Neutrophil Depletion Attenuates Placental Ischemia-Induced Hypertension in the Rat

Abstract

Preeclampsia is characterized by reduced placental perfusion with placental ischemia and hypertension during pregnancy. Preeclamptic women also exhibit a heightened inflammatory state and greater number of neutrophils in the vasculature compared to normal pregnancy. Since neutrophils are associated with tissue injury and inflammation, we hypothesized that neutrophils are critical to placental ischemia-induced hypertension and fetal demise. Using the reduced uteroplacental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in the rat, we determined the effect of neutrophil depletion on blood pressure and fetal resorptions. Neutrophils were depleted with repeated injections of polyclonal rabbit anti-rat polymorphonuclear leukocyte (PMN) antibody (antiPMN). Rats received either antiPMN or normal rabbit serum (Control) on 13.5, 15.5, 17.5, and 18.5 days post conception (dpc). On 14.5 dpc, rats underwent either Sham surgery or clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure (RUPP). On 18.5 dpc, carotid arterial catheters were placed and mean arterial pressure (MAP) was measured on 19.5 dpc. Neutrophil-depleted rats had reduced circulating neutrophils from 14.5 to 19.5 dpc compared to Control, as well as decreased neutrophils in lung and placenta on 19.5 dpc. MAP increased in RUPP Control vs Sham Control rats, and neutrophil depletion attenuated this increase in MAP in RUPP rats without any effect on Sham rats. The RUPP-induced increase in fetal resorptions and complement activation product C3a were not affected by neutrophil depletion. Thus, these data are the first to indicate that neutrophils play an important role in RUPP hypertension and that cells of the innate immune system may significantly contribute to pregnancy-induced hypertension.

Fig 1. Experimental Design.

Timing of injections, blood sampling and surgeries with days post conception (dpc) in timed pregnant Sprague Dawley rats. Necropsy and measurement of mean arterial pressure (MAP) occurred on 19.5 dpc.

Fig 2. Neutrophil depletion significantly attenuates placental ischemia-induced increase in mean arterial pressure (MAP).

Animals were treated with normal rabbit serum (Control) or antiPMN antibody from 13.5–18.5 dpc. The increase in MAP in RUPP Control (n = 14) compared to Sham Control (n = 5) was significantly inhibited by neutrophil depletion (RUPP antiPMN, n = 9). MAP did not differ between Sham antiPMN (n = 5) and Sham Control groups. Values represent mean ± SE of MAP measured 19.5 dpc. *p<0.05 for indicated comparison.

Fig 3. Neutrophil depletion does not affect placental ischemia-induced fetal resorptions.

Animals were treated with normal rabbit serum (Control) or antiPMN antibody from 13.5–18.5 dpc. The increase in fetal resorptions in RUPP Control (n = 14) compared to Sham Control (n = 6) was not affected by neutrophil depletion (RUPP antiPMN, n = 9). Fetal resorptions did not differ between Sham antiPMN (n = 6) and Sham Control groups. Values represent mean ± SE of the fraction of resorbed fetuses on 19.5 dpc of gestation. *p<0.05 for indicated comparison.

Fig 4. Neutrophil depletion does not affect placental ischemia-induced increase in C3a.

Animals were treated with normal rabbit serum (Control) or antiPMN antibody from 13.5–18.5 dpc. *p<0.05 for indicated comparison. The increase in serum C3a in RUPP Control (n = 10) compared to Sham Control (n = 4) was not affected by neutrophil depletion (RUPP antiPMN, n = 7). C3a did not significantly differ between Sham antiPMN (n = 4) and Sham Control groups. Values represent geometric mean ± SE of C3a units/ μl in serum obtained 19.5 dpc. Units of C3a are relative to a standard pool of yeast activated rat serum as described in Methods.

Fig 5. Treatment with antiPMN antibody 13.5–18.5 dpc reduces the percentage of circulating neutrophils.

Animals were treated with normal rabbit serum (Control) or antiPMN antibody as depicted by arrows. AntiPMN treatment significantly reduced circulating neutrophils compared to normal rabbit serum treatment. Values represent mean ± SE of % neutrophils in blood collected from 5–14 animals. Blood samples were taken prior to first injection (13.5 dpc) and prior to RUPP or Sham surgery (14.5 dpc), at 16.5 dpc, at 18.5 dpc with carotid cannulation and at necropsy (19.5 dpc).

Fig 6. Treatment with antiPMN antibody reduces lung myeloperoxidase (MPO) activity and reduces neutrophils in placenta.

Animals were treated with normal rabbit serum (Control) or antiPMN antibody from 13.5–18.5 dpc. *p<0.05 for antiPMN effect by analysis of variance. A). Lung MPO was used as an indicator of the number of neutrophils in the lung. AntiPMN treatment significantly reduced MPO compared to normal rabbit serum treatment. Values represent mean ± SE of Units of lung MPO activity per mg protein of lung homogenate in lungs collected from 5–14 animals on 19.5 dpc. B). AntiPMN treatment significantly reduced the number of neutrophils in placenta compared to normal rabbit serum treatment. Values represent mean ± SE of placental neutrophils per high-powered field in placenta collected from 3–6 animals on 19.5 dpc.