Prenatal Sildenafil treatment prevents postpartum neuroinflammation in the Dahl‐SS/Jr model of superimposed preeclampsia

Abstract

Superimposed preeclampsia is diagnosed in women with chronic hypertension who develop preeclampsia (proteinuria or end organ symptoms) after the 20th week of gestation. Superimposed preeclampsia can be detrimental to both the mother and fetus. Pregnant Dahl-SS/Jr rats display chronic hypertension and kidney dysfunction and exhibit many clinical findings of preeclampsia patients including hypertension, proteinuria, placental hypoxia, increased sFlt-1 & TNF-alpha, and fetal growth restriction. Sildenafil, a phosphodiesterase-5 inhibitor, has been studied as a potential therapeutic in preeclampsia patients, and we have shown that sildenafil treatment in the Dahl-SS/Jr improves maternal and fetal outcomes. Thus, we tested the hypothesis that the Dahl-SS/Jr rat model of superimposed preeclampsia will have increased neuroinflammation, evidenced by increased microglia density and frequency of activated microglia in the postpartum period, which will be attenuated by sildenafil treatment. Dahl-SS/Jr virgins (DV, n=8), prior pregnant (DP, n=9), and prior pregnant treated (DS, n=5) rats were used. DS rats received 50mg/kg/day of sildenafil from gestational day (GD)10 until delivery. DP and DS rats were mated again at 4.5 months old for a second pregnancy, receiving sildenafil (50mg/kg/day) as before. At 3.5 months postpartum, brains were harvested and processed for immunofluorescence staining. Anterior brain sections were cut into 20 µm thick sections and stained for microglia using ionized calcium-binding adapter molecule 1 (Iba-1). Z-stacks (0.5μm steps) were obtained using a confocal microscope, and microglia density, soma size, and morphology in the hippocampus and cortex were analyzed using Image J. Microglia morphology was quantified as follows: Type 1 microglia had small cell bodies and long, thin (ramified) processes. Type 2 microglia included those with asymmetrical cell body and long processes, Type 3 had rounded cell bodies and shorter, thicker processes, and Type 4 included those with round, enlarged cell bodies and short to no visible processes. In the hippocampus, DS rats had smaller microglia soma [DV: 65±2, DS: 58±5, DP: 68±2µm2; p= 0.06], with no difference in microglia density. Prenatal Sildenafil prevented hippocampal microglia activation with decreased type 3 activated microglia [DS: 85±4, DP: 95±2, DV: 86±4%, p= 0.01] as well as increased surveying type 2 microglia [DS: 13±4, DP: 5±2, DV: 12±4%, p= 0.01]. No significant microglia morphology change was found in the cortex. Our results support the hypothesis that superimposed preeclampsia induces postpartum hippocampal microglia activation which is prevented by prenatal sildenafil treatment. Ongoing and future studies will determine whether microglia activation occurs during pregnancy and whether postpartum neuroinflammation is associated with cognitive impairment.