Postischemic reperfusion causes smooth muscle calcium sensitization and vasoconstriction of parenchymal arterioles

Abstract

Background and purpose: Parenchymal arterioles (PAs) are high-resistance vessels in the brain that connect pial vessels to the microcirculation. We previously showed that PAs have increased vasoconstriction after ischemia and reperfusion that could increase perfusion deficit. Here, we investigated underlying mechanisms by which early postischemic reperfusion causes increased vasoconstriction of PAs.

Methods: Isolated and pressurized PAs from within the middle cerebral artery territory were studied in male Wistar rats that were either nonischemic control (n=34) or after exposure to transient middle cerebral artery occlusion (MCAO) by filament occlusion for 2 hours with 30 minutes of reperfusion (MCAO; n=38). The relationships among pressure-induced tone, smooth muscle calcium (using Fura 2), and membrane potential were determined. Sensitivity of the contractile apparatus to calcium was measured in permeabilized arterioles using Staphylococcus aureus α-toxin. Reactivity to inhibition of transient receptor potential melastanin receptor type 4 (9-phenanthrol), Rho kinase (Y27632), and protein kinase C (Gö6976) was also measured.

Results: After MCAO, PAs had increased myogenic tone compared with controls (47±2% versus 35±2% at 40 mm Hg; P<0.01), without an increase in smooth muscle calcium (177±21 versus 201±16 nmol/L; P>0.05) or membrane depolarization (-38±4 versus -36±1 mV; P>0.05). In α-toxin-permeabilized vessels, MCAO caused increased sensitivity of the contractile apparatus to calcium. MCAO did not affect dilation to transient receptor potential melastanin receptor type 4 or protein kinase C inhibition but diminished dilation to Rho kinase inhibition.

Conclusions: The increased vasoconstriction of PAs during early postischemic reperfusion seems to be due to calcium sensitization of smooth muscle and could contribute to infarct expansion and limit neuroprotective agents from reaching their target tissue.

Keywords: arterioles; brain infarction; calcium; no-reflow phenomenon; smooth muscle.

Figure 1. Effect of early post-ischemic reperfusion on myogenic tone and smooth muscle calcium in PAs

(A) Arteriolar diameter in response to pressure. Arterioles displayed myogenic vasoconstriction and were smaller actively after MCAO. (B) Percent tone at 40 mm Hg. Arterioles had increased tone that contributed to their smaller diameter. Passive diameters were not different between groups (not shown). (C) Smooth muscle calcium, measured using Fura 2 at 40 mm Hg, was not different in arterioles after MCAO. (D) Relationship between percent tone and smooth muscle calcium. Arterioles were more sensitive to calcium after MCAO. *p<0.05 vs Control; **p<0.01 vs. Control.

Figure 2. PA smooth muscle membrane potential and reactivity to TRPM4 inhibition after early post-ischemic reperfusion

(A) Percent tone and smooth muscle membrane potential (V_m) of pressurized PAs at 40 mm Hg. There was no difference in membrane potential of arterioles after MCAO despite increased myogenic tone. (B) Dilation in response to TRPM4 inhibition with 9-phenanthrol. TRPM4 inhibition caused equivalent dilations in Control and MCAO arterioles. *p<0.05 vs. Control.

Figure 3. Effect of early post-ischemic reperfusion on calcium sensitivity of smooth muscle in PAs

Arterioles permeabilized with S. aureus α-toxin were more sensitive to calcium after MCAO and had increased tone (A) and sensitivity to calcium (B). *p<0.05 vs. Control.

Figure 4. Reactivity of PAs to ROCK and PKC inhibition after post-ischemic reperfusion

Inhibition of ROCK with Y27632 caused dilation of both types of arterioles (A) that were less sensitive after MCAO. Inhibition of PKC with Gö6976 had little effect on either group of arterioles (B). *p<0.05 vs Control; **p<0.01 vs. Control.