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Contents lists available at ScienceDirect
NeuroToxicology
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Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats
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Dong a,1, Ming Ren b,1, Xiujie Wang a, Hongquan Jiang c, Xiang Yin c, Shuyu Wang c, Xudong Wang c, Honglin Feng c,*
Q1 Guangtao
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a b c
Department of Emergency Medicine, The First Affiliated Hospital of Harbin Medical University, Harbin, PR China Department of Neurology, The Affiliated Hospital of Weifang Medical University, Weifang, PR China Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 November 2014 Accepted 25 March 2015 Available online xxx
Approximately half of those who survive severe carbon monoxide (CO) poisoning develop delayed neurologic sequelae (DNS). Growing evidence supports the crucial role of free radicals in delayed brain injury associated with CO toxicity. Xanthine oxidase (XO) has been reported to play a pivotal role in the generation of reactive oxygen species (ROS) in CO poisoning. A recent report indicates that allopurinol both attenuated oxidative stress and possessed anti-inflammatory properties in an animal model of acute liver failure. In this study, we aimed to explore the potential of allopurinol to reduce the severity of DNS. The rats were first exposed to 1000 ppm CO for 40 min and then to 3000 ppm CO for another 20 min. Following CO poisoning, the rats were injected with allopurinol (50 mg/kg, i.p.) six times. Results showed that allopurinol significantly reduced neuronal death and suppressed expression of proinflammatory factors, including tumor necrosis factor-a, intercellular adhesion molecule-1, ionized calcium-binding adapter molecule 1, and degraded myelin basic protein. Furthermore, behavioral studies revealed an improved performance in the Morris water maze test. Our findings indicated that allopurinol may have protective effects against DNS caused by CO toxicity. ß 2015 Elsevier Inc. All rights reserved.
Keywords: Allopurinol Delayed neurologic sequelae Carbon monoxide poisoning Inflammation Myelin basic protein
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1. Introduction Q2
Carbon monoxide (CO) poisoning leads to an estimated 50,000 emergency room visits in the US annually (Hampson et al., 2012) and is one of the leading causes of death by poisoning. Approximately half of those who survive severe CO poisoning, characterized by loss of consciousness or carboxyhemoglobin (HbCO) greater than 25%, develop delayed neurologic sequelae (DNS) after a latency period of 2–40 days, even with treatment ˜ agas, 2004; Weaver et al., 2002). Hyperbaric oxygen (Kao and Nan (HBO) therapy is a widely accepted protocol for treating CO toxicity (Thom et al., 2006a; Weaver, 2009), but its efficacy is uncertain (Buckley et al., 2011; Hampson et al., 2012; Weaver, 2014). Growing evidence suggests that brain injury after acute
* Corresponding author at: No. 23, Youzheng Street, Nangang District, Harbin City, Heilongjiang Province, PR China. Tel.: +86 451 85555666; fax: +86 451 53605867. E-mail addresses: [email protected] (G. Dong), [email protected] (H. Feng). 1 These authors equally contributed to this work.
CO toxicity is related to oxidative stress, apoptosis, immunemediated injury and abnormal inflammatory responses (Piantadosi et al., 1997; Thom, 1990a; Thom et al., 2004, 2006a). Free radicals may have an initiating role in the damage cascade (Kao ˜ agas, 2004; Thom, 1990a; Thom et al., 2004). Several and Nan studies suggest that free radical scavengers, such as edaravone (Qingsong et al., 2013) and molecular hydrogen (Sun et al., 2011) ameliorate DNS after CO poisoning, supporting the concept that free radicals are involved in medicating the noxious effects of CO poisoning. Xanthine oxidase (XO), a form of xanthine oxidoreductase (XOR), may be the primary source of free radical generation during cerebral ischemia/reperfusion (Ono et al., 2009; Traystman et al., 1991). XO metabolizes hypoxanthine to xanthine, and then to uric acid, generating reactive oxygen species (ROS), including superoxide and hydrogen peroxide (Ono et al., 2009; Traystman et al., 1991), which are required for lipid peroxidation. Subsequently, XO has been reported to be a key component in generating ROS after CO poisoning (Thom, 1992). Furthermore, cognitive impairment associated with CO poisoning in rats was improved by depletion of XO with tungsten (Han et al., 2007).
http://dx.doi.org/10.1016/j.neuro.2015.03.015 0161-813X/ß 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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Allopurinol, a competitive and high-affinity XO inhibitor, has been used primarily for treating hyperuricemia and gout. Studies have also shown that allopurinol protects the brain from ischemia and reperfusion-induced injuries caused by oxidative stress in the brain (Isik et al., 2005), intestine (Sapalidis et al., 2013), kidney (Keel et al., 2013) and heart (Kang et al., 2006). Furthermore, XO inhibitors have anti-inflammatory effects in acute lung injury (Flaishon et al., 2006), acute pancreatitis (Martinez-Torres et al., 2009), multiple sclerosis (MS) (Honorat et al., 2013) and liver inflammation (Demirel et al., 2012). However, whether allopurinol can protect against encephalopathy due to CO toxicity is unclear. Therefore, in a model of CO poisoning, we investigated whether allopurinol reduces neurohistopathological lesion progression and inflammatory responses in the brain and improves the cognitive functioning.
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2. Materials and methods
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2.1. Animals
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Adult male Sprague-Dawley rats (280 20 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Animals were housed in a room maintained on a 12 h/12 h light/dark cycle with food and water available ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. Rats were randomly assigned to four different groups (N = 28 per group): air + vehicle (air exposed, vehicle-treated rats), air + allopurinol (air exposed, allopurinol-treated rats), CO + vehicle (CO exposed, vehicle-treated rats) and CO + allopurinol (CO exposed, allopurinol-treated rats).
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2.2. Catheter placement
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Rats in air + vehicle and CO + vehicle groups were anesthetized with 7% chloral hydrate (5 ml/kg, i.p.) 2 h before acute CO poisoning. Catheters were cannulated in the right femoral artery for blood pressure recording and blood gas sampling and were filled with heparinized normal saline (100 U/ml) when not in use.
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2.3. Drug preparation and experimental protocol
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Allopurinol (8 mg/ml) (Sigma–Aldrich Co., St. Louis, MO, USA) was dissolved in preheated saline containing 0.125 M NaOH and administered to animals (50 mg/kg). Doses were selected based on previous studies with allopurinol (50–200 mg/kg in rodents) (Ono et al., 2009; Thom, 1992). All solutions were freshly prepared on the day of testing. CO exposure was performed per a published protocol in 7-L Plexiglas chambers (Thom et al., 2004). CO, CO2 and O2 in the chamber were monitored. Vapor and carbon dioxide (CO2)-rich exhalation from the animals was absorbed by saline lime inside the chamber. Rats in the CO + vehicle and CO + allopurinol groups breathed 1000 ppm CO mixed with air for 40 min and then 3000 ppm CO mixed with air for 20 min. Animals lost consciousness after 3000 ppm CO exposure was initiated and quickly regained consciousness after they were removed from the chambers. Rats in the air + vehicle and air + allopurinol groups were placed in Plexiglas chambers as described above but breathed room air for 1 h. For rats in the air + allopurinol and CO + allopurinol groups, allopurinol was injected (i.p.) six times at 12-h intervals starting 1 h after CO exposure. During the same time points that allopurinol was administered to these rats, animals in the air + vehicle and CO + vehicle groups received an equivalent volume of preheated saline containing 0.125 M NaOH.
2.4. Arterial HbCO and monitoring of blood gases
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Blood samples (200 ml) were withdrawn from the right femoral artery of the rats through a catheter 60 min after CO exposure. HbCO, arterial blood gases and acid-base chemistry were measured with an ABL800 FLEX blood gas system (Radiometer Medical ApS., Denmark).
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2.5. Histologic and immunohistochemical studies
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Rats were euthanized with an overdose of 7% chloral hydrate then transcardially perfused with 100 ml of precooled normal saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Perfusion-fixed brains were removed and post fixed with 4% paraformaldehyde overnight. Paraffin embedded brains were sliced into 4 mm thick coronal sections then mounted onto slides for future use.
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2.6. Nissl staining
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For Nissl staining, 4 mm sections were deparaffinized, rehydrated and stained with thionine, followed by differentiation and dehydration before mounting. Images of the prefrontal cortex and the CA1 area of the hippocampus from each brain were captured using an Olympus DP73 microscope with three visual fields/per brain section selected. Intact pyramidal cells were counted by three investigators with Imaging-Pro-Plus (Leica Microsystems, Wetzlar, Denmark).
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2.7. Immunohistochemical staining
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For immunostaining, brain sections were sequentially treated with 0.3% H2O2 in PBS for 30 min, 1.5% normal goat serum and 1% bovine serum albumin for 30 min at room temperature. Then, sections were incubated with either mouse anti-ionized calciumbinding adapter molecule 1 (Iba1) antibody (1:400; Millipore, Temecula, CA) or rabbit anti-myelin basic protein (MBP) antibody (1:500; Abcam, Cambridge, UK) overnight at 4 8C and subsequently exposed to biotinylated goat anti-mouse or rabbit immunoglobulin G antibody and HRP-labeled Streptavidin (Beyotime Institute of Biotechnology, Jiangsu, China) for 30 min at room temperature. Immunostaining was visualized with 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 50 mM Tris–HCl buffer (pH 7.2). Sections were coverslipped and examined under a DP73, Olympus microscope.
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2.8. Quantitative analysis
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From every prefrontal cortex, hippocampus and corpus callosum three coronary sections (spaced 60-mm apart) (based on anatomical landmarks) were used for Nissl and three sections for immunohistochemical analysis, which was performed by three blinded researchers. In three microscopic fields (40 objective) in the prefrontal cortex, CA1 subregion and corpus callosum Nissl positive neurons or Iba1 positive cells were counted for each coronal brain section. Sections were evaluated under an Olympus DP73 microscope. Quantification of the myelinated area was performed by counting MBP-positive pixels in one series of sections using the threshold feature of Image-Pro Plus software (Media Cybernetics, Bethesda, MD). All images for each type of staining were taken with the same exposure time.
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2.9. ELISA
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Rats were euthanized with 7% chloral hydrate 3 days after CO exposure. Brains were removed. Fresh tissue from the cerebral
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cortices was dissected from bilateral frontal lobes and hippocampi immediately and homogenized in cold RIPA lysis buffer (containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium, deoxycholate and 0.5% SDS) with protease inhibitor cocktail (10 ml/ml; Pierce, Rockford, IL) and centrifuged for 10 min at 10,000 g at 4 8C. Supernatants were then collected and kept at 80 8C until further testing. Protein homogenate was quantified with a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). TNF-a, intercellular adhesion molecule-1 (ICAM-1) and highmobility group box 1 (HMGB1) protein were measured with a commercial ELISA kit (Uscn Life Science Inc., Wuhan, China) according to kit instructions.
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2.10. Western blotting
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To measure the levels of MBP, an aliquot (40 mg) of total protein from the samples was loaded on 5–20% SD polyacrylamide gels and separated by electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes then incubated with anti-MBP (1:500; Abcam, Cambridge, UK) rabbit polyclonal antibody overnight at 48C. Membranes were rinsed with 0.1% Tween 20 in phosphate-buffered solution (PBS-T) and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000; Beyotime Institute of Biotechnology, Jiangsu, China) for 2 h at room temperature. Immunoreactivity was measured with ECL. Optical density of bands was measured with ImageJ software (National Institutes of Health).
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2.11. Behavioral tests
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To measure cognitive impairment after CO toxicity, a Morris water maze (MWM) test was performed at 4 weeks in a 150-cmdiameter circular pool filled with water (20.0 1 8C). The pool was virtually separated into four quadrants (NW, SW, NE and SE). A transparent platform was submerged 1.5 cm below the water at a fixed location, i.e. southwest quadrant (SWQ) in the pool. A digital video recorder was mounted on the room ceiling and connected to a computer. Swimming paths of rats were tracked and recorded. Mean swimming speeds were calculated by using ZH-maze (Version 3.2) software (Zhenghua Biology Instrument, Anhui, China). Rats were adapted to the water for 120 s and then placed on the platform for 60 s before the acquisition test (d 0). In the acquisition phase, animals were subjected to two trials per day for 4 continuous days. During each trial, the rats were released from four random starting positions and allowed to swim for 120 s. Once the rats located the platform, the rats were allowed to rest for 30 s. If a rat did not find the platform within 120 s, it was guided to the platform and allowed to rest for 30 s. Probe trials were performed on d 5 to test rat memory retention. For this study, the platform was removed and rats were allowed to swim for 120 s in the pool. The amount of time spent in the SWQ was recorded, where the platform had been located during acquisition phase.
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2.12. Statistical analysis
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Statistical analysis was performed by using GraphPad Prism 6.02 software (La Jolla, CA). Mean escape latency and mean
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swimming speed data in the water maze test were analyzed by two-way analysis of variance (ANOVA) followed by a Student– Newman–Keuls post hoc test. Data of time of duration in the SWQ, Nissl staining, Iba1 positive cells, TNF-a, ICAM-1, HMGB1 levels and data presented as percentages were analyzed with one-way ANOVA followed by Student–Newman–Keuls post hoc test. P values of 0.05). (C) Representative swimming tracks of rats in probe trial are demonstrated. The blue square represents the start position and the red square represents the end position. (D) The time spent in southwest quadrant (SWQ), which is an indicator of the ability of spatial reference memory, was recorded. Data are presented as means SEM. **P < 0.01 compared with air + vehicle group; #P < 0.05 compared with CO + vehicle group (n = 7 for each group).
Fig. 2. Allopurinol was found to suppress carbon monoxide (CO)-induced elevation of tumor necrosis factor-a (TNF-a) levels, intercellular adhesion molecule-1 (ICAM-1) and high-mobility group box 1 (HMGB1) protein levels in the cortex and hippocampus of rats. Rat brains were removed and protein levels of TNF-a, ICAM-1 and HMGB1 were determined by ELISA. Allopurinol partially suppressed TNF-a (A, D), ICAM-1 (B, E) and HMGB1 expression (C, F) 3 days after CO poisoning. Data are expressed as means SEM. **P < 0.01 compared with air + vehicle group; #P < 0.05, ##P < 0.01 compared with CO + vehicle group (n = 7 for each group).
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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differences found among treatment groups in terms of swimming speeds during the 4 days of acquisition (Fig. 1B).
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3.3. Allopurinol reduced TNF-a, ICAM-1 and HMGB1 protein
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Allopurinol may affect the levels of inflammatory factors after CO exposure. For this reason, the protein levels of TNF-a, ICAM-1
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and HMGB1 were here measured using ELISA. TNF-a, ICAM-1 and HMGB1 protein levels in the cortex and hippocampus of the CO + vehicle group were significantly increased 3 days after CO exposure compared with those in the air + vehicle group. Treatment with allopurinol reduced tissue concentration of all 3 proteins in the cortex and hippocampus, compared with CO + vehicle treated rats. Allopurinol treatment had no effect in air treated animals (Fig. 2).
Fig. 3. Allopurinol reduced neuronal death induced by CO poisoning. Rat brains were removed 1 week after CO exposure and stained with Nissl dye. (A) Upper two panels and lower two panels show the Nissl staining in the cortex and CA1 subregion of the hippocampus, respectively. Damaged areas induced by CO inhalation have fewer Nisslpositive neurons. (B, C) The undamaged Nissl-positive neurons in the cortex and hippocampal subregion CA1 were quantified. Data are means SEM. **P < 0.01 compared with air + vehicle; ##P < 0.01 as compared with CO + vehicle group (n = 7 for each group).
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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3.4. Allopurinol treatment protected against neuronal death
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Representative Nissl staining 1 week after CO exposure revealed neuronal loss in the cerebral cortex and the hippocampal CA1 region compared with the air + vehicle group (Fig. 3A). Allopurinol reduced neuronal loss in the cortex and hippocampus as shown in the CO + allopurinol group (Fig. 3A). The extent of neuronal degeneration was quantified in the cerebral cortex and the hippocampal subregions CA1 region (Fig. 3B and C). There were significantly fewer neurons in these parts of the brains of animals exposed to CO than in those of air + vehicle animals, whereas the neurons in the cerebral cortex and hippocampus of the CO + allopurinol group were relatively spared when compared to the CO + vehicle group.
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3.5. Allopurinol treatment results in reduced microglia activation
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Microglial activation was quantified in the brain 4 weeks after MWM using anti-Iba1 antibody staining. In the CO + vehicle group, Iba-1 immunoreactivity was significantly increased and Iba-1
immunoreactive microglia cells were distinctly hypertrophied (Fig. 4A–C) compared with those from the air + vehicle group. However, in the CO + allopurinol group, Iba-1 immunoreactivity declined (Fig. 4A–C) compared with the CO + vehicle group.
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3.6. Allopurinol treatment results in reduced demyelination
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Using MBP (major central myelin protein) immunohistochemical staining and Western blotting, we observed demyelination in the brain of CO + vehicle-treated animals. Large plaques of demyelination were detected in the corpus callosum and hippocampus of CO exposed rats at peak toxicity (week 4 after CO exposure) when compared with normal rats (Fig. 5A and C). As expected, demyelination was dramatically reduced by allopurinol treatment compared to the CO + vehicle group (Fig. 5A and C). Western blotting confirmed that MBP protein was significantly decreased (Fig. 5B and D) in the corpus callosum and hippocampus of CO exposed rats compared with corresponding controls. However, MBP protein was significantly increased in the CO + allopurinol group compared with the CO + vehicle group (Fig. 5B and D).
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Fig. 4. Allopurinol significantly suppressed activation of microglia in the rat brain exposed to CO. Brain sections were immunostained with mouse anti-Iba1 antibody. (A) CO induced significant increases in the number of Iba1-positive cells, reflecting microglial activation, in the hippocampus and corpus callosum at weeks 4 after CO exposure. Allopurinol administration partially suppressed the increase of Iba1-positive cells induced by CO exposure. (B, C) Iba1-positive cells were measured as described in Methods. Increased Iba1 immunopositive cells in brains of CO + vehicle animals compared with those of the air + vehicle group were noted. Allopurinol significantly reduced the activation of microglia induced by CO exposure. Data are means SEM. **P < 0.01 compared with air + vehicle group; ##P < 0.01 compared with CO + vehicle group (n = 7 for each group).
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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Fig. 5. Allopurinol reduced the demyelination induced by CO in the corpus callosum and hippocampus in rat brains 4 weeks after CO exposure. Brain sections were prepared and immunostained with an anti-myelin basic protein (MBP) antibody. Proteins from fresh brain tissue were used for Western blotting with the same antibody. (A) MBPpositive myelinated fibers in the corpus callosum and hippocampus decreased after CO poisoning. However, allopurinol suppressed demyelination induced by CO exposure as shown by immunostaining. (B) MBP protein decreased markedly in both the corpus campus and hippocampus after CO poisoning, compared with air + vehicle group. Treatment with allopurinol suppressed MBP protein degradation induced by CO. (C) MBP-positive-myelinated fibers were quantified using Image-Pro Plus software. (D) MBP to actin protein ratio is shown as means SEM. **P < 0.01 compared with air + vehicle; ##P < 0.01, as compared with CO + vehicle group (n = 7 for each group).
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4. Discussion
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In the present study, we report for the first time that allopurinol is neuroprotective against DNS caused by CO poisoning. Allopurinol treatment initiated 1 h after CO exposure reduced neuronal death in the cerebral cortex and hippocampus, inhibited Iba1
expression, attenuated proinflammatory TNF-a, ICAM-1 and HMGB1 cytokines levels, reduced demyelination, and alleviated cognitive deficits in a rat behavioral model. Xanthine dehydrogenase (XDH) and XO are inter-convertible forms of XOR. Under ischemia and reperfusion, ATP is degraded to ADP, AMP, adenosine, inosine and hypoxanthine, and XDH is
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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increasingly converted to XO to oxidize hypoxanthine to xanthine, and then xanthine to uric acid and oxygen radicals (Traystman et al., 1991). Like cerebral ischemia-reperfusion, tissue hypoxia also occurs after CO poisoning with subsequent degradation of purine molecules, triggering the conversion of XDH to XO. This indicates that the XO pathway may be a source of the free radicals after CO poisoning (Thom, 1992), which may contribute to destruction of the cell membrane by initiating lipid peroxidation (Thom, 1990a,b). MBP modifications can occur due to reactions with lipid peroxidation products and may trigger a lymphocytic immunologic response, as well as lead to microglial activation that, in turn, can lead to neuropathological effects contributing to DNS (Thom et al., 2004) development. Allopurinol, as a strong XO inhibitor and a free radical scavenger (Moorhouse et al., 1987), may inhibit the initial step of the XO cascade. Conversion of XD to reversible XO has been detected as early as 40 min after the initiation of CO exposure (1000 ppm) and it was found to last for at least 90 min after discontinuation of CO exposure (Thom, 1992). This may explain why administration of allopurinol 1 h after CO exposure only partially suppresses the development of DNS. In addition, CO hypoxia, free radicals and oxidative stress cause neuronal necrosis and apoptosis (Piantadosi et al., 1997). CO poisoning causes hypoxia because CO has 210 times the affinity to Hb as O2. CO hypoxia induces neuronal death, and CO itself also causes neuronal death independent of hypoxia (Uemura et al., 2003). For CO poisoning, CO-hypoxia-induced (equivalent to about 10% O2) neuronal injury was much less severe than direct CO injury (Uemura et al., 2003). In vitro, CO induced hypoxia-independent apoptosis in neural cells through the intrinsic mitochondrial pathway (Tofighi et al., 2006). In the current study, the O2 concentration in the chambers was maintained at 20% (see Supplementary Fig. 1). PaO2 values (93.57 0.90) in the CO + vehicle group were relatively stable (Table 1), so hypoxic hypoxia was properly controlled. Then, results showed that allopurinol was protective against CO induced neuronal death (Fig. 3) and these data agree with previous reports of neonatal hypoxia-ischemia models (PeetersScholte et al., 2003). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuro.2015.03.015. Inflammation induced by CO exposure is also critical to DNS after CO poisoning (Thom et al., 2004, 2006a,b). HMGB1 is reported to function as a novel proinflammatory cytokine-like factor after reperfusion in the pathophysiology of cerebral ischemia-reperfusion and is released from neurons either during the early phase (Qiu et al., 2008) or in a delayed manner after reperfusion (Kim et al., 2006). Early release of HMGB1 from neurons was reported to cause release of pro-inflammatory cytokine (TNF-a) and then to upregulate expression of intercellular ICAM-1 in brain endothelial cells (Qiu et al., 2008). Recent studies suggest that HMGB1 expression and/or release might be related to oxidative stress (Tang et al., 2007; Tsung et al., 2007). In our study, HMGB1 levels increased in the rat brain during early post-CO exposure (Fig. 2C and F) and allopurinol suppressed elevated HMGB1, TNF-a and ICAM-1 (Fig. 2), which may be related to allopurinol’s antioxidant activity after CO exposure. In addition, recent investigation indicates that extracellular HMGB1 was a proinflammatory cytokine, triggering microglial activation in the postischemic brain (Kim et al., 2006). Thus, how HMGB1 functions in inflammation after CO poisoning warrants additional investigation. Microglia are principal immune cells and resting resident macrophage-like cells throughout the brain parenchyma. As sensors of pathological events, they can be activated by pathological changes in the brain (Kreutzberg, 1996). Activated microglia can trigger neuronal apoptosis by releasing ROS (Banati et al., 1993), inflammatory cytokines and nitric oxide (Palluy and Rigaud, 1996). Microglial and macrophage ROS production have
been implicated in myelin and axonal impairment in MS (van Horssen et al., 2011). Also, activated microglia are involved in immunological responses after CO poisoning (Thom et al., 2004, 2006a). We found that allopurinol restrained microglial activation (Fig. 4), a hallmark of brain inflammation and reduced demyelination induced by CO exposure in the hippocampal CA1 region and in the corpus callosum in rats (Fig. 5). Thus, allopurinol alleviated DNS caused by CO via reducing the inflammatory response. However, it is not clear whether ROS generated by XO triggers inflammation or inflammation activates XO and causes the generation of ROS during CO poisoning. In a cell culture model of anoxia/re-oxygenation, XO was a major source of ROS in a microglial cell line (Widmer et al., 2007). Honorat et al. reported strong expression of XO in microglia and macrophages in an animal model of MS (Honorat et al., 2013). CD11b is an important surface marker of microglia and ROS promote microglial activation via nitric oxide, characterized by increased CD11b expression (Roy et al., 2008). It can be reasonably inferred that allopurinol interrupts the feed forward mechanism by which ROS and microglia activation mutually enhance each other. XO represents a novel therapeutic target for treating delayed encephalopathy, and allopurinol, which has been used extensively to treat gout, is a promising compound for this target. Our results indicate that allopurinol, a potent inhibitor of XO, may improve the outcomes of delayed encephalopathy.
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5. Conclusion
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Allopurinol alleviated neuronal damage, attenuated inflammation and demyelination, and reduced cognitive deficits in an animal model of severe CO poisoning.
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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Transparency document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgements
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This work was supported by grants from the National Natural Q3 Science Foundation of China (No. 81171186), the Health Department Project of Heilongjiang Province (No. 2013010) and the Natural Science Foundation of Heilongjiang Province (No. ZD201417).
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References
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Sapalidis K, Papavramidis TS, Gialamas E, Deligiannidis N, Tzioufa V, Papavramidis S. The role of allopurinol’s timing in the ischemia reperfusion injury of small intestine. J Emerg Trauma Shock 2013;6(3):203–8 [PubMed: 23960379]. Sun Q, Cai J, Zhou J, Tao H, Zhang JH, Zhang W, et al. Hydrogen-rich saline reduces delayed neurologic sequelae in experimental carbon monoxide toxicity. Crit Care Med 2011;39(4):765–9 [PubMed: 21200321]. Tang D, Shi Y, Kang R, Li T, Xiao W, Wang H, et al. Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1. J Leukoc Biol 2007;81(3):741–7 [PubMed: 17135572]. Thom SR. Antagonism of carbon monoxide-mediated brain lipid peroxidation by hyperbaric oxygen. Toxicol Appl Pharmacol 1990a;105(2):340–4 [PubMed: 2219124]. Thom SR. Carbon monoxide-mediated brain lipid peroxidation in the rat. J Appl Physiol 1990b;68(3):997–1003 [PubMed: 2341364]. Thom SR. Dehydrogenase conversion to oxidase and lipid peroxidation in brain after carbon monoxide poisoning. J Appl Physiol 1992;73(4):1584–9 [PubMed: 1447108]. Thom SR, Bhopale VM, Fisher D. Hyperbaric oxygen reduces delayed immune-mediated neuropathology in experimental carbon monoxide toxicity. Toxicol Appl Pharmacol 2006a;213(2):152–9 [PubMed: 16325878]. Thom SR, Bhopale VM, Fisher D, Zhang J, Gimotty P. Delayed neuropathology after carbon monoxide poisoning is immune-mediated. Proc Natl Acad Sci USA 2004;101(37):13660–5 [PubMed: 15342916]. Thom SR, Bhopale VM, Han ST, Clark JM, Hardy KR. Intravascular neutrophil activation due to carbon monoxide poisoning. Am J Respir Crit Care Med 2006b;174 (11):1239–48 [PubMed: 16931637]. Tofighi R, Tillmark N, Dare E, Aberg AM, Larsson JE, Ceccatelli S. Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro. Brain Res 2006;1098(1):1–8 [PubMed: 16777078]. Traystman RJ, Kirsch JR, Koehler RC. Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J Appl Physiol 1991;71(4):1185–95 [PubMed: 1757340]. Tsung A, Klune JR, Zhang X, Jeyabalan G, Cao Z, Peng X, et al. HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med 2007;204(12):2913–23 [PubMed: 17984303]. Uemura K, Hoshino S, Uchida K, Tsuruta R, Maekawa T, Yoshida K. Hypothermia attenuates delayed cortical cell death and ROS generation following CO inhalation. Toxicol Lett 2003;145(2):101–6 [PubMed: 14581162]. van Horssen J, Witte ME, Schreibelt G, de Vries HE. Radical changes in multiple sclerosis pathogenesis. Biochim Biophys Acta 2011;1812(2):141–50 [PubMed: 20600869]. Weaver LK. Clinical practice. Carbon monoxide poisoning. N Engl J Med 2009;360 (12):1217–25 [PubMed: 19297574]. Weaver LK. Hyperbaric oxygen therapy for carbon monoxide poisoning. Undersea Hyperb Med 2014;41(4):339–54 [PubMed: 25109087]. Weaver LK, Hopkins RO, Chan KJ, Churchill S, Elliott CG, Clemmer TP, et al. Hyperbaric oxygen for acute carbon monoxide poisoning. N Engl J Med 2002;347(14):1057–67 [PubMed: 12362006]. Widmer R, Engels M, Voss P, Grune T. Postanoxic damage of microglial cells is mediated by xanthine oxidase and cyclooxygenase. Free Radic Res 2007;41(2):145–52 [PubMed: 17364940].
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Contents lists available at ScienceDirect
NeuroToxicology
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Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats
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Dong a,1, Ming Ren b,1, Xiujie Wang a, Hongquan Jiang c, Xiang Yin c, Shuyu Wang c, Xudong Wang c, Honglin Feng c,*
Q1 Guangtao
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a b c
Department of Emergency Medicine, The First Affiliated Hospital of Harbin Medical University, Harbin, PR China Department of Neurology, The Affiliated Hospital of Weifang Medical University, Weifang, PR China Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 November 2014 Accepted 25 March 2015 Available online xxx
Approximately half of those who survive severe carbon monoxide (CO) poisoning develop delayed neurologic sequelae (DNS). Growing evidence supports the crucial role of free radicals in delayed brain injury associated with CO toxicity. Xanthine oxidase (XO) has been reported to play a pivotal role in the generation of reactive oxygen species (ROS) in CO poisoning. A recent report indicates that allopurinol both attenuated oxidative stress and possessed anti-inflammatory properties in an animal model of acute liver failure. In this study, we aimed to explore the potential of allopurinol to reduce the severity of DNS. The rats were first exposed to 1000 ppm CO for 40 min and then to 3000 ppm CO for another 20 min. Following CO poisoning, the rats were injected with allopurinol (50 mg/kg, i.p.) six times. Results showed that allopurinol significantly reduced neuronal death and suppressed expression of proinflammatory factors, including tumor necrosis factor-a, intercellular adhesion molecule-1, ionized calcium-binding adapter molecule 1, and degraded myelin basic protein. Furthermore, behavioral studies revealed an improved performance in the Morris water maze test. Our findings indicated that allopurinol may have protective effects against DNS caused by CO toxicity. ß 2015 Elsevier Inc. All rights reserved.
Keywords: Allopurinol Delayed neurologic sequelae Carbon monoxide poisoning Inflammation Myelin basic protein
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1. Introduction Q2
Carbon monoxide (CO) poisoning leads to an estimated 50,000 emergency room visits in the US annually (Hampson et al., 2012) and is one of the leading causes of death by poisoning. Approximately half of those who survive severe CO poisoning, characterized by loss of consciousness or carboxyhemoglobin (HbCO) greater than 25%, develop delayed neurologic sequelae (DNS) after a latency period of 2–40 days, even with treatment ˜ agas, 2004; Weaver et al., 2002). Hyperbaric oxygen (Kao and Nan (HBO) therapy is a widely accepted protocol for treating CO toxicity (Thom et al., 2006a; Weaver, 2009), but its efficacy is uncertain (Buckley et al., 2011; Hampson et al., 2012; Weaver, 2014). Growing evidence suggests that brain injury after acute
* Corresponding author at: No. 23, Youzheng Street, Nangang District, Harbin City, Heilongjiang Province, PR China. Tel.: +86 451 85555666; fax: +86 451 53605867. E-mail addresses: [email protected] (G. Dong), [email protected] (H. Feng). 1 These authors equally contributed to this work.
CO toxicity is related to oxidative stress, apoptosis, immunemediated injury and abnormal inflammatory responses (Piantadosi et al., 1997; Thom, 1990a; Thom et al., 2004, 2006a). Free radicals may have an initiating role in the damage cascade (Kao ˜ agas, 2004; Thom, 1990a; Thom et al., 2004). Several and Nan studies suggest that free radical scavengers, such as edaravone (Qingsong et al., 2013) and molecular hydrogen (Sun et al., 2011) ameliorate DNS after CO poisoning, supporting the concept that free radicals are involved in medicating the noxious effects of CO poisoning. Xanthine oxidase (XO), a form of xanthine oxidoreductase (XOR), may be the primary source of free radical generation during cerebral ischemia/reperfusion (Ono et al., 2009; Traystman et al., 1991). XO metabolizes hypoxanthine to xanthine, and then to uric acid, generating reactive oxygen species (ROS), including superoxide and hydrogen peroxide (Ono et al., 2009; Traystman et al., 1991), which are required for lipid peroxidation. Subsequently, XO has been reported to be a key component in generating ROS after CO poisoning (Thom, 1992). Furthermore, cognitive impairment associated with CO poisoning in rats was improved by depletion of XO with tungsten (Han et al., 2007).
http://dx.doi.org/10.1016/j.neuro.2015.03.015 0161-813X/ß 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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Allopurinol, a competitive and high-affinity XO inhibitor, has been used primarily for treating hyperuricemia and gout. Studies have also shown that allopurinol protects the brain from ischemia and reperfusion-induced injuries caused by oxidative stress in the brain (Isik et al., 2005), intestine (Sapalidis et al., 2013), kidney (Keel et al., 2013) and heart (Kang et al., 2006). Furthermore, XO inhibitors have anti-inflammatory effects in acute lung injury (Flaishon et al., 2006), acute pancreatitis (Martinez-Torres et al., 2009), multiple sclerosis (MS) (Honorat et al., 2013) and liver inflammation (Demirel et al., 2012). However, whether allopurinol can protect against encephalopathy due to CO toxicity is unclear. Therefore, in a model of CO poisoning, we investigated whether allopurinol reduces neurohistopathological lesion progression and inflammatory responses in the brain and improves the cognitive functioning.
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2. Materials and methods
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2.1. Animals
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Adult male Sprague-Dawley rats (280 20 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Animals were housed in a room maintained on a 12 h/12 h light/dark cycle with food and water available ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. Rats were randomly assigned to four different groups (N = 28 per group): air + vehicle (air exposed, vehicle-treated rats), air + allopurinol (air exposed, allopurinol-treated rats), CO + vehicle (CO exposed, vehicle-treated rats) and CO + allopurinol (CO exposed, allopurinol-treated rats).
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2.2. Catheter placement
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Rats in air + vehicle and CO + vehicle groups were anesthetized with 7% chloral hydrate (5 ml/kg, i.p.) 2 h before acute CO poisoning. Catheters were cannulated in the right femoral artery for blood pressure recording and blood gas sampling and were filled with heparinized normal saline (100 U/ml) when not in use.
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2.3. Drug preparation and experimental protocol
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Allopurinol (8 mg/ml) (Sigma–Aldrich Co., St. Louis, MO, USA) was dissolved in preheated saline containing 0.125 M NaOH and administered to animals (50 mg/kg). Doses were selected based on previous studies with allopurinol (50–200 mg/kg in rodents) (Ono et al., 2009; Thom, 1992). All solutions were freshly prepared on the day of testing. CO exposure was performed per a published protocol in 7-L Plexiglas chambers (Thom et al., 2004). CO, CO2 and O2 in the chamber were monitored. Vapor and carbon dioxide (CO2)-rich exhalation from the animals was absorbed by saline lime inside the chamber. Rats in the CO + vehicle and CO + allopurinol groups breathed 1000 ppm CO mixed with air for 40 min and then 3000 ppm CO mixed with air for 20 min. Animals lost consciousness after 3000 ppm CO exposure was initiated and quickly regained consciousness after they were removed from the chambers. Rats in the air + vehicle and air + allopurinol groups were placed in Plexiglas chambers as described above but breathed room air for 1 h. For rats in the air + allopurinol and CO + allopurinol groups, allopurinol was injected (i.p.) six times at 12-h intervals starting 1 h after CO exposure. During the same time points that allopurinol was administered to these rats, animals in the air + vehicle and CO + vehicle groups received an equivalent volume of preheated saline containing 0.125 M NaOH.
2.4. Arterial HbCO and monitoring of blood gases
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Blood samples (200 ml) were withdrawn from the right femoral artery of the rats through a catheter 60 min after CO exposure. HbCO, arterial blood gases and acid-base chemistry were measured with an ABL800 FLEX blood gas system (Radiometer Medical ApS., Denmark).
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2.5. Histologic and immunohistochemical studies
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Rats were euthanized with an overdose of 7% chloral hydrate then transcardially perfused with 100 ml of precooled normal saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Perfusion-fixed brains were removed and post fixed with 4% paraformaldehyde overnight. Paraffin embedded brains were sliced into 4 mm thick coronal sections then mounted onto slides for future use.
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2.6. Nissl staining
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For Nissl staining, 4 mm sections were deparaffinized, rehydrated and stained with thionine, followed by differentiation and dehydration before mounting. Images of the prefrontal cortex and the CA1 area of the hippocampus from each brain were captured using an Olympus DP73 microscope with three visual fields/per brain section selected. Intact pyramidal cells were counted by three investigators with Imaging-Pro-Plus (Leica Microsystems, Wetzlar, Denmark).
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2.7. Immunohistochemical staining
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For immunostaining, brain sections were sequentially treated with 0.3% H2O2 in PBS for 30 min, 1.5% normal goat serum and 1% bovine serum albumin for 30 min at room temperature. Then, sections were incubated with either mouse anti-ionized calciumbinding adapter molecule 1 (Iba1) antibody (1:400; Millipore, Temecula, CA) or rabbit anti-myelin basic protein (MBP) antibody (1:500; Abcam, Cambridge, UK) overnight at 4 8C and subsequently exposed to biotinylated goat anti-mouse or rabbit immunoglobulin G antibody and HRP-labeled Streptavidin (Beyotime Institute of Biotechnology, Jiangsu, China) for 30 min at room temperature. Immunostaining was visualized with 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 50 mM Tris–HCl buffer (pH 7.2). Sections were coverslipped and examined under a DP73, Olympus microscope.
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2.8. Quantitative analysis
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From every prefrontal cortex, hippocampus and corpus callosum three coronary sections (spaced 60-mm apart) (based on anatomical landmarks) were used for Nissl and three sections for immunohistochemical analysis, which was performed by three blinded researchers. In three microscopic fields (40 objective) in the prefrontal cortex, CA1 subregion and corpus callosum Nissl positive neurons or Iba1 positive cells were counted for each coronal brain section. Sections were evaluated under an Olympus DP73 microscope. Quantification of the myelinated area was performed by counting MBP-positive pixels in one series of sections using the threshold feature of Image-Pro Plus software (Media Cybernetics, Bethesda, MD). All images for each type of staining were taken with the same exposure time.
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2.9. ELISA
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Rats were euthanized with 7% chloral hydrate 3 days after CO exposure. Brains were removed. Fresh tissue from the cerebral
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cortices was dissected from bilateral frontal lobes and hippocampi immediately and homogenized in cold RIPA lysis buffer (containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium, deoxycholate and 0.5% SDS) with protease inhibitor cocktail (10 ml/ml; Pierce, Rockford, IL) and centrifuged for 10 min at 10,000 g at 4 8C. Supernatants were then collected and kept at 80 8C until further testing. Protein homogenate was quantified with a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). TNF-a, intercellular adhesion molecule-1 (ICAM-1) and highmobility group box 1 (HMGB1) protein were measured with a commercial ELISA kit (Uscn Life Science Inc., Wuhan, China) according to kit instructions.
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2.10. Western blotting
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To measure the levels of MBP, an aliquot (40 mg) of total protein from the samples was loaded on 5–20% SD polyacrylamide gels and separated by electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes then incubated with anti-MBP (1:500; Abcam, Cambridge, UK) rabbit polyclonal antibody overnight at 48C. Membranes were rinsed with 0.1% Tween 20 in phosphate-buffered solution (PBS-T) and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000; Beyotime Institute of Biotechnology, Jiangsu, China) for 2 h at room temperature. Immunoreactivity was measured with ECL. Optical density of bands was measured with ImageJ software (National Institutes of Health).
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2.11. Behavioral tests
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To measure cognitive impairment after CO toxicity, a Morris water maze (MWM) test was performed at 4 weeks in a 150-cmdiameter circular pool filled with water (20.0 1 8C). The pool was virtually separated into four quadrants (NW, SW, NE and SE). A transparent platform was submerged 1.5 cm below the water at a fixed location, i.e. southwest quadrant (SWQ) in the pool. A digital video recorder was mounted on the room ceiling and connected to a computer. Swimming paths of rats were tracked and recorded. Mean swimming speeds were calculated by using ZH-maze (Version 3.2) software (Zhenghua Biology Instrument, Anhui, China). Rats were adapted to the water for 120 s and then placed on the platform for 60 s before the acquisition test (d 0). In the acquisition phase, animals were subjected to two trials per day for 4 continuous days. During each trial, the rats were released from four random starting positions and allowed to swim for 120 s. Once the rats located the platform, the rats were allowed to rest for 30 s. If a rat did not find the platform within 120 s, it was guided to the platform and allowed to rest for 30 s. Probe trials were performed on d 5 to test rat memory retention. For this study, the platform was removed and rats were allowed to swim for 120 s in the pool. The amount of time spent in the SWQ was recorded, where the platform had been located during acquisition phase.
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2.12. Statistical analysis
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Statistical analysis was performed by using GraphPad Prism 6.02 software (La Jolla, CA). Mean escape latency and mean
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swimming speed data in the water maze test were analyzed by two-way analysis of variance (ANOVA) followed by a Student– Newman–Keuls post hoc test. Data of time of duration in the SWQ, Nissl staining, Iba1 positive cells, TNF-a, ICAM-1, HMGB1 levels and data presented as percentages were analyzed with one-way ANOVA followed by Student–Newman–Keuls post hoc test. P values of 0.05). (C) Representative swimming tracks of rats in probe trial are demonstrated. The blue square represents the start position and the red square represents the end position. (D) The time spent in southwest quadrant (SWQ), which is an indicator of the ability of spatial reference memory, was recorded. Data are presented as means SEM. **P < 0.01 compared with air + vehicle group; #P < 0.05 compared with CO + vehicle group (n = 7 for each group).
Fig. 2. Allopurinol was found to suppress carbon monoxide (CO)-induced elevation of tumor necrosis factor-a (TNF-a) levels, intercellular adhesion molecule-1 (ICAM-1) and high-mobility group box 1 (HMGB1) protein levels in the cortex and hippocampus of rats. Rat brains were removed and protein levels of TNF-a, ICAM-1 and HMGB1 were determined by ELISA. Allopurinol partially suppressed TNF-a (A, D), ICAM-1 (B, E) and HMGB1 expression (C, F) 3 days after CO poisoning. Data are expressed as means SEM. **P < 0.01 compared with air + vehicle group; #P < 0.05, ##P < 0.01 compared with CO + vehicle group (n = 7 for each group).
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differences found among treatment groups in terms of swimming speeds during the 4 days of acquisition (Fig. 1B).
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3.3. Allopurinol reduced TNF-a, ICAM-1 and HMGB1 protein
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Allopurinol may affect the levels of inflammatory factors after CO exposure. For this reason, the protein levels of TNF-a, ICAM-1
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and HMGB1 were here measured using ELISA. TNF-a, ICAM-1 and HMGB1 protein levels in the cortex and hippocampus of the CO + vehicle group were significantly increased 3 days after CO exposure compared with those in the air + vehicle group. Treatment with allopurinol reduced tissue concentration of all 3 proteins in the cortex and hippocampus, compared with CO + vehicle treated rats. Allopurinol treatment had no effect in air treated animals (Fig. 2).
Fig. 3. Allopurinol reduced neuronal death induced by CO poisoning. Rat brains were removed 1 week after CO exposure and stained with Nissl dye. (A) Upper two panels and lower two panels show the Nissl staining in the cortex and CA1 subregion of the hippocampus, respectively. Damaged areas induced by CO inhalation have fewer Nisslpositive neurons. (B, C) The undamaged Nissl-positive neurons in the cortex and hippocampal subregion CA1 were quantified. Data are means SEM. **P < 0.01 compared with air + vehicle; ##P < 0.01 as compared with CO + vehicle group (n = 7 for each group).
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3.4. Allopurinol treatment protected against neuronal death
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Representative Nissl staining 1 week after CO exposure revealed neuronal loss in the cerebral cortex and the hippocampal CA1 region compared with the air + vehicle group (Fig. 3A). Allopurinol reduced neuronal loss in the cortex and hippocampus as shown in the CO + allopurinol group (Fig. 3A). The extent of neuronal degeneration was quantified in the cerebral cortex and the hippocampal subregions CA1 region (Fig. 3B and C). There were significantly fewer neurons in these parts of the brains of animals exposed to CO than in those of air + vehicle animals, whereas the neurons in the cerebral cortex and hippocampus of the CO + allopurinol group were relatively spared when compared to the CO + vehicle group.
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3.5. Allopurinol treatment results in reduced microglia activation
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Microglial activation was quantified in the brain 4 weeks after MWM using anti-Iba1 antibody staining. In the CO + vehicle group, Iba-1 immunoreactivity was significantly increased and Iba-1
immunoreactive microglia cells were distinctly hypertrophied (Fig. 4A–C) compared with those from the air + vehicle group. However, in the CO + allopurinol group, Iba-1 immunoreactivity declined (Fig. 4A–C) compared with the CO + vehicle group.
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3.6. Allopurinol treatment results in reduced demyelination
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Using MBP (major central myelin protein) immunohistochemical staining and Western blotting, we observed demyelination in the brain of CO + vehicle-treated animals. Large plaques of demyelination were detected in the corpus callosum and hippocampus of CO exposed rats at peak toxicity (week 4 after CO exposure) when compared with normal rats (Fig. 5A and C). As expected, demyelination was dramatically reduced by allopurinol treatment compared to the CO + vehicle group (Fig. 5A and C). Western blotting confirmed that MBP protein was significantly decreased (Fig. 5B and D) in the corpus callosum and hippocampus of CO exposed rats compared with corresponding controls. However, MBP protein was significantly increased in the CO + allopurinol group compared with the CO + vehicle group (Fig. 5B and D).
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Fig. 4. Allopurinol significantly suppressed activation of microglia in the rat brain exposed to CO. Brain sections were immunostained with mouse anti-Iba1 antibody. (A) CO induced significant increases in the number of Iba1-positive cells, reflecting microglial activation, in the hippocampus and corpus callosum at weeks 4 after CO exposure. Allopurinol administration partially suppressed the increase of Iba1-positive cells induced by CO exposure. (B, C) Iba1-positive cells were measured as described in Methods. Increased Iba1 immunopositive cells in brains of CO + vehicle animals compared with those of the air + vehicle group were noted. Allopurinol significantly reduced the activation of microglia induced by CO exposure. Data are means SEM. **P < 0.01 compared with air + vehicle group; ##P < 0.01 compared with CO + vehicle group (n = 7 for each group).
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Fig. 5. Allopurinol reduced the demyelination induced by CO in the corpus callosum and hippocampus in rat brains 4 weeks after CO exposure. Brain sections were prepared and immunostained with an anti-myelin basic protein (MBP) antibody. Proteins from fresh brain tissue were used for Western blotting with the same antibody. (A) MBPpositive myelinated fibers in the corpus callosum and hippocampus decreased after CO poisoning. However, allopurinol suppressed demyelination induced by CO exposure as shown by immunostaining. (B) MBP protein decreased markedly in both the corpus campus and hippocampus after CO poisoning, compared with air + vehicle group. Treatment with allopurinol suppressed MBP protein degradation induced by CO. (C) MBP-positive-myelinated fibers were quantified using Image-Pro Plus software. (D) MBP to actin protein ratio is shown as means SEM. **P < 0.01 compared with air + vehicle; ##P < 0.01, as compared with CO + vehicle group (n = 7 for each group).
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4. Discussion
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In the present study, we report for the first time that allopurinol is neuroprotective against DNS caused by CO poisoning. Allopurinol treatment initiated 1 h after CO exposure reduced neuronal death in the cerebral cortex and hippocampus, inhibited Iba1
expression, attenuated proinflammatory TNF-a, ICAM-1 and HMGB1 cytokines levels, reduced demyelination, and alleviated cognitive deficits in a rat behavioral model. Xanthine dehydrogenase (XDH) and XO are inter-convertible forms of XOR. Under ischemia and reperfusion, ATP is degraded to ADP, AMP, adenosine, inosine and hypoxanthine, and XDH is
Please cite this article in press as: Dong G, et al. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.015
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increasingly converted to XO to oxidize hypoxanthine to xanthine, and then xanthine to uric acid and oxygen radicals (Traystman et al., 1991). Like cerebral ischemia-reperfusion, tissue hypoxia also occurs after CO poisoning with subsequent degradation of purine molecules, triggering the conversion of XDH to XO. This indicates that the XO pathway may be a source of the free radicals after CO poisoning (Thom, 1992), which may contribute to destruction of the cell membrane by initiating lipid peroxidation (Thom, 1990a,b). MBP modifications can occur due to reactions with lipid peroxidation products and may trigger a lymphocytic immunologic response, as well as lead to microglial activation that, in turn, can lead to neuropathological effects contributing to DNS (Thom et al., 2004) development. Allopurinol, as a strong XO inhibitor and a free radical scavenger (Moorhouse et al., 1987), may inhibit the initial step of the XO cascade. Conversion of XD to reversible XO has been detected as early as 40 min after the initiation of CO exposure (1000 ppm) and it was found to last for at least 90 min after discontinuation of CO exposure (Thom, 1992). This may explain why administration of allopurinol 1 h after CO exposure only partially suppresses the development of DNS. In addition, CO hypoxia, free radicals and oxidative stress cause neuronal necrosis and apoptosis (Piantadosi et al., 1997). CO poisoning causes hypoxia because CO has 210 times the affinity to Hb as O2. CO hypoxia induces neuronal death, and CO itself also causes neuronal death independent of hypoxia (Uemura et al., 2003). For CO poisoning, CO-hypoxia-induced (equivalent to about 10% O2) neuronal injury was much less severe than direct CO injury (Uemura et al., 2003). In vitro, CO induced hypoxia-independent apoptosis in neural cells through the intrinsic mitochondrial pathway (Tofighi et al., 2006). In the current study, the O2 concentration in the chambers was maintained at 20% (see Supplementary Fig. 1). PaO2 values (93.57 0.90) in the CO + vehicle group were relatively stable (Table 1), so hypoxic hypoxia was properly controlled. Then, results showed that allopurinol was protective against CO induced neuronal death (Fig. 3) and these data agree with previous reports of neonatal hypoxia-ischemia models (PeetersScholte et al., 2003). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuro.2015.03.015. Inflammation induced by CO exposure is also critical to DNS after CO poisoning (Thom et al., 2004, 2006a,b). HMGB1 is reported to function as a novel proinflammatory cytokine-like factor after reperfusion in the pathophysiology of cerebral ischemia-reperfusion and is released from neurons either during the early phase (Qiu et al., 2008) or in a delayed manner after reperfusion (Kim et al., 2006). Early release of HMGB1 from neurons was reported to cause release of pro-inflammatory cytokine (TNF-a) and then to upregulate expression of intercellular ICAM-1 in brain endothelial cells (Qiu et al., 2008). Recent studies suggest that HMGB1 expression and/or release might be related to oxidative stress (Tang et al., 2007; Tsung et al., 2007). In our study, HMGB1 levels increased in the rat brain during early post-CO exposure (Fig. 2C and F) and allopurinol suppressed elevated HMGB1, TNF-a and ICAM-1 (Fig. 2), which may be related to allopurinol’s antioxidant activity after CO exposure. In addition, recent investigation indicates that extracellular HMGB1 was a proinflammatory cytokine, triggering microglial activation in the postischemic brain (Kim et al., 2006). Thus, how HMGB1 functions in inflammation after CO poisoning warrants additional investigation. Microglia are principal immune cells and resting resident macrophage-like cells throughout the brain parenchyma. As sensors of pathological events, they can be activated by pathological changes in the brain (Kreutzberg, 1996). Activated microglia can trigger neuronal apoptosis by releasing ROS (Banati et al., 1993), inflammatory cytokines and nitric oxide (Palluy and Rigaud, 1996). Microglial and macrophage ROS production have
been implicated in myelin and axonal impairment in MS (van Horssen et al., 2011). Also, activated microglia are involved in immunological responses after CO poisoning (Thom et al., 2004, 2006a). We found that allopurinol restrained microglial activation (Fig. 4), a hallmark of brain inflammation and reduced demyelination induced by CO exposure in the hippocampal CA1 region and in the corpus callosum in rats (Fig. 5). Thus, allopurinol alleviated DNS caused by CO via reducing the inflammatory response. However, it is not clear whether ROS generated by XO triggers inflammation or inflammation activates XO and causes the generation of ROS during CO poisoning. In a cell culture model of anoxia/re-oxygenation, XO was a major source of ROS in a microglial cell line (Widmer et al., 2007). Honorat et al. reported strong expression of XO in microglia and macrophages in an animal model of MS (Honorat et al., 2013). CD11b is an important surface marker of microglia and ROS promote microglial activation via nitric oxide, characterized by increased CD11b expression (Roy et al., 2008). It can be reasonably inferred that allopurinol interrupts the feed forward mechanism by which ROS and microglia activation mutually enhance each other. XO represents a novel therapeutic target for treating delayed encephalopathy, and allopurinol, which has been used extensively to treat gout, is a promising compound for this target. Our results indicate that allopurinol, a potent inhibitor of XO, may improve the outcomes of delayed encephalopathy.
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5. Conclusion
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Allopurinol alleviated neuronal damage, attenuated inflammation and demyelination, and reduced cognitive deficits in an animal model of severe CO poisoning.
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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Transparency document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgements
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This work was supported by grants from the National Natural Q3 Science Foundation of China (No. 81171186), the Health Department Project of Heilongjiang Province (No. 2013010) and the Natural Science Foundation of Heilongjiang Province (No. ZD201417).
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