Efficacy And Mechanism Of Cannabinoid Receptor 2 Agonist Prevents Thrombin-induced Brain Edema In Rats

BackgroundSpontaneous intracerebral hemorrhage(ICH)is a devastating disease.It constitutes 10–15% of all strokes in the USA,Europe,and Australia and 20–30% of strokes in Asia.Approximately 2 million cases of ICH are reported annually worldwide.There is currently no effective treatment for ICH,and it has a 1-month mortality rate of 30 to 50%.Patients who survive typically have major neurological impairments.Cerebral edema is primarily responsible for secondary injury after ICH.Edema increases mass effect and intracranial pressure(ICP)following ICH,which may directly damage brain tissue and ultimately result in herniation.Edema is also directly toxic to neurons and glia by changing osmotic gradients and disrupting the blood-brain barrier(BBB).Multiple pathways,including cytotoxic injury due to coagulation factors and a robust inflammatory response,lead to edema formation,and thrombin is the primary molecule that mediates the development of acute cerebral edema after ICH.The inhibition of thrombin with agratroban or hirudin also reduces edema after ICH in rats.Thrombin infusions into the caudate-putamen of the rat brain induces a rapid increase in edemawithin several hours that peaks from the first to the third day,and the edema declines gradually over several weeks.The trend of cerebral edema changes in parallel with changes in BBB permeability.Thrombin activates many intracellular signaling cascades in brain cells.extracellular signal-regulated kinase(ERK),is one of MAP kinase that is activated in the brain after intracerebral infusions of thrombin.The endocannabinoid system,including endogenous ligands,cannabinoid receptors,and degrading enzymes,is an important pharmacological target in many neurological diseases.Cannabinoid receptor type 1(CB1R)and cannabinoid receptor type 2(CB2R)are the most-studied cannabinoid receptors.The psychoactive effects of cannabinoids are associated with CB1 R,which is predominantly expressed by neurons.The psychoactive effects of CB1 R agonists limit their therapeutic potential,which leaves CB2 R agonists as the practical option.CB2 R is primarily expressed in immune cells,and it mediates anti-inflammatory actions,immune cell migration,cytokine production and antigen presentation.The anti-inflammatory and neuroprotective effects of cannabinoids in the brain were studied in animal models of multiple sclerosis(MS)and Alzheimer’s disease(AD).These effects were observed using pharmacological ligands that act on CB1 R,CB2R or both receptors.We previously demonstrated that a specific CB2 R agonist(JWH133)attenuated brain edema in rat models of germinal matrix hemorrhage(GMH),but the underlying mechanisms are not known.Increased metalloproteinase(MMP)expression is a key mechanism for increased BBB permeability after ICH.MMPs disrupt BBB integrity and promote edema by degrading tight junction proteins,type IV collagen,laminin,and fibronectin.The present study investigated the effects of a CB2 R agonist in a rat model of thrombin-induced BBB damage and the role of MMPs in the neuroprotective process.Part ⅠThrombin Induced Brain edemaand To Investigate the Injury of Blood Brain BarryObjectiveThe rats were euthanized 24 h later for measurement of the brain water content and the and toinvestigate the injury of the Blood Brain Barry.MethodsSprague–Dawley(S-D)rats were randomized into Saline group and Thrombin group.Brain Edema was induced by a thrombin(20 U)injection in the right basal ganglia.The Saline group only received a needle insertion.24 h after surgery.The brain water content was measured using the wet-weight/dry-weight method.Blood–Brain Barrier Damage was investigate by Evans blue(EB)extravasation.Microglial activation and matrix metalloproteinase expression were examined by immunofluorescence and Western blot.Results1.At 24 hours after injection of thrombin,brain edema was increased in the right basal ganglia compared with Saline-operated controls(p<0.01),2.Evans blue dye leakage from vessels within the boundary of the injection site 24 h after surgery(p<0.05),which indicates the Blood–Brain Barrier Damage,3.Iba-11 is an indicator of microglial activation.No obvious microglial activation was expected in the Saline group.While many activated microglial cells were widely observed in the Thrombin group.MMPs degrade the TJ proteins of the BBB.Therefore,we examined MMP expression in each experimental group.Our results demonstrated that MMP9 increased obviously in the right basal ganglia compared with Saline-operated group(p<0.05).ConclusionFollowing an intracerebral infusion of thrombin,brain edema and Evans blue dye leakage was increased in the right basal ganglia,while many activated microglial cells were widely observed and the expression of MMP9 also up-regulation in the Thrombin group,which may involve the Blood–Brain Barrier Damage.Part II JWH133 Attenuation of Thrombin Induced Brain edema,Inhibition of Microglial Activation and Matrix Metalloproteinase ExpressionObjectiveTo investigate the efficacy and mechanism of JWH133 reduces thrombin induced brain edema.MethodsThe rats were treated with JWH133(1.5 mg/kg)or SR-144528(3.0 mg/kg)and vehicle were intraperitoneally(i.p.)injected 1 h after surgery.In order to investigate the mechanism that JWH133 reduces thrombin induced brain edema,animals were randomly assigned to the following groups: sham-operated(Sham group),thrombin+vehicle(Vehicle group),thrombin+JWH133(JWH group),and thrombin+SR144528+JWH133(SR+JWH group).All animals were sacrificed 24 h after surgery.Brain water content and Blood–Brain Barrier Damage was investigate by Evans blue(EB)extravasation.Microglial activation,ERKpathway,Matrix Metalloproteinase expression and tight junction(TJ)-related protein ZO-1 were examined by immunofluorescence and Western blot.Results1.JWH133 administration significantly decreased thrombin-induced brain edema(p<0.01)and Evans blue extravasation(p<0.05),2.JWH133 reduced the number of Iba-1positive microglia.Phosphorylation level of ERK was lower in the JWH-133-treated group compared to the vehicle group(p<0.05),it also decreased the number of P44/P42(+)/Iba-1-1(+)microglia(p<0.05),3.Treatment with JWH133 inhibited the elevated matrix metallopeptidase(MMP)-9 and matrix metallopeptidase(MMP)-12 activities(p<0.05),while JWH133 also upregulated the expression of the tight junction protein ZO-1 compared with the vehicle(p<0.05).However,a selective CB2 R antagonist(SR-144528)reversed these effects.ConclusionCB2R agonist JWH133 attenuated brain edema by preserving BBB integrity following an intracerebral infusion of thrombin.We also found that dephosphorylation of the ERK pathway and the suppression of MMPs such as MMP-9 and MMP-12 were likely involved in the process.