Building And Evaluation Of The Mouse Model Of Intracerebral Hemorrhage Using Autologous Blood Infusion Method With Different Blood Volume

Section 1 Evaluation of the mouse model of intracerebral hemorrhage using autologous blood infusion methodObjective:To build and evaluate the mouse model of intracerebral hemorrhage with 5ul,10ul or 20ul autologous blood injected into the right striatum using the double injection method.Methods:104 C57 male mice were randomly selected into group 5ul(N=28),group 10ul(N=32),group 20ul(N=30) or sham group(N=14).Anesthetised mouse were fixed at the stereotactic apparatus,then double injected with autologous blood using the microliter syringe.The autologous blood was obtained by cutting the tail.And there was no anticoagulant used in the microliter syringe.After the operation,the four groups each was ramdomly seperated into 3 subgroups named by 3 different time point(1^st day,2^nd day or 3^th day).Before that,the death rate of different group is counted.Each subgroup was evaluated right at the time point its named.Neurologic deficits evaluation includes forelimb placing test and corner turn test.Results:1.86 mice survived throughout the operations.2.There was no significant difference in death rate result of 4 groups.3.There was no significant difference of weights among all groups of mice.4.Forelimb placing test:The contralateral formlimb placing was significantly worse than the ipsilateral one in all the 4 groups(P＜0.01);the contralateral formlimb placing in group 5ul,group 10ul and group 20ul were significantly worse than in sham group.In subgroups of contralateral,5ul-2^nd was worse than other 5ul subgroups(P＜0.05).10ul-3^th,20ul-3^th and sham-1^st were the worst in their own groups,but the differences failed to reach significance level.There was no difference between 5ul-1^st and sham-1^st(p＞0.05).5. Corner turn test:Corner turn asymmetry in group 5ul,group 10ul and group 20ul were more apparent than in sham group(P＜0.05).The LI of 5ul-2^nd,10ul-3^th,20ul-3^th and sham-1^st were the highest in their own groups(P＞0.05).There was no difference between 5ul-3^th LI and sham-3^th LI(P＞0.05).Conclusions:The mouse model of intracerebral hemorrhage with apparent focal neurologic deficits can be successfully built by double infusion of autologous blood of 5ul,10ul and 20ul.With the increase of infusion volume,neurologic functions decrease but death rates change little.Injurys are worst in 5ul-2^nd,10ul-3^th,20ul-3^th and sham-1^st.Group 5ul injures less,recovers faster and has lower specificity.Section 2 Evaluation of hematoma formation,neurologic deficits and their relationshipObjective:To determine the hematoma volume of group 5ul,group 10ul and group 20ul.And to evalute their relationships with neurologic deficits.Methods:19 mice were ramdonly selected out of the survived mice,including group 5ul(N=6),group 10ul(N=7) and group 20ul(N=6).Three groups each was ramdomly seperated into 3 subgroups named by 3 different time point(1^st day,2^nd day or 3^th day).Each subgroup was evaluated right at the time point it was named.Neurologic deficits evaluation includes forelimb placing test and corner turn test.Then the brains were fixed and cut into 20um coronal sections using the cryostat.The region of hematoma was outlined in the H&E-stained brain section.Then the volume of hematoma was estimated.Results:1.Hematoma formation:All the mice tested formed localized hematoma in the right striatum.The volume follows:0.9868±0.2493mm³ in group 5ul,2.3254±0.3939mm³ in group 10ul,3.6465±0.6623mm³ in group 20ul.2.Relationship between neurologic deficits and hematoma volume:The hematoma volume of group 5ul,group 10ul and group 20ul increased gradually(P＜0.01).The forelimb placing deficits of group 10ul and group 20ul both were worse than group 5ul(P＜0.01).But no difference was found between group 10ul and group 20ul,which was inconsistant with hematoma results.Conclusions:Group 5ul and group 10ul both form hematoma of infusion volume,with related neurologic deficits changing.Group 20ul is found much backflow during modeling,then fails to form stable hematoma and neurologic deficits.Section 3 Evaluation of brain edema,neurologic deficits and their relationshipObjective:To estimate the brain edema of group 5ul,group 10ul,group 20ul and sham group.And to evalute their relationships with neurologic deficits.To compare hematoma volume with brain edema additionally.Methods:67 mice left were tested in this section,including group 5ul(N=18),group 10ul(N=19),group 20ul(N=18) and sham group(N=12).Four groups each was ramdomly seperated into 3 subgroups named by 3 different time point(1^st day,2^nd day or 3^th day).Each subgroup was evaluated right at the time point its named.Neurologic deficits evaluation includes forelimb placing test and corner turn test.Then the brain slices without hematoma were obtained to be weighted before and after being dried.Brain edema was measured by brain water content which was calculated as follows:(wet weight-dry weight)/wet weight.Results:1.Brain water content:The tissue around hematoma or injection point contained more water than the normal side in all groups(P＜0.05).Brain edema around hematoma in group 10ul and group 20ul were worse than in sham group(P＜0.05),and group 5ul had no difference with sham group(P＞0.05).In subgroups,5ul-2^nd,10ul-3^th and 20ul-3^th were the highest in their own groups(P＜0.05).They were higher than both their normal side contrast and the sham subgroups(P＜0.05).But there was no difference among 3 of them(P＞0.05).All the normal side contrasts showed no difference(P＞0.05).2.Relationship between neurologic deficits and brain edema:Neurologic deficits,in group 5ul,group 10ul and group 20ul were more apparent than in sham group(P＜0.01),and increased gradually.Brain water content of tissue surrounding injury in sham group,group 5ul and group 10ul(P＜0.01) increased gradually,but decreased in group 20ul compared with group 10ul(P＜0.05),which was inconsistant with neurologic deficits changing.3.Brain edema around injury increased in correspondence with hematoma volume in group 5ul and group 10ul,but not in group 20ul.Conclusions:Group 10ul and group 20ul show worse brain edema in tissue surrounding hematoma than sham group.Group 5ul fails to show difference in brain edema with sham group.Brain edema becomes worst in 5ul-2^nd,10ul-3^th,20ul-3^th and sham-1^st.With the increase of brain water content,neurologic functions decrease gradually in sham group,group 5ul and group 10ul.Brain water content increase in group 5ul and group 10ul in consistant with hematoma volume.Group 20ul fails to show related neurologic deficits and brain edema because of the unstable hematoma formation.