Microthrombi Correlates With Infarction and Delayed Neurological Deficits After Subarachnoid Hemorrhage in Mice

Abstract

Background and purpose: Delayed neurological deficits are a devastating consequence of subarachnoid hemorrhage (SAH), which affects about 30% of surviving patients. Although a very serious concern, delayed deficits are understudied in experimental SAH models; it is not known whether rodents recapitulate the delayed clinical decline seen in SAH patients. We hypothesized that mice with SAH develop delayed functional deficits and that microthrombi and infarction correlate with delayed decline.

Methods: Adult C57BL/6J mice of both sexes were subjected to endovascular perforation to induce SAH. Mice were allowed to survive for up to 1 week post-ictus and behavioral performance was assessed daily. Postmortem microthrombi, large artery diameters (to assess vasospasm), and infarct volume were measured. These measures were analyzed for differences between SAH mice that developed delayed deficits and SAH mice that did not get delayed deficits. Correlation analyses were performed to identify which measures correlated with delayed neurological deficits, sex, and infarction.

Results: Twenty-three percent of males and 47% of females developed delayed deficits 3 to 6 days post-SAH. Female mice subjected to SAH had a significantly higher incidence of delayed deficits than male mice with SAH. Mice that developed delayed deficits had significantly more microthrombi and larger infarct volumes than SAH mice that did not get delayed deficits. Microthrombi positively correlated with infarct volume, and both microthrombi and infarction correlated with delayed functional deficits. Vasospasm did not correlate with either infarction delayed functional deficits.

Conclusions: We discovered that delayed functional deficits occur in mice following SAH. Sex differences were seen in the prevalence of delayed deficits. The mechanism by which microthrombi cause delayed deficits may be via formation of infarcts.

Keywords: adult; animals; humans; mice; subarachnoid hemorrhage.

Figure 1

Neurobehavioral Performance by Males (A) and Females (B). Individual performance is shown for SAH mice with DND (males n=6, females n=9). SAH mice without DND are plotted as mean and SD (males n=18-20/time-point, females n=10/time-point). Sham (n=8) performance is 23.5 (SD=0.70) for males and 23.8 (SD=0.30) for females. Time to delayed deficits after SAH in males (C) (with DND n=6, without DND n=20, p<0.0001) and females (D) (with DND n=9, without DND n=10, p=0.0413).

Figure 2

Microthrombi and Infarction Correlate with DND. A. Representative Images of Microthrombi (arrowheads) in MSB Staining. Bar=50 μm. B. Microthrombi Counts. C. Representative Images of Infarction after SAH. D. Infarct Volumes. Microthrombi counts and infarct volumes were assessed on Day 7 for Sham and SAH without DND; for SAH mice with DND, these measures were assessed on Day 5.7 (SD=1.37) for males and Day 6 (SD=1.05) for females. Sham n=8/sex, SAH without DND n=10/sex, SAH with DND n=6 males and n=9 females. * p<0.05 vs Sham, # p<0.05 vs SAH without DND.

Figure 3

Large Artery Vasospasm Does Not Correlate with DND. A. Representative Images of Brain Vasculature. Bar=0.5 mm. B. Vessel Diameters from Vessel Painted Brains. Sham n=5/sex, SAH without DND n=5/sex, SAH with DND n=4 males and n=5 females. C. Vessel Diameters from H&E Slices. Sham n=8/sex, SAH without DND=10/sex, SAH with DND n=6 males and n=9 females. Vessel diameters were assessed on Day 7 for Sham and SAH without DND; for SAH mice with DND, it was measured on Day Day 5.7 (SD=1.37) for males and Day 6 (SD=1.05) for females. * p<0.05 vs Sham.