The Role And Mechanism Of CCL12 In Aging Aggravating Brain Injury After Intracerebral Hemorrhage

Background and objects:Intracerebral hemorrhage?ICH?is a common disorder characterized by high morbidity,morality and disability especially in China.ICH is mainly found in middle-aged and elderly people,and as age grows,the clinical prognosis of patients with ICH is signifi cantly worse,suggesting that age is an important reason for the aggravation of brain injury after ICH.Although remarkable progress has been made in the study of the pathophysiological mechanism of ICH in recent years,thousands of therapeutic drugs that are effective in animal models have been shown to be ineffective when translated into clinical translational studies.One of the most important reasons for these outcomes is that most of the basic research subjects are young experimental animals,while the majority of ICH patients are middle-aged or elderly individuals.Therefore,elucidating the mechanisms by which aging aggravates brain injury has great therapeutic significance for ICH.Intriguingly,we and others have shown that transplanting plasma from young individuals can improve brain biofunctions in old mice and even ameliorate acute brain injury in old rodents with ICH and ischemic stroke.These results strongly suggest that circulating factors can significantly influence the process of normal agin g in the brain and diseased brain.Recently the levels of circulating factors,such as Chemokine C-C motif ligand 2?CCL2?,CCL11,CCL12,CCL19,haptoglobin and?2-microglobulin are found markedly elevated in old mice.Among them,CCL2,CCL11,CCL19,haptoglobin and?2-microglobulin have been shown to be significantly involved in the progression of normal aging and aging-related acute brain injuries.CCL12 is a chemokine that is also known as monocyte chemotactic protein 5?MCP-5?which is mainly secreted by peripheral immune cells such as monocytes and neutrophils.Moreover,our preliminary experiments showed that there are most significanct differences in plasma CCL12 levels between young and old mice.CCL12 plays an important role in the inflammatory response and can recruit peripheral immune cells to damaged areas.Therefore,we put forward hypothesis that aging upregulates CCL12 expression of plasma after ICH,which could aggravate ICH-induced brain injury by recruiting more peripheral inflammatory cells into the brain.Materials and methods:1.Construction of ICH model:we constructed ICH model of young?3-month old?WT?old?18?20-month old?WT and old CCL12^-/-mice.Then we obtained the blood and brain samples at 12 h,1 d,3 d,5 d and 7 d after ICH,and the blood sample were centrifuged to collect plasma,all the samples were freeze to-80?.2.Construction of parabiosis model:we constructed WT-WT,WT-CCL12^-/-,CCL12^-/--CCL12^-/-parabionts,then constructed ICH model after 15 d.Then we collect plasma and brain samples at 1 d after ICH,all the samples were freeze to-80?.3.Measurement of CCL12:the plasma CCL12 levels were detected at each time points after ICH using ELISA kits.And the brain CCL12 levels were detected at each time points after ICH by PCR and WB.4.Detection of brain injury:the neurological deficit scores,survival rates and brain water content were detected after ICH.And inflammatory chemokines were detected using ELISA kits.Using HE,TUNEL,FJB and Nissl stainings to evaluate the state of neurons around the hematoma.5.Detection of the recruitment of peripheral inflammatory cells:the infiltr ation of inflammatory cells around the hematoma were detected using immunofluorescence and immunohistochemistry after ICH.Then we extracted inflammatory cells from the brain,and the number of infiltrating inflammatory cells was detected by flow cytometry after ICH.Results:1.Plasma and brain CCL12 levels are markedly higher in old mice than in young mice after ICH.We further confirmed that the levels of plasma and brain CCL12 in old mice were significantly higher than that in young mice,and we found that CCL12 levels was further up-regulated after ICH.2.Peripheral CCL12 can enter the brain of mice through blood-brain barrier.We found that CCL12 protein was detected in the plasma and brain tissues of heterochronic CCL12^-/-mice,but CCL12 mRNA was absent in the brain tissues of heterochronic CCL12^-/-mice.What's more,the levels of CCL12 between the heterochronic mice were close to eachother.These results suggest that CCL12 can enter the brain through BBB.3.CCL12 aggravates ICH-induced brain injury.We found the degree of damage in the brain after ICH—as determined by neurological defict scores?NDS?,mortality rates,brain water content?BWC?,levels of inflammatory factors,numbers of degenerative and apoptotic neural cells and surviving neurons were significantly attenuated in old CCL12^-/-mice compared to that observed in old WT mice.These effects were reversed in old CCL12-treated mice.These results suggest that up-regulated CCL12 aggravate brain injury after ICH.4.CCL12 aggravates ICH-induced brain injury via recruitment of macrophages and T lymphocytes.We found that the number of perihematomal macrophages and T cells was significantly decreased in CCL12^-/-mice compared to old WT mice.In contrast,the effects were reversed in CCL12-treated old mice.Conclusions:The results show that the expression of plasma CCL12 is up-regulated in old ICH mice,which aggravates ICH-induced brain injury via recruitment of macrophages and T cells.This study identifies a mechanistic target for the treatment of ICH to reduce the infiltration of inflammatory cells after ICH.