| Objective(s):Cerebral blood flow(CBF)reduction after subarachnoid hemorrhage(SAH)is the pathological basis for poor prognosis of SAH.Transient receptor potential melastatin-4(TRPM4)plays an essential role in cerebral artery myogenic tone maintenance and CBF regulation under physiological conditions.However,its role in CBF reduction after SAH is unclear.In this study we aim to test whether TRPM4 contributes to CBF reduction after SAH in vivo and clarify the involved mechanism.Methods:120 clean-grade SD rats were randomly divided into the in vitro experimental group(48 rats)and the in vivo experimental group(72 rats).The in vitro experimental group was divided into sham group(24 rats)and SAH group(24 rats),and then sub-grouped for 3 time points(day 3,day 5 and day 7)(8 rats/group);the in vivo experimental group was divided into sham group(18 rats),SAH group(18 rats),sham+9-Phe(9-Phe,TRPM4 specific blocker)group(18 rats)and SAH+9-Phe group(18 rats),then sub-grouped for 3 time points(day 3,day 5 and day 7)(8 rats/group).Rat SAH model was established by stereotaxic injection of 0.2mL autologous nonheparinized arterial blood at the suprasellar cistern.The physiological saline was continuously pumped into the rat lateral ventricle of the SAH group and the sham group by a programmed subcutaneous pump,the concentration of 0.03 mmol/L of 9-Phe was continuously pumping into the rat lateral ventricle of sham+9-Phe group and the SAH+9-Phe group.Day 3,day 5,and day 7 after the establishment of the SAH model,isolation of the in vitro experimental group rat cerebral artery myocytes.TRPM4 expression and translocation in cerebral artery myocytes were detected by immunofluorescence and immunoblotting.TRPM4 macroscopic currents in cerebral artery myocytes were determined by whole-cell patch-clamp.Myogenic tone of cerebral arteries was studied by pressurized myography.Cortical and global CBF of the in vivo experimental group were measured via laser-Doppler flowmetry and fluorescent microspheres,respectively.Results:TRPM4 was expressed in cerebral artery myocytes from both SAH and sham groups.At day 3,day 5,and day 7 after SAH,total TRPM4 protein expression in cerebral artery myocytes was higher in SAH group compared to sham group(31.1± 2.3%versus 21.2 ± 1.4%,P<0.01;32.9 ± 4.5%versus 23.2 ± 2.7%,P<0.01;32.1 ± 2.5%versus 23.1 ± 1.9%,P<0.01).TRPM4 membrane translocation in cerebral artery myocytes was also greater in SAH group relative to sham group(61.4± 3.0%versus 31.4 ± 2.5%,P<0.01;64.8 ± 4.0%versus 44.3 ± 2.4%,P<0.01;63.9± 3.6%versus 43.2 ± 2.6%,<0.01).TRPM4 macroscopic current density recorded with Vm ranging from-80 to+50 mV in cerebral artery myocytes was increased in SAH group compared to sham group(P<0.01).In the presence of 9-Phe,macroscopic currents in both SAH and sham groups were almost completely inhibited.The myogenic tone of SAH group was higher than sham group(46.9 ± 1.8%versus 40.4 ± 1.5%,P<0.05;46.4±1.6%versus 40.1±1.8%,P<0.05;44.4±1.2 versus 40.1 ± 1.9%,P<0.05).After perfusion of 9-Phe,the myogenic tone of SAH group(15.2 ± 1.6 versus 46.9± 1.8,P<0.01;16.0 ± 1.2 versus 46.4 ± 1.6,P<0.01;16.2 ± 1.8 versus 44.4±1.2,P<0.01)and sham group decreased significantly(15.8±1.3%versus 40.4 ± 1.5%,P<0.01;16.0 ± 1.2%versus 40.1 ±1.8%,P<0.05;16.6 ± 1.5%versus 40.1 ±1.9%,<0.01).The decrease of myogenic tone in SAH group was significantly higher than sham group(31.7±1.8%versus 24.6±1.5,P<0.01;30.5±1.6%versus 24.1±1.8%,P<0.01;28.2±1.2%versus 23.5±1.9%,P<0.01).At day 3,day 5,and day 7 after SAH,cortical CBF in SAH group were lower than sham group(100.6 ± 16.0 PU versus 209.1±15.4 PU,P<0.01;90.5±15.8 PU versus 213.1 ± 16.1 PU,P<0.01;98.4 ± 16.7 PU versus 210.1 ± 15.9 PU,P<0.01).In the presence of 9-Phe,cortical CBF of the SAH+9-Phe group were greater than SAH group(235.9±15.7 PU versus 100.6 ±16.0 PU,P<0.01;239.9 ± 15.7 PU versus 90.5 ± 15.8 PU,P<0.01;231.5 ±15.0 PU versus 98.4 ± 16.7 PU,P<0.01),cortical CBF of the sham+9-Phe group were greater than sham group(259.9±13.7 PU versus 209.1±15.4 PU,P<0.01;263.2±12.7 PU versus 213.1±16.1 PU,P<0.01;260.9 ± 13.0 PU versus 210.1±16.0 PU,P<0.01);Global CBF in the SAH group were lower than sham group(73.3±5.4 ml·100g-1·min-1 versus 125.2±12.1 ml·100g-1·min-1,P<0.01;61.2±6.3 ml·100g-l·min-1 versus 130.7±8.5 ml·100g-1·min-1,P<0.01;70.8± 6.0 ml·100g-1·min-1 versus 127.1±8.0 ml·100g-1·min-1,P<0.01).In the presence of 9-Phe,global CBF of the SAH+9-Phe group were greater than SAH group(151.5±10.0 ml·100g-1·min-1 versus 73.3 ± 5.4 ml·100g-1·min-1,P<0.01;157.3±9.8 ml·100g-1·min-1 versus 67.2 ± 6.3 ml·100g·1-·min-1,P<0.01;153.2 ± 5.4 ml·100g-1·min-1 versus 70.8 ± 6.0 ml·100g-1.min-1,P<0.01),global CBF of the sham+9-Phe group were greater than sham group(166.7±9.1 ml·100g-1·min-1 versus 125.2 ± 12.1 ml·100g-1·min-1,P<0.01;168.2 ± 10.9 ml·100g-1·min-1 versus 130.7±8.5 ml·100g-1·min-1,P<0.01;169.0±8.2 ml·100g-1·min-1 versus 127.1±8.0 ml·100g-1·min-1,P<0.01).Conclusion(s):SAH has an inducing effect on TRPM4 activity,while increased TRPM4 activity contributes to CBF reduction after SAH. |
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