Establishing A Model For Recurrent Intracerebral Hemorrhage In Mice And Studying The Mechanism Of Dopaminergic Neuron Injury In The Remote Midbrain

Background:Intracerebral hemorrhage(ICH)is the second most common stroke subtype.It is associated with a high mortality of up to 30%within one month of presentation.Recurrence and secondary brain injury(SBI)were the main factors affecting ICH’s long-term prognosis.In addition,the prognosis of recurrent ICH is worse than that of the first ICH.Establishing mouse ICH models has improved our understanding of the pathophysiology of ICH.Due to the lack of a recurrent ICH model,the study on recurrent ICH was confined to observing clinical patients,such as location,time,severity,and risk factors of the second ICH.We knew little about recurrent ICH’s pathological mechanism and behavioral characteristics eliminating the influence of primary diseases such as hypertension and diabetes.This study aimed to establish a double(d)-ICH model by sequentially injecting bacterial collagenase into the bilateral striatum to mimic recurrent ICH.Then we evaluated a series of behavioral tests,compared the hematoma volume and the injury volume,and analyzed the number of dopaminergic neurons middle brain,remote area from the primary hematoma.At the same time,we observed the morphological distribution of IBA1-positive cells around the hematoma and midbrain.Finally,we compared the data obtained from the single-ICH and double-ICH mice models and their controls.Methods:In stage one,we established the s-ICH model by injecting 0.35μl collagenase Ⅶ-S(0.075 U in 0.5 μl saline)into the left striatum.In stage two,21 days later,we injected the same dose of collagenase into the right striatum to mimic the recurrent ICH.After the operation,we assessed mice for brain damage,motor,cognition,and emotion-related behaviors,and the number of dopaminergic neurons in the midbrain.We also observed the morphological distribution of IBA1-positive cells around the hematoma and midbrain.Results:We successfully established and characterized a recurrent ICH mouse model using a two-stage collagenase injection in the bilateral striatum method.The hematoma and lesion volume did not differ between single-ICH and double-ICH on day 3.The hematoma was repeatable.The single-ICH and double-ICH mouse models had significant motor deficits.The wire hanging test,but not the 24-point neurologic deficit scoring system,was sensitive evaluating locomotor abnormalities in the double-ICH model,especially on days 3,7,and 21 post-ICH.The locomotor function recovered gradually,but the mice still had neurologic deficits on day 21 after single-ICH and double-ICH.Furthermore,the neurologic deficits of the double ICH mice were more severe than those of the single ICH mice at the same time points.Double-ICH mice had more severe cognitive impairment,less interest in exploration,and more severe depression-like behaviors than single-ICH mice.Furthermore,double-ICH mice had a slower average speed and shorter walking distance in the open field test compared to single-ICH mice.Despite our expectations,the number of dopaminergic neurons in the midbrain was not less in single-ICH and double-ICH mice than in their respective controls.The activation of Ibal-positive cells was found in the fresh hematoma three days after hemorrhage,the activation range of Ibal-positive cells around the hematoma in the double bleeding model group was wide and cell deformation was obvious.Three days after the operation,activation of Ibal-positive cells was found around the hematoma and the midbrain,remote from the striatum in both groups of the mouse model.It is speculated that inflammation after ICH is involved in the appearance of distant injury,especially in the dopaminergic nervous system.However,the source of these IBA1 positive cells needs to be further explored.Conclusion:We established a novel two-stage bilateral ICH model in the mouse striatum.The hematoma and lesion volume three days after operation were stable and reproducible.The double-ICH and single-ICH mouse models generated reproducible brain damage and subsequent abnormalities in locomotor,cognition,and emotion-related behaviors,and the behavior changes of the double-ICH model were severer than the single-ICH mice model.Damage to the dopaminergic nervous system may be responsible for these behavioral changes.Inflammation after ICH is involved in remote lesions,especially in the dopaminergic nervous system.This new ICH model closely mimics recurrent ganglionic ICH in clinics and should be a novel tool for investigating the pathogenesis and new therapeutic strategy of recurrent ICH.