Immune Mechanisms In Hemorrhagic Brain Injury

Background and Objective: Intracerebral hemorrhage(ICH)is a severe type of stroke with high disability and mortality.ICH is an enormous public health problem that imposes a significant economic burden on victims.Despite of emergent life-saving surgery,there's no effective pharcolgoical therapy for ICH patients,highlighting an emergent need for mechanistic studies of ICH injury to develop new treatment.The pathological events of ICH consist of primary injury caused by physical damage of hematoma and secondary injury induced by blood and clot compoents.Among risk factors of ICH,NAFLD leads to liver inflammation and primes the peripheral immune system.However,still unclear are to what extent and by what mechanisms NAFLD-associated alterations in immune system may influence ICH injury.After the rupture of blood vessel,blood components such as red blood cells,leukocytes and plasma proteins rapidly enter brain tissues.As a plasma protein,the potential effects of fibrin on CNS inflammation and ICH injury remains unclear.This study is therefore carried out address above questions.Methods:(1)A methionine-choline deficient(MCD)diet was fed for 4 weeks to produce the mice model of NAFLD before ICH induction.The effects of nonalcoholic fatty liver on ICH were evaluated in neurological score and brain water content.The methods of collagenase and autologous blood injection were both used.The change of immune cells and inflammatory factors were detected by flow cytometry and PCR.In addition,evans blue staining was used to check the integrity of BBB.Finally,mice were injected with anti-mouse Gr-1 monoclonal antibody to evaluate the role of monocytes and neutrophils in above process.(2)ICH was induced by collagenase method.The content of fibrinogen in brain tissue and peripheral blood was measured by ELISA.Hirudin was used to inhibit fibrin formation.We evaluated the long-term funtion of mice using modified Neurological Severity Score(mNSS),foot-fault test,and cylinder test.Flow cytometry was performed to determine the effects of hirudin on microglia polarization and other immune cells in the brain.PLX3397 was then used to deplete microglia in order to investigate the role of microglia in ICH.Results:(1)NAFLD aggravates short-term and long-term neurological deficits after ICH in mice,and also increases brain water content on day 3.NAFLD causes greater damage to the blood-brain barrier and mobilizes more peripheral immune cells infiltrating into brain,of which monocytes and neutrophils are the most obvious.The deleterious effects of NAFLD disappeared after the Gr-1 antibody administraion,proving monocytes and neutrophils are involved.(2)The fibrinogen content in brain is significantly increased after ICH,which is the basis for generating more fibrin.Hirudin can reduce the number of immune cells infiltrating into brain and improve long-term outcome after ICH by inhibiting fibrin formation.In ICH mice receiving hirudin,anti-inflammatory microglia-related factors(CD206,IL-10)increased significantly.After the microglia were depleted,the protective effect of hirudin disappeared.Conclusions: NAFLD mobilize peripheral monocytes and neutrophils that contribute to CNS inflammation and hemorrhagic brain injury.Inhibition of fibrin production can reduce neuroinflammation and improve the long-term neurological outcome after ICH.Our results provide new evidence regarding the immune components in the periphery and CNS that contribute to ICH injury.