The objective of this study was to determine the effects of various estrous synchronization protocols utilizing the five-day controlled internal drug releasing (CIDR) In Experiment 1, overall days to estrus were greater (P ≤ 0.01) in U and P compared with C. In Experiment 2, overall days to estrus were greater (P ≤ 0.02) in U and P compared with G. In summary, the 5 d CIDR reduces the number of days required to bring ewes into estrus when compared to untreated ewes. iv ACKNOWLEDGEMENTS

Problem:The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of preeclampsia including maternal hypertension, intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). While we have previously shown a 5-fold higher cytotoxicity of RUPP NKs versus normal pregnant NKs, their role in RUPP pathophysiology remains unclear.In this study, we tested the hypotheses that (1) adoptive transfer of RUPP-stimulated NKs will induce maternal hypertension and IUGR in normal pregnant control (Sham) rats and (2) adoptive transfer of Sham NKs will attenuate maternal hypertension and IUGR in RUPP rats. Method of Study:On gestation day (GD)14, vehicle or 5 × 10 6 RUPP NKs were infused i.v. into a subset of Sham rats (Sham+RUPP NK), and vehicle or 5 × 10 6 Sham NKs were infused i.v. into a subset of RUPP rats (RUPP+Sham NK; n = 12/group). On GD18, Uterine Artery Resistance Index (UARI) was measured. On GD19, mean arterial pressure (MAP) was measured, animals were sacrificed, and blood and tissues were collected for analysis.Results: Adoptive transfer of RUPP NKs into Sham rats resulted in elevated NK activation, UARI, placental oxidative stress, and preproendothelin expression as well as reduced circulating nitrate/nitrite. This led to maternal hypertension and IUGR. RUPP recipients of Sham NKs demonstrated normalized NK activation, sFlt-1, circulating and placental VEGF, and UARI, which led to improved maternal blood pressure and normal fetal growth. Conclusion:These data suggest a direct role for cNKs in causing preeclampsia pathophysiology and a role for normal NKs to improve maternal outcomes and IUGR during late gestation. K E Y W O R D S hypertension, natural killer cells, preeclampsia, pregnancyThe authors declare that all supporting data are available within the article and its Supporting Information. | Animals12-13 week old, timed-pregnant Sprague-Dawley rats were purchased from Envigo. The animals were delivered to the Center for Comparative Research at the University of Mississippi Medical Center on day 10 or 11 of their gestation and weighed approximately 250-260 g upon delivery. The animals were housed in a temperature-controlled room (23°C) with a 12:12-h light-dark cycle and maintained on a normal diet. Rats were randomly assigned to experimental groups. The rats were group housed until after surgery.All experimental procedures were carried out in accordance with the National Institutes of Health guidelines for use and care of animals.All protocols were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. | Reduction in uterine perfusion pressureOn day 14 of gestation (GD14), animals underwent either RUPP or Sham surgeries under 2% isoflurane anesthesia delivered by a vaporizer (Ohio Medical Products). The RUPP surgery was performed to induce placental ischemia as previously described. 23,24 The animals received carprofen immediately following surgery and 24 h post-surgery...

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many characteristics of preeclampsia (PE) such as hypertension (HTN), intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). We have previously shown that natural killer (NK) cell depletion in RUPP rats improves PE pathophysiology and that RUPP NKs have a 5-fold increase in cytotoxicity vs normal pregnant NKs. In this study, we tested the hypotheses that (1) RUPP stimulated NKs play a direct role in causing HTN and IUGR in pregnant rats and (2) normal pregnant control (Sham) NKs attenuate HTN and IUGR in RUPP rats. NKs were isolated from the placentas of Sham and RUPP rats on gestation day (GD) 19. On GD14, vehicle or 5x10 6 RUPP NKs were infused i.v. into a subset of Sham rats, and vehicle or 5x10 6 Sham NKs were infused i.v. into a subset of RUPP rats. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound; GD19, cNKs were quantified via flow cytometry and MAP, fetal weight, and blood were acquired. Plasma VEGF and sFlt-1 were measured via ELISA. Placental cNKs (% gated) increased from 3±1% in Sham to 19±5% in RUPP and 21±4% in Sham+RUPP NK (p<0.05 vs Sham), and decreased to 3±1% in RUPP+Sham NK (p<0.05 vs RUPP). Circulating cNKs also followed this trend. MAP increased from 102±1 mmHg in Sham to 130±2 mmHg in RUPP and 121±2 mmHg in Sham+RUPP NK (p<0.05 vs Sham), and was blunted to 113±1 mmHg in RUPP+Sham NK (p<0.05 vs RUPP). Fetal weight decreased from 2.4±0.04 g in Sham to 2.1±0.07 g in RUPP and 2.1±0.03 g in Sham+RUPP NK (p<0.05 vs Sham), and this was normalized to 2.3±0.03 g in RUPP+Sham NK (p<0.05 vs RUPP). UARI increased from 0.56±0.05 in Sham to 0.75±0.06 in RUPP and 0.76±0.05 in Sham+RUPP NK (p<0.05 vs Sham), and decreased to 0.64±0.05 in RUPP+Sham NK (p<0.05 vs RUPP). Circulating sFlt-1 increased from 76±15 pg/mL in Sham to 1391±424 pg/mL in RUPP (p<0.05 vs Sham), 780±256 pg/mL in Sham+RUPP NK, and decreased to 67±8 pg/mL in RUPP+Sham NK (p<0.05 vs RUPP). Furthermore, circulating VEGF decreased in RUPP and Sham+RUPP NK compared to Sham (p<0.05), and increased in RUPP+Sham NK (p<0.05 vs RUPP). These data demonstrate a direct role for cNKs to mediate vascular dysfunction in PE and for normal NKs to promote positive maternal and fetal outcomes.

The R educed U terine P erfusion P ressure (RUPP) rat model of placental ischemia mirrors many of the hallmark features of preeclampsia (PE) such as hypertension, intrauterine growth restriction (IUGR), and immune activation, which includes increased cytolytic natural killer cells (cNKs). We have previously demonstrated that depletion of NK cells in RUPP rats improves PE pathophysiology. In this study we tested the hypothesis that RUPP-stimulated NKs have increased cytotoxicity and play a direct role in inflammation, oxidative stress, hypertension, and intrauterine growth restriction in pregnant rats. NK cells were isolated from the placentas of normal pregnant (NP) and RUPP rats on gestation day (GD) 19 and assessed for cytotoxic activity. RUPP NK cells demonstrated a 5-fold increase in cytotoxic activity vs NP NK cells. On GD 12, 5x10 6 NK cells from RUPP rats were infused into a subset of NP rats. On GD 19, circulating and placental total NK cells and cNKs were quantified via flow cytometry in NP, RUPP, and NP+RUPP NK rats. MAP, fetal and placental weights, placental reactive oxygen species (ROS), and placental cytokines were also measured. Placental cNK cells (%gated) were significantly increased in RUPP (18.5±3.6%) and NP+RUPP NK (16.1±3.6%) compared to NP (4.1±0.8%) controls. Circulating cNKs followed a similar trend. MAP increased from 100 mmHg in NP to 126 mmHg in RUPP, and 119 mmHg in NP+RUPP NK (p<0.05 vs NP). Fetal weight decreased from 2.4±0.03 g in NP to 2.0±0.04 g in RUPP and to 2.1±0.02 g in NP+RUPP NK (p<0.05 vs NP). Placental weights followed a similar trend. Placental cNK cytokines were significantly increased in RUPP and in NP recipients of RUPP NK cells: TNF-α: NP- 23±3.1, RUPP- 62.3±7.8 pg/mg, NP+RUPP NK-43.9±5.7 pg/mg (p<0.05 vs NP); IFNγ: NP- 3.3±0.7, RUPP- 7.6±1.2 pg/mg, NP+RUPP NK-6.6±1.9 pg/mg (p<0.05 vs NP). Placental VEGF decreased from 6.5±1.3 pg/mg in NP to 2±0.8pg/mg in RUPP and 2.9±0.6 pg/mg in NP+RUPP NK. Circulating levels followed a similar trend. Placental ROS significantly increased from 27.3±4.6 RLUs/min/mg in NP to 63.1±5.7 RLUs/min/mg in RUPP and 84±17.6 RLUs/min/mg in NP+RUPP NK. These data demonstrate a direct role for cNKs in causing PE pathophysiology and identifies cNKs as a novel therapeutic target for treatment of PE.

Preeclampsia (PE) is a multisystem pregnancy disorder characterized by maternal hypertension (HTN), intrauterine growth restriction (IUGR), and immune activation, including increased cytolytic natural killer cells (cNKs). The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many of these characteristics, and we have previously demonstrated that depletion of NK cells (NKs) in RUPP rats improves maternal and fetal outcomes. In this study we tested the hypotheses that (1) RUPP stimulated NKs play a direct role in causing maternal HTN and IUGR in pregnant rats and (2) normal pregnant control (Sham) NKs attenuate maternal HTN and IUGR in RUPP rats. NKs were isolated from the placentas of Sham and RUPP rats on gestation day (GD) 19 and assessed for cytotoxicity. RUPP NKs demonstrated a 5‐fold increase in cytotoxicity vs Sham NKs (p<0.05 vs Sham). On GD14, vehicle or 5x106 RUPP NKs were infused i.v. into a subset of Sham rats (Sham n=8, Sham+RUPP NK, n=7), and vehicle or 5x106 Sham NKs were infused i.v. into a subset of RUPP rats (RUPP n=8, RUPP+ Sham NK, n=7). On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound. On GD19, cNKs were quantified via flow cytometry, MAP, fetal and placental weights, and placental oxidative stress (ROS) were measured. Placental cNKs (%gated) were 3±1% in Sham and increased to 19±5% in RUPP and 21±4% in Sham+RUPP NK (p<0.05 vs Sham). This was reduced to 3±1% in RUPP+Sham NK (p<0.05 vs RUPP). Similarly, circulating cNKs (% gated) were 5±1% in Sham, increased to 20±3% in RUPP and 21±3% in Sham+RUPP NK (p<0.05 vs Sham) and decreased to 5±1% in RUPP+Sham NK (p<0.05 vs RUPP). MAP increased from 102±1 mmHg in Sham to 130±2 mmHg in RUPP and 121±2 mmHg in Sham+RUPP NK (p<0.05 vs Sham), but this was blunted to 113±1 mmHg in RUPP+Sham NK (p<0.05 vs RUPP). Mean fetal weight decreased from 2.4±0.04g in Sham to 2.1±0.07g in RUPP and 2.1±0.03g in Sham+RUPP NK (p<0.05 vs Sham); however, this was normalized to 2.3±0.03g in RUPP+Sham NK (p<0.05 vs RUPP). Similarly, placental weight decreased from 0.56±0.02g in Sham, to 0.47±0.02g in RUPP and 0.45±0.02g in Sham +RUPP NK (p<0.05 vs Sham) and increased to 0.53±0.01g in RUPP+Sham NK (p<0.05 vs RUPP). Placental ROS (RLUs/min/mg) was 35±2 in Sham, increased to 63±6 in RUPP and 70±12 in Sham +RUPP NK (p<0.05 vs Sham), and remained elevated at 60±5 in RUPP+Sham NK (p<0.05 vs Sham). UARI was 0.59±0.03 in Sham (n=3), 0.73±0.03 in RUPP (n=3; p=0.15 vs Sham), and 0.61±0.02 in RUPP+Sham NK (n=5; p>0.05 vs RUPP). UARI increased to 0.76±0.05 in Sham+RUPP NK (n=5; p<0.05 vs Sham). In conclusion, we observed that infusion of RUPP NKs led to HTN, IUGR, increased placental ROS, and increased UARI in pregnant rats, while Sham NK infusion improved the pathophysiology observed in RUPP rats. These data demonstrate a direct role for cNKs in causing PE pathophysiology and a role for normal NKs to promote positive maternal and fetal outcomes. Our study identifies cNKs as a therapeutic target in PE and suggests ...