Study on mass transfer of ethyl acetate in polymer adsorbent by experimental and theoretical breakthrough curves

Received: 29 November 2018  DOI: 10.1111/bcpt.13233

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  Accepted: 7 February 2019

ORIGINAL ARTICLE

Fasudil alleviates brain damage in rats after carbon monoxide poisoning through regulating neurite outgrowth inhibitor/ oligodendrocytemyelin glycoprotein signalling pathway Li Wang1,2 

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Jinglin Wang  

Jianghua Xu3 

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Jiyou Tang  

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Department of Neurology, Qianfoshan Hospital Affiliated to Shandong University, Jinan Shandong, China

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Department of Integration of Chinese and Western Medicine, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai Shandong, China

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Department of neurology, Yantai YEDA Hospital, Yantai Shandong, China

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Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan Shandong, China

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The First Affiliated Hospital of Shandong, University of Traditional Chinese Medicine, Jinan Shandong, China

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Emergency Centre, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai Shandong, China Correspondence Qin Li and Mingjun Bi, Department of Integration of Chinese and Western Medicine, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai Shandong, China. Emails: [email protected]; [email protected] Funding information National Natural Science Foundation of China, Grant/Award Number: 81571283; key project of research and development of Shandong province, Grant/Award Number: 2018GSF118215; Natural Science Foundation of Shandong, Grant/Award Number: ZR2016HL21 and ZR2017LH068; Traditional Chinese Medicine Science and Technology Development Project in Shandong, Grant/Award Number: 2015-420 and 2017-388

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Dadong Guo4  2

Yong Zou  

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Xudong Zhou5  6

Mingjun Bi  

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Wenwen Jiang2  2

Qin Li

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Abstract Carbon monoxide (CO) poisoning can lead to many serious neurological symptoms. Currently, there are no effective therapies for CO poisoning. In this study, rats exposed to CO received hyperbaric oxygen therapy, and those in the Fasudil group were given additional Fasudil injection once daily. We found that the escape latency in CO poisoning group (CO group) was significantly prolonged, the T1/Ttotal was obviously decreased, and the mean escape time and the active escape latency were notably extended compared with those in normal control group (NC group, P < 0.05). After administration of Fasudil, the escape latency was significantly shortened, T1/Ttotal was gradually increased as compared with CO group (>1 week, P < 0.05). Ultrastructural damage of neurons and blood‐brain barrier of rats was serious in CO group, while the structural and functional integrity of neuron and mitochondria maintained relatively well in Fasudil group. Moreover, we also noted that the expressions of neurite outgrowth inhibitor (Nogo), oligodendrocyte‐myelin glycoprotein (OMgp) and Rock in brain tissue were significantly increased in CO group, and the elevated levels of the three proteins were still observed at 2 months after CO poisoning. Fasudil markedly reduced their expressions compared with those of CO group (P < 0.05). In summary, the activation of Nogo‐OMgp/Rho signalling pathway is associated with brain injury in rats with CO poisoning. Fasudil can efficiently down‐ regulate the expressions of Nogo, OMgp and Rock proteins, paving a way for the treatment of acute brain damage after CO poisoning. KEYWORDS CO poisoning, cognitive function, Fasudil, Nogo, OMgp, Rock

Wang and Xu contributed equally as co‐first authors.

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152    wileyonlinelibrary.com/journal/bcpt © 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)

Basic Clin Pharmacol Toxicol. 2019;125:152–165.

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|   IN T RO D U C T ION

Carbon monoxide (CO) poisoning is the most common cause of accidental poisoning in France with approximately 4000 cases and in the United States with 50 000 patients per year as reported by the French CO poisoning surveillance system and centres for disease control and prevention of United States.1 Consequences may be severe and include transient neurological symptoms, acute brain injury and delayed encephalopathy after acute CO poisoning (DEACMP). Currently, little is known regarding the deficits underlying these neuropathology conditions. It has been demonstrated that the concentration of carboxyhaemoglobin (HbCO) in arterial blood is closely related not only to the concentration and duration of CO inhalation but also to the difference of detection methods and patients’ physical condition.2-4 Some studies also reported that extensive demyelination could be detected in brain in many patients with CO poisoning, which determined the occurrence, the severity and the prognosis of DEACMP.5,6 Nevertheless, the pathogenesis is poorly understood. Neurite outgrowth inhibitor (Nogo) and oligodendrocyte‐ myelin glycoprotein (OMgp) are the most important myelin‐derived axon growth inhibitor of Nogo/NgR signalling pathway, the activity changes of which directly affect the regeneration and remodelling of nerve cell axons. In addition, these factors have been regarded as the major target antigens of autoantibodies causing demyelinating diseases and involved the development of demyelinating diseases in central and peripheral nervous system.7,8 Recent studies suggest that these myelin‐associated inhibitory factors such as myelin‐associated glycoprotein (MAG), OMgp and Nogo, can directly activate the downstream molecules Rho‐Rock by binding to Nogo receptor/Lingo‐1/p75 receptor complex present in cytomembranes. Activated Rho‐Rock can then phosphorylate the myosin light chain and initiate a series of events that cause neurite retraction and collapse of the growth cone in the central nervous system (CNS).9 Our previous studies showed that CO poisoning could activate Nogo/NgR signalling pathway‐related molecules (including Nogo, NgR1, MAG), and the levels of these molecules are related to brain damage and demyelination lesions following CO intoxication. However, whether they are the specific biomarkers for the occurrence and development of DEACMP is still unclear. Thus, the effective therapies are further limited by major gaps in our understanding of the fundamental processes that improve rehabilitate and prevent neurogenesis of brain damage in adults.10 Fasudil is currently the only clinically approved Rock inhibitor11 and has been widely used to alleviate symptoms of a range of CNS disorders, including subarachnoid haemorrhage,12 spinal cord injuries,13 cerebral stroke,14 Parkinson's disease,15 experimental autoimmune encephalomyelitis,16

neuropathic pain and epilepsy. It has been reported that Fasudil could improve stroke protection and blood flow after cerebral ischaemia by increasing nitric oxide (NO) production and endothelium‐derived NO synthase (eNOS) expression,17 activating astrocyte secretion of granulocyte‐colony stimulating factor (G‐CSF), inhibiting glutamate induced neurotoxicity,18 and mobilizing endogenous NSCs in vivo and differentiating the C17.2 neural progenitor cell line in vitro.19 In this study, we aimed to explore the influences of Nogo‐OMgp/Rho pathway‐related molecules on brain injury, clarify the efficacy and the mechanism of Fasudil hydrochloride (Fasudil) in rats after CO exposure, so as to provide a new therapeutic strategy for the patients with CO poisoning.

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M ATERIAL S AND M ETHO D S

2.1  |  Animals and groups

A total of 120 adult, healthy, male Sprague‐Dawley rats (230 ± 20 g, SPF degree) were granted by Jinan Pengyue experimental animal Co., Ltd. [Jinan, China; animal assurance number: SCXK(LU) 20140007]. All animals were housed in a temperature‐ controlled environment with a 12‐hour light/ dark cycle for 7 days with free access to food and water prior to the experiment. All experiments were required to minimize the pain and discomfort in accordance with the program guidelines for the Care and Use of Laboratory Animals of the National Institute in China (animal welfare assurance number: 14‐0027) and the Protection Principles of Vertebrate Animals Used for Experimental and other Scientific Purposes of China in the Use of Animals in Toxicology. The study was also conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies.20 Rats enrolled in the experiment were divided into three groups randomly: a normal control group (NC group), a CO poisoning group (CO group) and a Fasudil treatment group (Fasudil group). Every group included 40 rats. Firstly, rats in CO group and Fasudil group were exposed to 1000 ppm CO for 40 minutes. and then to 3000 ppm for another 20 minutes. in a hyperbaric oxygen chamber (DWC 450‐1150, Shanghai, China) to establish an acute CO poisoning model as described previously,21,22 while those in NC group were permitted to breathe fresh air simultaneously. Then, the three groups were designed into four subgroups at random according to the duration of intervention: a 1‐day subgroup, a 1‐week subgroup, a 1‐month subgroup and a 2‐month subgroup, respectively. Each subgroup included 10 rats. Carboxyhaemoglobin (HbCO) concentration in femoral artery blood of rats was measured immediately after CO exposure by a Blood Gas Analyzer (Rapid Lab, Bayer HealthCare, Germany). Rats with coma and high HbCO concentration (>40%) were considered as the successful model of acute severe CO poisoning. As a result, we found that the HbCO concentration in CO group and Fasudil group was

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F I G U R E 1   Schematic diagram of consecutive brain slices in rat. A: About 2.5 mm in front of the bregma. B: About 0.5 mm behind the bregma; Yellow square marker represents the selected position in cortex for the experiment. C: About 1.5 mm behind the bregma. D: About 3.5 mm behind the bregma; Yellow square marker represents the selected position in hippocampal tissue for the experiment. E: About 5 mm behind the bregma

up to 57.8 ± 6.3%, and consistent with the diagnostic criteria of acute severe CO poisoning, while it was only 0.6 ± 0.4% in NC group. Core body temperature was maintained at 36~37°C in an incubator with proper humidity during the whole experiment. The vital signs of rats were detected hourly after CO exposure.

2.2  |  Treatment strategy

Fasudil was provided friendly by Tianjin Hongri pharmaceutical limited company (batch number: 1603231). After successful establishment of the model, all rats suffered hyperbaric oxygen (HBO) therapy within 10 minutes. after recovering consciousness from CO exposure in animal oxygen chamber once a day afterwards until they were killed. The oxygen concentration was maintained between 95% and 99% during HBO therapy. Rats in Fasudil group were given Fasudil hydrochloride by intraperitoneal injection at the dosage of 15 mg/kg/d once a day until the rats were killed; meanwhile, those in CO and NC groups received the same volume of normal saline.

2.3  |  Preparation and evaluation of neurological behaviour Prior to formal neurobehavioural test, all rats received training in water maze for 4 days, and the score of each rat was recorded. Those with significantly lower scores than the average were considered as cognitive impairment and were therefore excluded from the experiment. At the same time, rats eligible for the cognitive functional criteria were supplemented to test in the following experiment.

2.4  |  Morris water maze task

The spatial learning and memory of all rats enrolled in the present study was detected by a Morris water maze task (Shanghai Soft Information Technology Co., Ltd., Model

number: XR‐XM101). A platform was submerged into a tub (diameter = 150 cm; height = 50 cm; depth = 30 cm) of opaque water. Five rats in each group were placed into the water from different locations at the beginning of each trial, and four trials per session twice a day for 4 days before neurological behaviour test were performed. The water entry point, escape latency, swimming route and swimming time (T1 and T total) of rats were recorded by EthoVision XT 9 Software Analysis System. The average escape latency and the ratio of T1/Ttotal were calculated on day 1, week 1, month 1 and month 2 after exposure to CO, respectively. All scores were independently obtained from two assistants simultaneously in a double‐blind manner, and data were expressed as the average values of eight trials in 4 days for each rat in different groups.

2.5  |  Shuttle box experimental score

Five rats in each group received the Shuttle Box experimental test in a shuttle box (model number: 10080116012) to evaluate the learning and memory ability. Before Shuttle Box test, rats were permitted to access the shuttle room for 5 minutes. and finished twice behaviour trainings per day to acclimatize to the environment till 1 week. The parameters were set as follows: the stimulation current 1.2 mA, the intermittent time 20 seconds, the electric shock time 5 seconds and cycle 50 times. Experimental animals finished the shuttle test during the buzzer phase named active avoidance response (AAR), while it was called passive avoidance response (PAR) in the electrical stimulation phase. The AAR time and the electric shock times were calculated by the EthoVision XT 9 Software Analysis System on days 1, 7, month 1 and month 2 after CO exposure under the same experimental conditions. The active escape latency was expressed as the ratio of AAR time to (50－electric times).

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2.6  |  Transmission electron microscopy (TEM) analysis Five rats in each group were deeply anaesthetized with 4% phenobarbital intraperitoneally. The whole hippocampus was removed from the skull on ice, and the selected hippocampus tissue (as shown in Figure 1D) was immediately cut into several small pieces about 1 mm × 1 mm × 1 mm, continuously fixed in a 2.5% Glutaraldehyde phosphate buffer (pH 7.2) for 48 hours and 1% Osmiumoxide for 1 hour, then embedded in epoxy resin, finally cut into 60 nm specific ultrathin sections. With double staining of saturated uranyl acetate and citrate lead, the sections were put on copper nets (200 mesh) and were observed under a transmission electron microscope (JEM‐100CX2, Japan).

2.7  |  Immunohistochemical staining

Nogo and OMgp monoclonal antibodies were purchased from Santa Cruz company, and the SABC immunohistochemistry kit and DAB staining kit were granted by Wuhan Boster biological engineering Co. Ltd (Wuhan, China). Five rats in each group were deeply anaesthetized with 4% phenobarbital intraperitoneally at 1 day, 1 week, 1 month, 2 months, respectively, fixed by 200 mL of normal saline and 4% poly Formaldehyde solution from cardiac perfusion, respectively, and decapitated at the given time‐points mentioned above. The brain tissues were taken out of the skull and cut into consecutive coronal paraffin sections of 6‐μm thickness for immunohistochemical detection. All procedures were strictly performed in accordance with the instructions (Nogo dilution 1:150, OMgp dilution 1:100). After DAB staining, those with brown granular in cytoplasm or nucleus under a 400‐fold light microscope were considered as Nogo‐ or OMgp‐positive cells. Under a 400‐ fold optical microscope, four consecutive slices in each rat were selected, and four non‐overlapping fields of cortex in each slice were randomly observed (as shown in Figure 1B). The absorbance (A) values of each field were established and calculated by Leica Qwin image processing and analysis system (Leica Corporation, Germany). Some slices were treated 0.01 mol/L phosphate buffer (PBS, pH7.2, containing 1:200 blocking serum of non‐immunized animals) instead of monoclonal antibody as the negative control background to calibrate the A values of target proteins simultaneously.

2.8  |  Immunofluorescence staining

Rock monoclonal antibody was purchased from Santa Cruz company. All procedures were strictly performed in accordance with the instructions. Paraffin sections were probed with primary anti‐Rock (diluted 1:100)

for 2 hours at 37°C, biotin‐conjugated secondary antibody (1:150) and SABC‐FITC (1:100) for 30 minutes at 37°C, respectively. With laser excitation of 488 nm and reception of 510‐530 nm wavelength, Rho positive cells were observed under a 400‐fold fluorescence microscope, while those that did not express Rho could not be detected under the same conditions. Under a 400‐fold fluorescence microscope, four non‐overlapping views in cortex were observed at random from four serial slices for each rat (visual field shown in Figure 1B), and the A values of Rock protein expressions in each view were determined with Leica Qwin image processing and analysis system.

2.9  |  Western blotting detection

Five rats in each group were deeply anaesthetized with 4% phenobarbital at the corresponding time‐points as described above. The total protein was extracted from the brain tissue with RIPA lysis buffer on ice after rapid decapitation, added in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) prepared in advance, and transferred onto Polyvinylidene fluoride (PVDF) membrane by semidry method. The PVDF membranes were then immersed and incubated in Tris‐buffered saline with Tween‐20 (TBST) containing 10% skimmed milk at room temperature for 1 hour, monoclonal antibodies diluents (Nogo 1:500, OMgp 1:600 and Rock 1:500) at 4°C overnight, and the second antibodies diluents labelled by HRP (1:1000) at 25°C for 2 hours The Bio‐Rad 2000 gel imaging system was used to analyse the optical density (OD) value of target proteins. The Western blot procedure of each target protein was repeated three times for each brain sample. The average of the three OD values in Western blots represents the expression of target protein in brain tissue sample of the rat, and the average OD value of five rats in each group represents the expression level of target protein in this group. The OD value of β‐actin (dilution 1:500) was measured as an internal control in the same specimen simultaneously. The relative OD values of target protein expressions were normalized after measuring ratio with β‐actin and finally including this in the statistical analysis.

2.10  |  Statistical analysis

Statistical analysis was performed using SPSS 19.0 statistical software. All data were expressed as mean ± standard deviation (mean ± SD). Analysis of covariance (ANOVA) and the least significant difference (LSD) were run to determine the statistical difference for all analysis unless otherwise specified. The correlation between two variables was evaluated by linear regression analysis. A P‐value <0.05 was considered significant.

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|   R ES U LTS

3.1  |  Neurobehavioural scores of rats 3.1.1  |  Changes of advanced cognitive function Morris water maze test is mainly used for the detection of spatial or position learning and memory in rats. In the present study, the average escape latency of NC group rats was rapidly decreased at 1 week after swimming training in maze and kept at a lower score between 1 week and 2 months. The percentage of the first quadrant residence time (T1/Ttotal) was relatively stable and did not fluctuate significantly with the prolongation of the training time, indicating that the rats had stabilized memory on the platform. For the positioning navigation and space exploration, we observed that the average escape latency was significantly prolonged, and the T1/Ttotal was notably decreased in CO group in comparison to that of NC group (P < 0.05, Figure 2). This result suggests that CO intoxication can impair learning and memory function in rats. Meanwhile, we also noted that after the administration of Fasudil, the escape

F I G U R E 2   Comparison of average escape latency and 1st quadrant swimming time in each group (n = 5, one‐way ANOVA and LSD). The average escape latency was significantly prolonged, and the T1/Ttotal was notably decreased in CO group in comparison to that of NC group at the same time‐points (P < 0.05). After the administration of Fasudil, the escape latency was significantly shortened, T1/Ttotal was gradually increased as compared with CO group (P < 0.05). A, Comparison of average escape latency in each group. B, Comparison of 1st quadrant swimming time in each group

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latency was significantly shorter than that of CO group at the same time‐point (>1 weeks), T1/Ttotal was gradually increased, accompanied by a statistical difference between Fasudil and CO groups (P < 0.05). These data indicated that Fasudil can obviously improve advanced cognitive function in rats after CO poisoning, and the therapeutic efficacy of Fasudil is extremely significant at the late stage of CO poisoning (from 1 week to 2 months).

3.1.2  |  Alternations of simple memory in rats in different groups Shuttle box test is usually employed to assess classical conditioned reflex. Compared with NC group, the AAR time and active escape latency in CO group were notably prolonged, and there were significant differences from 1 day to 2 months after CO poisoning (P < 0.05, Figure 3). This result indicates that CO exposure can not only impair advanced cognitive activity but also damage simple memory function in rats. Moreover, we also found that the AAR time and active escape latency of rats in Fasudil group were slightly longer than those in NC group (P < 0.05), but shorter than those in CO group

F I G U R E 3   Alternations of AAR time and active escape latency in each group (n = 5, one‐way ANOVA and LSD). The AAR time and active escape latency in CO group were notably prolonged, and there were significant differences from 1 d to 2 mo after CO poisoning compared with NC group (P < 0.05). After the injection of Fasudil, the AAR time and active escape latency of rats were slightly longer than those of NC group (P < 0.05), but shorter than those in CO group (P < 0.05). A, Changes of AAR time in different groups. B, Changes of active escape latency in different groups

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(P < 0.05), suggesting that Fasudil can significantly improve the simple cognitive ability of rats with CO poisoning.

3.1.3  |  Ultrastructural observation in brain tissue in rats Under a transmission electron microscope, the exterior contour of neurons in NC group was clear, with big and round nucleus and uniform chromatin, and several dendrites of varying lengths and shapes extended to the different layers of brain. The double nucleus membrane was clear and complete. Mitochondria were abundant and scattered in cytoplasm with structural integrity. Endothelial cells that form blood‐brain barrier (BBB) were arranged neatly. The neurites, dendrites of neurons and gliacytes in hippocampus in NC group were interwoven and formed complex network membranous structure of neuropil. In the synaptic zone, the presynaptic and posterior membranes were very clear, and the presynaptic vesicles were mostly round and distinct. The vesicles were dense and of uniform size (Figure 4A). After CO poisoning, the outlines of presynaptic and posterior membrane were not clear; parts of them were discontinuous with the duration of poisoning time in CO group (Figure 4B). Neurons were

swelled, nucleus chromatin was condensed and marginalized, mitochondria were swelled and the number of mitochondria was markedly decreased. The structure of inner and outer membrane in most mitochondria was not complete, mitochondrial crista were disrupted, matrix staining was light, and vacuoles could be seen everywhere. The ultrastructure of BBB was obviously destroyed. The presynaptic vesicles were accumulated with unequality in size and shape. Partial synaptic vesicles were deformed, ruptured or fused. The postsynaptic membranes were swollen, dense material in cytoplasmic surface of posterior membranes became thick, and the synaptic gap was blurry. These results indicated that CO poisoning could directly damage the ultrastructure of synapses and mitochondria of neurocytes. By contrast, after the treatment of Fasudil, the double‐deckered nuclear membrane was clear, the swelling degree of mitochondria was relatively light, only a small number of mitochondrial spines were irregularly arranged. The damage degree of ultrastructure in BBB was slighter than that of CO group. The synapse structure of nerve cells was relatively complete (Figure 4C), suggesting that Fasudil can efficiently alleviate the ultrastructural damage of neurons and BBB induced by CO poisoning.

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F I G U R E 4   Ultrastructural changes of hippocampal neurons in rats in each group at 1 wk (TEM, n = 5, A1,B1,C1: scale bar = 5 μm; A2,B2,C2: scale bar = 500 nm). Under a transmission electron microscope, the exterior contour of neurons in NC group was clear, with big and round nucleus and uniform chromatin. Mitochondria were abundant and scattered in cytoplasm with structural integrity. The neurites, dendrites of neurons in hippocampus were interwoven and formed complex network membranous structure of neuropil. The vesicles were dense and uniform size (A). After CO poisoning, neurons were swelled, nucleus chromatin were condensed and marginalized, mitochondria were swelled and the amounts of mitochondria were markedly decreased, and vacuoles could be seen everywhere (B). By contrast, after treatment with Fasudil, the swelling degree of mitochondria in neurons was relatively light, only a small number of mitochondrial spines were irregularly arranged and little , BBB; , mitochondria; , vesicles; , synapses in neurite vacuole were detected nerve endings (C).

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3.2  |  Expressions of Nogo and OMgp proteins in rats after CO poisoning Under a 400‐fold optical microscope, a small amount of Nogo‐ and OMgp‐positive cells with irregular shape and different size were expressed in brain tissue in NC group rats. Some of them had short processes scattered in different regions of brain tissue. Nogo protein was widely expressed in neurons and oligodendrocytes in the central nervous system, whereas OMgp protein was mainly distributed on the surface of oligodendrocytes and myelin layer adjacent to axon, and also located in neurons. After CO exposure, the amount of Nogo‐ and OMgp‐positive cells was obviously increased at 1 day in CO group. A large number of Nogo‐ and A1

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OMgp‐positive cells were found in brain tissues in rats at 1 week after CO poisoning. Their absorbance (A) values were also increased, accompanied by significant differences as compared with that of NC group at the same time‐point (Figures 5 and 6, P < 0.05). The expressions of Nogo‐ and OMgp‐positive proteins were gradually decreased at 1 month and 2 months as compared with that at 1 week after CO poisoning (P < 0.05). This result suggests that CO poisoning can activate the Nogo‐OMgp/NgR1 signalling pathway, which is closely related to the growth and plasticity of axons and participate the occurrence and development of brain demyelination lesions induced by CO poisoning. After the treatment with Fasudil, the amount of Nogo‐ and OMgp‐positive cells was significantly reduced, accompanied by a statistical

F I G U R E 5   Expressions of Nogo protein in cortex in each group at different time‐points (Immunohistochemistry, n = 5, arrow: Nogo‐positive cells, scale bar = 20 μm, ANOVA and LSD). Under a 400‐fold light microscope, cells with brown granular in cytoplasm or nucleus identified by the black arrow were Nogo‐ or OMgp‐positive cells. A small amount of Nogo‐positive cells with irregular shape and different size scattered in different brain regions in NC group rats (A1‐D1). The Nogo‐positive cells were increased obviously at 1 wk and 1 mo in CO poisoning group (A2‐D2). After the treatment with Fasudil, the amount of Nogo‐positive cells was significantly reduced compared with that in CO group at the same time‐point (A3‐D3, P < 0.05). (A1) 1 d of NC group; (B1) 1 wk of NC group; (C1) 1 mo of NC group; (D1) 2 mo of NC group; (A2) 1 d of CO group; (B2) 1 wk of CO group; (C2) 1 mo of CO group; (D2) 2 mo of CO group; (A3) 1 d of Fasudil group; (B3) 1 wk of Fasudil group; (C3) 1 mo of Fasudil group; (D3) 2 mo of Fasudil group. (E) the OD value of Nogo protein in brain tissues in each group at different time‐points

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F I G U R E 6   Expressions of OMgp protein in cortex in each group at different time‐points (Immunohistochemistry, n = 5, arrow: OMgp‐positive cells, scale bar = 20 μm, ANOVA and LSD). Only a small amount of OMgp‐positive cells could be detected in brain tissue in NC group rats (A1‐D1). Along with the duration of poisoning time, the number of OMgp‐ positive cells was gradually increased in CO group (A2‐D2). After the administration of Fasudil, the expression of OMgp‐positive cells was significantly reduced as compared with in CO group at the same time‐point (A3‐D3, P < 0.05). (A1) 1 d of NC group; (B1) 1 wk of NC group; (C1) 1 mo of NC group; (D1) 2 mo of NC group; (A2) 1 d of CO group; (B2) 1 wk of CO group; (C2) 1 mo of CO group; (D2) 2 mo of CO group; (A3) 1 d of Fasudil group; (B3) 1 wk of Fasudil group; (C3) 1 mo of Fasudil group; (D3) 2 mo of Fasudil group. (E) the OD value of OMgp protein in brain tissues in each group at different time‐points

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difference as compared with that in CO group at the same time‐point (P < 0.05). The evidence strongly suggests that Fasudil can effectively inhibit the activities of Nogo and OMgp after CO poisoning, playing an important protective role against brain damage after CO intoxication.

3.3  |  Expressions of rock protein in rats after CO poisoning Using a 400‐fold fluorescence microscope, we found that the Rock‐positive cells scattered in various districts in brain

tissue, mainly appeared in neurons of cerebral cortex, hippocampus. The number of Rock‐positive cells and the A values was dramatically increased in rats in CO group at 1 day, peaking at 1 week and 1 month, and then slightly decreasing till 2 months after CO exposure. There were significant differences between CO and NC groups at the selected time‐points (Figure 7, P < 0.05). After Fasudil injection, the expression of Rock protein was obviously decreased and kept a lower level as compared with that of CO poisoning group at the same time‐points (P < 0.05). This result was consistent with that of the Western blot analysis (Figure 8).

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F I G U R E 7   Expressions of Rock protein in cortex in each group (Immunofluorescence staining, n = 5, scale bar = 40 μm, ANOVA and LSD). Using a 400‐fold fluorescence microscope. The number of Rock‐positive cells were observed and their A values were dramatically increased in rats in CO group at 1 d, peaked at 1 wk and 1 mo, and then slightly decreased till 2 mo after CO exposure. There were significant differences between CO and NC groups at the selected time‐points (P < 0.05). After Fasudil injection, the expression of Rock protein was obviously decreased and kept at a lower level as compared with that of CO poisoning group at the same time‐points (P < 0.05). (A1) 1 d of NC group; (B1) 1 wk of NC group; (C1) 1 mo of NC group; (D1) 2 mo of NC group; (A2) 1 d of CO group; (B2) 1 wk of CO group; (C2) 1 mo of CO group; (D2) 2 mo of CO group; (A3) 1 d of Fasudil group; (B3) 1 wk of Fasudil group; (C3) 1 mo of Fasudil group; (D3) 2 mo of Fasudil group. (E) the OD value of Rock protein in brain tissues in each group

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3.4  |  Relationship among Rock, Nogo and OMgp proteins in brain tissue In order to clarify the relationship among Nogo, OMgp and their downstream molecule Rock, we used linear regression analysis. As mentioned above, a small amount of Nogo‐ and OMgp‐positive cells was detected in brain tissue in NC group. The two proteins in brain tissue increased gradually from 1 day to 1 week, and then decreased till 2 months in CO poisoning group. Linear regression analysis showed the

positive correlation between the expression levels of Rock and Nogo proteins (R2 = 0.8803, Figure 9A). The number of Rock‐positive cells and the A value were dramatically increased in brain tissue at 1 day, and maintained a high level till 2 months after CO poisoning. It seems that the variation tendency of Rock in brain tissue was slightly different from those of NOGO and OMgp along with the duration of CO exposure. Linear regression analysis showed the correlation between Rock and OMgp proteins in Figure 9B (R2 = 0.7024, Figure 9B).

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F I G U R E 8   Relative expressions of Nogo, OMgp, and Rock proteins detected by Western blot in brain tissue in each group (n = 5, ANOVA and LSD). (A) The expressions of Nogo, OMgp, and Rock proteins in each group; (B) A value of Nogo in brain tissue in each group; (C) A value of OMgp in brain tissue in each group; (D) A value of Rock in brain tissue in each group. Lane 1: Fasudil group at 1 d; lane 2: Fasudil group at 1 wk; lane 3: Fasudil group at 1 mo; lane 4: Fasudil group at 2 mo; Lane 5: CO group at 1 d; lane 6: CO group at 1 wk; Lane 7: CO group at 1 mo; lane 8: CO group at 2 mo; Lane 9: NC group

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CO poisoning can lead to a number of neurophysiological and neuropathological changes, and cognitive dysfunction is the most common clinical manifestation. Many researchers focus on the learning and memory ability of animals by means of shuttle box test and water maze and address the relationship between neurotransmitter system and drug interaction. In the present study, we found that the escape time and active escape latency of rats were significantly increased, and the T1/Ttotal was notably decreased combined with shuttle box test and water maze test, suggesting that CO exposure can not only impair advanced cognitive activity but also damage simple memory function. It is well known that hippocampus is a major brain area controlling learning and memory. Many diseases and pathological conditions, such as depression, Alzheimer's disease, cerebral ischaemia,23-25 CO poisoning22 and sleep deprivation, can damage the integrity of hippocampal ultrastructure, which is closely related to the synaptic plasticity and the decline in learning and memory function. Synaptic plasticity plays a very important role in this process.26 By a mouse model with synapsin caveolin‐1(SynCav1) administration, the

Egawa study demonstrated that SynCav1 augmented synaptic plasticity by increasing total number of synapses, presynaptic vesicles (PSVs) per bouton, splitting synapse boutons and myelination, all of which were gross anatomical and microscopic indicators of structural neuroplasticity. Combined with the measurable electrophysiological changes, these ultrastructural alterations in hippocampus improved learning and memory, suggesting that caveolin‐1 (Cav‐1) may be an attractive molecular target to repair brain function in the context of neurodegenerative diseases.27 Zhao and colleagues found that the thickness of the postsynaptic density (PSD) decreased in hippocampus in an animal model after exposure to Arsenic, whereas the width of the synaptic cleft widened notably, and protein expressions of both PSD‐95 and synaptophysin (SYP) decreased significantly.28 In the present study, TEM analysis also showed some morphological and structural changes of synapsis in CO group including the less number of synapses and synaptic vesicles, the little thickness of PSD and the small curvature of the synaptic interface. However, there was no statistical difference between CO group and Fasudil group, and this result was slightly different from the data of Egawa and Zhao. We considered that the spatial structure changes of

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F I G U R E 9   Relationship among the expressions of Nogo, OMgp and Rock proteins (Linear regression analysis). (A) Correlation between the expressions of Rock and Nogo proteins; (B) Correlation between the expressions of Rock and OMgp proteins

pre‐ and postsynaptic membrane proteins, the maladjustment of neurotransmitters as well as the disorder of gene sequences, could directly impair synaptic function, although some ultrastructural changes might not be identified by common transmission electron microscopy. The changes in morphology and structure may lag behind the alternations in function of some synapses at the given time‐point we observed. In the central nervous system, mature oligodendrocytes had the characteristics of forming the insulated myelin structure, accelerating the efficient transmission of electroneurographic signals, maintaining and protecting the physiological function of neurons, whereas the immature oligodendrocytes lacked such kind of properties.29,30 As the central nervous system is subjected to adverse stimuli, oligodendrocyte precursor cells rapidly accumulate at the lesion area, further producing a variety of myelin‐derived axon growth inhibitors, resulting in the regeneration of CNS. Nogo is the most intense regeneration inhibitor of nerve fibre found at present, and is highly expressed in the oligodendrocytes of CNS. Therefore, it has significant inhibitory

function for the repairment and regeneration of CNS, and the continuous inhibition of Nogo‐A on the growth of nerve fibres may be one of the important causes of nerve dysfunction. Studies have shown that the axon NgR expression of nerve cells gradually increased and peaked at 24 hours and then began to decrease after cerebral ischaemia. The downstream effectors of Nogo/NgR signalling pathway, such as RhoA, Rock and other proteins and their mRNA, had consistent changes. Thus, NgR might play a role in maintaining the stability of neuronal circuit and the plasticity of axon after cerebral ischaemia, especially in the late stage. Increased expression of NgR may be a compensatory response to cerebral ischaemia, playing an important role in the reconstruction of neuronal circuits and axon regeneration or remodelling.31-33 Moreover, our results showed that the expression of Nogo protein significantly increased in the early stage after CO intoxication, then gradually decreased at 1 month, and remained at a relatively constant level at 2 months after CO poisoning. This time‐point coincided with the formation of extensive demyelination and delayed encephalopathy in

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rat brain tissue after CO poisoning, suggesting that the increased expression and activation of Nogo protein may be related to brain damage and the extensively demyelination following CO intoxication. Oligodendrocyte‐myelin glycoprotein (OMgp) is a glycosylphosphatidylinositol (GPI)‐anchored protein. OMgp also plays an important role in inhibition of axonal regeneration in CNS via combining with NgR, Nogo and MAG.34,35 Studies showed that OMgp could inhibit neurite axon outgrowth and induce growth cone collapsing by binding to the NgR/P75/

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TROY/Lingo‐1 receptor complex, and then activate RhoA pathway (Figure 10). Thus, NgR was indispensable in the inhibition of axonal growth mediated by OMgp.36,37 OMgp is expressed by mature oligodendrocytes and mainly located on the cell membrane, existing not only in the central nervous system but also in the peripheral nervous system.38 However, OMgp plays a slightly different role in injured immature and mature CNS,35,39 suggesting that OMgp may have other functions unrelated to neural regeneration. Our study showed that OMgp expressions in brain tissue in CO group

F I G U R E 1 0   Schematic diagram of Nogo‐OMgp/Rho signalling pathway. RhoA can be activated by a number of inhibitory molecules, like

Nogo, MAG and OMgp, activating a trimeric receptor complex comprising NgR1, LINGO‐1, and p75NTR or TROY, finally leading to the high Rock activity. Through phosphorylation of different molecular substrates, Rock activity affects a multitude of cellular functions, for example, cytoskeleton modulation, protein synthesis, autophagy, apoptosis, glial cell function, microglial activation and synaptic function. In the context of neurodegeneration and nerve regeneration in the central nervous system, the increased activity of Rock has been associated with rather detrimental effects, whereas inhibition of Rock activity has frequently resulted in attenuation of pathology and functional improvement

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were significantly higher than those in NC group at the same time‐point. The increased expression of OMgp protein was still observed at 2 months after CO poisoning. After treatment with Fasudil, the cognitive ability of the rats subjected to CO poisoning was markedly improved, and the levels of Nogo and OMgp proteins in brain tissues were significantly reduced. This result implies that CO poisoning can activate Nogo and OMgp, whereas Fasudil can efficiently protect neurocytes by down‐regulating the expressions of Nogo and OMgp proteins. Rock is a member of the Rho‐GTPases belonging to the Ras superfamily9 and is also a downstream effector of Nogo/ NgR signalling pathway. Many myelin‐derived axon growth inhibitors, such as Nogo MAG and OMgp, exert physiological effects through Rho A and Rock (Figure 10). It is demonstrated that the increased Rock activity could mediate neuronal apoptosis in ischaemic brain injury in rats via increased PTEN activity and decreased activation of Akt.40 Accumulation of Rock is involved in a variety of neuronal functions including inhibition of axonal regeneration and neuronal differentiation, the proliferation of tumour cells9,41-43 as well as growth cone collapse and stress fibre formation. Fasudil, a clinically approved Rock inhibitor, could reduce the neurological deficits scores, decrease cerebral infarct area, inhibit neuronal apoptosis,44 trigger axon initiation and increase size and motility of growth cone filopodia during neuronal maturation.40 Currently, Fasudil has also been tested in numerous clinical trials for many vascular pathologies, such as subarachnoid haemorrhage, Raynaud's syndrome and arterial hypertension.45,46 As we expected in the present study, Fasudil can obviously down‐regulate the expressions of Nogo, OMgp and Rock proteins, efficiently protect brain tissue against CO toxicity.

5 

|   CO NC LU S ION S

Based on an animal model of acute CO poisoning, we found that CO exposure can not only impair advanced cognitive activity but also damage simple memory function in rats. Fasudil can significantly improve cognitive ability, down‐ regulate the expressions of Nogo, OMgp and Rock proteins, inhibit the activation of Nogo‐OMgp/Rho signalling pathway, and maybe become a new treatment strategy for patients with acute CO poisoning in clinical practice. ACKNOWLEDGEMENTS This work was funded by the National Natural Science Foundation of China (No.: 81571283), the key project of research and development of Shandong province (No.: 2018GSF118215), the Natural Science Foundation of Shandong (No.: ZR2016HL21, ZR2017LH068), and the Traditional Chinese Medicine Science and Technology Development

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Project in Shandong (No.: 2015‐420, 2017‐388). We are thankful to Dr. Wu‐Lian and Yueheng‐Zhang in Binzhou Medical college for constant support during this study. CONFLICT OF INTEREST The authors declare that they have no competing financial interests. AUTHORS’ CONTRIBUTIONS QL and MB conceived and designed the experiments; LW, JX, XZ and WJ performed the experiments; JW and JT analysed the data; YZ and MB contributed reagents/materials/ analysis tools; LW and QL wrote the paper. All authors read and approved the manuscript. R E F E R E NC E S 1. Centers for Disease Control and Prevention (CDC). Carbon monoxide‐related deaths ‐ United States, 1999‐2004. MMWR Morb Mortal Wkly Rep. 2007;56(50):1309‐1312. 2. Nakamura T, Setsu K, Takahashi T, et al. Chronic exposure to carbon monoxide in two elderly patients using a kotatsu, a traditional Japanese charcoal‐based heater. Psychogeriatrics. 2016;16(5):323‐326. 3. Harlan N, Weaver LK, Deru K. Inaccurate pulse CO‐oximetry of carboxyhemoglobin due to digital clubbing: case report. Undersea Hyperb Med. 2016;43(1):59‐61. 4. Kulcke A, Feiner J, Menn I, Holmer A, Hayoz J, Bickle P. The accuracy of pulse spectroscopy for detecting hypoxemia and coexisting methemoglobin or carboxyhemoglobin. Anesth Analg. 2016;122(6):1856‐1865. 5. Geraldo AF, Silva C, Neutel D, Neto LL, Albuquerque L. Delayed leukoencephalopathy after acute carbon monoxide intoxication. J Radiol Case Rep. 2014;8(5):1‐8. 6. Fujiwara S, Yoshioka Y, Matsuda T, et al. Relation between brain temperature and white matter damage in subacute carbon monoxide poisoning. Sci Rep. 2016;6:36523. 7. Yang Y, Liu Y, Wei P, et al. Silencing Nogo‐A promotes functional recovery in demyelinating disease. Ann Neurol. 2010;67(4):498‐507. 8. Pourabdolhossein F, Mozafari S, Morvan‐Dubois G, et al. Nogoreceptor inhibition enhances functional recovery following lysolecithin‐induced demyelination in mouse optic chiasm. PLoS ONE 2014;9(9):e106378. 9. Liu J, Gao HY, Wang XF. The role of the Rho/ROCK signaling pathway in inhibiting axonal regeneration in the central nervous system. Neural Regen Res. 2015;10:1892‐1896. 10. Li Q, Cheng Y, Bi M, et al. Effects of N‐Butylphthalide on the expressions of Nogo/NgR in rat brain tissue after carbon monoxide poisoning. Environ Toxicol Phar. 2015;39(2):953‐961. 11. Jia XF, Ye F, Wang YB, Feng DX. ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro. Neural Regen Res. 2016;11:983. 12. Satoh S, Takayasu M, Kawasaki K, et al. Antivasospastic effects of hydroxyfasudil, a Rho‐kinase inhibitor, after subarachnoid hemorrhage. J Pharmacol Sci. 2012;118:92‐98. 13. Impellizzeri D, Mazzon E, Paterniti I, Esposito E, Cuzzocrea S. Effect of fasudil, a selective inhibitor of Rho kinase activity, in the

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How to cite this article: Wang L, Xu J, Guo D, et al. Fasudil alleviates brain damage in rats after carbon monoxide poisoning through regulating neurite outgrowth inhibitor/oligodendrocytemyelin glycoprotein signalling pathway. Basic Clin Pharmacol Toxicol. 2019;125:152–165. https://doi.org/10.1111/bcpt.13233