Effects Of Cannabinoid Receptor Ⅱ Induced Neuroprotection After Germinal Matrix Hemorrhage And The Involved Mechanisms

Background and purposeNeonatal cerebral hemorrhage is one of the detrimental brain injuries,which act as germinal matrix hemorrhage（GMH）mostly.GMH is defined as that matured vascular vessels cannot sustain the pressure of out sides,lick birth canal squeeze,and rupture.More and more GMH newborns survived because of the elevated perinatal medicine,with detrimental sequalae,lick post-hemorrhagic hydrocephalus（PHH）,cerebral atrophy and cerebral palsy and even epilepsy induced by the inflammatory scar.GMH and the followed sequalae are such social-medical problems that exploring the pathological process and therapeutic target may make significant changes.The severity of GMH correlated with embryonic period.Studies reported that 15 percent newborns within 37 weeks’ embryonic period were mature,and 50% out of that suffered GMH between the 24 th and 30 th week in embryonic state.Germinal matrix is the bleeding site of GMH,where consists neuronal progenitor cells（NPCs）,neurons and astrocytes.Because of the closed location,bleeding concerning subventricular zoon bec ome the standard of GMH categories,10% subventricular captured was defined as GMH State I and 50% subventricular occupation was defined as State II,50% subventricular occupation with ventricular enlargement was state III and state IV GMH was companied with severe obstructive hydrocephalus.John et al.reported collagenase induced GMH model of rat at the first time and the model mimicked human neonatal brain injury in pathological condition and cognitive sequalae,which proved a prominent model for basic research of therapeutic target on GMH.Neuroinflammation is an innate immunity state of nervous system and the consequence of activation of self-defense system.While,several studies proved that overexpressed neuroinflammation was the source of cytokine,like TNF-α,IL-6 and so on,which played key roles in secondary brain injury.After rupture of vascular vessels,components of blood cells lysis,lick hemoglobin,thrombin,iron and so on a ctivated the innate immune cells in central nervous system（CNS）-MICROGLIA.Microglia consists 15-25% of cells in the CNS and the key cell modulating secondary inflammatory brain injury induced by hemorrhagic stroke,with dynamic state of phenotype accordi ng to the situation around.Generally,microglia was defined as M0 state,M1 state and M2 state.M0 microglia was accepted as the silent sate,supervising the situational changes around in the CNS;M1 microglia was the activated state and changed into macrophages without or few brunches,that was also called cytotoxic phenotype.When injury occurred in brain,M0 microglia was activated and began to migrate to the injured site and changed into M1 gradually under the guide of chemokines,which was to swallow the debris of infiltrated blood.In the process,M1 microglia secreted many inflammatory cytokines,like TNF-α and IL-6,contributing to Neuroinflammation.As reported previously,M2 microglia is also one type of activated microglia.In physiological status,when M0 microglia supervised the overexpress of neuronal axons or disorder arrangement of neuronal cells,it activated into M2 microglia,which contacted the target neuron cells and pruning the unnecessary parts.While in pathological situation,M2 microglia may contribute to neuronal circuit improvement via direct contact or indirect secreting brain derived nutrition factors（BDNF）or insulin-like growth factors（IGF）.All in all,it is valuable to research in microglial phenotype transformation following hemorrhagic stroke and model of GMH is the suitable choice in evaluating microglia and neuron cross-talk.Endocannabinoid system（ECS）exist extensively in animals,participates in physiological pathological react,like metabolism,heart associated disease,cancer and nervous disease.ECS has special ligand systems of anandamide（Narachidonoylethanolamine,AEA）and 2-arachidonoylglycerol（2-AG）and special receptors of cannabinoid receptor1/2（CB1/2）.CB1/2 are G protein receptor in the membrane of cells.Generally,CB1 was associated with nervous system addiction and disease of neurodegenerative disease,like senile dementia,Alzheimer’s disease and Parkinson’ disease.Recently,studies reported the neuroprotection of CB2 R in neuroinflammation and found that upregulation of CB2 R in microglia showed positive effects on neuroinflammation induced by ischemic stroke in mice,but the precise mechanisms are unknown.In the study,we used collagenase Ⅶ to induce GMH in S-D pups postnatal day seven（P7）mimicking the pathological characters in human pups.Iconographic techniques,behavior testing and cryobiological techniques were used in the study.Targeted to CB2 R,we aimed to elaborate the role of activation of CB2 R and how could it regulating microglia under condition of GMH.Moreover,the molecular mechanisms are revealed in the process.The meanings of the study are: 1,searching for a new target in regulating microglia-induced neuroinflammation;2,providing a novel idea and methods for clinical treatment of GMH and the followed sequelae.Part Ⅰ Minocycline Attenuates Neuroinflamation and brain edema in neonatal rats of GMH by activating cannabinoid receptor 2ObjectiveThe current study investigates whether minocycline reduces cortical reactive microglia and protects the brain from injury in a rat model of collagenase-induced GMH by regulating the activity of CB2 R.Methods and materialsThis study was divided into two parts.First,rat pups postnatal seven（P7）were given a 0.3 U injection of collagenase type VII into the right matrix germinal brain region and minocycline or vehicle was intraperitoneally（i.p.）administered.The pups were euthanized 24 hours after GMH,and their brains were used for detection of CB2 R m RNA expression by real-time RT-PCR.Co-expression of CB2 R in microglia was detected by immunofluorescence.Second,after GMH and minocycline treatment,the rat pups were intraperitoneally injected with AM630（1 mg/kg,a selective CB2 R antagonist）or JWH133（1.5 mg/kg,a selective CB2 R agonist）and their vehicle.After euthanization at different time points,the pups underwent magnetic resonance imaging,and brain edema was measured by the wet-weight/dry-weight method.In addition,activation of microglia was measured by immunohistochemistry,and inflammatory cytokines were measured by ELISA.ResultsGMH and minocycline administration 24 hours after surgery l ed to increased CB2 R m RNA expression and increased CB2 R expression in microglia.Minocycline significantly reduced brain edema,lateral ventricular volume and accelerated cortical thickness.Quantification of cytokines,including IL-6,TNF-α and TGF-β,and assessment of microglia activation,proliferation in minocycline treated pups showed reduced inflammatory brain damage after GMH.All these neuroprotective effects of minocycline were prevented by AM630.JWH133 were used to strengthen the hypothesis,whic h showed the identical neuroprotective effects with minocycline.ConclusionOur study demonstrates,the first time,that minocycline attenuates inflammation-induced brain injury in a rat model of GMH,and activation of cannabinoid receptor 2 was partially involved in these processes.Part Ⅱ CB2 R activation attenuates microglial accumulation and brain injury following experimental GMH in rat pups via ERK dephosphorylationObjectiveHere,we investigated the effects of Cannabinoid receptor2（CB2R）agonist on microglia proliferation and the possibly involved mitogen-activated protein kinase（MAPK）family pathway in collagenase-induced GMH rat model and in thrombin-induced rat microglia cells.Methods and materialsA total of 98 rat pups post-natal day 7（P7）were subjected to colleganase-induced GMH.JWH-133（specific cannabinoid receptor2 agonist）or AM630（specific cannabinoid receptor2 antagonist）and their vehicle were administered intraperitoneally（i.p）at 1h and 6h following GMH.The brain edema,neuronal degeneration,inflammatory response and possibly involved phosphorylated extracellular signal-regulated kinase（p-ERK）protein level around hemorrhage were investigated 24 h following injury.In vitro,primary cultured microglia were treated with JWH-133 by different concentration（1.0,2.5,4μM）and UO126（1μM,inhibitor of ERK pathway）following thrombin（20U/ml）stimulation.P-ERK protein expression in microglia was determined by immunohistochemistry.Effects of CB2 R activation on MAPK family（P38,ERK）protein expression were detected by Western blot.Tumor necrosis factor（TNF）-α and inducible nitric oxide synthase（i NOS）protein were detected by ELISA and Microglial proliferation were detected by 5-Ethynyl-2’-deoxyuridine（EDU）assay.ResultWe demonstrated that activation of CB2 R played a key role in attenuating brain edema,neuronal degeneration,microglial accumulation and p-ERK protein level 24 h following GMH.Interestingly,administration of 1mg/kg AM630 reversed the neuroprotective effects of JWH-133 and inhibited ERK dephosphorylation induced by JWH133.In vitro,Western blot analysis and immunostaining indicated that ERK and P38 phosphorylatio n in microglia stimulated by thrombin were decreased after JWH-133 treatment,which was concentration dependent.Microglia proliferation（EDU + microglia）,inflammatory and oxidative stress response were attenuated by UO126 24 h after thrombin stimulation,which was prevented by AM630.ConclusionThe present study suggests that activation of CB2 R might attenuate inflammation-induced brain edema and neuronal degeneration after GMH in rats by reducing microglia proliferation and partially through a mechanism involving ERK,P-38 dephosphorylation in microglia/macrophage.Part Ⅲ CB2 R selective agonist restored GMH induced damage of neuronal circuit after GMH via up-regulating of CX3CR1 in microgliaObjectiveWe’re to prove the hypothesis that a cannabinoid receptor2（JWH133）accelerates the CX3CR1+ microglia secreting neurotrophic factors and restores damaged neuronal circuit.Methods and materialsGMH was induced in rat pups postnatal day 7（p7）by stereotactic injection of collagenase-Ⅶ and JWH133 was administered by intraperitoneal（i.p.）.In addition,CX3CR1 sh RNA（NM₁33534.1）was used to test the potential mechanism conducting neurotrophic microglia transformation.In vivo Diffusion tensor imaging（MRI-DTI）,immunohistochemistry,western blotting,proliferation detection and q RT-PCR were used to study the lone-term effects of JWH133 on the dialogue of microglia and neuronal circuit after GMH.ResultsLong-term treatment with JWH133 promoted direct contact of microglia with neuronal cells by double-labelling.The FA value of DTI in the internal zone around the hematoma revealed the positive effects of JWH133 on neural restoration.Moreover,JWH133 increased brain derived neurotrophic factor（BDNF）,but not minocycline,a microglia inhibitor.3 days after JWH133 treatment,we detected reduced expression of biomarkers for neural progenitor cells（NPCs）in pups pre-injected in the lateral ventricular with CX3CR1 sh RNA.ConclusionOverall,this study provides evidence that the CB2 R agonist promote a neurotrophic phenotype of microglia,beyond merely alleviating microglial proliferation and inflammation.Moreover,JWH133-induced CX3CR1+ microglia restore damaged neuronal circuit,which represent a novel therapeutic strategy following GMH in clinic.