History of pre-eclampsia negatively impacts stroke severity postpartum in rats

Abstract

Background: Preeclampsia (PE) is a serious hypertensive disorder of pregnancy that has lifelong deleterious effects, including increased risk of stroke postpartum (PP). Here we determined if previous PE exacerbates ischemic injury in the PP period and investigated underlying mechanisms including oxidative stress and collateral perfusion.

Methods: Female Sprague-Dawley rats were studied at 4-9 months PP, after either a normal pregnancy (NormP-PP n = 7) or experimental PE (ePE) induced via high cholesterol diet during gestation (ePE-PP n = 9). Animals underwent transient middle cerebral artery occlusion (tMCAO) for 2 hours with 1 hour reperfusion. Dual-site laser Doppler flowmetry measured changes in cerebral blood flow (CBF) in the MCA and collateral territories. Ischemic injury was measured by 2,3,5-triphenyl tetrazolium chloride staining. Circulating 8-isoprostane, 3-nitrotyrosine (3-NT), and oxidized low-density lipoprotein (oxLDL) were measured by enzyme-linked immunosorbent assays. In separate groups of animals, NormP-PP (n = 10) and ePE-PP (n = 9) that were 3-4 months PP, isolated pial collateral vessels, leptomeningeal anastomoses (LMAs), and mesenteric arteries were studied using pressure myography.

Results: Previous ePE pregnancy worsened stroke outcome in the PP state, significantly increasing infarction in ePE-PP vs. NormP-PP animals (40.6 ± 7.6% vs. 13.7 ± 6.5%; p <0.01) and edema (5.1 ± 2.0% vs. 2.6 ± 0.4%; p < 0.01), despite comparable changes in CBF in both MCA and pial collateral territories during ischemia and reperfusion. When infarction was analyzed as a function of perfusion deficit, ePE-PP animals had greater sensitivity to ischemia. Pial collaterals had increased pressure-induced myogenic tone vs. NormP-PP rats. Percent tone at 80 mmHg for ePE-PP vs. NormP-PP was 15.5 ± 1.6% vs. 8.6 ± 1.9% (p <0.01). In addition, ePE-PP animals had significantly elevated circulating 8-isoprostane and 3-NT, but not oxLDL, after tMCAO (*p<0.05 and **p<0.01, respectively).

Conclusions: We found worsened stroke outcome after ePE pregnancy that was related to increased sensitivity to ischemia, increased pial collateral tone, and elevated levels of oxidative stress markers. Thus, the pathologic effects of ePE persisted PP and negatively impacted stroke outcome.

Keywords: collateral flow; ischemic stroke; oxidative stress; postpartum; preeclampsia.

Figure 1. Study design, infarction and edema after transient middle cerebral artery occlusion (tMCAO) in normal pregnancy - postpartum (NormP-PP) and experimental preeclampsia (ePE)-PP animals.

(A) Diagram showing the study design and stratification of groups to tMCAO vs. pressure myograph. (B) Percent infarction in NormP-PP (white bars) and ePE-PP (gray bars) animals. (C) Percent edema in NormP-PP (white bars) and ePE-PP (gray bars) animals. ePE-PP animals had significantly larger infarct and greater edema than NormP-PP animals. (D) Representative 2,3,5-triphenyl tetrazolium chloride -stained brain slices from both groups of animals. Infarct, white tissue on the image, was seen in the ipsilateral hemisphere of both groups and was greater in ePE-PP animals. *p <0.05 by unpaired Student’s t-test.

Figure 2. Percent change in cerebral blood flow (CBF) in middle cerebral artery (MCA) and pial collateral territories during transient MCA occlusion (tMCAO).

Both experimental preeclampsia postpartum (ePE-PP) and normal pregnancy postpartum (NormP-PP) groups experienced similar perfusion deficit in both MCA and pial collateral territories during ischemia and reperfusion. (A) Schematic showing the location of probe 1 measuring changes in MCA flow (+4 mm right of midline and −2 mm posterior to Bregma) and probe 2 measuring changes in pial collateral blood flow (+3 mm right of midline and +2 mm anterior to Bregma). (B) Graph showing the initial % decrease in CBF in response to filament occlusion comparing MCA and pial collateral vascular territories. The decrease in CBF was not different between vascular territories in response to filament insertion, nor was there any difference in CBF changes between groups. (C) and (D) graphs showing the percent change in MCA territory CBF and pial collateral CBF calculated from pre-stroke baseline over time, respectively. There were no significant differences between NormP-PP and ePE-PP animals. ACA, anterior cerebral artery.

Figure 3. Flow response of primary (circle of Willis) and secondary (pial collateral) collaterals prior to and during transient middle cerebral artery occlusion (tMCAO).

(A) Representative tracing of MCA cerebral blood flow (CBF) (top red line), pial collateral CBF (middle blue line), and blood pressure (bottom black line). Shown are the changes during primary collateral recruitment after common carotid artery (CCA) occlusion and initial filament insertion. (B) Graph showing the average changes in MCA CBF in normal pregnancy - postpartum (NormP-PP) (open circles) and experimental preeclampsia (ePE)-PP (closed circles). MCA CBF decreased in response to ligating the CCA, that recovered due to collateral flow. (C) Graph showing the time to reach maximum recovery of collateral flow in each group after CCA ligation. There was a trend for ePE-PP animals to have slower recovery of CBF after ligation. (D) The % change in pial collateral blood flow every minute for the first 20 minutes of ischemia in both NormP-PP and ePE-PP. Pial collateral flow increased in response to filament insertion and remained elevated. There was no difference between groups in pial collateral recruitment during tMCAO. The insert shows the area under the curve for the first 3 minutes that was lower in ePE-PP vs. NormP-PP. BP, blood pressure.

Figure 4. Relationship between infarction and perfusion deficit in middle cerebral artery (MCA) and pial collateral territories.

(A) Graph showing correlation between infarction and MCA perfusion deficit over the first 20 minutes of ischemia. In both groups of animals, there was a positive linear correlation between infarction and perfusion deficit, demonstrating that the greater ischemia, the greater infarction. Experimental preeclampsia - postpartum (ePE-PP) animals had a leftward shift and increased slope suggesting these animals had worse ischemic tolerance. (B) Graph showing a significant linear correlation between infarction and leptomeningeal anastomose (LMA) perfusion deficit over the first 20 minutes of ischemia. There was also a significant positive correlation between infarction and LMA perfusion deficit in both groups of animals, demonstrating that the worse LMA flow, the greater the infarction. Compared to normal pregnancy-postpartum (NormP-PP), ePE-PP animals had a leftward shift in the correlation curve demonstrating higher sensitivity of infarction to pial collateral perfusion deficit.

Figure 5. Plasma concentration of markers of oxidative stress.

Graphs showing plasma levels of (A) 8-isoprostane, (B) 3-nitrotyrosine (3-NT), and (C) oxidized low-density lipoprotein (oxLDL) in both groups of animals. Experimental preeclampsia - postpartum (ePE-PP) animals had significantly higher plasma concentration of 8-isoprostane and 3NT (*p <0.05, **p <0.01 by unpaired Student’s t-test). There was no significant difference in plasma concentration of oxLDL between groups. NormP, normal pregnancy.

Figure 6. Active and passive lumen diameters and percent myogenic tone of leptomeningeal anastomoses (LMAs).

(A) Lumen diameters as a function of intravascular pressure in LMAs from normal pregnancy - postpartum (NormP-PP) dams. Both the active (open circles) and passive (open squares) states are shown for comparison. There was no difference in active and passive diameters in LMAs from NormP-PP animals. (B) Lumen diameters as a function of intravascular pressure in LMAs from experimental preeclampsia-postpartum (ePE-PP) dams. Both the active (closed circles) and passive (closed squares) states are shown for comparison. Active diameters of LMAs from ePE-PP animals were significantly smaller than passive diameters, demonstrating these vessels were vasoconstricted. **p <0.01 by multiple Mann-Whitney tests. (C) Percent tone of LMAs from NormP-PP and ePE-PP animals. ePE-PP animals demonstrated significantly higher myogenic tone than NormP-PP animals beginning at 80 mmHg and trended toward significance beginning at 60 mmHg. *p <0.05 by unpaired Mann-Whitney test. (D) Comparison of active diameters of LMAs between ePE-PP and NormP-PP. Vessels from ePE-PP animals were nonsignificantly smaller, at all pressures, due to the increased myogenic tone.