The Role Of Mass Effect On The Secondary Brain Injury After Intracerebral Hemorrhage

Intracerebral hemorrhage(ICH)is associated with high disability and mortality rate,and there is no clinically effective treatment.The research about the injury mechanisms of ICH has great significance to the exploitation of new treatment strategies.Mass effect induced by hematoma after ICH is the main cause of primary brain injury,which will chronically accompany the occurrence and development of secondary brain injury including erythrolysis and iron deposition.Previous research had demonstrated that mass effect directly caused irreversible damage to the neural network structure of brain tissue through compressing local tissue deformation.However,the regulation mechanism and space-time effect of hematoma mass effect on secondary injury after ICH remain unclear.Based on this,this paper focused on the clinical problems of ICH,using the technical means of biomechanics,neuroscience and materials science,and established an animal model used for mass effect research through synthesizing injectable thermo-sensitive Poly(N-isopropylacrylamide)(PNIPAM)hydrogel.Compared with the autologous blood injection model,this model had significantly reduced the impact of hematoma degradation products and formed similar mechanical compression on the surrounding brain tissues.In addition,the role of mass effect on erythrolysis and iron deposition after ICH was studied,and the time threshold,dose-effect relationship and mechanical signal transduction pathways of erythrolysis and iron deposition induced by mechanical regulation were investigated.Furthermore,this paper proposed potential measures to intervene the erythrolysis and iron deposition induced by mass effect.The main contents and conlusions of present study are as follow:(1)Establishment of an animal model suitable for the study of mechanical mass effect of hematoma.The thermo-sensitive hydrogel,which was similary with the physical properties of hematoma and the space-time evolvement of ICH pathology,was designed and formed mechanical mass effect through precise injecting into the basal ganglia area.Studies had shown that PNIPAM hydrogel had the similar topography and pore structure to the blood clot.The same volume of PNIPAM or blood injected ICH model produced similar Young's modulus increase,lesion volumes,and mechanically activated ion channel Piezo-2 upregulation in brain tissues(p > 0.05).Meanwhile,slight iron deposition,neuronal cell death and brain edema were observed in the PNIPAM hydrogel model compared to the blood model(p < 0.05).In addition,the PNIPAM hydrogel displayed good biocompatibility and stability in vivo via subcutaneous implantation.These results demonstrated that PNIPAM hydrogel cerebral infusion could used to simulate the mass effect of hematoma after ICH.This model could not only form a stable and controllable mass effect similar to hematoma,but also minimize the interference of blood,which was an ideal model for the study of mass effect after ICH.(2)The space-time effect of mass effect on the erythrolysis after ICH.Based on the established thermo-sensitive hydrogel model,the co-injection of a fixed amount of autologous blood(50 ?L)and different volumes of PNIPAM hydrogel(0,50,100 and150 ?L)was used to study the effect of mass effect on erythrocyte hemolysis.Meanwhile,the mass effect volume and the ICH time threshold of erythrolysis induced by mass effect were also evaluated.Firstly,the microscopic observation of erythrocytic morphology in the hematoma found that mass effect formed by 150 ?L PNIPAM hydrogel induced significant morphology abnormal of erythrocyte at 6 h after ICH,and reduced the erythrocytic surface area and diameter(p < 0.05).With the increase of mass effect,the surface area of erythrocyte was significantly reduced,while the content of hemoglobin in the brain tissues was significantly increased.The released hemoglobin was quantitatively evaluated by a polynomial concerning with the mass effect,the volume of hematoma and the time of ICH.The hemoglobin release mediated by mass effect increaseed more rapidly in the early stage of ICH.Mass effect also caused significant upregulation of the hemoglobin phagocytosis-related protein CD163 and the heme degradation rate-limiting enzyme HO-1.Meanwhile,mass effect aggravated the secondary brain injury after ICH,including iron deposition,cell death,brain edema,microglia activation,and neuronal deficits.These results illustrated that mass effect accelerated erythrolysis and hemoglobin release after ICH,and promoted the upregulation of metabolic proteins related to blood degradation products,and aggravated secondary brain injury.The protective role of trehalose against erythrolysis.Trehalose with different concentrations(0,50,100,and 150 mg/m L)were used to treat the 50 ?L autologous blood and 150 ?L PNIPAM hydrogel co-injected ICH model.It was found that trehalose could alleviate the brain injury by inhibiting early erythrolysis after ICH,which significantly reduced the content of hemoglobin in brain tissues,brain water content,iron deposition,HO-1 expression and cell apoptosis,and improved the prognosis of neurological function.Trehalose could effectively improve the secondary brain injury caused by mass effect,and was an effective intervention to inhibit the erythrolysis after ICH.(3)The effect of mass effect on iron deposition and ferroptosis after ICH.Focusing on the iron overload after ICH,the effect of mass effect on iron metabolism and ferroptosis after ICH was researched through co-injection of ferrous chloride solution and PNIPAM hydrogel(0,50,100,and 150 ?L)into the basal ganglia,and the potential pathways associated with these processes mediated by mass effect was explored.The detection of neuronal iron deposition suggested that mass effect aggravated the intracellular iron deposition at the early stage(6 h)after ICH under the iron overload microenvironment.Moreover,mass effect increased the expression of mechanosensitive ion channel Piezo-2,p ERK1/2,and iron uptake-related proteins(DMT1 and Tf R).Mass effect also downregulated the expression of iron death marker protein GPX4 and upregulated the expression of 4-HNE.These indicated that mass effect could regulate intracellular iron homeostasis by activating Piezo-2 and its downstream p ERK1/2 pathway,thereby affecting intracellular iron deposition and ferroptosis.Validation of the key pathway of iron metabolism mediated by mass effect.The p ERK1/2 inhibitor was used to intervene the ferrous chloride solution and 150 ?L PNIPAM hydrogel co-injected ICH model.The p ERK1/2 inhibitor PD98059 significantly decreased mass effect induced p ERK1/2 and iron uptake-related proteins(DMT1 and Tf R)upregulation,and increased the expression of GPX4.These demonstrated that the intervention of p ERK1/2 pathway could reverse the iron deposition and ferroptosis caused by mass effect.In conclusion,this paper was based on the clinical needs of ICH,focusing on the role of mass effect on the occurrence and development of secondary brain injury,explored the relationship between mechanics and erythrocyte hemolysis and iron deposition.Which clarified the brain injury process under mechanical-biochemical coupled condition after ICH.It had great significance for further understanding the brain injury mechanism of ICH and the development of new treatment strategies.