Placental ischemia (PI), a prenatal stressor, affects ∼1 in 10 human pregnancies worldwide and is associated with several pregnancy complications such as preeclampsia, placental abruption, and intrauterine growth restriction (IUGR). Both human and animal IUGR fetuses have an increased risk of developing hypertension (HTN) in adulthood, with males having a higher risk. Furthermore, multiple studies suggest that changes in brain function and molecular markers may contribute to HTN development. However, the alterations in brain mitochondrial dysfunction (MtDys), oxidative stress (OS), and epigenetic changes (miRNAs) in forebrain and midbrain collectively have not been investigated. Thus, we hypothesize that the sex difference in high blood pressure (HBP) is due to changes in miRNAs, brain MtDys, and increased OS in IUGR males (M) but not IUGR females (F). To test this hypothesis, IUGR and control (CON) M and F Sprague Dawley rats were evaluated at 16-18 wk (adulthood). IUGR adults were generated from PI dams, and CON adults from normal pregnant dams. Results identified 11 differentially expressed miRNAs in IUGR versus CON, with let-7d-3p miRNA being upregulated in IUGR M. IUGR M also displayed HBP, MtDys [decreased adenosine triphosphate (ATP)], and OS (∼50% increase in hydrogen peroxide). Conversely, mitochondrial G protein elongation factor (GFM-1), a protein regulated by let-7d-3p, and electron transport chain (ETC) proteins were increased with no changes in ATP production in IUGR F. In summary, our data suggest that increases in let-7d-3p will inhibit the compensatory increase in GFM-1 and ETC proteins needed to prevent HBP and cerebral OS in IUGR M. However, unchanged let-7d-3p may increase GFM-1 and ETC proteins in IUGR F to inhibit brain MtDys, OS, and HBP. Findings from this study provide insights into the mechanisms linking epigenetic changes to brain MtDys and OS along with HTN in adults born IUGR.NEW & NOTEWORTHY Adult IUGR male rodent offspring exposed to placental ischemia in utero have elevated mean arterial blood pressure with increased brain miRNA let-7d-3p expression, mitochondrial dysfunction, and oxidative stress, while adult IUGR females do not. Moreover, our results suggest that brain epigenetic changes may contribute to mitochondrial dysfunction and oxidative stress, eventually leading to hypertension. In conclusion, lifetime health begins in utero, and patients and healthcare providers should be aware of the consequences that prenatal stressors have on long-term health.

AbstractOne third of epilepsy patients are resistant to treatment with current anti‐seizure medications. The ketogenic diet is used to treat some forms of refractory epilepsy, but the mechanism of its action has not yet been elucidated. In this study, we aimed to investigate whether the hydroxycarboxylic acid receptor 2 (HCA2), a known immunomodulatory receptor, plays a role in mediating the protective effect of this diet. We demonstrate for the first time that selective agonists at this receptor can directly reduce seizures in animal models. Agonists also reduce network activity in rodent and human brain slices. Ketogenic diet is known to increase circulating levels of endogenous HCA2 agonists, and we show that the effect of ketogenic diet in reducing seizures in the 6 Hz seizure model is negated in HCA2‐deficient mice. Our data support the potential of HCA2 as a target for the treatment of epilepsy and potentially for neurodegenerative diseases.

Intrauterine growth restriction (IUGR) occurs when the fetus doesn’t receive adequate nutrients. The leading cause of IUGR is placental ischemia, often seen in preeclampsia (PE). Adult IUGR offspring have an increased risk of HTN and oxidative stress (OS), with sex differences. Studies have shown that IUGR males have HTN, cerebrovascular dysfunction (CVD), and cerebral OS at 17 wks; IUGR females are NT with limited CVD and no significant OS. Our study aims to characterize the sex differences in timing and changes of cerebral OS in IUGR offspring. HTN and OS are both present in adult IUGR males, however, the order and timing is unknown. We hypothesize that OS leads to HTN. To test this, we examined the changes in brain OS in 12 wk IUGR and control (CON) males (before HTN), and 17 wk IUGR males (which have HTN) vs CON males. We investigated the same time points in NT females. We divided pups from normal pregnant and PE Sprague-Dawley rats into groups based on pregnancy status and sex. Pups were aged to 12 and 17 wks. We collected brains to measure OS via western blot (Manganese superoxide dismutase) and colorimetric assays: hydrogen peroxide (H 2 O 2 ) and total antioxidant capacity (TAC). 12 wk IUGR and CON offspring were NT with no changes between groups or sex. There were no changes in cerebral H 2 O 2 , TAC, and MnSOD for IUGR offspring compared to their CON. At 17 wks, IUGR males had elevated mean arterial pressure (MAP; 143.0 ± 1.7 vs 133.0 ± 2.7 mmHg; p < 0.01). IUGR females were NT. Cerebral H2O2 levels in IUGR males were elevated (20.5 ± 8.0 vs 3.7 ± 1.0 uM/ mg Protein; p < 0.05) and unchanged in IUGR females. TAC in IUGR males was decreased (157.7 ±21.9 vs 234.9 ± 22.0 mM Trolox/mg Protein; p < 0.05) and unchanged in IUGR females. MnSOD was elevated in females with no differences between groups (150.4 ± 3.8 vs 110.9 ±7.1 IU/Protein/CON%; p < 0.002). All 12 wk rats were NT with no changes in cerebral OS. At 17 wks, IUGR males were HTN with elevated cerebral OS, with no changes in females. The lack of increase in OS at 12 wks, suggest that it’s not a precursor to HTN in males, but rather OS is a downstream effect from HTN. Future studies will look at factors that contribute to HTN and CVD in in IUGR offspring. Studies will also examine why 17 wk female IUGR are protected from HTN and cerebral OS.

Background: Intrauterine growth restriction (IUGR) often times results from placental ischemia, which restricts the distribution of nutrients and oxygen to meet the metabolic demand of the growing fetus. IUGR offspring (OS) have an increased risk for developing cerebrovascular diseases (CVD) later in life. Factors for development of CVDs are HTN and inflammation. Our lab has shown 17 week IUGR male rats from preeclamptic pregnancies have HTN and elevated inflammation via systemic IL-17, while our females show no change. The role of inflammation to cause HTN in IUGR offspring in a sex specific manner is unknown and the major focus of this study which examines the changes in cerebral inflammation IUGR offspring at 12 weeks. We hypothesize that cerebral inflammation in IUGR males will be elevated, while no change in female IUGR offspring will be observed. Methods: IUGR offspring originate from preeclamptic Sprague Dawley dams. OS weaned for 3 weeks and were separated by sex and IUGR status. At 12 weeks, the rats underwent carotid catheterization to measure BP. Brains were collected to measure pro-inflammatory cytokines, such as TNF-α and IL-17 via ELISAs. Results: At 12 weeks, all OS were normotensive. Brain TNF-α increased in female IUGR (17,366± 1,500 vs 8,116± 1,341 pg/mL, p<0.05) and was elevated in male IUGR (20,845±7571 vs 8,516±1712 pg/mL, ns). Brain IL-17 increased in male IUGR (13,580± 945 vs 9,620± 945 pg/mL, p<0.05) and was elevated in female IUGR (13,143± 1613 vs 10,930± 696 pg/mL, ns). Systemic IL-17 was increased in IUGR male OS (112±16.8vs100±3.9 IU/Protein/CON %, ns). Conclusion: At 12 weeks, brain TNF-α and IL-17 were elevated in IUGR males and females. The differences in expression of each cytokine between the sexes suggest that IL-17 contributes to the increase in BP, especially in males. Understanding the timing of these developments could help to elucidate the role of inflammation in the development of CVD with or without HTN. This study is clinically relevant as it provides a sex specific inflammatory target for therapeutics, to help prevent hypertension and CVD in IUGR offspring. In the future, we will evaluate the role of inflammation in IUGR offspring in a sex specific manner.

Herbal medicines have been used to treat epilepsy for centuries. Cannabis derived cannabanoids have received attention for their anti-epileptic activity. This study investigates the acute anti-epileptic potential of terpenes found in the Cannabaceae family of plants.

Preeclampsia (PE) is characterized by new-onset hypertension in association with elevated natural killer (NK) cells and inflammatory cytokines, which are likely culprits for decreased fetal weight during PE pregnancies. As progesterone increases during normal pregnancy, it stimulates progesterone-induced blocking factor (PIBF). PIBF has been shown to decrease inflammation and cytolytic NK cells, both of which are increased during PE. We hypothesized that PIBF reduces inflammation as a mechanism to improve hypertension in the preclinical reduced uterine perfusion pressure (RUPP) rat model of PE. PIBF (2.0 µg/mL) was administered intraperitoneally on gestational day 15 to either RUPP or normal pregnant (NP) rats. On day 18, carotid catheters were inserted. Mean arterial blood pressure (MAP) and samples were collected on day 19. MAP in NP rats ( n = 11) was 100 ± 2 mmHg and 105 ± 3 mmHg in NP + PIBF rats ( n = 8) and 122 ± 1 mmHg in RUPP rats ( n = 10), which improved to 110 ± 2 mmHg in RUPP + PIBF rats ( n = 11), P < 0.05. Pup weight was 2.4 ± 0.1 g in NP, 2.5 ± 0.1 g in NP + PIBF, 1.9 ± 0.1 g in RUPP, and improved to 2.1 ± 0.1 g in RUPP + PIBF rats. Circulating and placental cytolytic NK cells, IL-17, and IL-6 were significantly reduced while IL-4 and T helper (TH) 2 cells were significantly increased in RUPP rats after PIBF administration. Importantly, vasoactive pathways preproendothelin-1, nitric oxide, and soluble fms-Like tyrosine Kinase-1 (sFlt-1) were normalized in RUPP + PIBF rats compared with RUPP rats, P < 0.05. Our findings suggest that PIBF normalized IL-4/TH2 cells, which was associated with improved inflammation, fetal growth restriction, and blood pressure in the RUPP rat model of PE.

Preeclampsia, new onset hypertension in pregnancy, affects ~5%-10% of the world's population. Preeclampsia is the leading cause of morbidity and mortality for both the mother and fetus. As of today, there is no cure for this disease except for delivery of the fetal-placental unit. The exact causation and onset of the disease are unknown. However, recent studies have shown a strong correlation between mitochondrial dysfunction and preeclampsia. Circulating mitochondrial DNA, elevated reactive oxygen species, angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA), activated natural killer cells, and upregulated inflammatory responses all contribute to mitochondrial dysfunction and the pathophysiology of preeclampsia. This review summarizes the current literature of both experimental and clinical observations that support the hypothesis that mitochondrial dysfunction contributes to the pathophysiology of preeclampsia and may be a precursor to the disease onset. This review will also address the use of therapies to improve mitochondrial dysfunction in preeclampsia.

We have shown Preeclampsia (PE) to be a progesterone deficient state of pregnancy associated with increased TH1 lymphocytes, cytolytic natural killer (NK) cells, inflammatory cytokine, vasoactive pathways resulting in endothelial function and hypertension. Healthy normal pregnancy (NP) is associated with elevations in progesterone and TH2 lymphocytes, and uterine NK cells favoring immunotolerance toward the fetus. Importantly, NP lymphocytes express progesterone receptors, which stimulate Progesterone Induced Blocking Factor (PIBF) upon binding to progesterone, thus PIBF increases during NP. Therefore we hypothesize that PIBF blockade causes a proinflammatory hypertensive phenotype similar to PE. Rabbit anti‐PIBF IgG (0.50 mg/mL) was administered i.p. on gestational day 15 (GD 15) to NP rats, on GD 18 carotid catheters were inserted and on GD 19 mean arterial blood pressure (MAP) and samples were collected. MAP in NP rats (n=7) was 99+ 3 mmHg, which increased to 113+4 mmHg in NP+ anti‐PIBF (n=6), p<0.05. Plasma TNF‐α was 35+8 pg/mL in NP rats and increased to 84+21 pg/mL in NP+ Anti‐PIBF (n=4), p<0.05. Circulating total NK cells were 67+ 11 in NP rats (n=4), which decreased to 36+4 in NP+ Anti‐PIBF, while cytolytic NK cells were 0.6 + 0.2 in NP and increased to 3.0+1 in NP+ Anti‐PIBF, p<0.05. Circulating nitric oxide was 44+ 11 µM in NP rats (n=5), which decreased to 21+1 µM in NP+ Anti‐PIBF (n=6), p<0.05, while, renal cortex preproendothelin‐1 increased 15 fold in NP+ Anti‐PIBF (n=6) compared to NP rats (n=5). In order to determine if PIBF supplementation improves a PE phenotype during pregnancy, PIBF (2.0 µg/mL) was administered i.p. on GD 15 to the reduced uterine perfusion pressure (RUPP) a rat model of PE and compared to control RUPP and NP rats. On GD19, MAP and samples were collected. MAP in NP rats (n=11) was 100+ 2 mmHg, 105 + 3 in NP+PIBF (n=8), 122+ 1 in RUPP rats (n=10), which improved to 110+2 mmHg in RUPP+PIBF rats (n=11), p<0.05. PIBF lowered circulating and placental cytolytic NK cells in RUPPs from 15+6, 2.4+1% gate to 3+2, 0.4+0.1% in RUPP+PIBF(p<0.05, n=4‐8). Moreover PIBF supplementation increased circulating IL‐4 and TH2 to 40±8 pg/mL, 2+0.6 % gate in RUPP+PIBF (n=4‐8 p<0.05) compared to9 ±2 pg/mL, 0.5+ 0.1 % gate in RUPP. Importantly, vasoactive pathways preproendothelin‐1 and nitric oxide were normalized in RUPP+PIBF rats compared to RUPP rats, p<0.05. Collectively, these data demonstrate an important role for PIBF to control the inflammatory milieu thereby normalizing vasoactive pathways and maintaining healthy blood pressures during pregnancy.

Two important clinical features of preeclampsia (PE) are hypertension and fetal growth restriction. The reduced uterine perfusion pressure (RUPP) preclinical rat model of PE exhibits both of these features. Moreover, RUPP and PE women have elevated vasoconstrictor peptide endothelin-1 (ET-1) and inflammation. Interleukin-2 (IL-2) is a cytokine that regulates NK cell activity and is elevated in miscarriage, PE, and RUPP rats. The objective of this study was to examine a role for IL-2 in NK cell activation, fetal growth restriction, and hypertension during pregnancy by either infusion of IL-2 or blockade of IL-2 (basiliximab) in normal pregnant (NP) and RUPP rats. On gestational day 14, NP and RUPP rats received low (LD), middle (MD), or high dose (HD) IL-2 (0.05, 0.10, or 0.20 ng/ml) IP or basiliximab (0.07 mg per rat) by IV infusion. On day 19, blood pressure (MAP), pup weights, and blood were collected. Basiliximab had no effect on blood pressure, however, significantly lowered NK cells and may have worsened overall fetal survival in RUPP rats. However, IL-2 LD (102 ± 4 mmHg) and IL-2 HD (105 ± 6 mmHg) significantly lowered blood pressure, ET-1, and activated NK cells compared to control RUPPs (124 ± 3 mmHg, p < 0.05). Importantly, IL-2 in RUPP rats significantly reduced fetal weight and survival. These data indicate that although maternal benefits may have occurred with low dose IL-2 infusion, negative effects were seen in the fetus. Moreover, inhibition of IL-2 signaling did not have favorable outcome for the mother or fetus.

Women with preeclampsia (PE) have a greater risk of developing hypertension, cardiovascular disease (CVD), and renal disease later in life. Angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE during pregnancy and up to 2-year postpartum (PP), and in the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of AT1-AA with a specific 7 amino acid peptide binding sequence (‘n7AAc’) improves pathophysiology observed in RUPP rats; however, the long-term effects of AT1-AA inhibition in PP is unknown. Pregnant Sprague Dawley rats were divided into three groups: normal pregnant (NP) (n = 16), RUPP (n = 15), and RUPP + ‘n7AAc’ (n = 16). Gestational day 14, RUPP surgery was performed and ‘n7AAc’ (144 μg/day) administered via osmotic minipump. At 10-week PP, mean arterial pressure (MAP), renal glomerular filtration rate (GFR) and cardiac functions, and cardiac mitochondria function were assessed. MAP was elevated PP in RUPP vs. NP (126 ± 4 vs. 116 ± 3 mmHg, p < 0.05), but was normalized in in RUPP + ‘n7AAc’ (109 ± 3 mmHg) vs. RUPP (p < 0.05). PP heart size was reduced by RUPP + ’n7AAc’ vs. RUPP rats (p < 0.05). Complex IV protein abundance and enzymatic activity, along with glutamate/malate-driven respiration (complexes I, III, and IV), were reduced in the heart of RUPP vs. NP rats which was prevented with ‘n7AAc’. AT1-AA inhibition during pregnancy not only improves blood pressure and pathophysiology of PE in rats during pregnancy, but also long-term changes in blood pressure, cardiac hypertrophy, and cardiac mitochondrial function PP.

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy associated with chronic inflammation, mitochondrial (mt) dysfunction and fetal demise. Natural Killer cells (NK cells) are critical for the innate immune response against tumors or infection by disrupting cellular mt function and causing cell death. Although NK cells can be stimulated by Tumor necrosis factor alpha (TNF-alpha), we don't know the role of TNF-alpha on NK cell mediated mt dysfunction during PE. Our objective was to determine if mechanisms of TNF-alpha induced hypertension included activation of NK cells and multi-organ mt dysfunction during pregnancy. Pregnant rats were divided into 2 groups: normal pregnant (NP) (n = 18) and NP + TNF-alpha (n = 18). On gestational day 14, TNF-alpha (50 ng/ml) was infused via mini-osmotic pump and on day 18, carotid artery catheters were inserted. Blood pressure (MAP) and samples were collected on day 19. TNF-alpha increased MAP (109 +/- 2 vs 100 +/- 2, p < 0.05), circulating cytolytic NK cells (0.771 +/- 0.328 vs.0.008 +/- 0.003% gated, <0.05) and fetal reabsorptions compared to NP rats. Moreover, TNF-alpha caused mtROS in the placenta (12976 +/- 7038 vs 176.9 +/- 68.04% fold, p < 0.05) and in the kidney (2191 +/- 1027 vs 816 +/- 454.7% fold, p < 0.05) compared to NP rats. TNF-alpha induced hypertension is associated fetal demise, activation of NK cells and multi-organ mt dysfunction which could be mechanisms for fetal demise and hypertension. Understanding of the mechanisms by which TNF-alpha causes pathology is important for the use of anti-TNF-alpha therapeutic agents in pregnancies complicated by PE.

Cannabis has received attention for its potential anti-seizure activity, however studies of cannabis have predominantly focused on the plant’s cannabinoid constituents. This study aims to assess the anti-seizure potential of α-pinene and linalool, two monoterpenes commonly found in the Cannabaceae family of plants [1].

Preeclampsia (PE) is characterized by new onset hypertension during pregnancy and is associated with oxidative stress, placental ischemia, and autoantibodies to the angiotensin II type I receptor (AT1-AA). Mitochondrial (mt) dysfunction in PE and various sources of oxidative stress, such as monocytes, neutrophils, and CD4 + T cells, have been identified as important players in the pathophysiology of PE. We have established the significance of AT1-AA, TNF-α, and CD4 + T cells in causing mitochondrial (mt) dysfunction in renal and placental tissues in pregnant rats. Although the role of mt dysfunction from freshly isolated intact placental mitochondria has been compared in human PE and normally pregnant (NP) controls, variations among preterm PE or term PE have not been compared and mechanisms contributing to mt ROS during PE are unclear. Therefore, we hypothesized PE placentas would exhibit impaired placental mt function, which would be worse in preterm PE patients than in those of later gestational ages. Immediately after delivery, PE and NP patient's placentas were collected, mt were isolated and mt respiration and ROS were measured. PE patients at either < or >34 weeks gestational age (GA) exhibited elevated blood pressure and decreased placental mt respiration rates (state 3 and maximal). Patients delivering at >34 weeks exhibited decreased Complex IV activity and expression. Placental mtROS was significantly reduced in both PE groups, compared to NP placental mitochondria. Collectively, the study demonstrates that PE mt dysfunction occurs in the placenta, with mtROS being lower than that seen in NP controls. These data indicate why antioxidants, as a potential target or new therapeutic agent, may not be ideal in treating the oxidative stress associated with PE.

Electrical neurostimulation is effective in treating neurological disorders, but associated recording artefacts generally limit applications to ‘open-loop’ stimuli. Since light does not prevent concurrent electrical recordings, optogenetics enables real-time, continuous ‘closed-loop’ control of brain activity. Here we show that closed-loop optogenetic stimulation with excitatory opsins (CLOSe) affords precise manipulation of neural dynamics, both in vitro , in brain slices from transgenic mice, and in vivo , with anesthetised monkeys. We demonstrate the generation of oscillations in quiescent tissue, enhancement or suppression of endogenous patterns in active tissue, and modulation of seizure-like bursts elicited by 4-aminopyridine. New network properties, emergent under CLOSe, depended on the phase-shift imposed between neural activity and optical stimulation, and could be modelled with a nonlinear dynamical system. In particular, CLOSe could stabilise or destabilise limit cycles associated with seizure oscillations, evident from systematic changes in the variability and entropy of seizure trajectories that correlated with their altered duration and intensity. Furthermore, CLOSe was achieved using intracortical optrodes incorporating light-emitting diodes, paving the way for translation of closed-loop optogenetics towards therapeutic applications in humans.

Preeclamptic (PE) women have placental ischemia, hypertension (HTN), mitochondrial (mt) dysfunction, increased mt-reactive oxygen species (ROS), cytolytic natural killer (NK) cells, and pro-inflammatory cytokines, such as tumor necrosis factor (TNFα). The reduced uterine perfusion pressure (RUPP) preclinical rat model of PE, is a well-established model to study mechanisms of hypertension in response to placental ischemia during pregnancy. Previous studies from our lab have shown that TNFα is elevated in RUPP rats and that blockade of TNFα, via etanercept (ETAN) decreases TNFα and HTN. However, the effect of TNFα blockade on NK cell activation, mt protein/function, and mtROS is unknown. We hypothesized that ETAN would decrease blood pressure, NK cell activation, mtROS, and improve mt function in RUPP rats. Rats were divided into 4 groups: normal pregnant (NP) (n=41), RUPP (n=34), RUPP+low dose (LD) ETAN (0.4 mg/kg) (n=11), RUPP+high dose (HD) ETAN (0.8 mg/kg) (n=19). LD was used previously, while HD is clinically used to treat chronic inflammatory diseases. Gestational day 14, RUPP surgery was performed and ETAN administered subcutaneous on day 18. Day 19, conscious blood pressure (MAP), blood and tissues were collected, and mt were isolated. Flow cytometry was used for quantification of NK cells. MAP was elevated in RUPP vs. NP (119 ± 1 vs. 102± 1 mmHg, p<0.05) which was reduced to 110 ± 2 in RUPP + LD ETAN (p<0.05 vs. RUPP) and 116 ± 1 in RUPP + HD ETAN. Activated NK cells were increased in circulation (5.9 ± 1.8 vs. 4.0 ± 1.4 %), placenta (4.8 ± 1.3 vs. 1.9 ± 0.7 %), and kidneys (3.1 ± 0.5 vs. 0.8 ± 0.5%) of RUPP vs. NP (p<0.05). Both LD (2.1 ± 0.7%, 1.7 ± 0.7%, 0.8 ± 0.5%) and HD (0.2 ± 0.1%, 0.4 ± 0.1%, 0.8 ± 0.2%) ETAN decreased activated NK cells in circulation, placenta, and kidney respectively (p<0.05 vs RUPP). Placental (0.79± 0.02 vs 1±0.05 fold) and renal (0.67± 0.05 vs 1±0.06 fold) mtROS was reduced with LD ETAN vs RUPP (p<0.05). ETAN treatment lowers blood pressure and reduces circulating, placental, and renal activated NK cells and mtROS in placental ischemic rats. Therefore ETAN could be a potential therapeutic for improving HTN, inflammation, and mitochondrial function during complicated pregnancies. Supported: AHA18CDA34110264, HL130456, R01HD067541, & P20GM121334

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity in the U.S. While the pathogenesis remains unclear, PE is characterized by new onset hypertension associated with progesterone deficiency, elevated cytolytic natural killer cells (cNK), inflammation, and endothelial dysfunction. Progesterone is essential in the initiation and maintenance of pregnancy. We have shown that progesterone supplementation regulates endothelial function and suppresses endothelin‐1 (ET‐1) and stimulates nitric oxide (NO) in response to placental ischemia during pregnancy. In addition, progesterone signals the synthesis and release of progesterone induced blocking factor (PIBF) from lymphocytes in order to regulate the proinflammatory balance of early pregnancy. However the role of PIBF in PE pathology is not well examined. This study was designed to test the hypothesis that inhibition of PIBF causes inflammation and increases markers of endothelial dysfunction and hypertension during pregnancy. Rabbit anti‐PIBF IgG (0.25, low dose‐LD or 0.50 mg/mL, high dose‐HD) was administered intraperitoneal on gestation day (GD) 15 to normal pregnant Sprague Dawley (NP) rats, on GD 18 carotid catheters were inserted and on GD 19 blood pressure (MAP) and samples were collected. MAP in NP rats (n=7) was 99± 3 mmHg, which increased to 116± 2 in NP+ anti‐PIBF LD (n =10) and 113±4 mmHg in NP+ anti‐PIBF HD (n=6), p<0.05. Neither placental weight nor pup weight was affected by PIBF blockade. Plasma TNF‐alpha levels were 35±8 pg/mL in NP rats and increased to 84±21 pg/mL in NP+ Anti‐PIBF HD (n=4), p<0.05. Circulating total NK cells were 67± 11 in NP rats (n=4), which decreased to 36±4 in NP+ Anti‐PIBF HD however, cytolytic NK cells were 0.6 ± 0.2 in NP which were increased to 3.0±1 in NP+ Anti‐PIBF HD, p<0.05. Importantly, circulating NO levels were 44± 11 μM in NP rats (n=5), which significantly decreased to 21±1 μM in NP+ Anti‐PIBF (n=6) HD, p<0.05. Moreover, renal cortex PPET‐1 levels increased 15 fold in NP+ Anti‐PIBF (n=6) HD compared to NP rats (n=5). Collectively, our study demonstrates that inhibition of PIBF causes hypertension, increased TNF alpha while causing signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signaling pathways for the maintenance of endothelial function and inflammation during healthy pregnancy.Support or Funding InformationThis work was supported by National Institutes of Health grants RO1HD067541‐06, P20GM121334 and AHA 19CDA34670055

Pre-eclampsia complicates approximately 8-10% of all pregnancies and is associated with chronic inflammation, increased TNF-α and cytolytic Natural killer cells and mitochondrial (mt) dysfunction which is indicative of tissue respiratory efficacy. We have previously shown that infusing TNF-α into pregnant rats increased vasoconstrictor ET-1, mean arterial pressure (MAP) and decreased renal hemodynamics, however we don't know its effects on NK cells or renal and placental mt function. The objective of our study was to determine the relationship between TNF-α and NK cell mediated mt oxidative stress, measured by reactive oxygen species (ROS) and respiration. Pregnant rats were divided into 2 groups: normal pregnant (NP) (n=19) and NP +TNF-α (n=18). On gestational day 14, TNF-α (50ng/ml) was infused via mini-osmotic pump and on gestational day 18, carotid artery catheters were inserted to perform to blood pressure measurements on day 19 in conscious rats, and blood and placentas were collected. Placental and renal mt were isolated and used for mt assays. Mt respiration and ROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader. MAP was elevated in NP+TNF-α vs. NP rats (108 ± 1, n=18 vs. 100 ± 1 p< 0.05). There was a significant increase in circulating NK cells (n=6, p< 0.05) which is associated with an increase in mt ROS in both the placenta (n=16, 3874 ± 3663%, p=0.2) and kidney (n=16,of NP rats with TNF-α compared to control rats. No changes in respiration were observed. Although there were no changes in cellular respiration, TNF-α is associated with activation of cytolytic natural killer cells which promotes mt oxidative stress and hypertension. No change in mt respiration in TNF-α infused rats could suggest a potential protective mechanism which warrants further investigation.

BackgroundPreeclampsia (PE) is a pregnancy disorder that is characterized by an increase in blood pressure, angiotensin II type1 receptor agonistic autoantibodies (AT1‐AA), and neurological complications. The pre‐clinical RUPP (reduced uterine perfusion pressure) rat model of PE displays many of the clinical pathologies associated with PE, such as increased blood pressure, AT1‐AAs, blood brain barrier (BBB) permeability, and impaired cerebral blood flow (CBF) autoregulation.HypothesisWe hypothesized that specific inhibition of AT1‐AA, using the epitope binding 7 amino acid peptide sequence (7AA), will improve blood pressure, BBB integrity, and CBF autoregulation in RUPP rats.MethodsSprague Dawley rats, were divided into 3 groups: normal pregnant (NP) (n=19), RUPP (n=19), and RUPP+AT1‐AA inhibition (7AA) (n=21). RUPP surgery was performed on gestational day (GD) 14 and 7AA (2μg/μl) was administered via osmotic minipumps. GD 19, mean arterial pressure (MAP) was determined and BBB permeability was assessed using the in‐vivo imaging system (IVIS). CBF was measured by laser Doppler flowmetry through a 4 mm x 4 mm cranial window under anesthesia. MAP was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF.ResultsMAP was increased in RUPP vs NP (124±3 vs 100±1 mmHg, p<0.05) and decreased in RUPP+7AA vs RUPP (105±2 vs 124±3 mmHg, p<0.05) rats. Using IVIS, Texas Red/FITC green fluorescent imaging was decreased in RUPP+7AA vs RUPP (0.2±0.001 vs 0.6±0.001, p<0.05), indicating improvement in BBB permeability with AT1‐AA inhibition. CBF autoregulation was impaired in RUPP vs NP rats at 140 mmHg (142±7 vs 119±4%, p<0.05) and 160 mmHg (157±10 vs 129±6%, p<0.05) (both pressures are within the normal CBF autoregulatory range). AT1‐AA inhibition prevented impaired CBF at 140mmHg (125±5 RUPP+7AA vs 142±7% RUPP, p<0.05) and 160 mmHg (125±9 RUPP+7AA vs 157±10% RUPP, p<0.05). Furthermore, the increases in CBF at pressures above the autoregulatory range, 180 mmHg (122±10 vs 198±17%, p<0.05) and 190 mmHg (123±11 vs 208±19%, p<0.05), was prevented in RUPP+7AA vs RUPP, respectively.ConclusionAT1‐AA inhibition improved blood pressure, BBB permeability, and CBF autoregulation in the pre‐clinical RUPP rat model of PE, indicating that AT1‐AA inhibition may be a potential therapy used to improve neurological complications during PE.Support or Funding InformationSupported: AHA18CDA34110264

BackgroundAdult offspring (AOS) born from hypertensive mothers have an increased risk of developing hypertension, stroke, and neurological complications later in life. Women with preeclampsia (PE) and the pre‐clinical RUPP (reduced uterine perfusion pressure) rat model of PE have an increase in blood pressure, impaired cerebral blood flow (CBF) autoregulation, and cerebrovascular dysfunction during pregnancy. Studies have shown that blood pressure is elevated early in male AOS and later in female AOS born from RUPP dams. However, no study has examined the cerebrovascular function in AOS from RUPP dams.HypothesisWe hypothesize that AOS from RUPP dams will exhibit impaired cerebral vascular myogenic responses and CBF autoregulation.MethodsMale and female Sprague Dawley rats were born from normal pregnant (NP) and RUPP dams. After 3 weeks of weening, OS rats were separated by sex and groups determined by their mother. OS rats were aged to 17 weeks and mean arterial pressure (MAP) and body weight (BW) was recorded. At 22–23 weeks of age, myogenic responses were determined by changes in vascular diameter of the middle cerebral artery in response to increases in intraluminal pressure. At 29–30 weeks, CBF autoregulation was measured by laser doppler flowmetry through a 4 mm x 4 mm cranial window under anesthesia. MAP was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF.ResultsAt 17 weeks, BW was smaller in male AOS born from RUPP vs NP (381±10 vs 414±3g, p<0.05). No change was observed in female AOS BWs. MAP was elevated in male AOS RUPP vs NP (143±2 vs 133±3 mmHg, p<0.05) at 17 weeks, with no change in female AOS. At 22–23 weeks, the myogenic response was impaired and tone was increased in male AOS RUPP vs NP (p<0.05). At 29–30 weeks, CBF autoregulation was impaired in male OS RUPP vs NP rats at 120 mmHg (129±12% vs 103±4), at 140 mmHg (149±21 vs 107±12%), and at 160 mmHg (172±31 vs 108±15%) respectively. Interestingly, CBF autoregulation was also impaired in female AOS RUPP vs NP rats at 140 mmHg (130±14 vs 110±18%) and 160 mmHg (175±2 vs 99±8%, p<0.05) in the absence of hypertension.ConclusionA decrease in BW, increase in blood pressure, and impairments of CBF autoregulation was observed in male RUPP AOS. In contrast, female RUPP AOS exhibited no changes in body weight or blood pressure, but showed a marked impairment in CBF autoregulation, suggesting that elevated MAP is not a prerequisite or required for impaired CBF autoregulation in females. Future studies are warranted to examine the mechanisms underlying CBF autoregulation impairment and their associated sex differences in AOS born from hypertensive pregnancies associated with placental ischemia.Support or Funding InformationSupported: AHA18CDA34110264