Efficacy of DL-3 n-butylphthalide on psychiatric,behavioural and cognitive functions in delayed encephalopathy after acute carbon monoxide poisoning

Background The main hazards of carbon monoxide (CO) poisoning are its serious clinical effects,high incidence and mortality of toxicity.Acute brain injury and delayed encephalopathy after acute carbon monoxide poisoning (DEACMP) are the most common neurological complications.CO poisoning can cause neurological,psychiatric,behavioural and cognitive disorders,including cortical blindness,parkinsonism,amnesia,personality changes,dementia,and mental disorders such as anxiety and depression.This study was performed to assess the efficacy of DL-3 n-butylphthalide (NBP) and dexamethasone (DXM) and hyperbaric oxygen (HBO) on psychiatric,behavioural and cognitive functions in DEACMP. Patients and methods A total of 179 patients with DEACMP were recruited and assigned to the combined-therapy group(receiving NBP and DXM 5 mg/day plus HBO therapy) or the control group (HBO therapy as monotherapy).The behavioural and cognitive functions in patients were evaluated by the Mini-Mental State Examination (MMSE),the Montreal cognitive assessment (MoCA) scale and the Barthel index of activities of daily living (ADL) after the treatment at 1 month,3 months,and 1 year respectively.The psychiatric changes in patients were evaluated by the Hamilton Anxiety Scale (HAMA) and Hamilton Depression Scale (HAMD) after the treatment at 3 months and 1 year respectively. Results At 1 month,3 months and 1 year after the treatment,the MMSE,MoCA and ADL scores were all significantly higher in the combined therapy group than those in the control group.At 3 months and 1 year after the treatment,the HAMA and HAMD scores were all significantly lower in the combined therapy group than those in the control group.There were no significant alterations in blood glucose,blood lipids,or liver or kidney function during the whole treatment session.Some patients experienced loss of appetite,mild headache and minor skin irritations.However,these patients recovered by and needed no additional or special treatment. Conclusion These results indicated that NBP and DXM combined with HBO for the treatment of DEACMP can significantly improve the psychiatric,behavioural and cognitive functions of patients and is very safe.


Introduction
The main hazards of carbon monoxide (CO) poisoning are its serious clinical effects,high incidence and mortality of toxicity.CO affects nearly all tissues and organs.In fact,acute brain injury and DEACMP are the most common neurological complications [1,2] .CO poisoning can cause neurological,psychiatric,behavioural and cognitive disorders,including cortical blindness,parkinsonism,amnesia,personality changes,dementia,and mental disorders such as anxiety and depression.Depression,anxiety and dementia were the most common neuropsychiatric complication experienced after CO poisoning,and they have a significant negative impact on patients rehabilitation,functional recovery,quality of life,and even survival rate which impose a heavy economic and emotional burden on families and society.
Currently,the specific mechanisms of DEACMP are still unclear.A number of factors have been found to be associated with the development of DEACMP,such as ischaemia,hypoxia,immune dysfunction,cytotoxic injury,neurotransmitter dysregulation,and reperfusion injury [3][4][5] .HBO was often recommended by previous studies to treat patients with acute CO poisoning [6,7] .Researchers have also reported that HBO has significant effects on DEACMP [8][9][10] .It is difficult to control the progression of DEACMP in a timely and effective manner only using HBO.Therefore,the exploration of more effective drugs is still the focus of current research.
The main active component of NBP is dl-3-N-butylphthalide,which was originally isolated 4 from celery seeds.NBP is a lipid-soluble drug with good blood-brain barrier permeability [11] .In animal models of cerebral apoplexy,NBP was mainly used for inhibiting platelet aggregation,reducing thrombosis,improving microcirculation,reducing the volume of cerebral infarction,resisting oxidative stress,protecting mitochondrial function,reducing neuronal apoptosis,alleviating inflammatory reaction,mediating autophagy of nerve cells,promoting neurogenesis and targeting in several pathophysiological mechanisms,which are mainly reflected in its obvious neuroprotective effect [12][13][14][15][16][17][18][19][20][21][22] .NBP After further studies on its application,it was found that the beneficial effects of NBP are beyond those of apoplexy.Especially in neurodegenerative disease fields,it was found that NBP and its ramifications have unique function and effects in AD,vascular dementia (VaD),PD,ALS and DEACMP treatment [23][24][25][26] .
In addition,relevant clinical studies have shown that short-term NBP and HBO have a good clinical effect on the recovery of the cognitive function of DEACMP patients [27,28] .Relevant studies have also shown that DXM and HBO also have a good clinical effect on the recovery of cognitive function of DEACMP patients [29] .
Therefore,we conducted a study to investigate the effects of NBP and DXM combined with HBO on the treatment of psychiatric,behavioural and cognitive functions in DEACMP 5 patients and to test the efficacy and safety of this treatment.This study provides a basis for the clinical application of NBP and DXM for CO poisoning.

Recruited patients
The Institutional Review Board of Heze municipal hospital reviewed and approved the purpose and design of our work.Informed consent in written or oral form was formally obtained from all participants,and our was conducted in accordance with the Declaration of Helsinki.This clinical trial was performed between October 2013 and October 2018.

experimental methods
In this single-centre trial and retrospective cohort study,the included patients were assigned into two groups: the combined therapy (91,53.2%) and control groups (80,46.8%).Conventional treatments,including using antibiotics,performing sputum suction,reducing or avoiding respiratory tract infection,maintaining unobstructed airways,maintaining stable blood pressure and blood glucose,regulating water electrolytes,and preventing urinary tract infection,were provided to all patients.
Patients meeting the following criteria were included:(1) patients with a history of CO exposure;(2) patients with a Mini-Mental State Examination (MMSE) score of no more than 24 [30] ; (3)  to the previous study that used DXM to treat CO-poisoned patients [31] .
Researchers followed up and assessed the patients at 1 month,3 months,and 1 year.

Primary outcome
The psychiatric outcomes of patients were evaluated by the HAMA and HAMD after the treatment at 3 and 6 months.The cognition and movement changes of patients were evaluated by the MMSE,MoCA and ADL before and after the treatment at 1 month,3 months and 1 year.The total ADL score was set to 100,and the higher the score,the better the abilities of daily life of the patients.Moreover,the total MMSE and MoCA scores were both set to 30,and higher scores indicated stronger cognitive ability of the patients.Lastly,the degrees of HAMA and HAMD were both set to moderate to severe (the score of HAMA 21 and the score of HAMD 20),and higher scores indicated worse psychiatric disorder of the patients.Combined with the application of MoCA,MMSE can initially screen for various cognitive disorders,improving the detection rate of cognitive impairment,increasing the sensitivity and specificity,and reducing the false positive rate and false negative rate.

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The potential adverse events were also recorded and analysed to assess the acceptability of the combined application of NBP,DXM and HBO therapy.

statistical analysis
All statistical analyses were performed using SPSS 22.0 statistical software,and all the continuous variables are expressed as the mean ± standard deviation.Student's t-test and the chi-squared test were applied to investigate the differences between the two groups in regard to demographic and baseline clinical variables.We performed an analysis of covariance (ANCOVA) to assess the effect of the treatment modalities on the posttreatment ADL,MoCA,MMSE,HAMA and HAMD scores.All procedures were 2-tailed,and a value of P < 0.05 was considered statistically significant for all tests.

Demographic characteristics
A total of 179 patients with DEACMP were recruited at the start of the study.During the treatment,5 cases in the control group and 3 cases in the combined-therapy group were lost to follow-up.Finally,171 patients completed the entire treatment.There were 80 (46.8%) and 91 (53.2%) patients in the control and combined therapy groups,respectively.There were no significant differences in any baseline data,such as age,sex,educational level,profession,body mass index (BMI),CO exposure time,carboxyhemoglobin levels,latent phase,coma time,Glasgow coma scale,or Acute Physiology and Chronic Health Evaluation II (APACHE II).The detailed information is shown in Table 1.

Treatment efficacy
At 1 month,3 months,and 1 year after the treatment,the MMSE scores were all significantly higher in the combined therapy group than in the control group ( Table 2.

Adverse Events.
There were no significant alterations in blood glucose,blood lipids,or liver and kidney function during the whole treatment session,partly because of the low dose and the short time of DXM use.In the combined-therapy group,three patients experienced loss of appetite,three patients reported mild headache,and two patients had minor skin irritations.In the control group,two patients had loss of appetite,and two patients reported mild headache.However,these patients recovered by themselves and needed no additional medications or special treatment.

Discussion
In our study,we found that our therapy could significantly increase the ADL,MoCA,MMSEscores and decrease the HAMA and HAMD scores of patients with DEACMP after acute-phase treatment,which could significantly improve cognitive function and decrease the psychological disorder in patients with DEACMP.In addition,the ADL score 9 increased significantly in the combined-therapy group,suggesting that the addition of NBP and DXM to the course of disease management may have a better effect in protecting and restoring nervous function.In addition,the addition of NBP and DXM did not significantly increase the incidence of adverse events.Based on the above results,NBP and DXM combined with HBO in the treatment of DEACMP can achieve better clinical efficacy,and it is expected to become a potential new treatment method for DEACMP patients but requires further exploration.
DEACMP is the most common neurological complication after acute CO poisoning and is also a serious disease worldwide.The main manifestations are advanced intelligence loss,mental behaviour abnormalities,and dyskinesia,which seriously affect the quality of life of patients.The language,memory, and visual function of DEACMP patients rapidly decline in a very short term,and personality abnormalities and other cognitive functions(including judgement,generalization,and multiability in analysing and solving problems) appeared at a later stage in severe cases and were often accompanied by emotional disorders and pyramidal and extrapyramidal system dysfunction.At present,the detailed pathogenesis of DEACMP is not clear.Convincing evidence suggests that the main pathological mechanism of DEACMP may be the atrophy of the hippocampi after CO poisoning [32] and extensive demyelinating lesions of white matter in the brain tissue [33] .The pathogenesis of delayed neuronal death in the hippocampal CA1 subarea has been proven,including inflammation,increased excitatory amino acids,abnormal neurotransmitters,mitochondrial function inhibition,oxidative stress in mitochondria,apoptosis,lipid peroxidation and adaptive immune response [34][35][36] .Gorman et al reported that after CO poisoning,ischaemia and hypoxia,oxygen free radicals and peroxides in brain cells increased significantly,and lipid peroxidation in the cell membrane was enhanced,leading to demyelination of nerve cells and secondary neuronal cell death [33,37] .Other researchers reported that DEACMP is immune-mediated and that delayed synergistic mediated neuropathology is associated with an adaptive immune response to chemically modified myelin basic protein [38] .Most scholars believe that CO poisoning might lead to structural and functional changes of myelin basic proteins,the abnormal expression of myelin-derived axonal growth inhibitors(such as Nogo,NgR,and Ogmp),and the imbalanced expression of cytokines and inflammatory molecules in the central nervous system,which may be an important factor of generalized demyelination lesions in white matter of brain tissue [39,40] .In addition,Ischiropoulos et al.reported that CO poisoning altered nitric oxide (NO) levels and NO synthase activity,suggesting that NO may be involved in brain tissue damage [41] .DEACMP occurs due to the interaction of many factors.
NBP is a new type of anti-cerebral ischaemia drug.Its main active ingredient is dl-3-Nbutylphthalide,which is a fat-soluble substance that can pass through the blood-brain barrier.The mechanism of its effect on DEACMP may be correlated with its unique pharmacological effects:(1) selectively inhibiting the physiological and pathological process mediated by arachidonic acid and its metabolites,reversing microvasospasm to inhibit platelet aggregation and reduce thrombosis;(2) directly acting on the mitochondria in nerve cells of cerebral ischaemia areas,enhancing the fluidity of the mitochondrial membrane,improving the activity of complex IV enzyme in the respiratory chain,enhancing the activity of antioxidant enzymes,reducing the generation of oxygen-free radicals,and thereby increasing the content of ATP and phosphocreatine in brain cells;(3) maintaining the complete structure of microvessels,increasing the number of microvessels,promoting collateral circulation in cerebral ischaemia areas,improving microcirculation,and significantly increasing regional cerebral blood flow;(4) inhibiting the release of glutamate,reducing the intracellular calcium concentration,resisting oxidative stress,and thereby alleviating the inflammatory response and cell apoptosis after cerebral ischaemia.Therefore,NBP reveals a significant neuroprotective effect,alleviating nerve damage and reducing the disability or fatality rates in patients with DEACMP;and (5) NBP can promote axon and synapse formation and nerve regeneration in the subventricular area after cerebral ischaemia and hypoxia;upregulate TREK-1(TWIK-related K + channel 1,TREK-1) in the biforamen area;affect the proliferation of neural stem cells and astrocytes [42] ;significantly promote the proliferation of hippocampal cells after stroke;improve the survival rate of new cells;regulate the differentiation of new cells into mature neurons;improve the memory function of specific nerve cells;and effectively reverse or improve cognitive impairment.
At present,we have found that DXM can alleviate inflammation by protecting the myelin sheath from inflammation.Atalay et al [43] reported that DXM might affect brain tissue lipid peroxidation while treating CO-poisoned rats.Another animal study [44] indicated that early DXM treatment might have a positive effect on preventing DEACMP after 18 days of treatment.HBO therapy is beneficial to the recovery of damaged brain cells by increasing blood oxygen levels in brain tissue.It is suggested that NBP and DXM combined with HBO are effective for the treatment of DEACMP.
The limitations of our study are:(1) its retrospective design;(2) its non-random nature; (3) the sample size is relatively small;(4) all patients were from the same city;and (5) only the short-term efficacy of the combination of NBP,DXM and HBO was examined.Therefore,further studies are needed to confirm the efficacy and safety of NBP and DXM combined with HBO in the treatment of DEACMP by prospective,randomized,multicentre,large sample studies with long-term longitudinal follow-up.

Conclusion
Our results indicated that NBP and DXM combined with HBO in the treatment of DEACMP may improve the psychiatric,behavioural and cognitive functions of patients without significant side effect.Our study should be viewed as a potential new therapy.

Abbreviations
Our results indicated that NBP and DXM combined with HBO in the treatment of DEACMP may improve the psychiatric,behavioural and cognitive functions of patients without significant side effect.Our study should be viewed as a potential new therapy.

Consent for publication
Not applicable. work, and acquisition of data. LY and HL participated in the study design, data collection, analysis of data and preparation of the manuscript. YC carried out the study design, the analysis and interpretation of data and drafted the manuscript. All authors read and approved the final manuscript. mail:kundan0@126.com).

Disclosure
The authors declare that they have no competing interests in this section.   Efficacy of NBP on functions in DEACMP.sav