The Strategy And Mechanism Of Dopaming/D2 Receptor System Preservation In Early Brain Injury After Intracerebral Hemorrhage

IntroductionSpontaneous intracerebral hemorrhage (ICH) is a fatal stroke subtype that accounts for approximately 20%-30% of all strokes. Intracerebral hemorrhage (ICH) is the result of small vessel bleeds within the brain parenchyma and subsequent formation and expansion of the hematoma, associated with high rates of mortality and morbidity. Currently, there is no effective therapy to improve survival rate or treatment to improve the quality of life for survivors. Thus, the amelioration of ICH-indued early brain injury is a promising therapeutic approach to improve clinical outcomes.Multiple studies have described the compromised structural and functional integrity of blood-brain-barrier (BBB) surrounding the hemorrhage. Increasing evidence indicates that BBB disruption plays a critical role in the pathogenesis of secondary injury after ICH, leading to the failure of normal mechanism controlling passage of substances into the brain, and subsequent perihematomal cerebral edema formation. Thus, the amelioration of BBB dysfunction is a promising therapeutic approach to improve clinical outcomes and potential therapeutic candidates can be gained from the endogenous mediators regulating BBB integrity.Numerous studies have demonstrated that pronounced inflammatory reaction play an important role in the secondary brain injury after ICH, including microglial activation and neutrophil infiltration. After ICH, various stimuli could activate microglia and initiate inflammatory response, such as thrombin and glutamate, which activate microglia and subsequently release pro-inflammatory cytokines and chemokines to enhance neuroinflammation. Therefore, the strategy based on inhibition of microglia activation might provide therapeutic potential for ICH.As a neurotransmitter, dopamine acts via two receptor isoforms (D1-like and D2-like receptor, DRD1 and DRD2) and both of them were observed an increased expression in the pre-infarct area after ischemic stroke in the previous study, indicating the involvement of both D1 and D2 receptor in neuroprotection after stroke. However, increasing evidence indicated the importance of dopamine D2 receptor and its angonists in regulating endothelial cell barrier integrity and vasorelaxation in the peripheral tissues. In tumor vasculature, it has been recently shown that DA acts through D2 receptors to stabilize the morphology of blood vessels and inhibit leakiness of tumor vasculature; and in the subsequent study, Myrthala confirmed that the anti-angiogenic effect of DA is mediated by dopamine receptor 2 (DR2) in tumor vessels. More importantly, in translational medicine, it was indicated that dopamine D2 receptor-specific agonists can be an option for the treatment of angiogenesis and vascular stability therapy in progress of tumor and vascular hyperpermeability related lung edema in IPS-induced lung injury models. Moreover, the DRD2-/-mice exhibit significant increased BBB permeability in acute lung injury model, which robustly suggest the importance of DRD2 on vascular permeability modulation. However, in the central nervous system, the essential role of endogenous dopamine system for the maintenance of BBB structure and function has not been established.The dopamine D2 receptor, a member of the rhodopsin-like heptahelical receptor family, is an important target for anti-Parkinsonian drugs that ameliorate the motor deficits. Recent studies have demonstrated the presence of dopamine receptors in immune cells and in glial cells. Increasing evidence indicated a protective role for DRD2 agonists in regulating immune functions and inflammatory reaction, which is probably through inhibiting activated T cell proliferation and cytokines secretion. Additionally, GLC756, a novel mixed dopamine DIR antagonist and D2R agonist was shown to inhibit the release of TNF-α from activated mast cells. Another specific DRD2 agonist, Quinpirole. suppressed neuroinflammation and protected against brain injury in a MPTP-induced mouse model of Parkinson’s disease. However, the expression and immune function of DRD2 in central nervous system after experimental stroke have not been systemically studied.Interestingly, both previous and our own work have revealed a long-lasting up-regulation of dopamine secretion in experimental ICH models. However, whether the regional dopamine alterations is relative to neuroprotection in response to hemorrhagic injury remains unknown. Therefore, in the present study, we sought to determine physiological role of endogenous Dopamine-D2 receptor system in maintaining BBB integrity and the potential role and mechanisms of activation of dopamine D2 receptor in collagenase and autologous blood-induced ICH models. Our study will explore the potential therapeutic utility of DRD2 agonists in clinical ICH patients.Material and Methods1. ICH model was induced by intrastriatal infusion of bacterial collagenase or autologous blood.2. L-DOPA combined with or without D1 or D2 receptor antagonist (SCH23390 or Spiperone) and two D2 receptor agonist Quinpirole and Ropinirole were administered by daily intraperitoneal injection. Drd2 and CRYAB in vivo RNAi were performed 24 hours before ICH insult; the Gβγ subunit inhibitor(Gallein) and ERK1/2 inhibitor (U0126) was delivered with Quinpirole. Post-assessment included neurological function tests, brain edema, Evans blue extravasation, zymography assay, western blot, and immunohistochemistry assay.3. DRD2 agonists Quinpirole treatment(5 mg/kg) were administrated by daily intraperitoneal injection starting at 1 hour post-ICH. DRD2 and CRYAB in vivo knockdown was performed 48 hour before ICH insult. Behavioral deficits and brain water content, western blots, immunofluorescence staining, co-immunoprecipitation assay and proteome cytokine array were evaluated.Results1. The striatal DA system is significantly increased after experimental ICH, and DRD2 was Present in Astrocytes and Endothelial Cells.2. L-dopa preserved BBB integrity through D2 receptor, not D1 receptor.3. DRD2 in knockout Aggravated Neurobehavioral Deficits and BBB Permeability Post-ICH4. DRD2 Agonism Improved Neurobehavioral Functions, Reduced Brain Edema and Preserved BBB Integrity both at 24 and 72 hours following ICH.5. Drd2 tightly controls alphaB-crystallin Expression and Drd2 Activation Enhanced the Phosphorylation of alphaB-crystallin on serine-45 Post-ICH.6. CRYAB in vivo knockdown abolished the Protective Effects of DRD2 Activation on BBB Integrity at 24 hours Following ICH.7. The Gβγ/ERK1/2/pSer45-alphaB Crystallin signaling cascades is involved in the protection of DRD2.8. Endogenous DRD2 and CRYAB expression were increased after ICH.9. DRD2 knockdown aggravated the neurobehavioral deficits and the pronounced cytokines expression.10.DRD2 activation by Quinpirole and Ropinirole ameliorated neurological outcome, brain edema, IL-1β and MCP-1 expression, as well as microglia/macrophages activation in the perihematomal region.11. CRYAB in vivo Knockdown Abolished the Protective Effect of Quinpirole on Neuroinflammation Inhibition Following ICH.12. Quinpirole enhanced cytoplasmic binding activity between CRYAB and NF-κB, and decreased nuclear NF-κB expression.13. Similar therapeutic benefits were observed using autologous blood injection model and intranasal delivery of Quinpirole.Conclusion1. Our findings uncover a distinct role of endogenous Dopamine-D2 receptor system in maintaining BBB integrity via Gβγ/ERK1/2/pSer45-CRYAB signaling cascades following ICH. Importantly, this study also indicates that DA or D2 receptor-specific agonists can be an attractive choice to improve the efficacy of therapies for ICH and other diseases with abnormal cerebral microvasculature.2. Dopamine D2 receptor elevation occurred after ICH in brain tissues, and exogenous DRD2 agonists inhibited neuroinflammation and improved neurological outcome, which was probably mediated by aB-crystallin and enhanced cytoplasmic binding activity with NF-κB. This novel observation indicated an endogenous physiology role of glial dopamine D2 receptor on microglia innate immunity and neuroinflammation following ICH.3. Blood brain barrier plays a vital role in maintaining the hemostasis of central nervous system. Endogenous Dopamine-D2 receptor system play a critical role in maintaining BBB integrity and attenuate neouro-inflammation after ICH. DRD2 agonists strategies could significantly alleviate the early brain injury after ICH, and eventually improve the outcomes of ICH patients. In conclusion, the clinical translational researches based on dopamine D2 recptor system may provide a new way to treat the ICH patients.