Effect And Mechanism Of CSF1R Inhibitor PLX3397 On Hypoxic-Ischemic Brain Damage In Neonatal Mice

Background and PurposeNeonatal hypoxic-ischemic encephalopathy(HIE)refers to perinatal asphyxia caused by partial or complete hypoxia,cerebral blood flow reduction or pause,resulting in fetal or neonatal brain damage,characteristic neuropathological and pathophysiological changes,and clinical symptoms of encephalopathy.The global incidence rate of neonatal HIE is variable from 1.9 to 26.5 per 1000 live births in developing or developed countries.In China,neonatal HIE occurs in 3 to 6 per 1000 live births,which was similar to that in developed countries(3 per thousand～5 per thousand).After HIE,about 15%to 20%of the children died in the neonatal period,and 20-30%of the survivors have epilepsy,cerebral palsy,blindness and mental retardation.This is a heavy burden to the patients and their families and even the society,which is a serious social healthy issue.Recently,accumulating pre-clinical and clinical investigations have demonstrated that inflammatory immune response plays a crucial role in the pathogenesis of neonatal hypoxic-ischemic brain injury,and neuroinflammation takes part in the brain injury induced by HIE.Neuroinflammation causes additional brain parenchymal injury by activating brain local innate immune cell and recruiting peripheral immune cells into the injured brain.There is not just an age differences in HIE induced neuroinflammation between neonatal HIE and adult HIE,one feature is that HIE can trigger the innate immune response within a few minutes in the neonatal HIE patients.Microglia is the main type of immune cells in CNS.The activated microglia are resident macrophages in the brain.Their activation is the first step in the inflammatory response of CNS diseases including HIE.The activated microglia in the acute phase after cerebral hypoxia/ischemia can release oxidative active substances and inflammatory mediators,including cytokines and chemokines,which can chemotactic the infiltration of peripheral immune inflammatory cells into the brain tissue.At the same time,they interact with the infiltrated leukocytes to secrete more oxidative and pro-inflammatory mediators in the brain.The occurrence of pro-inflammatory microenvironment in injured brain promotes the damage of brain tissue,and further aggravate neurodegeneration.Activated microglia play an essential role in the initiation of brain inflammation,which has been generally considered detrimental in brain damage at acute phase after cerebral hypoxia/ischemia but might be some of them shift to be beneficial for neurorepair at the delayed recovery phase,and thus is considered to be a potential therapeutic target.In recent years,some experimental approaches such as drug-induced removal of microglia provide a new method for the study of the pathological role of microglia in HI.The proliferation,differentiation and survival of microglia are highly dependent on the signal transduction of colony stimulating factor-1 receptor(CSF1R).Pexidartinib(PLX3397),an inhibitor of CSF1R,has been used to specifically remove microglia in experimental animal studies,and this approach has limited side effects.Furthermore,the cell number and function of microglia cells can be reversed and gradually returned to normal levels after stopping administration.Additionally,PLX3397 has been approved by FDA for clinical treatment of tenosynovial giant cell tumor(TGCT),indicating that PLX3397 is relatively safe.Therefore,we selected PLX3397 to inhibit the function of CSF1R in order to eliminate microglia in brain.The published experimental investigations have reported that PLX3397 can effectively remove microglia in the brain parenchyma of adult mice,but the expression level of CSF1R and the clearance efficiency and side effects of PLX3397 on microglia cells in newborn mice remain poorly known.The main purpose of this study is to explore the possible role and mechanism of microglia in the acute phase of HI in newborn mice.The First Part of Study:Investigate the efficiency of PLX3397,an inhibitor of CSF1R in clearing microglia in the brain of normal newborn miceObjective:The objective is to investigate the expression of CSF1R in microglia of newborn mice,and investigate the effect of CSF1R inhibitor PLX3397 on the clearance of microglia in the brain,and the effects on the growth and development of normal newborn mice.Method:1.Administration of PLX3397:At postnatal day 4(P4),both the male and female littermate pups of CD1(ICR)were randomly divided into 4 groups according to the administration concentration:Three groups were orally administered PLX3397(5,15 and 25 mg/kg·d)twice a day starting from P4 to the end of the experiment(P9),and the control group was given the same volume(0 mg/kg·d)of vehicle.2.After continuous administration for 5 days(P9),they were tested by cell analysis with flow cytometry that the expression of CSF1R in microglia and the absence of microglia in the brain of newborn mice.Observe the relationship between the dose of PLX3397 and the efficiency of removing microglia.At the same time,the body weight change and death rate of the two groups were counted.The follow-up experiments were performed with the dose of this administration procedure of PLX3397 over 90%of total brain microglia were deleted and it was observed the effect of this dose of PLX3397 on the growth and development of newborn mice.Results:1.Same as the adult mice,it was found that brain microglia expressed CSF1R in CD1(ICR)neonatal mice.2.The PLX3397 dose-range study showed that clearance effect was enhanced with the increase of dose(F=30.872,P<0.05).The dose of 25 mg/kg·d was the optimal dose in the clearance of brain microglial cells in the newborn mice.The oral administration of PLX3397 for five consecutive days can effectively reduce over 90%of microglia in the brain of normal neonatal mice and the remaining microglial cell number was(1.74±0.36)×103 in the brain of PLX3397 treatment group,in the vehicle group was(23.65±1.49)×103.(t=14.26,P<0.05).3.Effect of PLX3397 on the body weight of normal newborn mice:before administration,the body weight of PLX3397 treatment group was(3.43±09)g,vehicle group was(3.48±0.08)g,no difference was detected(t=0.42,P>0.05);After 5 days of continuous administration,the body weight of PLX3397 treatment group was(5.44±0.08)g,vehicle group was(5.50±0.13)g,no difference was detected(t=0.38,P>0.05).4.Effect of PLX3397 on the number of neurons in the brain of normal newborn mice:the number of neurons in PLX3397 treatment group was(242±10.5)/mm2,vehicle group was(243±11.5)/mm2,no difference as detected(t=0.11,P>0.05)。Conclusion:PLX3397 can effectively eliminate brain microglia without affecting the number of neurons in the brain of normal newborn mice.The Second Part of Study:Investigate the protective effect of CSF1R inhibitor PLX3397 on brain injury in newborn HI miceObjective:The first objective is to establish the animal model of HI in newborn mice.The second objective is to investigate the protective effect of CSF1R inhibitor PLX3397 on brain injury in the newborn HI mice.Method:1.Newborn mice were daily given PLX3397 orally for 7 days(postnatal day 4-11(P4-P11)).2.On the 9th day after birth,the left common carotid artery was permanently ligated and exposed to 10%oxygen for 30 min,resulting in moderate HI injury in the left cerebral hemisphere,the moderate HI neonatal mouse model.3.Brain tissue samples were collected 48 hours after HI,and TTC staining was used to determine the degree of hypoxic-ischemic brain damage.4.The neurobehavioral deficits of HI newborn mice were detected by angle test and cylinder test.Results:1.The effect of PLX3397 on the infarct volume of brain injury in HI newborn mice at 48 hours after HI:PLX3397 group was(15.37 0±17)%,vehicle group was(28.70 3±66)%.The brain infarction size was significantly decreased by PLX3397 treatment with about 46.4%reduction rate(t=3.92,P<0.05).2.Effect of PLX3397 on neurobehavioral deficits of newborn mice at 48 hours after HI:for the degree of unilateral asymmetry in turn angle test,PLX3397 group 7was 3.4±3,vehicle group was 90±2.6,P<0.05;for the degree of unilateral asymmetry in cylinder test,PLX3397 group was 59.17±2.47,vehicle group was 78.33±3.33,P<0.05。Conclusion:PLX3397 has significant protective effect on the acute brain injury in HI newborn mice.The Third Part of Study:Investigate the potential mechanism of CSF1R inhibitor PLX3397 on the improvement of brain injury in HI newborn miceObjective:The objective is to explore the potential mechanism of CSF1R inhibitor PLX3397 in improving brain injury in the moderate HI model of neonatal mice.Methods:1.The newborn mice were treated with PLX3397,and the experimental animal model of moderate HI was then established.2.Flow cytometry analysis was used to determine the number of infiltrating immune cells in the brain tissue.The protein expression of cytokines/chemokines in the brain tissue lysates were detected by proteome profilometer and mouse XL cytokine array.The mRNA level of related inflammatory factors was detected by real-time quantitative PCR.The apoptotic-like neuronal cell death was detected and measured by immunohistochemistry.Results:1.Compared with vehicle group,PLX3397 group significantly reduced the number of neutrophils(69.7%),macrophages(77.4%)and T cells(72.9%)in brain tissue of HI newborn mice at 48 hours after HI.2.Expression of pro-inflammatory cytokines/chemokines and other proteins:PLX3397 treatment group significantly reduced the expression of 15 pro-inflammatory cytokines/chemokines and other proteins in the brain tissue of mice at 48 h after HI(P<0.05).3.Neuronal apoptosis:the number of NeuN+cells,NeuN+and active caspase 3+double positive neuronal apoptosis cells in the brain injury area of neonatal mice were measured at 48 hours after HI.The number of NueN+cells in PLX3397 treatment group was 6423.60±681.18,vehicle group was 3790.40±656.80,(t=3.18,P=0.024,)PLX3397 treatment group significantly increased the NueN+cells;The number of apoptotic cells in PLX3397 group was 615.20%±156.84,vehicle group was 1205.00±15,PLX3397 treatment group significantly decreased the apoptotic-like neuronal cells(t=2.78,P=0.013).Conclusion:CSF1R inhibitor PLX3397 can inhibit neuroinflammation and HI injury by changing the composition of immune cells and immune microenvironment in the HI brain of newborn mice.