| Backgrounds:Hydrocephalus is an independent factor for the poor prognosis of patients with hemorrhagic stroke.Studies have shown that the blood components play a crucial role in the development of hydrocephalus after intracranial hemorrhage.Thrombin,an important blood component,and previous studies have been shown to play an important role in development of hydrocephalus after IVH.However,the specific pathological changes and underlying mechanisms of thrombin-induced hydrocephalus are still completely understood.In humans,gender is an important factor affecting the severity of brain injury secondary to hemorrhagic stroke,and the exact mechanism remains unknown.Studies suggested that estrogen could participate in the pathological process of various diseases through regulating neutrophil chemotaxis.This study is divided into three parts to explore the mechanism of thrombin-induced hydrocephalus:1.Based on the intraventricular injection of thrombin to establish hydrocephalus models in rats and mice,the severity of hydrocephalus was evaluated by imaging means,and the pathological changes were observed at the histological level.2.To investigate the effect of gender on thrombin-induced hydrocephalus and its mechanism in rats.3.To explore the role of neutrophils in thrombin-induced hydrocephalus and its gender differences in mice.Part 1.To establish intraventricular injection of thrombin-induced hydrocephalus models in rats and miceAims:To establish intraventricular injection of thrombin-induced hydrocephalus models in rats and mice,to evaluate ventricular volume,and to observe its histological changes.Methods:Firstly,adult male Sprague-Dawley(SD)rats of the experimental group received intraventricular injection of 3 units(U)of thrombin,and the control group rat received an equal volume of saline(50μl),to establish hydrocephalus rat model.Secondly,adult male CD-1mice of the experimental group received intraventricular injection of 1 U of thrombin,and the control group mice received an equal volume of saline(10μl)injection.All animals underwent magnetic resonance T2weighted imaging(T2WI)examination to evaluate the ventricular volume and periventricular white matter swelling,and then euthanized at 24h after injection,and obtained brains were made into fresh frozen sections for hematoxylin and eosin(H&E)staining and immunohistochemical staining.Results:In the rat hydrocephalus model:T2WI could detect significant ventriculomegaly in male SD rats(23.1±5.1 vs.8.5±0.8 mm~3,p<0.01)and periventricular white matter swelling(3.6±2.1 vs.1.2±0.8 mm~3,p<0.05)at 24h after unilateral intraventricular injection of 3 U thrombin.Histologically,intraventricular injection of thrombin resulted in severe ventricular wall destruction(26.3±8.1%vs.4.3±2.0%,p<0.01),and neutrophils infiltration was observed in the choroid plexus,ventricular wall,and the swollen area of the periventricular white matter in male rats.In the mouse hydrocephalus model:intraventricular injection of 1 U thrombin could induce hydrocephalus(5.16±1.43 vs.2.17±0.47 mm~3,p<0.01)and periventricular white matter swelling(0.7±0.8 vs.0 mm~3,p<0.05)at 24h in mice.Significant neutrophil infiltration(524±92 vs.0cells/mm2,p<0.01)and formation of neutrophil extracellular traps(298.5±101.4 vs.0 cells/mm2,p<0.01)were observed in the choroid plexus at 24 hours after thrombin injection in mice.Activated epiplexus cells were observed at 24h after thrombin injection in mice(Iba1-positive cells:10.98±1.98%vs.3.72±1.21%;p<0.01;CD68-positive cells:1.62±1.25%vs.6.54±1.52%;p<0.01).In addition,neutrophil infiltration was also observed in the swollen area of periventricular white matter(203±165 vs.0 cells/mm2,p<0.05)at 24h after thrombin injection.Conclusions:Intraventricular injection of thrombin resulted in ventricular enlargement and periventricular white matter swelling in male rats and male mice.Histologically,similar pathological changes could be observed in thrombin-induced both hydrocephalus models,providing a different model for further exploration of thrombin in the formation of hydrocephalus after hemorrhagic stroke.Part 2.The effect of gender on thrombin-induced hydrocephalus in rats Aims:To investigate the gender difference of thrombin-induced hydrocephalus and periventricular white matter,and exploring the underlying mechanisms in adult SD rats.Methods:Firstly,adult male and female rats received intraventricular injection of 5 U of thrombin,to evaluate the mortality rate at 24h after injection.Secondly,adult female rats of the experimental group received intraventricular injection of 3 U thrombin,and the control group received an equal volume of saline,and the T2WI data and fresh frozen sections of male rats in the first part were included as the male group in this part,to observe the changes of ventricle volume and periventricular white matter in different sexual rats.Thirdly,adult male rats the experimental group were pretreated with 17-βestradiol(5 mg/kg,intraperitoneal injection),and the control group was pretreated with an equal vehicle at 24h and 2h before intraventricular injection of 3 U thrombin,to verify whether the differences of thrombin-induced hydrocephalus in different sexual rats is mediated by estrogen.All rats were examined by T2WI and then were euthanized at 24h after injection,and obtained brains were made into fresh frozen sections for H&E staining and immunohistochemical staining.Results:Intraventricular injection of 5 U thrombin resulted in 100%and0%mortality in female and male rats,respectively.However,intraventricular thrombin injection could induce larger ventricle volume(36.1±12.5 vs.23.1±5.1 mm~3,p<0.05)and more severe white matter swelling(12.8±8.2 mm3 vs.3.6±2.1mm~3,p<0.01))in female rats,and more severe ventricular wall destruction(40.9±8.5%vs.26.3±8.1%,p<0.05),compared with male rats.In female rats,the number of infiltrated neutrophils in the choroid plexus and the swollen area of periventricular white matter was significantly higher than that in male rats(choroid plexus:938±313 vs.576±216/mm~2,p<0.01;white matter:1092±680 vs.281±284/mm~2,p<0.05).Compared with the vehicle group,intraventricular injection of 3 U thrombin resulted in larger ventricle volume(36.0±9.8 vs.23.4±3.5 mm~3,p<0.05),more severe periventricular white matter swelling(10.5±7.2 vs.2.5±3.2 mm~3,p<0.05),and more number of neutrophils infiltrating into the choroid plexus and the swollen area of white matter(choroid plexus:1142±646 vs.452±144 cells/mm~2,p<0.05;white matter:721±500 vs.205±249cells/mm~2,p<0.05)in male rats pretreated with 17-β-estradiol.Linear regression analysis showed that the number of neutrophils infiltrating into the choroid plexus was positively correlated with the ventricular volume(r=0.65,p<0.0005).And the number of neutrophils infiltrating into the swollen area of periventricular white matter was also positively correlated with the volume of the swollen area of periventricular white matter(r=0.84,p<0.0001).Conclusions:Intraventricular thrombin-induced hydrocephalus in rats differs by gender,and is more severe in female rats.Estrogen mediates sex differences in thrombin-induced hydrocephalus through the recruitment of neutrophils.Understanding gender differences in thrombin-induced brain injury may contribute to future individualized interventions for hemorrhagic stroke.Part 3.Neutrophils depletion attenuates thrombin-induced hydrocephalus in miceAims:To investigate the effect of neutrophil depletion in peripheral blood on thrombin-induced hydrocephalus in CD-1 mice.Methods:Firstly,male mice of the experimental group were treated with anti-Ly6G neutralizing antibody(0.5 mg/mouse,intraperitoneal injection)at 24h and 5 minutes before thrombin injection(1 U),and the control group was treated with an equal Lg G2A,to verify the effect of neutrophils on thrombin-induced hydrocephalus in male mice.Secondly,adult female mice of the experimental group received intraventricular injection of thrombin,the control group received equal volume of saline,and the magnetic resonance data and MPO positive cell data of male mice in the first part of the study were included,to verify the effect of thrombin-induced hydrocephalus whether sex difference exists in mice.Thirdly,female mice of the experimental group received anti-Ly6G neutralizing antibody at 24h and 5 minutes before intraventricular injection of thrombin,the control group received Ig G2A,to verify the effect of neutrophils on thrombin-induced hydrocephalus in female mice and to test whether sex differences in thrombin-induced hydrocephalus were abrogated after neutrophil depletion.All mice were examined by T2WI and then were euthanized at 24h after injection,and obtained brains were made for fresh frozen sections for H&E staining and immunohistochemical staining.Results:Peripheral blood neutrophils could be significantly depleted(36.17±6.24%vs.4.83±1.72%;p<0.01)at 24h after intraperitoneal injection of mouse anti-Ly6G neutralizing antibody in male mice.Neutrophil depletion attenuated ventricular enlargement in male mice at24h after thrombin injection(anti-Ly6G group:median=2.82 mm~3;Ig G2A control group:median=4.34 mm~3;p<0.01),the number of Iba1-positive cells(5.47±1.69%vs.9.77±1.76%;p<0.01),and the number of CD68-positive cells(1.62±1.25%vs.6.54±1.52%;p<0.01)in the choroid plexus were significantly decreased.However,neutrophil depletion failed to alleviate thrombin-induced periventricular white matter swelling in males(2.70±0.54%vs.4.62±1.26 mm~3;p>0.05).Intraventricular thrombin injection also resulted in significant ventricular enlargement(7.71±1.59 vs.2.36±0.31 mm~3;p<0.01)and marked neutrophil infiltration(Thrombin group:median=717.0 cells/mm~2;saline group:median=0 cells/mm~2;p<0.05)at 24h in female mice.The ventricle volume of female mice after thrombin injection was larger than that of male mice(7.71±1.59 vs.5.16±1.43 mm~3,p<0.05).Neutrophil depletion also attenuated thrombin-induced hydrocephalus in female mice(3.50±1.02 vs.7.07±2.01 mm~3,p<0.01).Furthermore,the difference in thrombin-induced ventricular enlargement between male and female mice disappeared at 24h after injection(3.50±1.02 vs.2.70±0.54 mm~3,p>0.05).Conclusions:Neutrophil depletion could attenuate thrombin-induced hydrocephalus and activation of choroidal inflammation in mice,but not periventricular white matter swelling.Furthermore,neutrophil depletion eliminated the gender differences in thrombin-induced hydrocephalus.Interventions targeting neutrophils may be a potential therapeutic strategy for the treatment of post-hemorrhagic hydrocephalus. |
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