Maternal Aspirin Treatment Improves Ischemic Stroke Outcome in Adult Male Offspring From Experimental Preeclamptic Dams

Abstract

Background: Preeclampsia, a serious hypertensive disorder of pregnancy, is associated with increased long-term risk of cardiovascular disease in adult offspring, particularly stroke. Although low-dose aspirin (LDA) is used prophylactically to prevent preeclampsia, its impact on offspring is unclear. This study investigated the effect of maternal LDA treatment during experimental preeclampsia (ePE) on adult first-generation (F1) offspring, including stroke outcome.

Methods: ePE was induced in pregnant Sprague-Dawley rats via a high-cholesterol diet starting on gestational day 7 and treated with LDA (1.5 mg/kg) or vehicle. Offspring were weaned and fed standard chow until transient middle cerebral artery occlusion at 12 to 18 weeks (3-hour ischemia and 1-hour reperfusion). Fetal and juvenile weights were taken at gestational day 20 and from weeks 10 to 13. Infarct and edema were quantified using 2,3,5-triphenyltetrazolium chloride staining. Multisite laser Doppler was used to measure cerebral hemodynamics, including cerebral blood flow autoregulation and collateral flow. Circulating proinflammatory and anti-inflammatory factors were measured via multiplex immunoassay.

Results: Male offspring from ePE dams (ePE-F1) had larger infarction and edema versus male offspring from normal pregnant dams (NormP-F1, 48%±6 versus 11%±4; P<0.01) and all female offspring. Maternal treatment with LDA was protective of male offspring (ePE+Asp-F1) that had reduced infarct and edema. Increased infarction in ePE-F1 males was associated with greater collateral perfusion deficit and elevated levels of TNF-α (tumor necrosis factor-alpha) and IL (interleukin)-1β that were prevented by maternal LDA treatment. There were no differences in infarct, edema, or perfusion deficit in female offspring.

Conclusions: Prenatal exposure to ePE worsened stroke severity and inflammation in male but not female offspring, which was largely mitigated by maternal LDA treatment, potentially due to an improved intrauterine environment. These findings highlight a sex-specific impact of prenatal preeclampsia exposure on long-term cerebrovascular health and suggest that maternal LDA may confer long-lasting protection to the offspring in addition to the mother.

Keywords: aspirin; cerebrovascular diseases; ischemic stroke; platelet activation; pre-eclampsia.

Figure 1:

Experimental design and offspring weights. A) Experimental design and timeline illustrating experimental groups and treatment time course. B) Graph showing fetal weights taken at e20. There were no differences between groups at this age. C) Graph showing female offspring weights taken twice each week between 10-13 weeks of age. ePE-F1 female rats were significantly smaller than both NormP-F1 and ePE+Asp-F1 female rats (row and column effect, *p<0.05, **p<0.001 by two-way repeated measures mixed-effect model with a Tukey’s posthoc test for multiple comparisons). D) Graph showing male offspring weights were not different between groups from 10-13 weeks of age.

Figure 2:

Effect of ePE and maternal LDA treatment on stroke outcome of offspring. A) Graph showing percent infarct (edema corrected) in all groups of offspring. Infarction was larger in ePE-F1 males compared to NormP-F1 male and ePE-F1 female rats. ePE+Asp-F1 males had larger infarct than ePE+Asp-F1 female rats. B) Representative TTC stained coronal sections for all groups of male offspring. C) Graph showing ePE-F1 males had significantly more edema than NormP-F1 and ePE+Asp-F1 males and ePE-F1 females. **p<0.001, ****p<0.0001 by one-way ANOVA with a posthoc Tukey’s test for multiple comparison. D) Representative TTC stained coronal sections for all groups of female offspring.

Figure 3:

Change in CBF during tMCAO in all groups of offspring. A) Diagram depicting Doppler probe placements on the skull. Probe 1 was placed over the expected MCA core infarct territory and probe 2 was placed over the pial collateral territory to measure LMA flow. B) Graph showing the initial drop of CBF in the MCA and pial collateral territory immediately following MCA occlusion normalized to baseline. ePE-F1 males had a larger percent drop in pial collateral CBF compared to NormP-F1 males. ePE+Asp-F1 males also had a large drop in collateral CBF that trended to be statistically different from NormP-F1 males (p=0.067). In female offspring, the change in CBF in the MCA territory was similar between groups and not different than males. ePE-F1 rats had a greater drop in pial collateral CBF compared to NormP-F1 female rats that trended to be statistically significant (p=0.066). Maternal LDA treatment did not improve collateral CBF in ePE+Asp-F1 females. *p<0.05 by one-way ANOVA with a posthoc Tukey’s test for multiple comparisons. Percent change in C) MCA CBF and D) Collateral CBF in all male groups normalized to filament insertion. ePE-F1 males had worse MCA flow and poorer collateral recruitment than the other groups of male offspring. Percent change in E) MCA and F) collateral flow in all groups of female offspring over the first 30 minutes of ischemia. The change in MCA and collateral CBF was similar between all female offspring groups. *p<0.05 vs. NormP, ‡p<0.05 vs. ePE+asp-F1 by two-way repeated measures ANOVA with a posthoc Tukey’s test for multiple comparisons.

Figure 4:

Collateral CBFAR, change in pial collateral CBF to an acute increase in blood pressure in response to phenylephrine infusion. Collateral CBF exhibited a transient increase in response to elevated blood pressure during phenylephrine infusion in A) NormP-F1 males, B) NormP-F1 females, C) ePE-F1 males, and D) ePE-F1 female rats which returned to or near baseline levels indicating intact CBFAR in the expected peri-infarct region. E) eEP+Asp-F1 males had a prolonged increase in CBFAR over the course of phenylephrine infusion which remained elevated following the termination of phenylephrine infusion, indicating impaired CBFAR. F) Collateral CBF increased and remained elevated in ePE+Asp-F1 females, suggesting impaired CBFAR to a lesser degree than ePE+Asp-F1 males. On and Off denote the start and stop of the phenylephrine infusion period.

Figure 5:

Circulating inflammatory factor levels after tMCAO. A) Graph showing levels of TNF-α in all groups of animals. TNF-α was elevated in ePE-F1 compared to NormP-F1 male rats. There was no difference between female offspring groups. B) Graph showing levels of IL-1ß in all groups of animals. IL-1ß was increased in ePE-F1 males compared to NormP-F1 males and ePE-F1-females. There was no difference between female offspring. C) Graph showing ICAM-1 levels in all groups of animals. ICAM-1 was elevated in NormP-F1 male rats compared to females. There were no differences between female groups. D) Graph showing circulating levels of IFN-γ in all groups. IFN-γ levels were elevated in ePE+Asp-F1 female rats compared to ePE+Asp-F1 males. There were no differences between females or other groups of offspring. *p<0.05 by Kruskal-Wallis test with posthoc Dunn’s test for multiple comparisons for all comparisons.