.3% in group A.
Transmission Electron Microscopy (TEM) Five animals in each group were deeply anesthetized by 10% chloral hydrate, and decapitated at 1, 2, 7 day after CO exposure and drug administration, respectively. A small amount of fresh brain tissue was taken out from left hemisphere, cut into small pieces of 1 mm 3 1 mm 3 1 mm, fixed in 2.5% glutaraldehyde for 24 h, post-fixed in osmic acid for 1 h, dehydrated with graded acetone, and finally embedded with epoxy resin Epon 812. Using the ultramicrotome (Leica EM UC6, Germany), the tissue was cut into 50-nm ultrathin slices, put on copper grid and stored at 4 C. The ultrathin slices was added a drop of 3% uranyl acetate-alcohol saturated solution (pH 3.5) in a clean petri dish, covered copper grid on the dye liquor for 30 min, rinsed in double-distilled water for 10 min three times, and then drained water. With the same method, the slices were dyed by 6% lead citrate dye liquor (pH 12) for 5min, drained water at room temperature for the ultra-structure observation under TEM (JEM-1200EX, Japan).
Cell Apoptosis Assay Five animals in each group were deeply anesthetized and decapitated at the given time points. Sequential paraffin
Environmental Toxicology DOI 10.1002/tox
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slices of 6 lm thickness at an interval of 100 lm were obtained for cell apoptosis assay. The procedure of cell apoptosis assay was performed according to the manufacture guidance of DendEnd TUNEL cell apoptosis kit (Santa Cruz Company). Some slides added Deoxyribonuclease I (usually called DnaseI) at a dose of 1 lg mL21 were regarded as positive control simple, and those treated without TdT were the negative ones. Under a 400-fold microscope, the apoptosis cells appeared brown granules in nucleus. The absorbance (A) value was determined at four random views in cortex and striatum from four serial slices in a blinded manner with Leica Qwin image processing and analysis system (Leica Company).
SABC-FITC (1:100) 37 C for 30 min, respectively. The sample was added with anti-Nrf-2 (1:100), the second antibody (1:100), SABC-CY3 (1:100), successively, and covered with waterborne mount. HO-1 and NRF-2 positive cells were observed in the same field of views with the excitation light of different wavelengths under a fluorescence microscope in order to determine the relationship between the two proteins. All slides were carried out under a dark circumstance throughout the procedures and washed fully in PBS, so as to avoid excessive residual or metamorphosis fluorescence.
Immunohistochemical Assay
Results were expressed as mean 6 standard error (x 6 s), and the numbers represented at least three separate experiments for each group. Statistical comparisons were made by analysis of variance (ANOVA) and the least significant difference (LSD) t test. A P value 0.05).
Environmental Toxicology DOI 10.1002/tox
To investigate the relationship between HO-1 and Nrf-2 proteins in brain tissue, we used double immunofluorescence labeling. HO-1 and Nrf-2 positive cells, with irregular shape, were scattered in many regions of brain tissue, and their expression levels were elevated after CO exposure under the immunofluorescence microscope. These positive cells mainly resided in neurons and astrocytes. In contrast to the location of the two proteins in the same field of view, we found that some but not all HO-1 positive cells showed Nrf2 immunoreaction. Obviously, many Nrf-2 positive cells did not appear HO-1 immunogenicity (Fig. 8). It offers a potent evidence that the two proteins can co-exist in a single neurocyte, also can be expressed in different cells, respectively. Although the expression of these two proteins could be detected in normal brain tissue, and gradually increased after CO poisoning, no correlation was found between HO-1 and Nrf-2 expressions by statistical analysis. The result suggests
Fig. 3. The apoptosis cells in brain tissue in rats after C0 exposure. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
EDARAVONE ATTENUATES BRAIN DAMAGE IN RATS
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binds to hemoglobin and produces HbCO which results in the displacement of oxygen and subsequently reduces the supply of oxygen available to the tissues of the body. Once ischemia and hypoxia occurred, the structure and function of certain vital organs were undoubtedly impaired. Briefly, neuronal structure was damaged, the oxidative phosphorylation of mitochondria was decreased or stopped, energy metabolism was disordered, electrolyte both inside and outside the cell membrane was imbalance, and thus a cascade of
Fig. 4. The expressions of HO-1 protein in each group under a 400-fold light microscope. CO poisoning could induce the expression of HO-1 protein. The amount of HO-1 positive cells and the A value increased in many brain areas at 1 and 7 day in group B. The overexpression of HO-1 protein were detected, and the A value of each view was also increased after Edaravone administration. Significant differences were found between the group B and group C (P < 0.01), suggesting that Edaravone play an important role in the activation of the HO-1/Nrf-2 pathway against oxidative stress damage. A: Group B in cortex at 1 day after CO exposure; B: Group C in cortex at 1 day after CO exposure; C: Group B in cortex at 7 day; D: Group C in cortex at 7 day; E: Group B in striatum at 1 day after CO exposure; F: Group C in striatum at 1 day after CO exposure; G: Group B in striatum at 7 day; H: Group C in striatum at 7 day (scale bar: 30 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
that HO-1 and Nrf-2 proteins may be activated successively by one or variety of pathways in brain tissue after CO poisoning.
DISCUSSION Cases of CO poisoning remain an important cause of death in the practice of pathologists who perform autopsies following deaths within the community. When people inhale CO, it
Fig. 5. The expressions of Nrf-2 protein in rats after CO exposure under a 400-fold light microscope. With the duration of CO exposure, the increased Nrf-2 positive cells were detected in group B at 1 and 7 day. As compared with group B, HO-1 positive cells in group C were obviously enhanced, and its A value of each view was also increased at the same time points both in cortex and striatum (P < 0.01). A: Group B in cortex at 1 day after CO exposure; B: Group C in cortex at 1 day after CO exposure; C: Group B in cortex at 7 day; D: Group C in cortex at 7 day; E: Group B in striatum at 1 day after CO exposure; F: Group C in striatum at 1 day after CO exposure; G: Group B in striatum at 7 day; H: Group C in striatum at 7 day (scale bar: 30 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
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Fig. 6. The A values of H021 in each group at different time points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 7. The A values of Nrf22 in each group at different time points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
reactions were stimulated, as a result, to lead to cell death and malfunction. As we showed under TEM, neurons in group B were irregular with chromatin condensation and marginalization, cytoplasm dissolution and vacuoles formation. Edaravone could effectively protect brain tissue from ultrastructural damage following CO poisoning. Neurons are non-renewable. The apoptosis or death of neurocytes directly affects the normal physiological function of the central nervous system. Preventing or reducing apoptosis can significantly improve neurological malfunction. CO exposure leads to diffuse hypoxic-ischemic encephalopathy and focal cortical injury that, during the acute stage of intoxication, is owing to transient vasogenic edema or frank necrosis, predominantly involving gray matter. By flow cytometry, Guratowska et al. (2010) found significantly increased apoptosis of the lymphocytes in research group compared to control individuals correlated with the CO poisoning severity, but did not depend on hypoxia. Decreased number of leukocytes caused by the cytotoxic effect of CO stimulated the release of the CD 341 to the peripheral blood. Brvar et al.’s (2010) study showed that CO poisoning induced ganglionic cell apoptosis. The percentage of apoptotic cells in rats exposed to CO was 32%. Tofighi et al. (2006) insisted that after exposed to CO, cells exhibited loss of mitochondrial membrane potential, release of cytochrome C into the cytosol, nuclei with chromatin condensation. Those cell features
were fully consistent with the characteristics of apoptosis. CO also triggered activation of caspase and calpain proteases. As we expected, apoptosis cells were significantly increased in group B. After the treatment of Edaravone, the amount of apoptosis cells was significantly decreased both in cortex and striatum, suggesting that Edaravone may prevent apoptosis process induced by CO poisoning. Reactive oxygen species/reactive nitrogen family (ROS/ RNS), highly reactive molecules, can damage nucleic acids and proteins, resulting in lipid peroxidation and inactivation of cell membrane receptors and enzyme, and ultimately affecting the function of cell. The human body has several mechanisms to counteract oxidative stress by producing antioxidants. HO-1/Nrf-2 is one of the important anti-oxidation pathway. HO-1 is an isoform in hemeoxygenase (HO) system, which is responsible for cellular heme degradation to biliverdin, iron, and carbon monoxide. It has been extensively studied mainly by its ability to respond to cellular stresses such as hemin, nitric oxide donors, oxidative damage, hypoxia, hyperthermia, and heavy metals (Ye et al., 2012; Wang et al., 2013; Zhang et al., 2014). Nrf-2 is a gene transcription factor that binds to the electrophile response element (EpRE) and triggers expression of various genes with antioxidant properties, including HO-1. Nrf-2 contributes significantly to cytoprotection against oxidative stress. The increase in mRNA expression of antioxidant enzymes
Fig. 8. The relationship of HO-1 and Nrf-2 expression in rats after CO poisoning. With the excitation light of different wavelengths under a fluorescence microscope, HO-1 positive cells appeared yellow-green light (A), while Nrf-2 positive cells showed red (B). HO-1 and Nrf-2 positive cells, with irregular shape, were scattered in many regions of brain tissue. Overlapping the two photographs of the same view, we found that some but not all HO-1 positive cells showed Nrf-2 immunoreaction, many Nrf-2 positive cells did not appear HO-1 immunogenicity (C). It offers a potent evidence that the two proteins can co-exist in a single neurocyte, also can be expressed in different cells, respectively. They may be activated successively by one or variety of pathways in brain tissue after CO poisoning. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
EDARAVONE ATTENUATES BRAIN DAMAGE IN RATS
was positively correlated with the Nrf-2 transcription in cortex and hippocampus. The deletion or activation barriers of Nrf-2 gene made the enhanced sensitivity of neurons to oxidative stress (Kobayashi et al., 2004; Genc et al., 2010). Nrf2 expression could be detected in MSCs. Transient expression of Nrf-2 protected MSCs against cell death and apoptosis triggered by hypoxic and oxidative stress conditions (Mohammadzadeh et al., 2012). Nrf-2 also enhanced the activity of SOD and HO-1. Ha et al. (2012) insisted that hypoxia induced HO-1 expression through PI3K/Akt/Nrf-2 signal pathways. Our results showed that the basal expression of HO-1 and Nrf-2 could be found in brain tissue of group A, indicating that the body could keep equilibrium of transcription and translation and degradation of the two proteins, maintain the balance of oxidant/ antioxidant system under physiological conditions. Nrf-2 and HO-1 expressions increased significantly in rats after CO poisoning, suggesting that Keapl-Nrf-2/ARE antioxidant system be activated in brain tissue following CO exposure. Meanwhile, we found that some but not all HO-1 positive cells showed Nrf-2 immunoreaction. Many Nrf-2 positive cells did not appear HO-1 immunogenicity. The expression level of the two proteins lacks correlation. The data demonstrated that HO-1 and Nrf-2 proteins might be activated successively by one or variety of pathways in brain tissue after CO poisoning. After the treatment of Edaravone, the expressions of Nrf-2 and HO-1 were increased obviously, suggesting that the neuroprotection of Edaravone on brain tissue from oxidative stress be partly mediated by Keapl-Nrf-2/ARE pathway via the upregulation of Nrf-2 and HO-1 expression.
CONCLUSION We successfully established the animal model of CO poisoning in a hyperbaric oxygen chamber by exposed to CO. As we expected, Edaravone can inhibit apoptosis, mediate the overexpression of Nrf-2 and HO-1protein, be in part associated with activation of the Keapl-Nrf-2/ARE against oxidative stress in rats following CO poisoning. Edaravone, as a novel neuroprotective treatment, may be designed for patients with acute CO poisoning.
REFERENCES Suput Brvar M, Luzar B, Finderle Z, D, Bunc M. 2010. The timedependent protective effect of hyperbaric oxygen on neuronal cell apoptosis in carbon monoxide poisoning. Inhal Toxicol 22: 1026–1031. Centers for Disease Control and Prevention (CDC). 2007. Carbon monoxide-related deaths—United States,1999–2004. MMWR Morb Mortal Wkly Rep 56:1309–1312. Hampson NB, Hauff NM. 2008. Risk factors for short-term mortality from carbon monoxide poisoning treated with hyperbaric oxygen. Crit Care Med 36:2523–2527.
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Hampson NB, Weaver LK. 2007. Carbon monoxide poisoning: A new incidence for an old disease. Undersea Hyperb Med 34:163–168. Han ST, Bhopale VM, Thom SR. 2007. Xanthine oxidoreductase and neurological sequelae of carbon monoxide poisoning. Toxicol Lett 170:111–115. Martı MJ, Garrabou G, Inoriza JM, Moren C, Oliu G, Miro O, Cardellach F. 2011. Mitochondrial injury in human acute carbon monoxide poisoning: the effect of oxygen treatment. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 29:32–51. Genc K, Egrimez MY, Genc S. 2010. Erythropoietin induces nuclear translocation ofNrf-2 and heme oxygenase-1 expression in SH-SY5Y cells[J]. Cell Biochem Funct 28:197–201. Guoping J, Jianping G, Yongshan X, Yuefeng M, Guanyu J, Weiqi Y. 2009. Structural and functional improvement of injured brain after severe acute carbon monoxide poisoning by stem cell-based therapy in rats. Crit Care Med 37:1416–1422. Guratowska M, Pach D, Pach J, Groszek B. 2010. The causes and consequences of the cellular death (apoptosis and necrosis) in the course of acute poisoning with carbon monoxide. Przegl Lek 67:566–570. Ha YM, Kim MY, Park MK, Lee YS, Kim YM, Kim HJ, Lee JH, Chang KC. 2012. Higenamine reduces HMGB1 during hypoxiainduced brain injury by induction of heme oxygenase-1 through PI3K/Akt/Nrf-2 signal pathways. Apoptosis 17:463–474. Kikuchi K, Takeshige N, Miura N, Morimoto Y, Ito T, Tancharoen S, Miyata K, Kikuchi C, Iida N, Uchikado H, Miyagi N, Shiomi N, Kuramoto T, Maruyama I, Morioka M, Kawahara KI. 2012. Beyond free radical scavenging: Beneficial effects of edaravone (Radicut) in various diseases (Review). Exp Ther Med 3:3–8. Kobayashi A, Ohta T, Yamamoto M. 2004. Unique function of the Nrf-2 keapl pathway in the inducible expression of antioxidant and detoxifying enzynmes[J]. Methods Enzymol 378:273–286. Kusuba Y, Taki K, Ohta A. 2012. Questionnaire results of hyperbaric oxygen therapy for acute carbon monoxide poisoning in Japan. Undersea Hyperb Med 39:639–645. Mohammadzadeh M, Halabian R, Gharehbaghian A, Amirizadeh N, Jahanian-Najafabadi A, Roushandeh AM, Roudkenar MH. 2012. Nrf-2 overexpression in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity. Cell Stress Chaperones 17:553–565. Tofighi R, Tillmark N, Dare E, Aberg AM, Larsson JE, Ceccatelli S. 2006. Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro. Brain Res 1098:1–8. Wang YF, Gu YT, Qin GH, Zhong L, Meng YN. 2013. Curcumin ameliorates the permeability of the blood-brain barrier during hypoxia by upregulating heme oxygenase-1 expression in brain microvascular endothelial cells. J Mol Neurosci 51:344–351. Ye Z, Guo Q, Xia P, Wang N, Wang E, Yuan Y. 2012. Sevoflurane postconditioning involves an up-regulation of HIF-1 and HO-1 expression via PI3K/Akt pathway in a rat model of focal cerebral ischemia. Brain Res 1463:63–74. Zhang M, Wang S, Mao L, Leak RK, Shi Y, Zhang W, Hu X, Sun B, Cao G, Gao Y, Xu Y, Chen J, Zhang F. 2014. Omega-3 fatty acids protect the brain against ischemic injury by activating Nrf-2 and upregulating heme oxygenase 1. J Neurosci 34:1903–1915.
Environmental Toxicology DOI 10.1002/tox
Emergency Centre, Yantai Yuhuangding Hospital Affiliated Hospital of Qingdao University Medical College, Yantai Shandong 264000, People’s Republic of China
2
Department of Clinical Medicine, Qingdao University Medical College, Qingdao Shandong 266003, People’s Republic of China
3
Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College, Qingdao Shandong 266003, People’s Republic of China
Received 24 August 2013; accepted 10 September 2014 ABSTRACT: Acute carbon monoxide (CO) poisoning is the most common cause of death from poisoning all over the world and may result in neuropathologic and neurophysiologic changes. Acute brain damage and delayed encephalopathy are the most serious complication, yet their pathogenesis is poorly understood. The present study aimed to evaluate the neuroprotective effects of Edaravone against apoptosis and oxidative stress after acute CO poisoning. The rat model of CO poisoning was established in a hyperbaric oxygen chamber by exposed to CO. Ultrastructure changes were observed by transmission electron microscopy (TEM). TUNEL stain was used to assess apoptosis. Immunohistochemistry and immunofluorescence double stain were used to evaluate the expression levels of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf-2) protein and their relationship. By dynamically monitored the carboxyhemoglobin (HbCO) level in blood, we successfully established rat model of severe CO poisoning. Ultrastructure changes, including chromatin condensation, cytoplasm dissolution, vacuoles formation, nucleus membrane and cell organelles decomposition, could be observed after CO poisoning. Edaravone could improve the ultrastructure damage. CO poisoning could induce apoptosis. Apoptotic cells were widely distributed in cortex, striatum and hippocampus. Edaravone treatment attenuated neuronal apoptosis as compared with the poisoning group (P < 0.01). Basal expressions of HO-1 and Nrf-2 proteins were found in normal brain tissue. CO poisoning could activate HO-1/Nrf-2 pathway, start oxidative stress response. After the administration of Edaravone, the expression of HO-1 and Nrf-2 significantly increased (P < 0.01). These findings suggest that Edaravone may inhibit apoptosis, activate the Keapl-Nrf/ARE pathway, and thus improve the ultrastructure damage and neurophysiologic changes C 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 372–379, 2016. following acute CO poisoning. V Keywords: CO poisoning; edaravone; ultrastructure changes; apoptosis; oxidative stress response; HO-1; Nrf-2
INTRODUCTION Correspondence to: Q. Li; e-mail: [email protected] Published online 28 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22052
Carbon monoxide (CO) inhalation is a relatively frequent cause of death, as indicated by the Centers for Disease Control and Prevention (Centers for Disease Control and
C 2014 Wiley Periodicals, Inc. V
372
EDARAVONE ATTENUATES BRAIN DAMAGE IN RATS
Prevention, 2007; Hampson et al., 2007). CO poisoning may result in neuropathologic and neurophysiologic changes, such as acute brain damage and delayed encephalopathy, which seriously affect the life quality of patients. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly elucidated. Hyperbaric oxygen is considered to be one of the most effective treatment, has been widely used in the patients of acute CO poisoning. However, there has been controversy over the effectiveness of hyperbaric oxygen treatment on CO poisoning (Hampson et al., 2008; Kusuba et al., 2012; Garrabou et al., http://www.ncbi.nlm.nih.gov/pubmed/214249752011). Some people insisted that hyperbaric oxygen could rapidly improve the clinical symptoms, reduce the mortality and the neurologic morbidity in mild and moderate patients. Others stated that hyperbaric oxygen had no positive effect on white matter damage after CO poisoning, and whether or not it could reduce neurological sequelae was still unclear. Hyperbaric oxygen therapy might be harmful, or at least no good for the nervous system complications induced by CO poisoning. It is imperative that novel effective neuroprotective strategy be designed for repairing the injured cerebral structure and improving neurologic function and the life quality of patients with acute severe CO poisoning. Edaravone, as a new free radical scavengers, has been widely used in patients and animal models with acute cerebral infarction. It can effectively eliminate active oxygen and cytotoxic hydroxy radical, inhibit lipid peroxidation, prevent the damage of vascular endothelial cell, suppress delayed neurocyte death (Kikuchi et al., 2012) and thus significantly improve the neurological symptoms of patients with cerebral infarction. Whether it has the same neuro-protective effects on brain damage after CO poisoning or not need to be further explored. The aim of this study was to evaluate the neuro-protection of Edaravone on the injured brain tissue after CO poisoning. We hypothesized that Edaravone might be comparably efficient in the pathologic and physiologic amelioration of brain damage in rat models.
MATERIALS AND METHODS Experimental Model and Group Total of 90 adult healthy male Sprague–Dawley rats, weighing (230 6 20) g and SPF grade, were obtained from Shangdong Academy of Medical Sciences, China. The laboratory animals were treated according to the animal ethics guidelines of the Chinese National Health and Medical Research Council (NHMRC). All rats were kept in an air-conditioned room with a 12-h light/dark cycle, and food and water available ad libitum. After being adapted to the environment for 7 days at the room temperature of (25 6 2) C, rats were randomly selected as a normal group (Group A, n 5 30), and the rest were exposed to CO. Rat model of CO poisoning was established as described previously (Han et al., 2007;
373
Guoping et al., 2009). Briefly, the rats were shut in a hyperbaric oxygen chamber and intoxicated with 1000 ppm CO (Shangdong Gas, China) for 40 min, followed with 3000 ppm CO for another 20 min until loss of consciousness. Then they were removed from the chamber to breathe in fresh air and retrieve consciousness. During the whole experiment, core body temperature of all rats were maintained at 36–37 C using a homeothermic blanket control unit (Shandong Apparatus, China). About 0.3 mL whole blood was drawn from the left femoral artery for carboxyhemoglobin (HbCO) assay after proper anesthesia via 3% pentobarbital intraperitoneal injection. A blood gas analyzer (RapidLab, Bayer HealthCare, Leverkusen, Germany) was used for HbCO detection. Rats with coma and high HbCO concentration (>40%) were regarded as the successful models of acute severe CO poisoning. Three halfway death cases were removed and not included in the experimental statistics. The rats from group A were given intraperitoneally the same dose of phosphate-buffered saline (PBS, pH 7.4) at 1 h after inhaling fresh air. Sixty poisoning rats were randomly subdivided into one of the following two groups: a poisoning control group (Group B, n 5 30), each was injected PBS intraperitoneally 2 h after CO exposure; a treatment group (Group C, n 5 30), each was administered Edaravone (10 mg kg21) intraperitoneally 2 h after CO poisoning, twice a day till decapitated. By dynamically monitored the level of HbCO, we found that the HbCO concentration in group B and group C was up to 58.9% 6 6.1%, and consistent with the standard of severe CO poisoning, whereas it was only 0.5% 6 0.3% in group A.
Transmission Electron Microscopy (TEM) Five animals in each group were deeply anesthetized by 10% chloral hydrate, and decapitated at 1, 2, 7 day after CO exposure and drug administration, respectively. A small amount of fresh brain tissue was taken out from left hemisphere, cut into small pieces of 1 mm 3 1 mm 3 1 mm, fixed in 2.5% glutaraldehyde for 24 h, post-fixed in osmic acid for 1 h, dehydrated with graded acetone, and finally embedded with epoxy resin Epon 812. Using the ultramicrotome (Leica EM UC6, Germany), the tissue was cut into 50-nm ultrathin slices, put on copper grid and stored at 4 C. The ultrathin slices was added a drop of 3% uranyl acetate-alcohol saturated solution (pH 3.5) in a clean petri dish, covered copper grid on the dye liquor for 30 min, rinsed in double-distilled water for 10 min three times, and then drained water. With the same method, the slices were dyed by 6% lead citrate dye liquor (pH 12) for 5min, drained water at room temperature for the ultra-structure observation under TEM (JEM-1200EX, Japan).
Cell Apoptosis Assay Five animals in each group were deeply anesthetized and decapitated at the given time points. Sequential paraffin
Environmental Toxicology DOI 10.1002/tox
374
LI ET AL.
slices of 6 lm thickness at an interval of 100 lm were obtained for cell apoptosis assay. The procedure of cell apoptosis assay was performed according to the manufacture guidance of DendEnd TUNEL cell apoptosis kit (Santa Cruz Company). Some slides added Deoxyribonuclease I (usually called DnaseI) at a dose of 1 lg mL21 were regarded as positive control simple, and those treated without TdT were the negative ones. Under a 400-fold microscope, the apoptosis cells appeared brown granules in nucleus. The absorbance (A) value was determined at four random views in cortex and striatum from four serial slices in a blinded manner with Leica Qwin image processing and analysis system (Leica Company).
SABC-FITC (1:100) 37 C for 30 min, respectively. The sample was added with anti-Nrf-2 (1:100), the second antibody (1:100), SABC-CY3 (1:100), successively, and covered with waterborne mount. HO-1 and NRF-2 positive cells were observed in the same field of views with the excitation light of different wavelengths under a fluorescence microscope in order to determine the relationship between the two proteins. All slides were carried out under a dark circumstance throughout the procedures and washed fully in PBS, so as to avoid excessive residual or metamorphosis fluorescence.
Immunohistochemical Assay
Results were expressed as mean 6 standard error (x 6 s), and the numbers represented at least three separate experiments for each group. Statistical comparisons were made by analysis of variance (ANOVA) and the least significant difference (LSD) t test. A P value 0.05).
Environmental Toxicology DOI 10.1002/tox
To investigate the relationship between HO-1 and Nrf-2 proteins in brain tissue, we used double immunofluorescence labeling. HO-1 and Nrf-2 positive cells, with irregular shape, were scattered in many regions of brain tissue, and their expression levels were elevated after CO exposure under the immunofluorescence microscope. These positive cells mainly resided in neurons and astrocytes. In contrast to the location of the two proteins in the same field of view, we found that some but not all HO-1 positive cells showed Nrf2 immunoreaction. Obviously, many Nrf-2 positive cells did not appear HO-1 immunogenicity (Fig. 8). It offers a potent evidence that the two proteins can co-exist in a single neurocyte, also can be expressed in different cells, respectively. Although the expression of these two proteins could be detected in normal brain tissue, and gradually increased after CO poisoning, no correlation was found between HO-1 and Nrf-2 expressions by statistical analysis. The result suggests
Fig. 3. The apoptosis cells in brain tissue in rats after C0 exposure. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
EDARAVONE ATTENUATES BRAIN DAMAGE IN RATS
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binds to hemoglobin and produces HbCO which results in the displacement of oxygen and subsequently reduces the supply of oxygen available to the tissues of the body. Once ischemia and hypoxia occurred, the structure and function of certain vital organs were undoubtedly impaired. Briefly, neuronal structure was damaged, the oxidative phosphorylation of mitochondria was decreased or stopped, energy metabolism was disordered, electrolyte both inside and outside the cell membrane was imbalance, and thus a cascade of
Fig. 4. The expressions of HO-1 protein in each group under a 400-fold light microscope. CO poisoning could induce the expression of HO-1 protein. The amount of HO-1 positive cells and the A value increased in many brain areas at 1 and 7 day in group B. The overexpression of HO-1 protein were detected, and the A value of each view was also increased after Edaravone administration. Significant differences were found between the group B and group C (P < 0.01), suggesting that Edaravone play an important role in the activation of the HO-1/Nrf-2 pathway against oxidative stress damage. A: Group B in cortex at 1 day after CO exposure; B: Group C in cortex at 1 day after CO exposure; C: Group B in cortex at 7 day; D: Group C in cortex at 7 day; E: Group B in striatum at 1 day after CO exposure; F: Group C in striatum at 1 day after CO exposure; G: Group B in striatum at 7 day; H: Group C in striatum at 7 day (scale bar: 30 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
that HO-1 and Nrf-2 proteins may be activated successively by one or variety of pathways in brain tissue after CO poisoning.
DISCUSSION Cases of CO poisoning remain an important cause of death in the practice of pathologists who perform autopsies following deaths within the community. When people inhale CO, it
Fig. 5. The expressions of Nrf-2 protein in rats after CO exposure under a 400-fold light microscope. With the duration of CO exposure, the increased Nrf-2 positive cells were detected in group B at 1 and 7 day. As compared with group B, HO-1 positive cells in group C were obviously enhanced, and its A value of each view was also increased at the same time points both in cortex and striatum (P < 0.01). A: Group B in cortex at 1 day after CO exposure; B: Group C in cortex at 1 day after CO exposure; C: Group B in cortex at 7 day; D: Group C in cortex at 7 day; E: Group B in striatum at 1 day after CO exposure; F: Group C in striatum at 1 day after CO exposure; G: Group B in striatum at 7 day; H: Group C in striatum at 7 day (scale bar: 30 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
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LI ET AL.
Fig. 6. The A values of H021 in each group at different time points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 7. The A values of Nrf22 in each group at different time points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
reactions were stimulated, as a result, to lead to cell death and malfunction. As we showed under TEM, neurons in group B were irregular with chromatin condensation and marginalization, cytoplasm dissolution and vacuoles formation. Edaravone could effectively protect brain tissue from ultrastructural damage following CO poisoning. Neurons are non-renewable. The apoptosis or death of neurocytes directly affects the normal physiological function of the central nervous system. Preventing or reducing apoptosis can significantly improve neurological malfunction. CO exposure leads to diffuse hypoxic-ischemic encephalopathy and focal cortical injury that, during the acute stage of intoxication, is owing to transient vasogenic edema or frank necrosis, predominantly involving gray matter. By flow cytometry, Guratowska et al. (2010) found significantly increased apoptosis of the lymphocytes in research group compared to control individuals correlated with the CO poisoning severity, but did not depend on hypoxia. Decreased number of leukocytes caused by the cytotoxic effect of CO stimulated the release of the CD 341 to the peripheral blood. Brvar et al.’s (2010) study showed that CO poisoning induced ganglionic cell apoptosis. The percentage of apoptotic cells in rats exposed to CO was 32%. Tofighi et al. (2006) insisted that after exposed to CO, cells exhibited loss of mitochondrial membrane potential, release of cytochrome C into the cytosol, nuclei with chromatin condensation. Those cell features
were fully consistent with the characteristics of apoptosis. CO also triggered activation of caspase and calpain proteases. As we expected, apoptosis cells were significantly increased in group B. After the treatment of Edaravone, the amount of apoptosis cells was significantly decreased both in cortex and striatum, suggesting that Edaravone may prevent apoptosis process induced by CO poisoning. Reactive oxygen species/reactive nitrogen family (ROS/ RNS), highly reactive molecules, can damage nucleic acids and proteins, resulting in lipid peroxidation and inactivation of cell membrane receptors and enzyme, and ultimately affecting the function of cell. The human body has several mechanisms to counteract oxidative stress by producing antioxidants. HO-1/Nrf-2 is one of the important anti-oxidation pathway. HO-1 is an isoform in hemeoxygenase (HO) system, which is responsible for cellular heme degradation to biliverdin, iron, and carbon monoxide. It has been extensively studied mainly by its ability to respond to cellular stresses such as hemin, nitric oxide donors, oxidative damage, hypoxia, hyperthermia, and heavy metals (Ye et al., 2012; Wang et al., 2013; Zhang et al., 2014). Nrf-2 is a gene transcription factor that binds to the electrophile response element (EpRE) and triggers expression of various genes with antioxidant properties, including HO-1. Nrf-2 contributes significantly to cytoprotection against oxidative stress. The increase in mRNA expression of antioxidant enzymes
Fig. 8. The relationship of HO-1 and Nrf-2 expression in rats after CO poisoning. With the excitation light of different wavelengths under a fluorescence microscope, HO-1 positive cells appeared yellow-green light (A), while Nrf-2 positive cells showed red (B). HO-1 and Nrf-2 positive cells, with irregular shape, were scattered in many regions of brain tissue. Overlapping the two photographs of the same view, we found that some but not all HO-1 positive cells showed Nrf-2 immunoreaction, many Nrf-2 positive cells did not appear HO-1 immunogenicity (C). It offers a potent evidence that the two proteins can co-exist in a single neurocyte, also can be expressed in different cells, respectively. They may be activated successively by one or variety of pathways in brain tissue after CO poisoning. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
EDARAVONE ATTENUATES BRAIN DAMAGE IN RATS
was positively correlated with the Nrf-2 transcription in cortex and hippocampus. The deletion or activation barriers of Nrf-2 gene made the enhanced sensitivity of neurons to oxidative stress (Kobayashi et al., 2004; Genc et al., 2010). Nrf2 expression could be detected in MSCs. Transient expression of Nrf-2 protected MSCs against cell death and apoptosis triggered by hypoxic and oxidative stress conditions (Mohammadzadeh et al., 2012). Nrf-2 also enhanced the activity of SOD and HO-1. Ha et al. (2012) insisted that hypoxia induced HO-1 expression through PI3K/Akt/Nrf-2 signal pathways. Our results showed that the basal expression of HO-1 and Nrf-2 could be found in brain tissue of group A, indicating that the body could keep equilibrium of transcription and translation and degradation of the two proteins, maintain the balance of oxidant/ antioxidant system under physiological conditions. Nrf-2 and HO-1 expressions increased significantly in rats after CO poisoning, suggesting that Keapl-Nrf-2/ARE antioxidant system be activated in brain tissue following CO exposure. Meanwhile, we found that some but not all HO-1 positive cells showed Nrf-2 immunoreaction. Many Nrf-2 positive cells did not appear HO-1 immunogenicity. The expression level of the two proteins lacks correlation. The data demonstrated that HO-1 and Nrf-2 proteins might be activated successively by one or variety of pathways in brain tissue after CO poisoning. After the treatment of Edaravone, the expressions of Nrf-2 and HO-1 were increased obviously, suggesting that the neuroprotection of Edaravone on brain tissue from oxidative stress be partly mediated by Keapl-Nrf-2/ARE pathway via the upregulation of Nrf-2 and HO-1 expression.
CONCLUSION We successfully established the animal model of CO poisoning in a hyperbaric oxygen chamber by exposed to CO. As we expected, Edaravone can inhibit apoptosis, mediate the overexpression of Nrf-2 and HO-1protein, be in part associated with activation of the Keapl-Nrf-2/ARE against oxidative stress in rats following CO poisoning. Edaravone, as a novel neuroprotective treatment, may be designed for patients with acute CO poisoning.
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Environmental Toxicology DOI 10.1002/tox
