C3/C3aR Inhibition Prevents Astrocyte-microglia Crosstalk And Improves Hydrocephalus After Germinal Matrix Hemorrhage In Rats

BackgroundGerminal matrix hemorrhage(GMH)is one of the most common and critical neurological diseases in neonates,which is defined as subependymal or periventricular germinal matrix hemorrhage in immature brain tissue.Studies have shown that neonatal,especially premature infants(<37 weeks)and low body weight(weight <1500g)neonatal cerebral germinal matrix vascular wall structure is not mature,birth canal injury or ischemia and hypoxia can cause vascular rupture to form GMH,the incidence of about 3.5‰.Children with GMH often suffer from many serious complications,such as cerebral palsy,learning disability,mental disorder,hydrocephalus and so on,in which post-hemorrhagic hydrocephalus(PHH)is the most common and most serious,will cause serious,long-term psychomotor and cognitive impairment,poor prognosis.Currently,ventricular shunt is the main clinical treatment strategy for hydrocephalus.About 15% premature infants with severe hydrocephalus receive hydrocephalus shunt surgery,including ventriculoperitoneal shunt(VPS)and lumbocipoperitoneal shunt(LPS).But ventricular shunt is not a long-term solution.Invasive cerebrospinal fluid(CSF)shunting,an empirical ?one-size-fits-all‘ approach with high morbidity due to frequent shunt obstructions and infections requiring surgical revision.A 10-year follow-up study suggested that 51% of patients with VPS needed to be recatheterized.Other treatments,such as serial lumbar punctures,ventricular taps,external ventricular drainage,ventricular access device,ventricular subgaleal shunt,endoscopic third ventriculostomy,and endoscopic coagulation of the choroid plexus are not satisfactory.In addition,patients with GMH hydrocephalus have special characteristics of the population,and the surgical treatment effect is not good and non-surgical treatments(continuous CSF extraction,diuretics,ventricular fibrinolytic therapy)for PHH are rarely effective.The lack of effecti ve treatment for hydrocephalus after GMH is mainly due to the complex and vague pathophysiology of its development and the lack of effective intervention targets.A noninvasive clinical approach will significantly improve the long-term quality of life of GMH patients.In the early stage,the research group studied the role and mechanism of neuroinflammation in the injury of nerve loop after GMH,and elucidated: 1)microglia cells are the "trigger" cells of neuroinflammation after GMH,which can achieve diff erent regulatory effects of neuroinflammation through phenotypic.2)Glibenclamide can up-regulate aquaporin 4(AQP4)expression and reduce PHH in rats.Our study found that neuroinflammation and PHH are the key injury-causing links of nerve dysfunction after GMH,and they promote each other,but the mechanism is not clear.What‘s more,the complement C3 is found to be activated in the cerebrospinal fluid of normal pressure hydrocephalus patients.As is known,C3 is mainly expressed in astrocytes and C3aR is mainly expressed in microglia.Therefore,further study on the influence of the interaction between the major inflammatory regulatory cells(microglia)and the key injur-causing cells(astrocytes)of hydrocephalus on the occurrence and development of hyd rocephalus after GMH through the C3/C3 a R signaling can provide a more comprehensive understanding of the brain injury mechanism after GMH,and provide new targets and strategies for the prevention and treatment of PHH.ObjectiveLateral ventricle dilatation,cognitive function,expression of periventricular inflammatory cytokines,expression of C3 and C3 a R were measured to investigate the effects of inhibition of C3/C3 a R mediated astrocytes-microglia crosstalk on hydrocephalus after cerebral hemorrhage in newborn rats.MethodsGMH was induced by collagenase ?-S injection into the ganglionic eminence in Sprague-Dawley(S-D)rat pups that were post-natal day 7(P7).First,we detected the expression of C3 and C3 a R over time(by immunofluorescence,WB and RT-PCR)after GMH.Then,C3aRA(selective C3aR antagonist)was administered intraperitoneally,and the effects of C3aRA on hydrocephalus(by MRI),the learning and memory function(by Morris Water Maze),and the inflammatory reaction(by detection of IL-1?,IL-6,TNF-?,TGF-?1 and aquaporin 4)after GMH were further explored.Results1.The expression of periventricular C3 and C3aR was up-regulated 3 days after GMH by immunofluorescence,WB and PCR.2.By MRI indicated,C3aR antagonist alleviated hydrocephalus at 28 days after GMH.3.C3aR antagonist improved cognitive function during the water maze tests on day 28 after GMH.4.WB and PCR showed that C3aR antagonist could decreased GMH-induced inflammatory.5.Through immunofluorescence and WB detection,C3aRA could reduce the expression of AQP4 in the acute phase and inhibit the mis-location of AQP4 in the chronic phase after GMH.ConclusionAfter GMH,astrocytes up-regulated and released C3,which bound to the C3aR on microglia to promote inflammation and further aggravate hydrocephalus.C3aRA may be a promising alternative for hydrocephalus after GMH.