| BackgroundIntracerebral hemorrhage(ICH)is a clinical common subtype of stroke,which is also a common type of acute cerebrovascular disease in middle-aged.The incidence of ICH is approximately 12-15 cases per a hundred thousand person-year.The main cause of ICH is hypertension.Because of its high morbidity and mortality,ICH is the one of the primary causes of death of urban and rural residents in China.ICH attacks dangerously,changes rapidly,and the most of survival patients have severe neurological and cognitive dysfunction.At present,there is no effective clinical treatment for ICH.Therefore,ICH is a heavy spiritual and economic burden to the family,society and whole country.ICH-mediated brain injury can be divided into primary and secondary injury.Surgical hematoma evacuation was thought to be an important treatment to ICH primary brain injury which is mediated by its mass effect.However,a randomized and double-blind controlled clinical trials with large sample published in Lancet in 2005 indicated that hematoma evacuation failed to improve the prognosis of patients with ICH.Secondary brain injury is mainly due to the toxic effects of blood and its metabolites on brain tissue.It has been confirmed that the metabolites and degradation products secondary to clot resolution following ICH are important pathogenic factors leading to secondary injury.Iron is the main degradation products of erythrocyte in the clot resolution,and then there will be large brain iron deposition in the brain tissue which could lead to acute cerebral edema and long-term neurological cognitive dysfunction.Iron chelator treatment,such as deferoxamine(DFX)and minocycline(Mino),could significantly attenuate cerebral edema,reduce neuronal cell death,and improve neurological dysfunction,which are mediated by brain iron overload.In recent years,stroke researchers focused on the mechanism and prevention of clot resolution-mediated brain iron deposition after ICH which was a key issue.However,the mechanism of clot resolution and its mediated brain iron deposition after ICH are still unclear.The mechanisms of clot resolution after ICH are complicated,the main pathophysiologic process is erythrocytolysis and erythrophagocytosis by microglia/macrophage.The characteristic pathological changes of erythrocytolysis are changes of membrane permeability and morphology.The formation and activation of membrane attack complex(MAC)plays a critical role in erythrocytolysis.MAC goes through the erythrocyte membrane,as a transmembrane hydrophilic channel,making the erythrocyte membrane loss of barrier effect.The morphology of erythrocyte transforms from the normal double concave disc shape into a spherical shape,finally leading to the red blood cell rupture and release of hemoglobin and other contents due to the osmotic pressure alternation.Microglia/macrophage expresses and actives a variety of surface phagocytic receptors,mediating the endocytosis of erythrocytes and degradation products after hemorrhagic stroke.The endocytic erythrocyte and hemoglobin could be degraded into iron ions,deposited in microglia/macrophages as hemosiderin.The hemosiderin dose not induce severe pathological response.While iron deposition extracellularly around the hematoma induces a series of secondary brain damage,including inflammation.Studies have proved that some intervention on mechanisms of clot resolution had neuroprotective effect,which is a potential therapeutic target for secondary brain injury after ICH.Iron chelator DFX was applied to acute iron poisoning and chronic iron accumulation disease,and has been proved that it had a neuroprotective effect on ICH-induced brain injury.Minocycline,as a class of broad-spectrum tetracycline antibiotics,has also been showed beneficial with its iron chelating effect.However,the effect of iron chelator treatment on clot resolution following ICH and the quantitative imaging method of brain iron deposition,still need further exploration.This study aims to observe the dynamic changes of clot resolution and brain iron deposition following ICH,to explore the possible mechanisms,and to investigate the quantitative imaging method of brain iron deposition.In addition,the effects of iron chelators,deferoxamine and minocycline,on clot resolution and brain iron deposition were further studied.MethodsPart 11.The ICH model was induced by an injection of autologous blood.Male piglets were randomly assigned into ICH 4-hour group,ICH 1-day group,ICH 3-day group and ICH 7-day group.The process of hematoma resolution and the erythrocyte morphology were examined on coronal sections of pig brain after hematoxylin and eosin(HE)staining.Hemoglobin and MAC contents in the clot were measured by Elisa kit.The expression of CD47(a regulator of erythrophagocytosis),CD 163(a hemoglobin scavenger receptor),and heme oxygenase-1(HO-1,a heme degradation enzyme)in the clot were measured by Western blot and immunostaining.The morphology of microglia/macrophages with erythrocyte swallowed,Iba-1 positive cells,and CD 163 positive cells and HO-1 positive cells were compared by HE staining and immunostaining.2.The ICH model was induced by an injection of autologous blood.Male piglets were treated with DFX or vehicle(saline),and then assigned into ICH + Vehicle 3-day group,ICH + DFX 3-day group,ICH + Vehicle 7-day group and ICH + Vehicle 7-day group.Hemoglobin and MAC contents in the clot were measured by Elisa kit.The expression of CD47,CD 163 and HO-1 were measured by Western blot and immunostaining.Part 21.The ICH model was induced by an injection of autologous blood.18-month-old male Fischer rats were randomly assigned into sham group,ICH 1-day group,ICH 3-day group,ICH 7-day group,ICH 14-day group and ICH 28-day group.The distribution image of brain iron deposition was exported through MR R*2 Mapping sequence scanning,and then compared with histological iron staining.The quantitative calculation of brain iron deposition content was based on standard iron concentration scanning.2.The ICH model was induced by an injection of autologous blood.18-month-old male Fischer rats were assigned into sham group and ICH groups,then ICH groups were treated with Mino or vehicle(saline),and assigned into ICH + Vehicle 7-day group,ICH + Mino 7-day group,ICH + Vehicle 28-day group and ICH + M:ino 28-day group.The neurological dysfunction was assessed by behavior test.The quantitative calculation of brain iron deposition content,and the measurement of brain swelling and brain atrophy were according to the MR scanning(T2、T2*,R2*Mapping sequence).The expression of Ferritin(iron storage protein),HO-1 and DARPP-32(a specific marker of GABAergic neurons in basal ganglia)were measured by Western blot and immunostaining.ResultPart 11.The HE staining showed that the hematoma dissolved gradually within 7 days after ICH in piglet.The erythrocyte at 4 hours were double-faced concave disc shape,then transformed to irregular shape with spike,and finally uniformly sized spherical cells.The diameter of RBC decreased gradually with a significant decrease at day 3 and stayed at small value at day 7.Elisa analysis showed the level of hemoglobin declined gradually over the time after the onset of ICH with a significant reduction at day 3,while MAC contents in the clot gradually increased with a marked accumulation at day 3.Erythrophagocytosis was observed in the hematoma edge at day 3,and hemosiderin deposition was identifiable in the hematoma edge at day 7.RBCs in the hematoma center started to be phagocytized by macrophages/microglia at day 7.The expression of CD 47 in hematoma reduced gradually after ICH with a significant reduction at day 3.It remained at low levels at day 7.CD 163 and HO-1 positive cells infiltrated into the hematoma from edge to center over the time after ICH.The morphological characteristics of the CD 163 and HO-1 positive cells in the clot were microglia/macrophage like.Western blot analysis showed the protein expression of HO-1 in hematoma increased gradually.2.The hemoglobin contents in hematoma were significantly higher in the deferoxamine-treated group than in the vehicle-treated group at day 3 after ICH.Deferoxamine also reduced the MAC content in the clot at day 3.The loss of CD47 in the clot was reduced significantly by deferoxamine treatment at day 3 and day 7.Deferoxamine treatment also caused a significant reduction in infiltrating CD 163 positive and HO-1 positive cells in the hematoma at day 3 and day 7.Western blot analysis showed that the protein expression of HO-1 was decreased in the hematoma in the deferoxamine-treated group at day 3 and at day 7.Part 21.MRI R2*mapping was used to determine brain iron content in perihematoma area after ICH in aged rats.The brain iron content of ipsilateral hemispheres in ICH model much higher than the content of contralateral hemisphere,and the content increased gradually over the time after ICH with a peak at day 14,and stayed at high levels at day 28.The positive region of Perls’ staining is roughly consistent with the distribution image of brain iron deposition exported by MRI R2*mapping.2.MRI scanning showed that Mino treatment reduced brain swelling at 3 days after ICH.Mino treatment also reduced the content of brain iron deposition around the hematoma and decrease the T2*lesion volume at day 7 and day 28.Mino treatment attenuated brain atrophy at day 28.Western blot and immunostaining showed that Mino treatment reduced the protein expressions of HO-1 at 7 days,DARPP-32 at day 28 and Ferritin at day 7 and 28.The Behavioral test suggested that Mino treatment improved neurological dysfunction at day 7 and 28.ConclusionPart 11.The hematoma dissolved gradually over the time after ICH with reduced Hb content in clot,which is significant at day 3.One of the factors associated with clot resolution is erythrocytolysis.The alternation of erythrocyte morphology and diameter may be related to the gradual stimulation of MAC in the complement system.Another factor associated with clot resolution is erythrophagocytosis.The endocytosis of red blood cell by microglia/macrophage and the infiltration of CD 163 positive and HO-1 positive cells may be associated with the degradation of red blood cell surface receptor CD47 in the hematoma.2.DFX treatment attenuated Hb degradation and reduces MAC content after ICH.DFX treatment also inhibited the degradation of CD47 protein and decreased the infiltration of CD 163 positive and HO-1 positive cells.DFX treatment slowed down hematoma clearance may be associated with the reduced erythrocytolysis and the inhibition of erythrophagocytosis by microglia/macrophage.Part 21.A gradual increase of brain iron deposition in perihematoma area occurred after ICH in aged rat.MRI R2*mapping was a reliable and noninvasive method of quantitative measure for brain iron content after ICH.2.Mino treatment reduced brain iron deposition content and protein expressions of iron metabolism marker,such a as ferritin and HO-1,after ICH in aged rats;Mino treatment also attenuated ICH-induced brain swelling,neuronal cell death,brain atrophy and neurological deficits.Mino treatment reduced secondary brain injury after ICH,which may be associated with attenuation of brain iron deposition. |
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