Placental Ischemic Rats Research Articles

Inhibition of the AT1R agonistic autoantibody in a rat model of preeclampsia improves fetal growth in late gestation.

Placenta ischemia, the initiating event in preeclampsia (PE), is associated with fetal growth restriction. Inhibition of the agonistic autoantibody against the angiotensin type 1 receptor AT1-AA, using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased blood pressure at gestational day (G)19 in the clinically relevant reduced uterine perfusion pressure (RUPP) model of PE. Thus we tested the hypothesis that maternal administration of 'n7AAc' does not transfer to the fetus, improves uterine blood flow and fetal growth, and attenuates elevated placental expression of miRNAs implicated in PE and FGR. Sham or RUPP surgery was performed at G14 with vehicle or 'n7AAc' (144 µg/day) administered via an osmotic pump from G14 to G20. Maternal plasma levels of the peptide on G20 were 16.28 ± 4.4 nM, and fetal plasma levels were significantly lower at 1.15 ± 1.7 nM (P = 0.0007). The uterine artery resistance index was significantly elevated in RUPP (P < 0.0001) but was not increased in 'n7AAc'-RUPP or 'n7AAc'-Sham versus Sham. A significant reduction in fetal weight at G20 in RUPP (P = 0.003) was not observed in 'n7AAc'-RUPP. Yet, percent survival was reduced in RUPP (P = 0.0007) and 'n7AAc'-RUPP (P < 0.0002). Correlation analysis indicated the reduction in percent survival during gestation was specific to the RUPP (r = 0.5342, P = 0.043) and independent of 'n7AAc'. Placental miR-155 (P = 0.0091) and miR-181a (P = 0.0384) expression was upregulated in RUPP at G20 but was not elevated in 'n7AAc'-RUPP. Collectively, our results suggest that maternal administration of 'n7AAc' does not alter fetal growth in the RUPP implicating its potential as a therapeutic for the treatment of PE.NEW & NOTEWORTHY The seven amino acid inhibitory peptide to the AT1-AA ('n7AAc') has limited transfer to the fetus at gestational day 20, improves uterine blood flow and fetal growth in the reduced uterine perfusion pressure model of preeclampsia (PE), and does not impair fetal survival during gestation in sham-operated or placental ischemic rats. Collectively, these findings suggest that maternal administration of 'n7AAc' as an effective strategy for the treatment of PE is associated with improved outcomes in the fetus.

IL‐25 Supplementation Induces M2 Macrophage Polarization, Reduces Blood Pressure, and Improves Fetal Weight in Placental Ischemic Rats

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction (IUGR), and increased macrophage activation. Macrophages can be broadly categorized into pro‐inflammatory M1 macrophages (M1s) and anti‐inflammatory M2 macrophages (M2s). The M1:M2 ratio is significantly increased in PE women. However, the precise contribution of increased M1s to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that supplementation of RUPP rats with IL‐25, a cytokine that drives macrophage polarization to anti‐inflammatory M2s, will increase M2s, decrease mean arterial pressure (MAP), and improve fetal growth. On gestation day (GD) 14, timed pregnant 10‐12 week old Sprague Dawley rats underwent the RUPP or Sham procedure and a mini‐osmotic pump infusing IL‐25 (0.5µg/day) or vehicle was implanted i.p., in both groups. Carotid catheters were placed on GD18. On GD19, MAP was measured, animals were euthanized, and blood and tissues were collected for analysis. Flow cytometry analysis was performed on placental and renal tissues. M2 macrophages (CD3‐/CD68+/CD163+) were markedly reduced in placentas of RUPP rats compared to Sham (0.2% vs. 4.2% of total macrophages) and in kidneys of RUPP rats vs. Sham (0.19% vs. 1.445% total of macrophages). IL‐25 supplementation elevated M2 populations in placental (3.985%) and renal (5.985%) tissues of RUPP. Likewise, IL‐25 treatment increased M2 macrophages in Sham placental (18.56%) and renal (12.99%) tissues. Increased MAP was observed in RUPP rats compared with Sham (123.5 mmHg vs. 106.5 mmHg, respectively) and was reduced in RUPP + IL‐25 (107.5 mmHg). No changes were observed in MAP of Sham rats treated with IL‐25. Fetal weights were reduced in RUPP vs. Sham (2.035g vs. 2.405g, respectively) and were normalized in RUPP after IL‐25 supplementation (2.425g). IL‐25 supplementation had no effect on fetal weight of Sham rats. Additional analyses will include assessment of blood and tissue cytokines and reactive oxygen species, and vascular function in all groups. In conclusion, IL‐25 supplementation increased placental and renal anti‐inflammatory M2 macrophage populations, reduced MAP, and improved fetal weights in RUPP recipients. These data suggest that macrophage activation is a key contributor to PE pathophysiology and may represent a therapeutic target to improve maternal and fetal outcomes in PE.

Interferon γ neutralization reduces blood pressure, uterine artery resistance index, and placental oxidative stress in placental ischemic rats.

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction, and increased cytolytic natural killer cells (cNKs), which secrete interferon γ (IFNγ). However, the precise role of IFNγ in contributing to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that neutralization of IFNγ in RUPPs will decrease placental reactive oxygen species (ROS) and improve vascular function resulting in decreased MAP and improved fetal growth. On gestation day (GD) 14, the RUPP procedure was performed and on GDs 15 and 18, a subset of normal pregnant rats (NP) and RUPP rats were injected with 10 μg/kg of an anti-rat IFNγ monoclonal antibody. On GD 18, uterine artery resistance index (UARI) was measured via Doppler ultrasound and on GD 19, mean arterial pressure (MAP) was measured, animals were euthanized, and blood and tissues were collected for analysis. Increased MAP was observed in RUPP rats compared with NP and was reduced in RUPP + anti-IFNγ. Placental ROS was also increased in RUPP rats compared with NP rats and was normalized in RUPP + anti-IFNγ. Fetal and placental weights were reduced in RUPP rats, but were not improved following anti-IFNγ treatment. However, UARI was elevated in RUPP compared with NP rats and was reduced in RUPP + anti-IFNγ. In conclusion, we observed that IFNγ neutralization reduced MAP, UARI, and placental ROS in RUPP recipients. These data suggest that IFNγ is a potential mechanism by which cNKs contribute to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE.

Perivascular adipose tissue (PVAT)‐mediated buffering of vasoconstriction in placental ischemia‐induced hypertensive rats

Preeclampsia is a pregnancy-specific hypertensive disorder diagnosed along with systemic endothelial and vascular dysfunction. A novel regulator of vascular tone is the perivascular adipose tissue (PVAT) that surrounds most blood vessels and functions to blunt vasoconstriction. However, it is unknown whether PVAT becomes dysfunctional in preeclampsia. Preeclampsia arises from placental ischemia to elicit release of pro-vasoconstrictive factors into the maternal circulation. In this study, it was hypothesized that placental ischemic rats have attenuated PVAT-mediated buffering of vasoconstriction. Placental ischemia was induced on gestational day 14 in Sprague-Dawley rats by conducting the Reduced Uterine Perfusion Pressure (RUPP) protocol. Conscious blood pressure measurements (carotid catheter) and wire myography were conducted on day 19. Mean arterial blood pressure was significantly elevated (P<0.05) in RUPP (N=6, 117±3 mmHg) vs. Pregnant (N=6, 97±3 mmHg) rats. In aortas, the maximum response (as % increase in force from baseline tension) to KCl-induced depolarization was not affected (P>0.05) by RUPP or the presence of PVAT: Pregnant+PVAT (43±9%) vs. Pregnant-PVAT (42±5%) and RUPP+PVAT (42±%7) vs. RUPP-PVAT (48±7%). Furthermore, phenylephrine (Phe, alpha1-adrenergic receptor agonist)-induced vasoconstriction was similar (P>0.05) in Pregnant+PVAT (69±11%) vs. Pregnant-PVAT (70±12%) and RUPP+PVAT (38±12%) vs. RUPP-PVAT (34±9%), with the 2-way ANOVA detecting a main effect only for RUPP (P<0.05). It was then assessed whether the endothelium controls PVAT function whereby denuding the endothelium did not impact (P>0.05) any of the respective Phe responses in Pregnant+PVAT-Endo (60±12%), Pregnant-PVAT-Endo (72±11%), RUPP+PVAT-Endo (30±15%), or RUPP-PVAT-Endo (22±7%). In smaller, third-order mesenteric arteries, direct depolarization by KCl responses were similar (P>0.05) between Pregnant+PVAT (176±19%), Pregnant-PVAT (149±17%), RUPP+PVAT (148±30%), and RUPP-PVAT (167±27%). As for Phe, the 2-way ANOVA identified a main effect (P<0.05) only for PVAT to blunt Phe-induced vasoconstriction in Pregnant+PVAT (259±38%) vs. Pregnant-PVAT (337±26%) and RUPP+PVAT (176±39%) vs. RUPP-PVAT (379±47%), with no difference for the influence of PVAT between RUPP and Pregnant groups. Removal of the endothelium further attenuated Phe responses in the PVAT groups: Pregnant+PVAT-Endo (174±66%) and RUPP+PVAT-Endo (-3±3%) vs. their respective groups without PVAT: Pregnant-PVAT-Endo (379±39%) and RUPP-PVAT-Endo (339±167%). In fact, the degree of this attenuation to Phe-induced vasoconstriction was the greatest (P<0.05) in the RUPP+PVAT-Endo (-104±3%) group compared all other groups: RUPP-PVAT-Endo (19±59%), Pregnant+PVAT-Endo (10±11%), and Pregnant-PVAT-Endo (10±11%). In conclusion, as it was found that removal of the endothelium allows for enhanced PVAT blunting of vasoconstriction in RUPP rats, these data indicate that placental ischemia drives endothelial dysfunction to subsequently disrupt PVAT function in small arteries during pregnancy.

Neutralization of Natural Killer Cell Associated Cytokines Improves Vascular Function and Reduces Blood Pressure in Placental Ischemic Rats

Women with preeclampsia (PE) have increased cytolytic NK cells (cNKs), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in the circulation and placenta. In previous studies, adoptive transfer of placental cNKs from the Reduced Uterine Perfusion Pressure (RUPP) model of PE into normal pregnant Sprague Dawley (SD) rats caused increased IFNγ and TNFα, and induced vascular dysfunction, hypertension (HTN), and restricted fetal growth (IUGR) similar to what is observed in PE patients. The purpose of this study was to examine the roles of IFNγ and TNFα in RUPP and PE pathophysiology. We hypothesized that neutralization of IFNγ or TNFα would attenuate PE-associated pathophysiology in RUPP rats. Pregnant SD rats underwent RUPP or Sham procedure on Gestation Day (GD) 14. On GD 15 and 18, a subset of Sham and RUPP rats received vehicle or 10μg/kg anti-IFNγ monoclonal antibody (IFNy-ab; n=9/group) i.p. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound and on GD19, mean arterial pressure (MAP), fetal and placental weight were measured, and blood and tissues were processed for analysis. MAP was 105±2 mmHg and 108±2 mmHg in Sham and Sham+IFNγ-ab respectively, was elevated to 125±1 mmHg in RUPP (p<0.05 vs Sham), and reduced to 112±2 mmHg in RUPP+ IFNγ-ab (p<0.05 vs RUPP). Fetal and placental weights were significantly reduced in RUPP and remained so in RUPP+IFNγ-ab (p<0.05 vs Sham). UARI was 0.6±0.02 in Sham and Sham+ IFNγ-ab, was elevated to 0.73±0.02 in RUPP (p<0.05 vs Sham), and was normalized to 0.6±0.02 in RUPP+ IFNγ-ab (p<0.05 vs RUPP). Both placental ROS and plasma soluble Fms-like Tyrosine kinase 1 (sFlt-1) were elevated in RUPP (p<0.05 vs Sham) and were normalized in RUPP+IFNγ-ab (p<0.05 vs RUPP). In separate experiments, pregnant SD rats underwent RUPP or Sham procedure on GD 14. On GD 15 and 18, a subset of Sham and RUPP rats received vehicle or 0.4 mg/kg of Etanercept (ETA), a soluble TNFα receptor (n=10/group) i.p. Analyses were performed as described above. MAP was elevated in RUPP (127±2 mmHg) compared to Sham (105±3 mmHg) and Sham+ETA (109±2 mmHg, p<0.05) and was normalized to 109±3 mmHg in RUPP+ETA (p<0.05 vs RUPP). Both fetal and placental weights were significantly reduced in RUPP rats (p<0.05 vs Sham) and were normalized in RUPP+ETA (p<0.05 vs RUPP). UARI was 0.6±0.02 in Sham and 0.6±0.01 in Sham+ETA. UARI was elevated in RUPP (0.72±0.02, p<0.05 vs Sham) and was normalized to 0.61±0.02 in RUPP+ETA (p<0.05 vs RUPP). Plasma total nitrate was significantly reduced in RUPP (p<0.05 vs Sham) and was normalized in RUPP+ETA (p<0.05 vs RUPP). Placental ROS was elevated in RUPP (p<0.05 vs Sham) and remained so in RUPP+ETA (p>0.05 vs RUPP). Plasma sFlt-1 was 92±34 pg/mL in Sham and 53±9 pg/mL in Sham+ETA, was elevated to 1039±211 pg/mL in RUPP (p<0.05 vs Sham), and was normalized to 145±33 pg/mL in RUPP+ETA (p<0.05 vs RUPP). The results of this study suggest that increased secretion of IFNγ and TNFα by cNKs mediate vascular dysfunction to cause maternal HTN and IUGR in RUPP and PE pathophysiology. IFNγ and TNFα are potential therapeutic targets for treatment in PE.

Angiotensin II type 1 receptor autoantibody blockade improves cerebral blood flow autoregulation and hypertension in a preclinical model of preeclampsia

ABSTRACT Introduction:Women with preeclampsia (PE) and reduced uterine perfusion pressure (RUPP) pre-clinical rat model of PE have elevated angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA) and cerebrovascular dysfunction. Methods:Sprague Dawley rats had RUPP surgery with/without AT1-AA inhibitor (‘n7AAc’144 μg/day) osmotic minipumps. Mean arterial pressure (MAP), CBF autoregulation, blood brain barrier (BBB) permeability, cerebral edema, oxidative stress, and eNOS were assessed. Results:‘n7AAc’ improved MAP, restored CBF autoregulation, prevented cerebral edema, elevated oxidative stress, and increased phosphorylated eNOS protein in RUPP rats. Conclusion:Inhibiting the AT1-AA in placental ischemic rats prevents hypertension, cerebrovascular dysfunction, and improves cerebral metabolic function.

Interleukin‐17 Impairs Cerebrovascular Function, Increases Blood‐Brain‐Barrier Permeability, and Induces Cerebral Edema in Pregnant Rats

Cerebrovascular events account for ~40% of all preeclamptic/eclamptic‐related deaths and can induce neurological complications. While placental ischemia can impair cerebrovascular function in women with preeclampsia (PE) and rats which develop a PE‐like syndrome, the placental‐derived factors mediating these effects are elusive. Although pro‐inflammatory cytokines, including Interleukin‐17 (IL‐17), are increased by placental ischemia, it is not known whether IL‐17 disrupts cerebrovascular function during pregnancy. Therefore, we tested the hypothesis that infusion of IL‐17 during pregnancy impairs cerebral blood flow (CBF) autoregulation and myogenic tone, while also promoting increased regional blood‐brain‐barrier (BBB) permeability and brain water content (BWC). Pregnant Sprague Dawley rats were infused with recombinant IL‐17 (150 pg/day) via mini‐osmotic pump (i.p.) from gestational days 14–19. Cerebral blood flow was measured by laser Doppler flowmetry and myogenic tone was assessed in isolated middle cerebral arteries (MCA) by pressure‐flow myography. BWC was determined by quantifying dry:wet weight ratio and BBB permeability was measured by Evans blue extravasation. As previously shown, IL‐17 increased MAP (112 ± 2.5 vs 99.7 ± 2.8 mmHg; p&lt;0.01) compared to pregnant control rats. CBF was significantly greater in IL‐17‐treated pregnant rats at 180 mmHg (187 ± 19 vs 149 ± 22%; p&lt;0.05) and 190 mmHg (215 ± 18 vs 160 ± 23%; p&lt;0.01) compared to pregnant control rats. Myogenic tone peaked at 125 mmHg in the MCA of IL‐17‐treated rats and declined thereafter, forming an inverted‐U curve in response to increases in intraluminal pressure. In contrast, myogenic tone peaked at 175 mmHg in control pregnant rats, forming a hyperbolic curve, which suggests that myogenic responses are blunted at high arterial pressures in IL‐17‐treated rats. BWC tended to increase in the cortex (80.5 ± 0.2 vs. 79.5 ± 0.4%, p = 0.054) and striatum (77.6 ± 0.8 vs. 75.1 ± 1.4%, p = 0.062), and was significantly increased in the posterior cerebrum (79.7 ± 0.4 vs. 77.8 ± 0.5%, p&lt; 0.01) and cerebrum (79.8 ± 0.3 vs. 78.8 ± 0.1%, p&lt; 0.01) of IL‐17‐treated rats. IL‐17 increased BBB permeability in the cortex only (0.017 ± 0.002 vs 0.012 ± 0.002, p&lt;0.05). These data suggest that increases in circulating IL‐17 during pregnancy could contribute to cerebrovascular dysfunction leading to impaired CBF control and myogenic responses, cerebral edema, and increased BBB permeability. IL‐17 may be a potential therapeutic target to prevent or ameliorate neurological impairment in preeclampsia. Future studies examining whether blockade of IL‐17 in placental ischemic rats could prevent cerebrovascular dysfunction may provide valuable insight into the role of cytokines on cerebrovascular health during pregnancy.Support or Funding InformationFunding: R01HL12186106, R01HL136684‐02, R01HL136684, P01HL051971, P20GM104357, 5U54GM115428, T32HL105324, and 19POST34450074.

Abstract P1110: Tumor Necrosis Factor Alpha Blockade Improves Natural Killer Cell Activation, Hypertension, and Mitochondrial Oxidative Stress in a Preclinical Rat Model of Preeclampsia

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&lt;0.05) which was reduced to 110 ± 2 in RUPP + LD ETAN (p&lt;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&lt;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&lt;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&lt;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, &amp; P20GM121334

TNFα Impairs Cerebral Blood Flow Autoregulation in Pregnant Rats

Neurological symptoms are often reported among preeclamptic patients and cerebrovascular events account for ~40% of all preeclamptic/eclamptic‐related deaths. While cerebral blood flow (CBF) autoregulation is impaired in preeclamptic (PE) women as well as in placental ischemic rats which develop a PE‐like syndrome, the underlying mechanisms responsible for abnormal CBF autoregulation are unclear. Placental ischemic rats have higher levels of circulating cytokines, and we previously reported that tumor necrosis factor – alpha (TNFα), at levels that mimic TNFα in human preeclampsia, increases blood brain barrier (BBB) permeability and cerebral edema in pregnant rats. Therefore, we tested the hypothesis that TNFα alters CBF autoregulation in pregnant rats. To test this hypothesis, normal pregnant Sprague Dawley rats were infused with TNFα (200ng/day) and cerebral blood flow was measured by laser Doppler flowmetry under anesthesia. Rats were intubated and ventilated in order to maintain constant blood gasses and mean arterial pressure was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF. TNFα was found to impair CBF autoregulation in pregnant rats where CBF was significantly increased in TNFα‐infused rats at 180 mmHg (225±13 vs 173±8) and 190 mmHg (253±17 vs 188±9) compared to normal pregnant rats with no significant difference in peak CO2 between the two groups at 180 mmHg (44.9±0.8 vs 45.4±0.9 mmHg) or 190 mmHg (43±0.8 vs 45.3±0.9). Autoregulatory index (percent change in blood flow divided by percent change in perfusion pressure) was also significantly increased in TNFα‐infused rats at 180 mmHg (1.56±0.17 vs 0.91±0.11) and 190 mmHg (1.7±0.07 vs 0.99±0.11) compared to normal pregnant rats. These data suggest that circulating TNFα from the ischemic placenta could contribute to cerebrovascular complications during pregnancy and postpartum. TNFα could serve as a novel pharmacological target to ameliorate CBF autoregulation in preeclampsia.Support or Funding InformationThis study was supported by National Institutes of Health Grants, HLHL51971, HL136684‐A0, and an NRSA Institutional Training Grant, T32HL105324.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Placental Ischemia Leads to Regional Astrocyte Activation: Role of TNFα

Preeclampsia, a hypertensive disorder of pregnancy, can quickly progress to eclampsia when seizure develops in the peri-partum period. We have shown that the preclinical rat model of preeclampsia, induced by surgical reduction of uterine perfusion pressure (RUPP), has reduced latency to drug induced seizures and cerebral edema. The increased seizure sensitivity was associated with increased cerebrospinal fluid (CSF) cytokines, an indicator of tissue inflammation and potential activation of neuroglial cells. Moreover, other labs have shown that treatment of brain slices with serum from pregnant women induced increased neuronal activity and neuro-inflammation through TNFα. Thus, we tested the hypothesis that placental ischemia induces regional astrocyte activation that can be reversed by pharmacological blockade of TNFα. On GD14, time pregnant rats were randomly assigned to normal pregnant (NP, n=6) or RUPP (n=6) groups and further assigned to no treatment or etanercept injection (0.8 mg/kg, sc) on GD18. RUPP rats underwent surgery on GD14 to induce placental ischemia by inserting silver clips around the abdominal aorta and each of the uterine artery branches. Normal pregnant rats did not undergo surgery. Brains were harvested on GD19 and processed for immunofluorescence staining. Anterior brain was cut into 20 μm thick sections and stained for glial fibrillary acidic protein (GFAP), an astrocyte marker. Z-stacks (0.5 μm steps) were obtained and percent area of GFAP positive astrocytes was analyzed using Image J in the anterior commissure, corpus callosum, cortex, and caudate putamen. Two-Way Analysis of Variance revealed a significant increase in the percent area of astrocytes in the caudate putamen in RUPP rats [RUPP: 5.05 ± 1.09%; NP: 1.32 ± 0.28% (P = 0.01)] which was reduced by etanercept (2.11 ± 0.85%). There were no significant changes in % area in the anterior commissure, corpus callosum, or cortex (P> 0.05). Increased astrocyte area in the caudate putamen was associated with higher number of astrocytes [NP: 7 ± 1; RUPP: 13 ± 1 (p<0.01)] which was unchanged by etanercept (12 ± 3 cells). Our results are consistent with the hypothesis that placental ischemia induces cytotoxic edema (cell swelling) in the anterior cerebrum through regional astrocyte activation (caudate putamen). Moreover, increased circulating TNFα in placental ischemic rats may contribute to astrocyte activation, and in turn induce the release of TNFα and other pro-inflammatory cytokines, increasing seizure susceptibility and cytotoxic edema. Additional analyses of astrocytic morphology are ongoing and the direct role of pro-inflammatory cytokines on seizure susceptibility during pregnancy are areas of future study. Support or Funding Information NIH K99/R00HL129192, COBRE Pilot Grant, P20GM104357, P01HL051971 Representative images of GFAP+ astrocytes in the Caudate Putamen (CP) taken at 60X. Graph shows % area covered by astrocytes for each group. Individual points represent average data per section (1–3 images - 2 sections per rat). NP-normal pregnant, RUPP - reduced uterine perfusion pressure. *p<0.05, **p<0.01. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Maternally Sequestered, Biopolymer-Stabilized Vascular Endothelial Growth Factor (VEGF) Chimera for Treatment of Preeclampsia.

BackgroundPreeclampsia is a hypertensive syndrome that complicates 3% to 5% of pregnancies in the United States. Preeclampsia originates from an improperly vascularized and ischemic placenta that releases factors that drive systemic pathophysiology. One of these factors, soluble fms‐like tyrosine kinase‐1, is believed to sequester vascular endothelial growth factor (VEGF), leading to systemic endothelial dysfunction and hypertension. With the goal of targeting soluble fms‐like tyrosine kinase‐1 while simultaneously preventing fetal exposure to VEGF, we fused VEGF to elastin‐like polypeptide, a biopolymer carrier that does not cross the placental barrier (ELP‐VEGF).Methods and Results ELP‐VEGF restored in vitro endothelial cell tube formation in the presence of plasma from placental ischemic rats. Long‐term administered ELP‐VEGF in pregnant rats accumulated in maternal kidneys, aorta, liver, and placenta, but the protein was undetectable in the pups when administered at therapeutic doses in dams. Long‐term administration of ELP‐VEGF in a placental ischemia rat model achieved dose‐dependent attenuation of hypertension, with blood pressure equal to sham controls at a dose of 5 mg/kg per day. ELP‐VEGF infusion increased total plasma soluble fms‐like tyrosine kinase‐1 levels but dramatically reduced free plasma soluble fms‐like tyrosine kinase‐1 and induced urinary excretion of nitrate/nitrite, indicating enhanced renal nitric oxide signaling. ELP‐VEGF at up to 5 mg/kg per day had no deleterious effect on maternal or fetal body weight. However, dose‐dependent adverse events were observed, including ascites production and neovascular tissue encapsulation around the minipump.Conclusions ELP‐VEGF has the potential to treat the preeclampsia maternal syndrome, but careful dosing and optimization of the delivery route are necessary.

Abstract P263: Interleukin-17 Infusion Promotes Cerebral Edema and Increased Blood Brain Barrier Permeability in Pregnant Rats

While cerebrovascular abnormalities such as cerebral edema and increased blood-brain barrier (BBB) permeability are a common complication of (pre)eclampsia, the exact mechanisms underlying these abnormalities remain unclear. One potential mechanism could be increased Interleukin-17 (IL-17), a pro-inflammatory cytokine that is increased in both preeclampsia patients and the rat model of placental ischemia created by reducing uterine perfusion pressure. While IL-17 has been linked to the pathogenesis of preeclampsia, its effect on cerebrovascular function during pregnancy is unknown. Thus, we tested the hypothesis that increasing circulating IL-17 during pregnancy promotes regional increases in brain water content and BBB permeability. Recombinant mouse IL-17 (150 pg/day) was infused via mini-osmotic pump (i.p) from gestational day 14 to 19 in pregnant rats (n = 5-7 per group). Brain water content was assessed using the dry weight to wet weight ratio and BBB permeability was assessed by quantifying Evans blue extravasation into the anterior cerebrum, hippocampus, cortex, striatum, posterior cerebrum, and cerebrum. As reported previously, IL-17 infusion resulted in a significant increase in mean arterial pressure (119 ± 3 vs. 104 ± 4 mmHg in normal pregnant group; p = 0.010). Water content tended to be increased in the cortex (80.5 ± 0.2 vs. 79.5 ± 0.4%, p = 0.054) and striatum (77.6 ± 0.8 vs. 75.1 ± 1.4%, p = 0.062) and was significantly increased in the posterior cerebrum (79.7 ± 0.4 vs. 77.8 ± 0.5%, p = 0.007) and cerebrum (79.8 ± 0.3 vs. 78.8 ± 0.1%, p = 0.005) of the IL-17 treated group. BBB permeability was increased only in the cortex (0.017 ± 0.002 vs 0.012 ± 0.002, p = 0.046) of rats receiving IL-17 infusion. These data suggest that increased IL-17 during pregnancy contributes to edema formation and increased BBB permeability in the cerebral cortex and may be a therapeutic target in preeclampsia. Future studies will determine whether reducing IL-17 levels in placental ischemic rats will prevent these cerebrovascular changes.

Magnesium Sulfate Prevents Placental Ischemia-Induced Increases in Brain Water Content and Cerebrospinal Fluid Cytokines in Pregnant Rats.

Magnesium sulfate (MgSO4) is the most widely used therapy in the clinic to prevent the progression of preeclampsia, a hypertensive disorder of pregnancy, to eclampsia. Eclampsia, manifested as unexplained seizures and/or coma during pregnancy or postpartum, accounts for ~13% of maternal deaths worldwide. While MgSO4 continues to be used in the clinic, the mechanisms by which it exerts its protective actions are not well understood. In this study, we tested the hypothesis that MgSO4 protects against placental ischemia-induced increases in brain water content and cerebrospinal fluid cytokines. To test this hypothesis, MgSO4 was administered via mini-osmotic pump (60 mg/day, i.p.) to pregnant and placental ischemic rats, induced by mechanical reduction of uterine perfusion pressure, from gestational day 14–19. This treatment regimen of MgSO4 led to therapeutic level of 2.8 ± 0.6 mmol/L Mg in plasma. MgSO4 had no effect on improving placental ischemia-induced changes in mean arterial pressure, number of live fetuses, or fetal and placental weight. Placental ischemia increased, while MgSO4 prevented the increase in water content in the anterior cerebrum. Cytokine and chemokine levels were measured in the cerebrospinal fluid using a multi-plex assay. Results demonstrate that cerebrospinal fluid, obtained via the cisterna magna, had reduced protein, albumin, interleukin (IL)-17A, IL-18, IL-2, eotaxin, fractalkine, interferon gamma, vascular endothelial growth factor (VEGF), and macrophage inflammatory protein (MIP)-2 following MgSO4 treatment. These data support the hypothesis that MgSO4 offers neuroprotection by preventing placental ischemia-induced cerebral edema and reducing levels of cytokines/chemokines in the cerebrospinal fluid.

Clinical Implications

Clinical Implications

Placental ischemia increases seizure susceptibility and cerebrospinal fluid cytokines.

Eclampsia is diagnosed in preeclamptic patients who develop unexplained seizures and/or coma during pregnancy or postpartum. Eclampsia is one of the leading causes of maternal and infant morbidity and mortality, accounting for ∼13% of maternal deaths worldwide. Little is known about the mechanisms contributing to the pathophysiology of eclampsia, partly due to the lack of suitable animal models. This study tested the hypothesis that placental ischemia, induced by reducing utero-placental perfusion, increases susceptibility to seizures, cerebrospinal fluid (CSF) inflammation, and neurokinin B (NKB) expression in brain and plasma. Pentylenetetrazol (PTZ), a pro-convulsive drug, was injected into pregnant and placental ischemic rats (40 mg/kg, i.p.) on gestational day 19 followed by video monitoring for 30 min. Seizure scoring was blindly conducted. Placental ischemia hastened the onset of seizures compared to pregnant controls but had no effect on seizure duration. Placental ischemia increased CSF levels of IL-2, IL-17, IL-18 and eotaxin (CCL11), had no effect on plasma NKB; however, PTZ increased plasma NKB in both pregnant and placental ischemic rats. NKB was strongly correlated with latency to seizure in normal pregnant rats (R2 = 0.88 vs. 0.02 in placental ischemic rats). Lastly, NKB decreased in the anterior cerebrum in response to placental ischemia and PTZ treatment but was unchanged in the posterior cerebrum. These data demonstrate that placental ischemia is associated with increased susceptibility to seizures and CSF inflammation; thus provides an excellent model for elucidating mechanisms of eclampsia-like symptoms. Further studies are required to determine the role of CSF cytokines/chemokines in mediating increased seizure susceptibility.

Placental ischemia-induced increases in brain water content and cerebrovascular permeability: role of TNF-α.

Cerebrovascular complications and increased risk of encephalopathies are characteristic of preeclampsia and contribute to 40% of preeclampsia/eclampsia-related deaths. Circulating tumor necrosis factor-α (TNF-α) is elevated in preeclamptic women, and infusion of TNF-α into pregnant rats mimics characteristics of preeclampsia. While this suggests that TNF-α has a mechanistic role to promote preeclampsia, the impact of TNF-α on the cerebral vasculature during pregnancy remains unclear. We tested the hypothesis that TNF-α contributes to cerebrovascular abnormalities during placental ischemia by first infusing TNF-α in pregnant rats (200 ng/day ip, from gestational day 14 to 19) at levels to mimic those reported in preeclamptic women. TNF-α increased mean arterial pressure (MAP, P < 0.05) and brain water content in the anterior cerebrum (P < 0.05); however, TNF-α infusion had no effect on blood-brain barrier (BBB) permeability in the anterior cerebrum or posterior cerebrum. We then assessed the role of endogenous TNF-α in mediating these abnormalities in a model of placental ischemia induced by reducing uterine perfusion pressure followed by treatment with the soluble TNF-α receptor (etanercept, 0.8 mg/kg sc) on gestational day 18. Etanercept reduced placental ischemia-mediated increases in MAP, anterior brain water content (P < 0.05), and BBB permeability (202 ± 44% in placental ischemic rats to 101 ± 28% of normal pregnant rats). Our results indicate that TNF-α mechanistically contributes to cerebral edema by increasing BBB permeability and is an underlying factor in the development of cerebrovascular abnormalities associated with preeclampsia complicated by placental ischemia.

Abstract 127: Agonistic Autoantibodies to Angiotensin II Type I Receptor Contributes Partly to Placental Ischemia-induced Cerebrovascular Abnormalities

Placental ischemia, a characteristic feature of preeclampsia, leads to impaired cerebral blood flow (CBF) autoregulation, cerebral edema, and increased blood-brain barrier (BBB) permeability; however, the placental factors that contribute to these cerebral abnormalities are not clear. Agonistic autoantibodies to the angiotensin II type 1 receptor (AT1-AA) are increased in preeclamptic patients as well as in a rat model of preeclampsia induced by placental ischemia. In this study, we tested the hypothesis that AT1-AA mediates placental ischemia-induced cerebrovascular abnormalities. To determine whether the AT1-AA contributes to impaired CBF autoregulation, we infused purified rat AT1-AA into normal pregnant rats from gestational day (GD) 12 to 19 via mini-osmotic pumps and measured CBF using laser Doppler flowmetry on GD 19. Autoregulatory index increased from 0.7 to 1.0±0.2 in the AT1-AA infused group over the range of 120 to 160 mmHg compared to pregnant controls (0.3 to 0.5±0.1 over the same range of pressures, p&lt;0.05) suggesting impaired CBF autoregulation. However, AT1-AA infusion did not affect brain water content at baseline blood pressures (104±2 mmHg in normal pregnant rats vs. 113±2 mmHg in AT1-AA infused rats, p&lt;0.01). To determine the role of endogenous AT1-AA in mediating placental ischemia-induced cerebrovascular abnormalities, losartan (5 mg/kg/day), an AT1 receptor antagonist, was administered in the drinking water from GD 14 to 19. Losartan reduced anterior brain water content from 79.6±0.2% in placental ischemic rats to 79.2±0.1% (compared to 79.1±0.1% in normal pregnant untreated rats) and BBB permeability from 0.06±0.01 in placental ischemic rats to 0.03±0.03 (compared to 0.03±0.004 in normal pregnant untreated rats). These results indicate that impaired CBF autoregulation in response to placental ischemia is due, at least in part, to increases in circulating AT1-AA. While AT1-AA infusion, by itself, did not alter brain water content at baseline blood pressures, the beneficial effects of losartan in placental ischemic rats suggests that the renin-angiotensin system may interact with other placental factors to promote cerebral vascular changes common to preeclampsia.

Placental Ischemia Impairs Cerebral Blood Flow Autoregulation and Increases Blood‐Brain Barrier Permeability in Pregnant Rats

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia related deaths, and neurological symptoms are common among preeclamptic patients. We previously reported that placental ischemia, induced by reducing utero‐placental perfusion pressure, leads to impaired cerebrovascular myogenic reactivity and cerebral edema in the pregnant rat. Whether the impaired myogenic reactivity is associated with altered cerebral blood flow (CBF) autoregulation and the edema is due to altered blood‐brain barrier (BBB) permeability is unclear. Therefore, we tested the hypothesis that placental ischemia leads to impaired CBF autoregulation and a disruption of the BBB. CBF autoregulation, measured in vivo by laser Doppler flowmetry, was significantly impaired in placental ischemic rats compared to normal pregnant rats. Brain water content was increased in the anterior cerebrum of placental ischemic rats and BBB permeability, assayed using the Evans blue extravasation method, was also increased in the anterior cerebrum. The expression of the tight junction proteins: claudin‐1 was increased in the posterior cerebrum while zonula occludens‐1, and occludin, were not significantly altered in either the anterior or posterior cerebrum. These results are consistent with the hypothesis that placental ischemia mediates anterior cerebral edema through impaired CBF autoregulation and the associated increased transmission of pressure to microvessels, resulting in increased BBB permeability and cerebral edema.

Abstract W P389: Placental Ischemia Increases Seizure Susceptibility in Pregnant Rats

Eclampsia is diagnosed in preeclamptic patients who develop convulsions and/or unexplained coma during pregnancy or postpartum. It is one of the leading causes of maternal and infant morbidity and mortality accounting for about 13% of all maternal deaths worldwide. While several factors contributing to the pathogenesis of preeclampsia have been identified, little is known about the mechanisms contributing to the pathophysiology of eclampsia, partly due to the lack of suitable animal models of eclampsia. The aim of this study was to test the hypothesis that placental ischemia, induced by reducing utero-placental perfusion, increases susceptibility to seizures and cerebral edema. Pentylenetetrazol (PTZ), a pro-convulsive drug, was injected into pregnant and placental ischemic rats (40 mg/kg, i.p.) on gestational day 19 followed by video monitoring of rats for seizure activity for a duration of 30 minutes. Seizure scoring was blindly conducted. Results indicate the placental ischemic rats (n=8) have reduced latency to first seizure (264.5 ± 78.7s) compared to normal pregnant rats (n=7) (835.7 ± 246.6s; p &lt; 0.05). Placental ischemia increased brain water content in the cerebrum (78.8 ± 0.1%) compared to the normal pregnant group (78.4 ± 0.1%; p &lt; 0.05) while PTZ treatment significantly increased brain water content in both the pregnant (78.8 ± 0.1%; p &lt; 0.05) and placental ischemic group (78.9 ± 0.1%; p &lt; 0.01). These data demonstrate that placental ischemia is associated with increased susceptibility to seizures and edema formation; thus the rat model of placental ischemia represents an excellent model for studying mechanisms of eclampsia-like symptoms. Further studies are required to elucidate the exact mechanisms contributing to increased seizure susceptibility following placental ischemia.

Placental ischemia in pregnant rats impairs cerebral blood flow autoregulation and increases blood-brain barrier permeability.

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia‐related deaths, and neurological symptoms are common among preeclamptic patients. We previously reported that placental ischemia, induced by reducing utero‐placental perfusion pressure, leads to impaired myogenic reactivity and cerebral edema in the pregnant rat. Whether the impaired myogenic reactivity is associated with altered cerebral blood flow (CBF) autoregulation and the edema is due to altered blood–brain barrier (BBB) permeability remains unclear. Therefore, we tested the hypothesis that placental ischemia leads to impaired CBF autoregulation and a disruption of the BBB. CBF autoregulation, measured in vivo by laser Doppler flowmetry, was significantly impaired in placental ischemic rats. Brain water content was increased in the anterior cerebrum of placental ischemic rats and BBB permeability, assayed using the Evans blue extravasation method, was increased in the anterior cerebrum. The expression of the tight junction proteins: claudin‐1 was increased in the posterior cerebrum, while zonula occludens‐1, and occludin, were not significantly altered in either the anterior or posterior cerebrum. These results are consistent with the hypothesis that placental ischemia mediates anterior cerebral edema through impaired CBF autoregulation and associated increased transmission of pressure to small vessels that increases BBB permeability leading to cerebral edema.