| ObjectiveNeonatal cerebral hemorrhage and hypoxic-ischemic encephalopathy are the common types of perinatal brain injuries,and lead to severe long-term neurological and cognitive deficits,including cerebral palsy and mental retardation.Several factors are associated with preterm brain bleeds,including hemostasis,low Apgar scores,patent ductus arteriosus,vaginal delivery and infection,among which germinal matrix hemorrhage(GMH)is the predominant factor.GMH occurs due to the greater preterm vascular fragility of the germinal matrix region in association with multiple factors involved with cerebral blood flow fluctuation.Blood rupture into the ventricles,leading to the post-hemorrhagic hydrocephalus,and ultimately cerebral palsy and atrophy.The underlying pathophysiology of neonatal hypoxic-ischemic encephalopathy(HIE)is the prolonged interruption of blood supply to the brain,which causes insufficient oxygen and glucose delivery and triggers a cascade of biochemical events including elevation of intracellular calcium,induction of oxidative stress,accumulation of pro-inflammatory cytokines and cellular apoptosis,resulting in extensive brain damage,impairing motor and cognitive function.Considering GMH and HI have tremendous effects on immature brain,it is particularly important to investigate the therapeutic target and the involved pathological processes in GMH and HI.Hemorrhagic brain injuries divided into primary and secondary injury.Primary brain injury initiated within the first few hours after hemorrhge occures,which is resulted from the mechanical damage of hematoma to adjacent tissues.Secondary brain injury,such as astrogliosis,microgliosis and inflammatory responses,play a critical role in the neurological dysfunction,especially the inflammatory responses.Thus,suppressing inflammatory response could be an essential intervention to limit GMH-induced brain injury.Microglia play critical roles in the immune response after GMH.Traditionally,microglia are considered injurious in the past-hemorrhagic brain due to the production of inflammatory cytokines.However,emerging data show that microglia have a beneficial role in neonatal stroke and that depletion of microglia exacerbates neuroinflammation and brain injury in neonatal ischemic stroke.Furthermore,preclinical studies show that regulation of the immune response after GMH involves an M1 to M2 phenotype transformation in microglia,and that promoting M2 polarization inhibits the expression of pro-inflammatory cytokines.Oxygen and glucose in the plasma are the main energy sources of neonatal brains.The neonatal brain is more susceptible than the adult brain due to the underdeveloped microvasculature.Mounting evidence demonstrated that cysteinyl aspartate specific proteinase(caspase)-dependent apoptosis plays an important role in the evolution and propagation of HI-induced cerebral damages.Furthermore,pharmacological or chemical modifications of caspases protect the brain against cerebral ischemia as well as reduced the behavioral deficits in rodents.Chemerin is synthesized as a 163-amino acid precursor,and released by several tissues,including immune cells,liver,and spleen.Chemerin or Chemerin derived peptide has been demonstrated to involve in multiple physiological or pathological conditions,including cardiovascular diseases and peritonitis.Currently,three proteins have been identified as Chemerin receptors:the natural receptor,Chemerin Receptor 23(ChemR23),as well as two other receptors,chemokine CC motif receptor-like 2(CCRL2)and G protein-coupled receptor 1(GPR1).It has been well documented that Chemerin and Chemerin derived peptide could inhibit the production of inflammatory cytokines in macrophages,reduce neutrophil recruitment and promote phagocytosis of apoptotic cells.Moreover,activation of ChemR23 after myocardial ischemia-reperfusion injury showed an obvious suppression of cardiomyocytic apoptosis.However,the effect of neonatal stroke on Chemerin/ChemR23 signaling and therapeutic benefits of exogenous Chemerin in an animal model of GMH and HI remained unexplored.In the present study,GMH was induced by intraparenchymal injection of bacterial collagenase(0.3U)in P7 rat pups,while P10 rat pups were subjected to right common carotid artery ligation followed by 2.5h hypoxia to mimic HI in human infants.Behavior testing and cryobiological techniques were performed.This study was aimed to elaborate the neuroprotective roles of Chemerin/ChemR23 in neonatal stroke,as well as the potential mechanisms.The objectives of this study are:1.searching for a potential agent in attenuating GMH-induced neuroinflammation;2,providing a therapeutic target in ameliorating HI-induced neuronal apoptosis;3.offering a novel method for improving both of short-term and long-term outcomes in neonatal stroke.PART I EFFECTS OF CHEMERIN ON GERMINAL MATRIX HEMORRHAGE-INDUCEDNEUROINFLAMMATION AND THE PROTENTIONAL MECHANISMSObjective1.To explore the temporal expression of endogenous Chemerin and its natural ligands,ChemerR23,CCRL2 and GPR1,after GMH.2.To observe the effects of activation of ChemR23 via recombinant human Chemerin(rh-Chemerin)on the GMH-induced neurological impairments,as well as the microglial accumulation,proliferation and differentiation.3.To investigate whether Chemerin/ChemR23 exerted its anti-inflammatory roles via calmodulin-dependent protein kinase kinase 2(CAMKK2)/adenosine monophosphate-activated protein kinase(AMPK)/Nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway.Methods1.GMH was induced by intraparenchymal injection of bacterial collagenase(0.3U)in P7 rat pups.The pups were euthanized Oh,3h,6h,12h,24h,72h,5d,7d after GMH,and their brains were used for detecting protein and mRNA levels of endogenous Chemerin,ChemR23,CCRL2 and GPR1 by Western bloting(WB)and reverse transcription-polymerase chain reaction(RT-PCR).Colocalization of Chemerin and ChemR23 on microglia,astrocytes and neurons were detected by immunofluorescence.2.Pups were randomly assigned to receive rh-Chemerin(3?g/kg/day,9?g/kg/day or 27?g/kg/day)or saline via intraperitoneal or intranasal administration at 1h post-GMH and then once daily for 3 days(short-term study)or 7 days(long-term study).Righting reflex and negative geotaxis were performed to investigate the effects of rh-Chemerin on the short-term neurological impairments from 24h to 72h after GMH;WB were used to test the optimal delivery route and dosage of rh-Chemerin;Ki-67,5-bromo-2-deoxyuridine(BrdU),CD11b/c(marker of activated microglia)and CD206(marker of M2 microglia)were performed to investigate the microglial accumulation,proliferation and differentiation with different interventions at 72h after GMH;RT-PCR was applied to measure the mRNA level of Arginase-1(marker of M2 microglia)at 72h after GMH;Water maze,foot-fault and rotarod were performed to analyze the long-term effects of rh-Chemerin on GMH-induced motor and cognitive impairments at 4 weeks after GMH;Nissl's staining were used to measure the cortical thickness and ventricular volume at 4 weeks after GMH;Myeloperoxidase(MPO)and Ionized calcium binding adapter molecule 1(Iba1)staining were applied to explore the number of neutrophils and microglia on choroid plexus at 3d,14d,28d after GMH.3.ChemR23 small interfering RNA(siRNA)or liposome-encapsulated specific ChemR23/CAMKK2/AMPK inhibitors were administered through intracerebroventricular injection 24h prior to GMH.Immunofluorescence was applied to confirm that liposomes were engulfed by activated microglia;WB were used to measure the protein expression of Chemerin/ChemR23/CAMKK2/AMPK/Nrf2,as well as interleukin-1 beta(IL-1 beta),interleukin-6(IL-6)and tumor necrosis factor alpha(TNF alpha)at 72h after GMH.Results1.The temporal expression of endogenous Chemerin and its receptors after GMH(1)Western blot analysis and RT-PCR data showed that endogenous Chemerin was significantly increased at 24 h(p<0.05),and continuously upregulated till 7 days after GMH compared to pups without GMH.Accordingly,ChemR23 was also significantly increased from 3 days to 7 days relative to pups without GMH(p<0.05).No changes were observed in expression of GPR1 and CCRL2 after GMH.(2)Double immunofluorescence staining showed that Chemerin and ChemR23 were abundantly expressed in microglia,neurons and astrocytes surrounding the lateral ventricle of pups with GMH.By contrast,Chemerin and ChemR23 seemed to have weaker expression levels in the microglia,neurons and astrocytes around the periventricular area of pups without GMH.2.The effects of rh-Chemerin on GMH-induced neurological and morphological impairments(1)All three doses of rh-Chemerin treated groups showed a significantly improved short-term neurological function(p<0.05).Intranasal administration delivered significantly more rh-Chemerin into the brain tissue compared to intraperitoneal administration(p<0.05).(2)In the water maze test,rh-Chemerin-treated animals performed significantly better than Vehicle-treated animals,as evidenced by the significantly less swimming distance(p<0.05),shorter escape latency(p<0.05)and more duration(p<0.05)in the defined quadrant;In the foot fault test,the performance was significantly better in rh-Chemerin-treated group compared to the Vehicle controls(p<0.05).Moreover,rh-Chemerin treatment significantly reduced the falling latency at both of the 5rpm and lOrpm acceleration compared to Vehicle controls(p<0.05).Besides,GMH significantly slowed normal growth from 14d to 28d after hemorrhage,as demonstrated by decreased body weight in the Vehicle group compared to the Sham group(p<0.05).(3)Nissl's staining showed that the ventricular volume was significantly reduced in rh-Chemerin-treated pups compared to the Vehicle group(p<0.05).White matter volume was significantly restored in rh-Chemerin treated pups(p<0.05).Rh-Chemerin treated pups had significantly less cortical loss(p<0.05).Since ventriculomegaly could be associated with cerebrospinal fluid overproduction after brain hemorrhage.Immunofluorescence showed that rh-Chemerin treatment significantly reduced the MPO immunoreactivity in choroid plexus at 3d,14d,and 28d post GMH(p<0.05).There was no evident difference in the overall number of Iba1+ cells between different experimental groups at 3d,14d,and 28d post GMH.3.The effects of rh-Chemerin on the production of pro-inflammatory cytokines,as well as the microglial accumulation,proliferation and differentiation(1)GMH induced IL-1 beta,IL-6,and TNF alpha in Vehicle-treated pups,whereas the levels of IL-1beta,IL-6 and TNF alpha were significantly reduced in rh-Chemerin-treated pups(p<0.05).However,knockdown of ChemR23 significantly reversed the inhibitory effect of rh-Chemerin on the expression of IL-1 beta,IL6 and TNF alpha relative to rh-Chemerin-treated and scramble siRNA groups(p<0.05).ChemR23 expression was significantly decreased after the administration of ChemR23 siRNA at 72h after GMH compared to rh-Chemerin-treated pups with or without Scramble siRNA group.(2)Compared to the Sham group,the number of Iba1+ and CD11b/c+microglia increased significantly in both Vehicle and rh-Chemerin treated pups 72h post-GMH(p<0.05).In addition,the number of activated microglia was significantly more in rh-Chemerin group compared to Vehicle group(p<0.05).(3)The number of Ki67+/Iba1+ and BrdU+/Iba1+ microglia were significantly increased in pups with rh-Chemerin compared to the Vehicle group.However,the number of Ki67+/Iba1+ and BrdU+/Iba1+ cells in the Vehicle group was comparable with Sham group.(4)No CD206+ microglia were detected in the Sham group,and Vehicle-treated pups showed a slight tendency towards having more CD206+ microglia.By contrast,rh-Chemerin treatment significantly increased CD206+ microglia compared to Sham and Vehicle groups(p<0.05).Rh-Chemerin also markedly increasing the anti-inflammatory mediator,Arginase-1,expression at 72h after GMH(p<0.05).4.The potential protective mechanisms of rh-Chemerin in GMH-induced neuroinflammation(1)The ratio of phosphorylated CAMKK2(p-CAMKK2)to CAMKK2 and phosphorylated AMPK(p-AMPK)to AMPK,as well as the expression of Nrf2 were significantly increased,and continuously increased up to 7 days after GMH compared to Sham group(p<0.05).Rh-Chemerin treatment further increased the phosphorylation of CAMKK2 and AMPK,and Nrf2 expression compared to Vehicle-treated pups(p<0.05).However,ChemR23 knockdown significantly suppressed the phosphorylation of CAMKK2 and AMPK,as well as the expression of Nrf2 compared to rh-Chemerin-treated and scramble siRNA groups(p<0.05).(2)Fluorescently labeled liposomes were engulfed almost exclusively in activated Iba1+ microglia rather than in GFAP+ and NeuN+ cells at 72h after GMH.The addition of Lipo-Alpha-NETA,Lipo-STO-609 or Lipo-Dorsomorphin to rh-Chemerin did not affect ChemR23 expression.The phosphorylation of CAMKK2 was significantly reduced in Lipo-Alpha-NETA and Lipo-STO-609 treated pups compared to rh-Chemerin with or without Lipo-PBS treated pups(p<0.05).In addition,Lipo-STO-609 inhibited phosphorylation of AMPK(p<0.05).However,Lipo-dorsomorphin had no effect on phosphorylation of CAMKK2.Administration of Lipo-Dorsomorphin significantly reduced the phosphorylation of AMPK and expression of Nrf2(p<0.05).Administration of all three liposome-encapsulated inhibitors significantly increased the expression of IL-1 beta,IL-6 and TNF alpha compared to rh-Chemerin with or without Lipo-PBS treated pups(p<0.05).ConclusionCumulatively,these data showed that rh-Chemerin ameliorated GMH-induced inflammatory response by promoting ChemR23/CAMKK2/AMPK/Nrf2 pathway,and M2 microglia may be a major mediator of this effect.Thus,rh-Chemerin can serve as a potential agent to reduce the inflammatory response following GMH.PART II EFFECTS OF CHEMERIN ON HYPOXIA-ISCHEMIA-INDUCED NEURONAL APOPTOSIS AND THE PROTENTIONAL MECHANISMSObjective1.To explore the temporal expression of endogenous Chemerin and its natural ligands,ChemerR23,CCRL2 and GPR1,after HI insults.2.To observe the effects of rh-Chemerin on the HI-induced short-term and long-term neurological impairments,as well as the neuronal apoptosis and oxidative stress.3.To determine whether Chemerin/ChemR23 exerted its anti-apoptosis via CAMKK2/AMPK/Nrf2 signaling pathway.Methods1.HI was induced by the ligation of right common carotid artery followed by 2.5h of hypoxia in P10 rat pups.The pups were euthanized Oh,6h,12h,24h,72h after HI,and their brains were used for detecting expression of endogenous Chemerin,ChemR23,CCRL2 and GPR1 by WB.Co-expression of Chemerin and ChemR23 in neurons were detected by immunofluorescence.2.After HI,pups were randomly assigned to receive rh-Chemerin(3?g/kg/day,9?g/kg/day or 27?g/kg/day)or saline via intranasal administration at 1h post-HI and then once daily for 1 day(short-term study)or 7 days(long-term study).Righting reflex and negative geotaxis were performed to investigate the effects of rh-Chemerin on the short-term neurological impairments;TTC staining was performed to evaluate the infarct volume at 24h after HI;Fluoro Jade C,TUNEL and immunofluorescence staining were conducted to estimate the neuronal apoptosis at 24h post HI;4-HNE and DHE staining were performed to analyze the generation of ROS;Water maze,foot-fault and rotarod were applied to evaluate the long-term effects of rh-Chemerin on HI-induced motor and cognitive impairments at 4 weeks after HI;Nissl's staining were used to measure the brain tissue loss at 4 weeks after HI.3.ChemR23 siRNA were administered through intracerebroventricular injection 24h prior to HI.Specific ChemR23/CAMKK2/AMPK inhibitors were delivered through intraperitoneal injection at 30min post HI.WB were conducted to measure the protein expression of Chemerin/ChemR23/CAMKK2/AMPK/Nrf2,as well as Cleaved Caspase 3/Caspase 3 and Bax at 24h post HI.Results1.The temporal expression of endogenous Chemerin and its receptors after HI(1)Western blot analysis showed that Chemerin and ChemR23 were significantly upregulated in a time-dependent manner and peaked 72h after HI(p<0.05).GPR1 and CCRL2 expression were slightly increased after HI,but did not reach statistically significance.(2)Double immunofluorescence staining revealed the increased Chemerin and ChemR23 signals in the peri-infarct area(ipsilateral cortex)compared to Sham pups and the contralateral control following HI.Furthermore,a robust increase in Chemerin and ChemR23 expression in cortical neurons following HI were detected.2.The effects of rh-Chemerin on HI-induced neurological and morphological impairments(1)Vehicle group exhibited marked neurological deficits(p<0.05),and these deficits were significantly improved by rh-Chemerin treatment(p<0.05).Body weight of rh-Chemerin(9?g/kg)treated pups was comparable to that of Sham pups.(2)Rh-Chemerin improved cognitive impairments in spatial learning and memorizing compared with Vehicle,as demonstrated by less swim distance to find the platform(p<0.05),less escape latency(p<0.05)and more time spent in probe quadrant(p<0.05).In the foot fault test,the performance was significantly better in rh-Chemerin-treated group compared to the Vehicle controls(p<0.05).Moreover,rh-Chemerin significantly reduced total foot-faults,especially in the left lateral,compared with Vehicle pups(p<0.05).Moreover,rh-Chemerin significantly improved rotarod latency at both of the 5rpm and lOrpm acceleration compared with Vehicle(p<0.05).(3)Nissl's staining revealed that the HI significantly induced brain weight and tissue loss(p<0.05),however,rh-Chemerin restored histologically defined injury at 4 weeks after HI(p<0.05).3.The effects of rh-Chemerin on the generation of pro-apoptotic cytokines and ROS at 24h after HI(1)Western blot results confirmed that rh-Chemerin significantly suppressed the expression of the pro-apoptotic proteins,Cleaved Caspase 3 and Bax(p<0.05).In vivo knockdown of ChemR23,which decreased ChemR23 expression by?2.0-fold,had a significantly positive regulation of Cleaved Caspase 3 and Bax(p<0.05).(2)Fluoro-Jade C staining that the number of Fluoro-Jade C + cells was markedly reduced in rh-Chemerin-treated pups than in HI pups(p<0.05).Likewise,density of TUNEL+ neurons were lesser in rh-Chemerin treated pups than HI pups in the peri-infarct area(p<0.05).Furthermore,ChemR23 siRNA reversed rh-Chemerin's protective effect as shown by the increased Fluoro-Jade C+cells and TUNEL+ neurons compared with rh-Chemerin treated group and scramble siRNA control group(p<0.05).Additionally,we observed Fluoro-Jade C signals were greater in the ipsilateral hippocampus than the contralateral one.In contrast to pups with HI,TUNEL and Fluoro-Jade C+ neurons were scarce in Sham group.(3)Immunofluorescence staining revealed that rh-Chemerin significantly attenuated the relative fluorescent intensity of DHE staining and the number of 4-HNE+ neurons in pups with HI compared with Vehicle treated pups and ChemR23 siRNA group(p<0.05).4.The potential mechanisms of rh-Chemerin in HI-induced neuronal apoptosis(1)The ratio of p-CAMKK2 to CAMKK2 and p-AMPK to AMPK,as well as the expression of Nrf2,were significantly increased at 24h after HI compared to Sham group(p<0.05).Rh-Chemerin treatment further increased the phosphorylation of CAMKK2 and AMPK,and Nrf2 expression compared to Vehicle-treated pups(p<0.05).However,ChemR23 knockdown significantly suppressed the phosphorylation of CAMKK2 and AMPK,as well as the expression of Nrf2 compared to rh-Chemerin-treated and scramble siRNA groups(p<0.05).(2)ChemR23 expression in ipsilateral hemisphere was unaffected at 24h after HI in Alpha-NETA,STO-609,and Dorsomorphin treated pups when compared with rh-Chemerin treated pups(p<0.05).The phosphorylation of CAMKK2 was significantly reduced at 24h after treating with Alpha-NETA and STO-609,while there was no significant difference between Dorsomorphin and rh-Chemerin treated pups.Administration of Dorsomorphin significantly reduced the phosphorylation of AMPK and expression of Nrf2(p<0.05),whereas Dorsomorphin exerted no significant effect on either the expression of ChemR23or phosphorylation of CAMKK2.Administration of Alpha-NETA,STO-609 and Dorsomorphin significantly increased the expression of Cleaved Caspase 3 and Bax compared with rh-Chemerin + DMSO treated pups(p<0.05).(3)TTC staining showed that ChemR23siRNA or specific inhibition of ChemR23/CAMKK2/AMPK significantly reversed the neuroprotective roles of rh-Chemerin(p<0.05),as demonstrated by the enlarged infarct area.Additionally,all four interventions significantly down-regulated body weight compared with Sham pups(p<0.05).ConclusionCollectively,the data provided evidence that intranasal administration of rh-Chemerin attenuated neuronal apoptosis at least in part via activating ChemR23/CAMKK2/AMPK signaling pathway.Rh-Chemerin could represent a therapeutic target for treatment of neonatal hypoxic-ischemic encephalopathy. |
PDF Full Text Request