The Research About Neuroprotective Effects Of Dexmedetomindine On Secondary Injury After Intracerebral Hemorrhage And Related Meabanisms

PurposeIntracerebral Hemorrhage(ICH)is a kind of stroke,and it is a common clinical disease worldwide.During intracerebral hemorrhage,the expanding hematoma in the brain parenchyma can cause damage to normal anatomical structures and lead to increased intracranial pressure.The disease is more common in young and middle-aged people,with slightly more men than women,and its incidence is increasing year by year and showing a trend of youthfulness.Recent data show that the annual incidence of stroke in China is 246.8/100,000,and the mortality rate is 114.8/100,000.Among them,ICH accounts for 15.8%of the total stroke,but the death rate is as high as 30-40%,surviving patients with a disability rate of up to 70%,would with serious complications such as paralysis,aphasia,epilepsy,dementia,affecting the quality of life.Brain damage related to ICH can be divided into primary and secondary types due to different mechanisms.ICH primary brain injury is caused by the mechanical occupying effect of hematoma to brain parenchymal damage,including hematoma compression,and intracranial hypertension.Secondary inj ury of ICH is thought to be mainly metabolites(including thrombin,hemoglobin,heme and iron overload)produced during hemolysis and decomposition,which produces strong cytotoxicity to adjacent brain cells,producing inflammatory reaction and oxidation.stress,and intracellular signal cascade amplification,causing brain edema,blood-brain barrier disruption,etc.,eventually causing cell damage.Surgery can only relieve mechanical compression,and has no obvious improvement effect on secondary injury;currently there still be lack of effective treatment for secondary injury.Secondary injury occurs few minutes after the onset of ICH,and can last for several days or even months,which is an important factor leading to cell death and long-term neurological damage in the brain.Dexmedetomidine(Dex)is a highly selective a2AR agonist,as a commonly used adjuvant,exhibiting superior anti-anxiety,sedative and analgesic activity in clinical anesthesia practice.The pharmacological mechanism of Dex is characterized by inhibition of noradrenaline release(reducing excitability of the postsynaptic membrane)and hyperpolarization of neuronal cell membranes,and inhibition of intracellular cAMP by inhibiting adenylate cyclase activity and generation.Dex exerts a sedative effect with little effect on respiratory function and rarely has a negative impact on the circulatory system.Recent studies have suggested that Dex has neuroprotective effects,including inhibition of inflammatory response,anti-oxidative stress,inhibition of neuronal apoptosis(directly or indirectly),and release of catecholamines.In addition,many studies have used some animal models such as cerebral ischemia,subarachnoid hemorrhage,and spinal cord injury to explore the positive effects of Dex on the nervous system,but there is currently no study about Dex in the secondary injury model of intracerebral hemorrhage.As a common disease in patients with acute and severe disease,Dex may be used in both surgical anesthesia and sedation in ICH.Although studies have supported clinical doses of Dex are safe in patients with ICH,but the detail influence,especially on secondary injury,are still lacking.Based on this,we propose that Dex would exert neuroprotective effects in the secondary injury after cerebral hemorrhage.So,the purpose of this study is to explore the following:1.Does Dex have the protective effect of neurocytes in the secondary injury after cerebral hemorrhage?2.If Dex has neuroprotective effects in secondary brain injury after cerebral hemorrhage,which aspects does it affect?3.What the signal pathway be affected by Dex in the protection?4.Whether clinical patients can achieve results consistent with in vitro experiments when using Dex?MethodsCell experiment:SH-SY5Y cells were cultured and incubated with FeCl2 to mimic the state of iron overload in neurocytes after ICH,and an iron overload model of secondary injury was established.The control group was treated with serum-free DMEM,and the experimental group was treated with serum-free DMEM containing a certainconcentration of FeCl2/FeCl2+Dex.1.MTT method was used to detect cell viability,to explore the optimal concentration of FeCl2,and the effect of Dex on cell viability.2.Iron assay kit was used to measure the total iron concentration in the cells to verify the state of cellular iron overload.3.The intracellular ROS were labeled with a DCFH-DA probe to measure the oxidative stress level of the cells.4.Apoptotic cells were labeled with TUNEL staining,and the degree of apoptosis was quantitatively analyzed using the Annexin V probe.5.Western Blotting method to determine the expression levels of TNF-a,IL-1?,IL-6,bax,bcl,cleaved-caspase 3,caspase-3,etc.;and separate the nucleus and cytoplasm proteins,respectively,then WB analysis of NF-?B P65 expression level.6.Immunofluorescence staining to observe the distribution of NF-KB p65 in cells.Clinical Trial:Patients with intracerebral hemorrhage undergoing emergency hematoma evacuation surgery were enrolled in this experiment.Gender and age were not limited,ASA?? IV,Glasgow score>5 points.A total of 66 patients were automatically divided into Dex group(group D)and control group(group C)according to the order of operation time.Before induction of anesthesia,patients in group D were injected with 0.6 ?g·kg-1 dexmedetomidine(diluted to 15 ml)for 15 min,and then changed to 0.5 ?g·kg-1·h-1 until 30 min before surgery;The patients in the group C were continuously pumped with equal volume of normal saline.Record the patient's preoperative and intraoperative general data,including hematoma size,preoperative GCS score,surgical bleeding,intraoperative urine volume,and so on.Peripheral venous blood was collected at four time points:before the anesthesia(T1),surgery finish(T2),and postoperative 6 hours(T3).24 h after surgery(T4).Serum inflammatory factors(IL-1?,IL-6 and TNF-?),and S-100? and NSE levels were measured by ELISA.ResultsCell experiment:1.20?M Dex has no cytotoxic effect and protects FeCl2-treated SH-SY5Y cells:FeCl2<20?M will not have a significant effect on cells;more than 20?M FeCl2,with increasing concentration,cell viability can be decrease,the median lethal concentration of FeCl2 is?200 ?M.When the concentration of Dex was>40?M,the cell viability was weakened.If the cells were incubated with FeCl2 and Dex for 24 hours,Dex could protect the cell viability.Dex increased the resistance of the cells to 200?M FeCl2,and the effect was dose dependent.2.FeCl2 caused iron overload in SH-SY5Y cells and produced toxicity.Dex did not inhibit iron from entering cells:after incubation with high concentration of FeCl2 medium,SH-SY5Y cells showed iron overload.20 ?M Dex did not reduce the total amount of iron entering the cells.When FeCl2<20?M(incubated for 24h),it would not cause significant toxic effects on cells;at>20?M,cell viability decreased with increasing FeCl2 concentration.3.Dex reduced the expression of inflammatory cytokines:IL-1?,IL-6 and TNF-a protein expression and mRNA levels were significantly higher in the Dex+FeCl2 group after treatment with 200 ?M FeC12 for 24 hours.The levels of these cytokines were significantly lower than in the FeCl2 group.4.Dex inhibits the accumulation of ROSThe fluorescence of ROS was observed directly under fluorescence microscope.The ROS content of FeCl2 group was significantly higher than that of the control group.The ROS level of FeCl2+Dex group was lower than that of FeCl2 group.The fluorescence intensity of cytoflow detection was consistent with the observation under microscope.5.Dex inhibits apoptosisCompared with FeCl2+Dex,cells in the FeCl2 group showed more apoptosis in TUNEL staining.In the flow cytometry experiment,the apoptotic cells in the FeCl2+Dex group were significantly reduced compared with the FeCl2 group,slightly higher than the control group.6.Dex inhibits the expression of apoptosis-related proteinsThe expression of Bcl-2 was decreased and the expression of bax was increased in the FeCl2 group;Dex could inhibit these changes.The ratio of bax/bcl-2 was the lowest in the control group,and the ratio of Dex+FeCl2 was lower than that of FeC12 group.The expression of Caspase 3 in FeCl2 group was higher than that in the control group,but lower than that in Dex+FeCl2 group.On the other hand,FeCl2 group,the level of cleaved-caspase 3 was significantly higher than that of the control group,but still lower than that of the Dex+FeCl1 group.7.Dex down-regulates NF-?B signaling pathwayFeCl2 significantly down-regulated the expression level of NF-?B protein p65.When FeCl2 was combined with Dex,this down-regulation was weak.After incubation with FeCl2 for 24 hours,more NF-?B was localized in the nucleus,whereas NF-?B p65 was less translocated in the Dex+FeCl2 group.The level of nuclear NF-?B p65 in the FeCl2 group was highest in the three groups.Clinical trail1.Generally.There were 28 cases in group C,16 males and 12 females;25 patients in group D and 14 males;the average age,BMI index,GCS score and hematoma volume calculated from preoperative CT images were not statistically significant between the two groups.There were no significant differences in the operation time and bleeding volume between the group C and group D.The anesthesia time of the group D was slightly longer than that of the group D,but there was no statistically significant difference.The intraoperative urine volume of the group D patients was not significant.Significantly increased,about 2 times that of patients in group C.2.Serum inflammatory cytokines levels.Serum inflammatory cytokines were measuredby ELISA at four time points:pre-anesthesia(T1),complete(T2),postoperative 6h(T3),and postoperative 24h(T4).There were no significant differences in the levels of inflammatory factors TNF-a,IL-1? and IL-6 between the T1 and T2 groups.At 6 hours after surgery,the levels of inflammatory factors were increased in both groups compared with preoperative,but the levels of TNF-?,IL-1? and IL-6 in group D were significantly lower than those in group C.At the T4 time point,the levels of the three inflammatory factors were further elevated,but the D group was still lower than the C group.3.Serum NSE and S100? levels.There was no significant difference between the two groups of NSE and S100? serum levels and between groups before and after anesthesia.The NSE levels in group D and group C were significantly higher than those in group D at 6 hours after operation,but there was no significant difference between the groups.At the time of T4,the serum NSE levels of the two groups were further increased,but group D was significantly lower than group C.The level of S100? was significantly higher than that of T1and it was significantly lower in group D than in group C.The level of postoperative 24h was lower than that at 6h after operation,but it was still higher than that of group T1,and group D was still lower than group C.Conclusion1.Dex can inhibit the damage caused by iron overload on SH-SY5Y cells effectively,but does not reduce the total amount of iron entering the cells.2.Dex has antioxidant,anti-inflammatory and anti-apoptotic effects,inhibits nuclear translocation and activation of NF-kB p65 by inhibiting NF-kB signaling pathway,and exerts neuroprotective effects.3.The clinical dose of Dex in patients with intracerebral hemorrhage during anesthesia can reduce the short-term peripheral inflammatory factor levels postoperation,improve the body's inflammatory response,and reduce neurocytes damage.Research meaning1.This study explored the effects of Dex on secondary brain injury after intracerebral hemorrhage,which is the first time at China or abroad;demonstrated the neuroprotective effect of Dex,and explored related mechanisms,providing a scope for the application of Dex and the management of post-cerebral hemorrhage.2.In clinical trails,direct comparison analysis of patients with and without Dex during anesthesia for intracerebral hemorrhage demonstrates the additional role of Dex in patients with severe disease-specific surgery.There is no relevant report in China.