RESUMO
Objective:To investigate the role of neurite outgrowth inhibitor (Nogo)-oligodendrocyte myelin glycoprotein (Omgp)/Ras homologous (Rho)-Rho-associated coiled-coil forming protein kinase (Rock) signaling pathway in acute brain injury of carbon monoxide (CO) poisoning rats and treatment feasibility with Rho kinase inhibitor hydrochloride fasudil.Methods:According to random number table method, 135 healthy male SD rats were divided into three groups: a normal control group, a CO poisoning group and a fasudil treatment group ( n=45). Rat models of acute severe CO poisoning were established in the CO poisoning group and fasudil treatment group by inhalation method in a hyperbaric oxygen chamber. All rats received hyperbaric oxygen therapy for two weeks. Rats in the farsudil treatment group were intraperitoneally injected with hydrochloride farsudil for intervention (15 mg/[kg·d], once a d for 2 weeks), while those in the CO poisoning and normal control groups received the same volume of normal saline. The ultrastructures of rat brain tissues were observed by transmission electron microscopy one week after modeling. Staining intensities of Nogo- and OMgp-positive cells were detected by immunohistochemistry, and those of Rock-positive cells were analyzed by immunofluorescence one d, one week, one month and two months after modeling. The protein expressions of Nogo, OMgp and Rock in brain tissues were detected by Western blotting one d, one week, one month and two months after modeling. Results:In the CO poisoning group, the ultrastructures of brain tissues and blood-brain barrier were damaged obviously, and the changes in nucleus, mitochondria and synaptic structure were obvious; while fasudil treatment could effectively maintain the integrity of ultrastructures and functions of brain tissues, and reduce brain edema. One d, one week, one month and two months after modeling, the staining intensities of Nogo, OMgp and Rock positive cells and protein expression levels of Nogo, OMgp and Rock in the CO poisoning group were significantly higher than those in the normal control group at the same time point ( P<0.05); the staining intensities of Nogo, OMgp and Rock positive cells and protein expression levels of Nogo, OMgp and Rock in the fasudil treatment group were significantly lower than those in the CO poisoning group at the same time point ( P<0.05). Conclusion:The activation of Nogo-OMgp/Rho-Rock signaling pathway related molecules (Nogo, OMgp and Rock) is closely related to acute brain injury caused by CO poisoning; hydrochloride fasudil can effectively down-regulate the protein expressions of Nogo, OMgp and Rock, therefore obviously alleviate brain injury after CO poisoning.
RESUMO
Objective To explore the effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier (BBB) in rats with acute carbon monoxide (CO) poisoning. Methods A total of 144 adult healthy male Sprague-Dawley (SD) rats were randomly divided into normal control group, CO poisoning group, and NBP treatment group, with 48 rats in each group. The acute CO poisoning model was reproduced in hyperbaric oxygen chamber, and all model rats were given hyperbaric oxygen therapy once daily. The rats in the normal control group were free to breathe fresh air. The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death. The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil. Four rats were sacrificed from each group at 1, 3, 7, 14 days after model reproducing, respectively. The changes in ultrastructure of BBB were observed under transmission electron microscope. The expressions of ZO-1 and claudin-5 proteins were determined by immunofluorescence staining and Western Blot. The localization of the two target proteins was observed by immunofluorescence double staining. The correlation between the two proteins was analyzed by linear regression. Results The ultrastructure of BBB was normal in normal control group, some ZO-1 and a large number of claudin-5 positive cells were observed. The ultrastructure of BBB was seriously injured, ZO-1 and claudin-5 positive cells in brain tissue were significantly decreased, and the expressions of ZO-1 and claudin-5 proteins in brain tissue at 1 day after poisoning in CO poisoning group were significantly lower than those of normal control group (ZO-1 protein:3.38±0.30 vs. 24.50±5.62, claudin-5 protein: 11.38±0.93 vs. 46.35±6.88, both P < 0.05), and although gradually restored, they were maintained at relatively lower levels until 14 days as compared with those in normal control group (ZO-1 protein: 10.35±0.80 vs. 24.63±3.57, claudin-5 protein: 32.35±3.11 vs. 46.43±7.20, both P < 0.05). NBP treatment could significantly alleviate the ultrastructure injury of BBB induced by acute CO poisoning, the amount of ZO-1 and claudin-5 positive cells in brain tissue were significantly increased, as well as the expressions of ZO-1 and claudin-5 proteins were significantly increased, which were significantly higher than those of CO poisoning group from 1 day and 3 days on, respectively (1-day ZO-1 protein: 7.57±0.69 vs. 3.38±0.30, 3-day claudin-5 protein:20.46±1.42 vs. 11.43±0.86, both P < 0.05), and which showed an increase tendency with time prolongation. The results of immunofluorescence double staining showed that ZO-1 and claudin-5 proteins could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed the positive correlation between the expressions of ZO-1 and claudin-5 proteins in brain tissue of rats with acute CO poisoning (R2= 0.917, P = 0.022). Conclusion NBP could markedly improve the ultrastructure and functional integrity of BBB through up-regulating the expressions of ZO-1 and claudin-5 proteins, and then reduce brain damage caused by CO poisoning.
RESUMO
Objective To explore the neuroprotective effect of targeted regulation Nrf2 gene on rats with brain injury caused by acute severe carbon monoxide ( CO ) poisoning. Methods A total of 180 healthy adult SD rats were divided into 4 groups at random:normal control group( NC group) ,CO poisoning group(CO group),lentivirus group(LV group) and Nrf2 gene therapy group(Nrf2 group),and 45 rats in each group. An acute CO toxic rat model was established by inhalation in a hyperbaric oxygen tank. The lentivirus group was directly injected with lentivirus dilution (4×106 TU/μl) into striatum with a microsy-ringe guided by a stereotactic apparatus,and the Nrf2 gene therapy group was administrated the same dose of recombinant Nrf2 gene lentivirus dilution,while rats in the normal control group and the CO poisoning group were received the same amount of normal saline. Five rats were taken and decapitated at day 1,day 7 and week 2 from each group,respectively. The mitochondrial membrane potential (MMP) of neurons in brain tis-sue was detected by JC-1 method,and the expressions of Nrf2 and GCLC proteins were observed by immuno-histochemistry and Western Blot. Results Compared with the NC group (cortex:(75. 3±6. 8);hippocam-pus:(76. 4±7. 1);striatum:(73. 8±7. 3)) at the same time point,the MMPs of neurons in CO group (cor-tex:(34. 5±6. 7);hippocampus:(30. 3±5. 6);striatum:(41. 5±6. 1) and LV group (cortex:(36. 8±6. 2);hippocampus:(30. 8±6. 0);striatum:(42. 7±6. 3)) were significantly decreased,and the difference was sig-nificant(P<0. 05). However,there was no significant difference between the CO poisoning group and the lentivirus group (P>0. 05). A small amount of Nrf2 protein (0. 22±0. 05) and GCLC protein (0. 24±0. 04) were expressed in the brain tissue of normal control rats. The expressions of Nrf2 protein (0. 31±0. 06,0. 31 ±0. 05) and GCLC protein (0. 30±0. 04,0. 31±0. 07) in CO group and LV group were slightly increased (P<0. 05). Similarly,there was no significant difference between the CO poisoning group and the lentivirus group (P>0. 05). The MMPs value of nerve cells in the Nrf2 group (cortex:(53. 3±5. 3);hippocampus:(56. 9±6. 1);striatum:(60. 6±6. 0)) also decreased,but it was significantly higher than that in the CO group and the LV group at the same time point (P<0. 05) . The expression of Nrf2 in brain tissue was signifi-cantly increased (0. 59±0. 05),and there was significant difference between CO group and LV group at the same time point (P<0. 05);GCLC protein increased slightly (0. 37±0. 06),but there was no statistical difference compared with CO poisoning group and lentivirus group (P>0. 05). Conclusion CO poisoning could induce oxidative stress and damage mitochondrial function of nerve cells. The active state of targeted regulation Nrf2 could significantly enhance the antioxidant capacity of rats and positively protect rats against brain injury induced by acute severe CO poisoning.
RESUMO
Objective To investigate the therapeutic effects of Xingzhi Yinao (XZYN) particles combined with hyperbaric oxygen therapy on cognitive impairment and motor dysfunction in patients with delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Methods Sixty-seven patients with DEACMP were admitted to the Affiliated Yantai Yuhuangding Hospital of Qingdao University from January 2011 to December 2015, and they were randomly divided into a control group (given conventional treatment such as inhalation of oxygen, cytidine diphosphate cholin and vitamin B, 19 cases), a hyperbaric oxygen (HBO) treatment group (given conventional treatment + hyperbaric oxygen therapy once a day, 24 cases) and a XZYN particles treatment (XZYN group, given conventional treatment, hyperbaric oxygen and XZYN particles, 24 cases), the therapeutic course being 2 months in the three groups. Before and after treatment for 1 and 2 months, the cognitive function and motor function of the patients were evaluated by the use of activity of daily living (ADL) scale, Montreal cognitive assessment (MoCA) scale, and mini-mental state examination (MMSE) scale; the severity of cerebral white matter injury was assessed by age related white matter changes (ARWMC) scale; and the electromyographic evoked potential was used to detect the amplitude and latency of P300 to assess the severity of cognition impairment and prognosis. Results With the prolongation of therapeutic time, after treatment, the neurological function scores of ADL, MoCA, MMSE and amplitude of P300 were increased, while ARWMC was decreased and the latency of P300 was shortened gradually in the three groups, and the changes of above indexes after treatment for 2 months in XZYN group were more significant than those in either HBO group or control group[ADL score: 70.2±8.3 vs. 60.5±8.1, 23.0±6.1, MoCA score: 26.1±3.1 vs. 22.2±2.7, 18.2±3.6, MMSE score:25.9±4.1 vs. 22.4±3.5, 18.1±4.5, ARWMC score: 7.0±2.1 vs. 8.7±2.2, 15.2±3.3, latency of P300 (ms):332.9±20.4 vs. 352.5±23.6, 381.7±30.3, amplitude of P300 (μV): 6.5±1.6 vs. 5.6±1.3, 4.1±1.5, all P < 0.05]. Conclusion The hyperbaric oxygen therapy combined with XZYN particles for treatment of patients with DEACMP can significantly improve their cognitive and motor functions and ameliorate the severity of cerebral white matter injury.
RESUMO
Objective To evaluate the neuroprotective effect of edaravone on brain tissues after acute carbon monoxide (CO) poisoning and explore its mechanism.Methods Ninety Sprague-Dawley rats were randomly selected as normal control group,poisoning group and treatment group (n=30).Rats in the poisoning group and treatment group were established animal models of acute CO poisoning with hyperbaric chamber method and received hyperbaric oxygen therapy.The rats in the treatment group were given intraperitoneal injection ofedaravone (10 mg/kg) additionally.TUNEL and real time-PCR were used to detect the cell apoptosis and their apoptosis-related gene expressions (Bcl-2 and Bax mRNA) in brain tissues 1,2 and 7 d after CO poisoning in each group.Immunohistochemistry and Western blotting were used to observe the positive cells and protein changes of heme oxygenase-1 (HO-1) and nuclear factor erythrocyte two related factors-2 (NRF-2).Results The apoptosis cell number in the poisoning group and the treatment group was significantly increased as compared with that in the normal control group (P＜0.05);that in the treatment group was relatively fewer than that in the poisoning group with significant differences (P＜0.05).As compared with those in the normal control group,the Bcl-2 and Bax mRNA expressions in the poisoning group were obviously up-regulated with significant difference (P＜0.05);the Bcl-2 mRNA level was significantly increased,the Bax mRNA level was signficantly down-regulated,and the ratio ofBcl-2 mRNA/Bax mRNA was notablyly increased in the treatment group as compared with those in the poisoning group (P＜0.05).The positive cells and HO-1 and NRF-2 proteins in the brain tissues of the poisoning group were significantly higher than those in the normal group (P＜0.05);those in the treatment group were obviously increased as compared with those in the poisoning group (P＜0.05).Conclusion Edaravone might be partly associated with activation of NRF-2/HO-1 pathway to counteract oxidative stress damage,inhibit neuronal apoptosis,and play a neuroprotective role in brain damage after acute CO poisoning.
RESUMO
Objective To investigate the expressions of neurite outgrowth inhibitor (Nogo),myelin-associated glycoprotein (MAG) and Nogo receptor-1 (NgR1),and neuro-protective effect of N-Butylphthalide (NBP) in rat brain tissues following acute CO poisoning.Methods Sixty Sprague-Dawley rats were randomly divided into normal group,CO poisoning group and NBP treatment group (n=20).Rats in CO poisoning group and NBP treatment group were induced acute CO poisoning in hyperbaric chamber and received hyperbaric oxygen therapy.Meanwhile,rats in the NBP treatment group were subjected to oral NBP 6 mg/100 g twice a day additionally.The expressions ofNogo,MAG and NgR1 were investigated in rat brain tissues by immunohistochemistry and double immunofluorescence staining 1 day,3 days,1 week and 4 weeks after CO exposure.Results With the prolongation of CO poisoning,the levels ofNogo,MAG and NgR1 in brain tissues of the CO poisoning group were gradually increased,and their expressions could still be detected at 4 weeks after CO poisoning.NBP treatment group had significantly reduced Nogo and NgR1 protein levels,and statistical differences were noted as compared with those in the CO poisoning group at each time point (P＜0.05).The level of MAG in NBP treatment group was slightly lower than that in CO poisoning group without statistical difference (P＞ 0.05).Conclusions The levels ofNogo,MAG and NGR1 proteins may be associated with brain injury and demyelination induced by CO poisoning.NBP can downregulate the levels of Nogo and NgR1 proteins (but not MAG),and may play a neuro-protective role in brain damage after acute CO poisoning.