Long-term impairment of neurovascular coupling following experimental subarachnoid hemorrhage

Abstract

CO₂-reactivity and neurovascular coupling are sequentially lost within the first 24 h after subarachnoid hemorrhage (SAH). Whether and when these impairments recover is not known. Therefore, we investigated the reactivity of pial and intraparenchymal vessels by in vivo two-photon microscopy one month after experimental SAH. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. One month later, cerebral blood flow following CO₂-challenge and forepaw stimulation was assessed by laser Doppler fluxmetry. Diameters of pial and intraparenchymal arterioles were quantified by in vivo two-photon microscopy. One month after SAH, pial and parenchymal vessels dilated in response to CO₂. Neurovascular coupling was almost completely absent after SAH: vessel diameter did not change upon forepaw stimulation compared to a 20% increase in sham-operated mice. The current results demonstrate that neurovascular function differentially recovers after SAH: while CO₂-reactivity normalizes within one month after SAH, neurovascular coupling is still absent. These findings show an acute and persistent loss of neurovascular coupling after SAH that may serve as a link between early brain injury and delayed cerebral ischemia, two distinct pathophysiological phenomena after SAH that were so far believed not to be directly related.

Keywords: Subarachnoid hemorrhage; in vivo; mice; neurovascular coupling; two-photon microscopy.

Figure 1.

Experimental design and SAH induction. (a) Schematic representation of the experimental design for both SAH and sham-operated animals. Neurovascular coupling and endothelium-dependent response were assessed one month after the surgical procedure. (b) Cerebral blood flow and (c) intracranial pressure recordings starting 5 min before SAH or sham-operated induction and continuing for subsequent 15 min. A sudden decrease in CBF and sharp increase in ICP confirms SAH inductions. Mean ± SD; n = 10 in sham and n = 9 in SAH group.

Figure 2.

Parenchymal arterioles respond normally to hypercapnia one month after SAH. (a) Representative two-photon microscopy images of pial (top panels) and parenchymal (bottom panels) arterioles of sham and SAH mice before and during 10% CO₂ inhalation. (b) Pial and (c) parenchymal artery diameter quantification during hypercapnia in mice subjected to sham surgery (white symbols) or SAH (grey symbols). Arteriolar dilation in response to hypercapnia relative to baseline was verified in pial and parenchymal arterioles in sham-operated mice. Parenchymal arterioles in SAH mice also dilated in response to hypercapnia. Mean ± SD. 12 to 24 arterioles in (b) and 12 to 15 arterioles in (c) in n = 7 to 8 mice per group.

Figure 3.

Two-photon imaging of arteriolar diameter in response to discrete stimulation one month after SAH. (a) Representative two-photon microscopy images of parenchymal arterioles in sham (top panels) and SAH mice (bottom panels) at baseline and after stimulation. (b) Arteriolar dilation in response to discrete stimulation was verified in sham-operated mice, but found to be impaired after SAH; 17 to 30 arterioles in n = 7 to 8 mice per group.

Figure 4.

Two-photon imaging of arteriolar diameter in response to a continuous stimulation one month after SAH. (a) Representative two-photon microscopy images of parenchymal arterioles in sham operated (top panels) and SAH mice (bottom panels) at baseline and after sensory stimulation. (b) Arteriolar dilation in response to continuous stimulation was verified in sham-operated mice, but found to be attenuated in SAH mice. SAH mice showed a trend to dilation relative to baseline. Mean ± SD; 10 to 13 arterioles in 7 to 8 mice per group.

Figure 5.

Scatterplot of vessel dilation vs. baseline diameter. Vessel dilation at every time point across stimulation modalities plotted against baseline vessel diameter for both sham- and SAH-operated mice.

Figure 6.

Changes in CBF response to neurovascular coupling one month after SAH. Continuous electrical stimulation at the forepaw leads to an increase in CBF in sham-operated mice (white symbols) of approximately 20% compared to baseline while in the SAH group (grey symbols) the response was lower but not significantly different. Mean ± SD; n = 7 to 8 per group.