Experimental subarachnoid hemorrhage causes early and long-lasting microarterial constriction and microthrombosis: an in-vivo microscopy study

Abstract

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) is characterized by a severe, cerebral perfusion pressure (CPP)-independent reduction in cerebral blood flow suggesting alterations on the level of cerebral microvessels. Therefore, we aimed to use in-vivo imaging to investigate the cerebral microcirculation after experimental SAH. Subarachnoid hemorrhage was induced in C57/BL6 mice by endovascular perforation. Pial arterioles and venules (10 to 80 μm diameter) were examined using in-vivo fluorescence microscopy, 3, 6, and 72 hours after SAH. Venular diameter or flow was not affected by SAH, while >70% of arterioles constricted by 22% to 33% up to 3 days after hemorrhage (P<0.05 versus sham). The smaller the investigated arterioles, the more pronounced the constriction (r(2)=0.92, P<0.04). Approximately 30% of constricted arterioles were occluded by microthrombi and the frequency of arteriolar microthrombosis correlated with the degree of constriction (r(2)=0.93, P<0.03). The current study demonstrates that SAH induces microarterial constrictions and microthrombosis in vivo. These findings may explain the early CPP-independent decrease in cerebral blood flow after SAH and may therefore serve as novel targets for the treatment of early perfusion deficits after SAH.

Figure 1

(A) Schematic drawing of the location of the cranial window used for intravital microscopy. (B) Schematic drawing of the experimental protocol used in the present study. Anesthesia was discontinued after subarachnoid hemorrhage (SAH) and mice were reanesthetized at different time points after SAH for intravital fluorescence microscopy.

Figure 2

(A) Microscopic overview (magnification × 100) of the middle cerebral artery (MCA) territory 3 hours after subarachnoid hemorrhage (SAH). A black halo surrounding some arterioles indicates the presence of subarachnoid blood (white arrows). (B) Pearl string-like microarteriolar spasm (solid arrow) in the cerebral microcirculation 3 hours after SAH (magnification × 250). A microthrombus occludes the proximal side of the spasm (upper dotted arrow). (C) The proportion of spastic vessels expressed as percent of all vessel segments of the ipsilateral MCA at different time points after SAH (n=6 to 8 per each time point).

Figure 3

(A) Change of vessel diameter in sham-operated animals and at different time points after subarachnoid hemorrhage (SAH) (n=6 to 8 per animal per time point). Histograms showing the distribution of the degree of microvasospasms (B) 3 hours, (C) 6 hours, and (D) 72 hours after SAH.

Figure 4

Severity of microarteriolar spasm depending on the baseline diameter of the affected vessels (A) 3 hours, (B) 6 hours, and (C) 72 hours after subarachnoid hemorrhage (SAH). The smallest arterioles are most severely affected by microvasospasm.

Figure 5

(A) Example of a microvessel carrying a large thrombus (solid arrows) between two microvasospasms (dotted arrows). (B) Percentage of nonspastic (open bar) and spastic (striped bar) vessels carrying microthrombi 3 hours after subarachnoid hemorrhage (SAH). (C) Correlation between the number of microthrombi and microvasospasm severity. The more pronounced microvasospasms were the more microthrombi could be found in the corresponding vessel segments.