| Background and ObjectiveIntracerebral hemorrhage(ICH)is a hemorrhage of the brain parenchyma caused by the rupture of cerebral blood vessels.It is the fatal subtype of the common types of stroke,accounting for almost 10-15% of all strokes and affecting approximately 2million people in the world every year.The prognosis of intracerebral hemorrhage is worse than that of ischemic stroke,the 30-day mortality rate is 43-51%,and most patients have neurological dysfunction associated with the hemorrhage area.The body and local brain tissues can undergo a series of related pathophysiological changes after intracerebral hemorrhage,including hematoma formation,space-occupying effect caused by hematoma,changes in local cerebral blood flow,blood-brain barrier destruction,cerebral edema and neurological deficits.The mechanism of brain damage after intracerebral hemorrhage mainly includes primary damage caused by hematoma formation and secondary damage caused by hematoma pathological reaction.After intracerebral hemorrhage,the blood clot degrades into products including hemoglobin,heme and iron,causing brain damage.This process can last for several days or even weeks.As one of the main degradation products of hemoglobin,iron ions cause brain edema and cell death after intracerebral hemorrhage through the activation of a series of oxygen free radicals and inflammatory reactions,causing secondary brain damage.Iron overload can cause brain damage through a variety of ways,such as lipid peroxidation and free radical formation.Deferoxamine,as an iron chelator,is clinically mainly used to treat acute iron poisoning and chronic iron overload caused by transfusion dependent anemia.Since deferoxamine has a strong affinity for Fe3+,it can quickly penetrate the blood-brain barrier after systemic administration,quickly and effectively combine with iron ions to form a stable and non-toxic water-soluble complex to discharge,reduce the iron content of the hematoma site,and improve the brain secondary damage caused by bleeding.On the one hand,deferoxamine can increase the transcription of heme oxygenase-1(HO-1)by reducing the hydroxyl radical activity of free iron.On the other hand,deferoxamine can bind to iron through the Fenton/Haber-Weiss reaction to prevent the formation of hydroxyl radicals.In previous animal experimental models,deferoxamine has been shown to reduce hemoglobin-related edema,neuronal death,neurological deficits and brain atrophy after intracerebral hemorrhage.Minocycline(Mino)is a semi-synthetic second-generation tetracycline derivative that has been used since the 1970 s.It has anti-inflammatory and bactericidal effects and has been widely used in clinics as an antibiotic.Minocycline can easily pass through the blood-brain barrier because of its high lipophilicity and high tissue penetration properties,thereby acting as a neuroprotective agent.Minocycline can not only inhibit the activation and proliferation of microglia and matrix metalloproteinases,but also play a neuroprotective effect by chelating iron ions.Although it has been confirmed that these two drugs have a certain neuroprotective effect after cerebral hemorrhage,whether the combination of deferoxamine and minocycline can enhance this effect has not been reported in the literature.In this study,we used collagenase to construct the ICH model in rats,aiming to observe the neuroprotective effects of deferoxamine,minocycline,and the combination of the two drugs in the acute phase after intracerebral hemorrhage in rats,and provide new clinical treatment Ideas and experimental basis.Methods1.96 adult(about 8 weeks)male SD rats will be randomly divided into 4 groups:ICH control group(Vehicle group,24 rats),deferoxamine group(DFX group,24 rats),Minocycline group(Mino group,24 rats),deferoxamine combined with minocycline group(D+M group,24 rats).Each group was further assigned into three subgroups according to the administration time(1d,2d,3d,),with 8 rats in each subgroup.2.In each group,0.5 μl collagenase was inμected into rat striatum to prepare Ⅶintracerebral hemorrhage model.After operation,neurobehavioral score was used to determine whether the model was successful.3.The administration method of the treatment group: The deferoxamine group was administered by intraperitoneal inμection(dose 100 mg/kg,concentration 50mg/kg)2 hours after the model preparation,and then every 12 hours until they were executed at the corresponding time point.The minocycline group was given intraperitoneal inμection(dose 45mg/kg,concentration 45mg/kg)2 hours after the model was prepared,and then the dose was halved and administered every 12 hours(dose 22.5mg/kg,concentration 45mg/kg))until the corresponding time point is executed.The combined treatment group was given the above two interventions at the same time,and the vehicle group was given intraperitoneal inμection of the same dose of normal saline.4.Each group of rats will undergo behavioral tests at various time points before and after surgery,including forelimb-placing test and corner turn test.5.Rats in each group were killed at the set time points(1d,2d,3d).6.The brain damage of each group of rats at different time points after intracerebral hemorrhage was detected and histopathological evaluation was performed.The brain damage area was assessed by HE staining.Perls staining was used to assess iron deposition and the number of hemosiderin cells after ICH.Fluoro-Jade C staining was used to observe the degeneration of nerve cells at different time points.Death cells at different time points was detected by TUNEL fluorescence staining.Iba1 immunofluorescence staining was used to evaluate the activation of microglia.7.Western-blot was used to detect the expression of ferritin and apoptotic protein in each group after intracerebral hemorrhage,including ferritin heavy chain(FTH),ferritin light-chain(FTL),apoptotic proteins include C-PARP,Bax and Bcl-2proteins.Results1.Statistical analysis of HE staining results showed that obvious brain damage occurred after intracerebral hemorrhage,and the area of brain damage was the largest on the third day.Image J analysis showed that at 1d,2d,3 day(s)after ICH,the area of brain damage in the deferoxamine treatment group,minocycline treatment group and combination treatment group was smaller than that of in the vehicle group(p<0.05).The area of brain damage in the combined medication group was smaller than that of each monotherapy group(p<0.05).2.Statistical analysis of Perls staining results showed that the number of iron-containing heme cells gradually increased after ICH.The number of heme cells in the deferoxamine group,the minocycline group and the combination treatment group was less than that in the vehicle group at 1,2,and 3 day(s)after ICH(p<0.05).The number of iron deposits in the combination therapy group was less than that of each monotherapy group(p<0.05).3.Statistical analysis of Fluoro-Jade C(FJC)staining results showed that at 1,2,and 3day(s)after ICH,compared with the vehicle group,the positive cells in the deferoxamine group,minocycline group and combination treatment group were significantly reduced(p<0.05).The number of positive cells in the combination therapy group was less than that in each monotherapy group(p<0.05).4.Statistical analysis of TUNEL staining results showed that multi-cell death mostly occurs in the area of brain damage and reached a peak at 3d after ICH.The number of cell deaths in the deferoxamine group,minocycline group and combination treatment group were significantly reduced compared with the vehicle group at 1,2,and 3day(s)after ICH(p<0.05).The number of dead cells in the combination group was less than that in each monotherapy group(p<0.05).5.Statistical analysis of Iba1 staining results showed that the number of microglia gradually increased at 1,2,and 3day(s)after ICH.The number of positive cells in the vehicle group was significantly higher than that in each treatment group(p<0.05);and the number of microglia in the combination group was less than that in the deferoxamine group and the minocycline group(p<0.05).6.Compared with the vehicle group,the expression of iron-related protein in the tissues around the hematoma in each treatment group,including FTH and FTL,were significantly lower at 1,2,and 3day(s)after ICH(p<0.05).In addition,the expression of iron-related protein in the combined treatment group was less than that in the monotherapy group(p<0.05).Compared with the Vehicle group,the expression of apoptosis protein C-PARP and pro-apoptotic protein Bax in DFX group,Mino group and DFX+Mino group were all decreased,which is increased in that of apoptosis-inhibiting protein Bcl-2,and the ratio of Bax/Bcl-2 decreased(P<0.01),the combined drug group significantly down-regulated the expression of C-PARP and Bax protein,and the protein expression of Bcl-2 was significantly up-regulated(P<0.01).7.Statistical analysis of neurological deficit examination and behavioral test results showed that compared with the vehicle group,each treatment group could reduce neurological deficits,including neurological function score,forelimb placement test and corner turn test at 2 and 3 days after ICH(p<0.05).The neurological function improvement was not obvious at 1 day after ICH,only the combination treatment group was significantly different from the vehicle group(p<0.05).Conclusions1.A series of pathophysiological changes occurred after intracerebral hemorrhage,mainly manifested as the increase of inflammatory cells around the hemorrhage area,the activation of microglia,the degeneration and death of neurons and so on.2.After intracerebral hemorrhage,deferoxamine,minocycline and the combined application of both drugs can reduce the area of brain damage after cerebral hemorrhage,improve neurological deficits,inhibit the activation of microglia,and reduce the number of degeneration and death of neurons,thereby improving the nerve function damage after ICH.3.After intracerebral hemorrhage,deferoxamine,minocycline and the combination of the two all reduce the expression of iron-related protein,and play a neuroprotective effect by chelating iron ions. |
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