The Effects Of Platelet-derived Growth Factor Receptor-β On Brain Injury After Intracerebral Hemorrhage And Its Molecular Mechanism

Part Ⅰ: The temporal expression of platelet-derived growth factor receptor-β and platelet-derived growth factor-BB after intracerebral hemorrhage in miceObjective: To investigate the temporal expression of platelet-derived growth factor receptor-β(PDGFR-β) and platelet-derived growth factor-BB(PDGF-BB) after intracerebral hemorrhage(ICH) model in mice.Methods: Forty-two mice were divided into 6 groups(Sham, and 3, 6, 12, 24, 72 hours after ICH). Seven mice in each group(n=7). Molecularly, the temporal expression of PDGF-BB and PDGFR-β was characterized by Western blot and immunofluorescence staining.Results: Western blot results indicated that PDGF-BB level was increased in the ipsilateral hemisphere 24 hours post ICH when compared to contralateral hemisphere(p<0.05) and sham mice(p<.05). PDGFR-β levels were increased(p<0.05) 6 hours post ICH and remained at the high levels until 24 hours(p<0.05). The double immunofluorescence staining revealed that PDGFR-β immunoreactivity was found on vascular smooth muscle cell(VSMC) around perihematoma area.Conclusions: The expression of PDGFR-β and PDGF-BB increased post ICH in mice and PDGFR-β expressed on the VSMC around perihematoma area. It point out that PDGFR-β maybe involved in the progress of brain injury and vascular smooth muscle cell phenotypic transformation after intracerebral hemorrhage in mice. Part II: Platelet-derived growth factor receptor-β inhibition can improve the brain injury after intracerebral hemorrhage in miceObjective: To investigate the Platelet-derived growth factor receptor-β small interfering RNA(PDGFR-β si RNA) and PDGFR-β inhibitor could improve the brain injury after intracerebral hemorrhage in mice using ICH model.Methods: One hundred and eight mice were divided into five groups including Sham(n=24),ICH(n=26),ICH + scramble si RNA(n=26), ICH + PDGFR-β si RNA(n=26), ICH+Gleevec(n=6). The PDGFR-β si RNAs mixture or scramble si RNA was administered intraventricularly 24 hours before ICH. A PDGFR-βantagonist, Gleevec, was administered(intraperitoneal injection) 1h following ICH. Samples for Western blot and immunostaining were collected 24 hours after ICH. Neurological deficits and brain edema were measured at 24 and 72 hours.Results: ICH mice had behavioral deficits compared to sham group in the Garcia test(p<.05). After PDGFR-β si RNA injection, the Garcia tests improved at both 24 and 72 hours compared to ICH or ICH+scramble si RNA mice(p<.05). Brain edema in the ipsilateral basal ganglia following PDGFR-β si RNA injection was also reduced at both 24 hours(p<0.05) and 72 hours(p<0.05) compared to ICH or ICH+scramble si RNA group. Western blot results showed that smooth non-muscle myosin IIB(SMemb),Intercellular adhesion molecule-1(ICAM-1) and myeloperoxidase(MPO) levels in the ipsilateral hemisphere significantly increased after ICH and were reduced after PDGFR-β si RNA injection when compared to ICH or ICH+scramble si RNA groups(p<.05). Representative pictures of immunostaining showed that SMemb and ICAM-1 immunoreactivity in VSMC in the perihematoma area 24 hours after ICH and PDGFR-β si RNA treatment reduced the number of MPO-positive cells in the perihematoma area compared to ICH or ICH+scramble si RNA mice.Conclusions: Platelet-derived growth factor receptor-β inhibition can improve the behavioral test, brain edema and improve the inflammation and vascular smooth muscle cell phenotypic transformation after intracerebral hemorrhage in mice. Showed that receptor-β inhibition could improve the brain injury after intracerebral hemorrhage in mice. Part Ⅲ: The molecular mechanism of platelet-derived growth factor receptor-β on regulating vascular smooth muscle cell phenotypic transformation and neuro-inflammation after intracerebral hemorrhage in miceObjective: To investigate the PDGFR-β activation regulate the vascular smooth muscle cell phenotypic transformation through the p38-MK2 pathway by using PDGFR-β si RNA,PDGFR-β inhibitor, recombinant PDGF-BB and mitogen-activated protein kinase-activated protein kinase 2(MAPKAPK2,MK2)inhibitor(KKKALNRQLGVAA).Methods: The samples of Sham(n=7),ICH(n=7),ICH + scramble si RNA(n=7), ICH + PDGFR-β si RNA(n=7) come from Part II. Another twelve mice were divided into two groups including PDGF-BB(n=6) and PDGF-BB + MK2 inhibitor(n=6). Recombinant PDGF-BB was injected into the right basal ganglia in na?ve mice with or without a MK2 inhibtor, KKKALNRQLGVAA. Samples for Western blot and immunostaining were collected 24 hours after ICH.Results: Western blot results showed that phosphorylated PDGFR-β levels were increased when compared to sham mice(p <0.05), whereas levels were attenuated by PDGFR-β si RNA injection compared to ICH and ICH+scramble si RNA group(p<.05). PDGFR-β si RNA injection also reduced the levels of phosphorylated p38 MAPK and phosphorylated MK2(p<.05) when compared to ICH or ICH+scramble si RNA groups. At 24 hours following recombinant PDGF-BB injection to the brain, ICAM-1 levels increased in the ipsilateral hemisphere compared to sham mice(p<.05). The level of ICAM-1 of PDGF-BB + MK2 inhibitor group decreased in the ipsilateral hemisphere compared to PDGF-BB group(p<.05). The double immunofluorescence staining showed that phosphorylated p38 and phosphorylated MK2 immunoreactivity were found in the VSMC.Conclusions: PDGFR-β activated by PDGF-BB and then activate the p38-MK2 pathway to regulate the vascular smooth muscle cell phenotypic transformation and inflammation after intracerebral hemorrhage in mice.