Hypertension Induced by Episodic Reductions in ...

Hypertension in Pregnancy, Early Online:1–13, 2010 Copyright © Informa UK Ltd. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2010.507853

Hypertension Induced by Episodic Reductions in Uteroplacental Blood Flow in Gravid Rat 1525-6065 1064-1955 LHIP Hypertension in Pregnancy Pregnancy, Vol. 1, No. 1, Jul 2010: pp. 0–0

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AorticetOcclusion Reho al. in Gravid Rat

John J. Reho,1,2 Jennifer Peck,2 Jacqueline Novak,3 and Rolando J. Ramirez1,2 1

Program in Integrated Biosciences, The University of Akron, Akron, OH, USA Department of Biology, The University of Akron, Akron, OH, USA 3 Department of Mathematics and Science, Walsh University, Canton, OH, USA 2

Background. The etiology of preeclampsia remains unclear. Animal modeling of preeclampsia has been useful; however, no model to date represents episodic changes in uteroplacental blood flow that may occur in preeclampsia. Objective. To develop a gravid rat model characterized by episodic reductions in uteroplacental blood flow. Method. Pregnant Sprague Dawley rats were used and subjected to SHAM, reduced uterine perfusion pressure (RUPP), or aortic occlusion on gestational Day 14. Aortic occlusion surgery consisted of implantation of a silastic vascular occluder around the abdominal aorta and silver clips around the uterine–ovarian arteries. Aortic occlusion animals were subjected to five consecutive days of occlusion (40% reduction) each session lasting 1 h. On Day 21, maternal mean arterial pressure (MAP) and fetal morphology were assessed. For isolated blood vessels, resistance-sized mesenteric arteries were harvested and mounted on a pressure arteriograph. Result. Occluder animals experienced a 10 mmHg rise in MAP as compared to SHAM (p < 0.05), and RUPP MAP was significantly increased as compared to control subjects (p < 0.05). Pups from Occluder animals exhibited a decrease in fetal weight as compared to SHAM (p < 0.05), but an increase in fetal weight as compared to RUPP (p < 0.05). Myogenic reactivity of second-order mesenteric arteries increased in Occluder animals as compared to SHAM (p < 0.05), but were similar to that of RUPP. Conclusion. Episodic reductions in uteroplacental blood flow play a crucial role in the altered vascular reactivity seen in Occluder animals and may represent a new model to investigate the mechanisms associated with episodic reductions in uteroplacental blood flow in pathological pregnancies. Keywords Preeclampsia, Vascular reactivity, Blood flow, Episodic, Aortic occlusion, Vasospasm.

INTRODUCTION In spite of the recent advances in our understanding of the pathologies associated with pregnancy, preeclampsia still remains one of the main causes of both maternal and fetal mortality throughout the world. Preeclampsia leads to high blood pressure, and renal and vascular dysfunction, as indicated by human studies (1). These facets of the disease appear to be related to injury deriving from a defective endothelium. Endothelial injury may cause

Address correspondence to Rolando J. Ramirez, Department of Biology, The University of Akron, Akron, OH 44325-3908, USA. E-mail: [email protected]


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increased peripheral resistance, vasospasm, and end organ underperfusion that occurs in preeclampsia. Although research efforts into this disease have been copious, preeclampsia still remains the “disease of theories” (2). Although the etiology of the disease remains elusive, a general consensus is that reductions in uteroplacental perfusion are a central component of the disease (3,4). Preeclampsia is a human disease, but proper modeling of this disease in animals is essential for elucidating its pathological mechanisms. Chronic infusion of L-NAME (nitric oxide synthase inhibitor) and overexpression of sFlt-1 (soluble VEGF receptor variant) have led to preeclampsia-like conditions in pregnant rats (5,6). In addition, a number of animal models have been developed to model the reduction of uteroplacental blood flow, which is thought to be central to the pathology of preeclampsia. Such examples include uterine artery banding (7), uterine artery ligation in pregnant baboon (8), and chronic reduction in uteroplacental perfusion (RUPP) via both the abdominal aorta and uterine–ovarian arteries in gravid rats (9,10). Granger and colleagues have shown that the surgical RUPP procedure reduces blood flow to the uteroplacental unit by ∼40% by placing silver clips around the abdominal aorta and uterine–ovarian arteries (10). As compared to SHAM-operated controls, RUPP animals display hypertension, fetal growth restriction, and several other similarities to preeclampsia (10,11). Though this model has identified numerous mechanisms that may be applicable to the pathology of preeclampsia, it represents a single disruption in uteroplacental blood flow. In the human equivalency, this would mean a reduction in uteroplacental blood flow by 40% that would last from the second trimester of pregnancy up to the time of delivery. It is likely that preeclamptic women experience episodic blood pressure and/or blood flow changes as they go about their daily lives similar to what is seen in normal pregnancy (12). Ambulatory blood pressure measurement studies in normal pregnant mothers have shown that daily blood pressure fluctuates; furthermore, blood pressure may differ on a day-to-day basis depending on the mother’s environment (13). In addition, as a result of altered vascular behavior, maternal vasospasm is a hallmark of preeclampsia, which could lead to episodic alterations in blood pressure and blood flow changes. Doppler velocimetry studies in preeclamptic mothers have described disruptions in uterine, umbilical, and placental blood flow as well as changes in feto–placental resistance (14). The changes in blood flow have been described as restrictions as well as reversals in blood flow (15,16). To date, the ramifications of episodic reductions in uteroplacental blood flow throughout gestation are not clear. Therefore one objective of this study was to see what impact episodic reductions would have on gestation. To do this we chose to employ aortic occlusion to accomplish episodic reductions in uteroplacental blood flow. This has been used previously in pregnant sheep (17) and in gravid dogs (18–20). Woods et al. showed that acute occluder inflation of the abdominal aorta led to increases in mean arterial blood pressure for the duration of the inflation in pregnant dogs and suggested that vasoconstrictor prostaglandins mediate this effect (18). Using a similar technique, we have placed a silastic vascular occluder around the abdominal aorta to acutely

Aortic Occlusion in Gravid Rat


change uteroplacental blood flow. This model may represent a pathology that is more comparable to the human condition. Therefore, the hypothesis to be tested is that episodic reductions in uteroplacental blood flow will result in maternal hypertension and fetal growth restriction when compared to SHAM operated controls. We further hypothesize that a contributing factor to the pathology of episodic reductions in uteroplacental blood flow is an alteration in vascular reactivity of the resistance-sized vessels. Our final hypothesis is that the pathology of this episodic reduction in blood flow will be different from that of gravid rats with chronic reductions in uteroplacental blood flow (i.e., RUPP).

METHODS Animals Eight- to twelve-week old virgin Sprague Dawley females (Hilltop Labs, Scottsdale, PA) were used for these studies. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Akron. Female rats were mated overnight with a male and the presence of sperm on a vaginal smear designated Day 1 of pregnancy (full term = 22–23 days). The rats were housed separately for the duration of their pregnancy. Three separate groups of animals were used for this study. Animals subjected to SHAM surgery (n = 7), animals subjected to RUPP surgery (n = 7), and animals that underwent aortic occlusion surgery (n = 12). Surgical Manipulation On Day 14 of gestation, animals were subjected to aortic occlusion, RUPP, or SHAM surgeries. RUPP has been previously described in detail elsewhere (10). Briefly, a midline incision was made and the uterine–ovarian arteries were isolated. A silver clip (0.106 mmID) was placed around each uterine–ovarian artery. Next, the abdominal aorta was isolated distal to the renal arteries and proximal to the iliac bifurcation. A silver clip (0.203 mmID) was placed around the aorta. SHAM animals underwent the same surgery but did not receive the silver clips. We modified the existing RUPP procedure to establish our aortic occlusion model by placing the silastic vascular occluder in place of the 0.203 mmID silver clip. We placed the vascular occluder (3 mm diameter; Harvard Apparatus, Holliston, MA, USA) above the bifurcation of the iliac arteries and below the renal arteries. As per the RUPP procedure, we placed silver clips (0.106 mmID) around both uterine–ovarian arteries. The aortic occlusion tubing was then tunneled through the back and exposed between the shoulder blades along the longitudinal axis of the animal. Aortic Occlusion Aortic occlusion occurred for five consecutive days starting on Day 16 of pregnancy lasting to Day 20. Animals were trained and placed in a clear plastic restraint in their home cages. The animals were given 30 min to equilibrate to the restraint apparatus. After the 30 min period, a 3-mL syringe with an 18-gauge Luer adapter (Clay Adams, Parsippany, NJ, USA) was attached to

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the end of the vascular occluder tube. A volume of 1.5 cc of air was pumped into the occluder and the pressure was held for 1 h. We chose 1.5 cc of air based on our preliminary studies demonstrating a 40% reduction in blood pressure measured by a femoral arterial catheter. Maternal/Fetal Pregnancy Outcomes Maternal body weights were determined prior to termination (Gestational Day 21). After termination and tissue collection, the uterus was excised and fetal pups were counted and weighed. Fetal resorptions were counted. Blood pressure was measured under sodium pentothal anesthesia (50 mg/kg IP; EJ Lily, Indianapolis, IN, USA) via a carotid artery catheter (0.015 in × 0.030 in OD). The catheter was attached to a pressure transducer through a Doppler BioAmp (Crystal Biotech, Cincinnati, OH, USA). Data were collected and analyzed using a computer data acquisition software (DATAQ, Akron, OH, USA). Pressure Arteriograph Mesenteries were excised from the animals and placed in ice cold HEPES buffer as previously described elsewhere (21–23). Small, resistance-sized, second-order mesenteric arteries were isolated and mounted within an isobaric arteriograph (Living Systems, Burlington, VT, USA) as previously described (11,22,23). Myogenic Reactivity After a stable tone was achieved at 60 mmHg, the arteries were preconstricted to 70–85% of their initial diameters with phenylephrine (PE). The intraluminal pressure was dropped to 20 mmHg, and the pressure was increased by 20 mmHg every 5 min to a maximum of 120 mmHg as described in detail previously (11,22,23). Diameters were measured at each pressure step with an electronic filar (Lasico, Los Angeles, CA, USA). After completion of the pressure steps, the arteries were washed and the nonspecific nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMA), was applied to the arteries (0.1 μmol/L) and allowed to incubate for 15 min, and then the myogenic reactivity experiment was repeated. Myogenic reactivity for each vessel was compared as a per cent change from the initial diameter at 20 mmHg, as noted in the following equation: ((DX − D20) / (D20)) × 100, where DX represents the diameter at a specific transmural pressure and D20 is the luminal diameter at 20 mmHg. A positive per cent change, as indicated by this equation, is representative of dilation, whereas a negative per cent change is representative of constriction. Increases in myogenic reactivity is identified as a net zero change or a negative per cent change from the initial diameter (11,22,23). Agonist Responses For methacholine (ME) responsiveness, small mesenteric arteries were preconstricted to 50% of their initial diameter with PE. After a stable constriction was achieved, the initial diameter was measured and increasing concentrations of ME (5 nmol/L–10 μmol/L) were added to the bath as previously described by our lab (11). Changes in diameter at each ME concentration were normalized and compared to the initial artery diameter as per cent maximum relaxation. The procedure was repeated in the presence of L-NMA afterward.



Constrictor responses to the a-adrenergic agonist PE were measured by administering cumulative concentrations of 0.1–10 μmol/L as previously described by our lab (11). Data were expressed as a normalized per cent maximum constriction at a particular PE concentration. Statistics Data were expressed as means ± SEM. Mean arterial blood pressure, maternal weight, and fetal morphology were analyzed using a one-way ANOVA with repeated measures and a Bonferroni post-hoc test. Mesenteric artery assays were analyzed using a two-way ANOVA with repeated measures and a Bonferroni post-hoc test. Student’s t-test was used where applicable, and statistical significance was accepted if p < 0.05.

RESULTS As reported by our lab (11) and others (10,24), RUPP animals (Table 1) exhibited a significant increase in mean arterial pressure (anesthetized) as compared to SHAM animals (p < 0.05), with the Occluder animals experiencing a small but significant increase in blood pressure as compared to SHAM (10%; p < 0.05). Maternal weight increased in SHAM animals (∼25%) as compared to both Occluder and RUPP mothers (p < 0.05). Fetal outcomes were also altered by the surgical procedures. Litter size was reduced for RUPP animals as compared to SHAM (p < 0.05), with fetal resorption being increased in both Occluder and RUPP mothers as compared to SHAM (p < 0.05). Fetal weights were significantly lower in RUPP mothers as compared to SHAM (p < 0.05) confirming the previous results from our lab (11). Interestingly, fetuses from Occluder mothers exhibited a significantly lower total weight as compared to SHAM (p < 0.05) and a significantly higher total weight as compared to RUPP (p < 0.01). Intraluminal pressure increases caused increased myogenic responsiveness of small mesenteric arteries from Occluder and RUPP mothers as compared to SHAM (p < 0.05; Figure 1). Investigating the role of nitric oxide in this pathway, nitric oxide synthase inhibition with L-NMA significantly increased myogenic reactivity of small mesenteric arteries from SHAM animals (p < 0.05) but had virtually no effect on mesenteric arteries from Occluder and RUPP mothers (Figure 2). This data would suggest a defective endothelial response to pressure increases in resistance caliber mesenteric vessels from both Occluder and RUPP instrumented animals. Endothelium-dependent vasodilation with methacholine was reduced in small mesenteric arteries from RUPP animals as compared to SHAM (p < 0.05). Methacholine dilation responses of Occluder animals were similar to that of SHAM-operated controls (Figure 3). Treatment of arteries with L-NMA (Figure 4) ameliorated any differences between the treatment groups. Apparently, Occluder and RUPP surgery had no effect on PE constriction (Figure 5) as responses were similar between all three treatment groups. However, there was a trend in the concentration of PE required to constrict the vessel to 50% of the maximum diameter (EC50) for RUPP as compared to Occluder and SHAM animals (p = 0.06): RUPP (EC50 = 0.6 ± 0.09 μmol/L), SHAM (EC50 = 1.1 ± 0.28 μmol/L), and Occluder (EC50 = 1.2 ± 0.27 μmol/L).

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Percent change from initial diameter at 20 mmHg

SHAM Occluder RUPP

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* *

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0 20

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–20

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Intraluminal pressure (mmHg)

Figure 1: Myogenic reactivity of small second-order mesenteric arteries from SHAM, Occluder, and RUPP animals. Resistance-sized mesenteric arteries from Occluder and RUPP animals exhibited a significant increase in myogenic reactivity as compared to SHAM (p < 0.05), as indicated by the decreased change in total diameter when intraluminal pressure was increased.

60 Occluder Occluder + NMA

40 20 0 20 –20

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Percent change from initial diameter at 20 mmHg

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(A) Percent change from initial diameter at 20 mmHg

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(C) Percent change from initial diameter at 20 mmHg


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Figure 2: Incubation of small second-order mesenteric arteries from SHAM, Occluder, and RUPP animals with the nitric oxide synthase inhibitor NG-methyl-L-arginine (L-NMA) led to a significant increase in myogenic reactivity from SHAM animals (C) (p < 0.05). L-NMA had no significant effect on mesenteric artery myogenic reactivity in Occluder (A) and RUPP (B) animals.

Aortic Occlusion in Gravid Rat 120

Percent maximum relaxation

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0 5 x 10–9 1 x 10–8 3 x 10–8 5 x 10–8 1 x 10–7 3 x 10–7 5 x 10–7 1 x 10–6 [ME]

Figure 3: Concentration response curve of small second-order mesenteric arteries from SHAM, Occluder, and RUPP animals in response to the endothelium dependent vasodilator methacholine. Mesenteric arteries from RUPP animals experienced a decrease in methacholine dilation as compared to SHAM and Occluder animals (*p < 0.05).

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SHAM Occluder RUPP

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0 5 x 10–9 1 x 10–8 3 x 10–8 5 x 10–8 1 x 10–7 3 x 10–7 5 x 10–7 1 x 10–6 [ME]

Figure 4: Incubation of small second-order mesenteric arteries from SHAM, Occluder, and RUPP with L-NMA and subsequent subjection of the arteries to methacholine dilation. No significant differences were found between the treatment groups.

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Figure 5: Constriction of small second-order mesenteric arteries from SHAM, Occluder, and RUPP animals with the selective a-adrenergic receptor agonist phenylephrine. No differences were found between the three treatment groups.

DISCUSSION The results of this investigation support our hypothesis that episodic reductions in uteroplacental blood flow in pregnant rats manifest a pathology similar to preeclampsia. Specifically, aortic occlusion animals exhibited an increase in mean arterial pressure (10%), demonstrated fetal growth restriction, and showed altered vascular behavior indicative of a hypertensive phenotype. To our knowledge, our study represents the first investigation utilizing repetitive episodic reductions in uteroplacental perfusion pressure in pregnant rats. Our model consisted of implantation of a silastic vascular occluder around the abdominal aorta along with silver clips (0.106 mmID) around the uterine–ovarian arteries to reduce the adaptive increase in blood flow to the uterine arcade (25). As determined by our preliminary studies, inflation of the vascular occluder with 1.5 cc of air reduced blood pressure by 40% via a femoral catheter. The animals were then subjected to five consecutive days of 40% reduction each session lasting for a period of 1 h during late pregnancy (gestational Day 16–Day 20). This model of aortic occlusion is similar to what has been shown in pregnant sheep (17) and in dogs (18). However, our model has been modified to study the long-term consequences of repetitive reductions in uteroplacental perfusion pressure on pregnancy outcomes and vascular reactivity. Maternal blood pressure, fetal growth, and vascular reactivity are facets of a myriad of factors that are affected by the pathological pregnancy disease preeclampsia. Animal modeling of this disease has been limited owing to the fact that it is a human disease. One of the most characterized animal models



of the preeclampsia pathology is the RUPP pregnant rat model, which involves a surgical procedure to reduce uteroplacental blood flow for a period of 7 days. This model has provided insights into various mechanisms that are likely occurring in response to reductions in uteroplacental blood flow in preeclamptic women. Previous data from our lab (11) and others (26) have shown that RUPP mesenteric arteries are altered in terms of vascular reactivity leading to a more constrictive phenotype versus control pregnant animals. It is speculated that this may be involved in the high maternal blood pressure seen in RUPP animals. This body of knowledge gained from the RUPP model has proved invaluable to our understanding as to the influence of uteroplacental perfusion on maternal/fetal physiology. However, the RUPP procedure leads to a severe form of the disease with large increases in MAP and a high number of reabsorbed fetuses. This, however, is not always the case in women with preeclampsia. In preeclampsia, blood pressure is variable among women with the presence of gestational proteinuria (1), and fetal growth restriction is seen in about one-third of all preeclamptic pregnancies (27). In the current study we wanted to evaluate if episodic reductions in uteroplacental blood flow could produce a preeclampsia-like condition in pregnant rats. Doppler velocimetry studies have confirmed that reductions and/or reversals in uterine blood flow occur in preeclamptic pregnancies (14–16). In addition, ischemia reperfusion injury is thought to contribute to the pathology of preeclampsia most likely via the disruption of normal vascular function by oxidative stress (28,29). Both would suggest that preeclamptic uteroplacental blood flow restrictions may be episodic in nature. There are a number of factors that could likely result in episodic restrictions in blood flow. The first is vasospasm, which is a hallmark characteristic of preeclampsia and involves detrimental alterations in vascular reactivity caused by an injured endothelium (30,31). Roberts and Lain suggests that blood flow may be reduced periodically in normal pregnant women on account of the routine of their daily lives (12). Accordingly, evidence of periodic changes in blood pressure of both normal and preeclamptic mothers resides in ambulatory blood pressure (13,32). Presumably the combination of the degree of episodic reductions in uteroplacental blood flow and the timing at which such episodic reductions are initiated, as well as the maternal response to these alterations, determine the progression of pregnancy to preeclampsia. With this in mind, we have chosen to modify the existing RUPP pathology to take into consideration these possible interrupted changes in uteroplacental blood flow. Episodic insults followed by periods of reduced perfusion may activate different cellular and molecular mechanisms as compared to chronic reductions in blood flow. Periodic occlusions in uterine blood flow in rodents have been found to activate mechanisms such as TNF a (33) and endothelin-1 (34). Harrison and colleagues have found that uterine arterial occlusion leads to drops in uterine blood flow, uterine contractions, and localized pH (35,36), and that this is reversed upon reperfusion. Accordingly, periodic uterine insults increase cyclooxygenase-2 production, which may ultimately affect vasoconstrictor prostanoids such a thromboxane (37). These changes caused by the episodic reductions in uteroplacental perfusion pressure may lead to more

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localized instances of endocrine stress, which may represent a different facet of the preeclamptic pathology that is not yet fully understood. Thus, changes in localized mechanisms associated with episodic reductions in uteroplacental blood flow may also ultimately affect vascular function especially through arteries integrally involved in blood pressure, that is, resistancesized arteries. In this investigation, we sought to elucidate whether episodic reductions in uteroplacental blood flow affected resistance-sized mesenteric arteries that aid in the regulation of localized blood flow, and ultimately blood pressure. Occluder animals exhibited a small but significant increase of 10 mmHg in MAP as compared to SHAM animals, with RUPP animals exhibiting a significant increase as compared to SHAM (p < 0.05). Thus, episodic reductions in uteroplacental blood flow can lead to hypertension. Myogenic reactivity is the active response of a blood vessel to changes in intraluminal pressure. This mechanism is an integrated response of the entire blood vessel (endothelium, smooth muscle, and extracellular matrix) (23). Alterations in myogenic responsiveness can be indicative of altered peripheral resistance regulation and thus may play a role in blood pressure regulation. We have previously correlated changes in resistance-sized mesenteric and renal artery myogenic responsiveness with alterations in blood pressure and renal function (38). Previous data from our lab have also shown that the RUPP pathology alters myogenic reactivity of small, resistance-sized mesenteric and renal arteries toward a constrictive phenotype (11), a finding confirmed in this study. This may be indicative of the increased blood pressure seen in this animal model by our lab and by others (10,11). Resistance-sized mesenteric arteries from aortic occlusion animals also exhibited increased myogenic reactivity as compared to SHAM animals (p < 0.05). Interestingly this increase in responsiveness was very similar to that in RUPP animals. The only other study similar to this model is that of Woods (1989), in which it was shown that mean arterial pressure increased as a result of an acute 1-h aortic occlusion in gravid dogs and that this was likely because of an increase in vasoconstrictor prostaglandins (18). This work represents the long-term ramifications of repetitive episodic perfusion pressure reductions; thus, the assumption can be made that the repetitiveness of our manipulation had a differential effect on isolated resistance caliber vessels thus leading to the increase in myogenic reactivity witnessed in this study. Previous results from our lab suggest that nitric oxide plays a prominent role in the regulation of the myogenic response in mesenteric arteries from pregnant rats (11,23). Indeed, nitric oxide is the preeminent vasodilator of rat and human arteries during pregnancy (39). Hence we investigated whether the changes in myogenic reactivity seen in our aortic occlusion animals were caused by changes in nitric oxide. Incubation of mesenteric arteries from all three groups of pregnant rats with the nitric oxide synthase inhibitor NG-methyl-L-arginine (L-NMA) ameliorated all differences in myogenic reactivity, as all groups responded to intraluminal pressure increases with vasoconstriction. This indicates a prominent role for nitric oxide in mediating the mechanism for myogenic reactivity seen in these animals. A role for nitric oxide in the vascular alterations associated with RUPP has been supported by our data (11) and a study by Alexander and colleagues, who found that dietary treatment of RUPP animals with L-arginine, the amino acid

Aortic Occlusion in Gravid Rat Table 1: Maternal and fetal pregnancy outcomes.

MAP (mmHg) Maternal body weight (g) Fetal number Fetal resorption Fetal weight (g)

SHAM (n = 7)

Occluder (n = 12)

RUPP (n = 7)

100.41 ± 2.03* 459.4 ± 3.62* 13.22 ± 1.18 0.67 ± 0.52* 4.16 ± 0.17*

109.24 ± 3.94 355.3 ± 7.25 10.42 ± 1.43 3.25 ± 0.93*** 3.61 ± 0.15***

122.04 ± 7.49** 339.7 ± 9.09** 6.67 ± 2.28** 7.00 ± 1.73** 2.85 ± 0.38**


*p < 0.05 SHAM vs. Occluder, **p < 0.05 SHAM vs. RUPP, ***p < 0.05 Occluder vs. RUPP.

precursor of nitric oxide, improved the pathology associated with RUPP (24). These data in combination with our myogenic reactivity data suggest that decreases in nitric oxide may be involved with the pathology seen in RUPP animals. Thus we sought to elucidate if agonist-induced vasodilation with methacholine was altered in our aortic occlusion animals. Methacholineinduced vasodilation was similar between Occluder and SHAM mesenteric arteries, and both increased as compared to RUPP (p < 0.05). These data indicate that agonist-induced stimulation of nitric oxide is intact in aortic occlusion animals as compared to RUPP. As expected, incubation of small mesenteric arteries with L-NMA ameliorated the differences in methacholineinduced dilation for all three groups of pregnant rats. Small mesenteric artery responses to the a-adrenergic receptor agonist PE were similar between all three groups of pregnant rats. This is in agreement with previous results from our lab showing no differences between RUPP and SHAM animals for PE constriction (11). Fetal growth restriction affects 7–15% of all pregnancies and may lead to many cardiovascular problems later in life for the offspring (40). Intrauterine growth restriction is present in one-third of all preeclamptic pregnancies (27). It is thought that reductions in uteroplacental blood flow can lead to fetal hypoxia, growth restriction, and possibly neurological damage and death. This may occur through deficits in placental nutrient transport and oxygen diffusion (41). Our RUPP animals experienced a significant lowering in fetal weight as compared to SHAM (31%), confirming the previous results from our lab (11) and others (24), along with a reduction in maternal weight (∼25%). Interestingly, pups from Occluder mothers were also growth-restricted as compared to SHAM (13% lower), but not to the degree of RUPP mothers (21% higher). This suggests that episodic reductions in placental blood flow may lead to transient reductions in placental transport. These deficits may have resulted in growth restriction seen in our Occluder pregnancy pups. Interestingly even though fetal weights were reduced, litter sizes were similar between Occluder and SHAM animals. In conclusion, our results show that episodic reductions in uteroplacental blood flow lead to increases in maternal blood pressure and instances of fetal growth restriction. Vascular reactivity is altered in aortic occlusion animals as compared to SHAM animals, as myogenic reactivity is increased in small mesenteric arteries. This indicates an integral role for episodic reductions in uteroplacental blood flow in the regulation of vascular reactivity and ultimately blood pressure. This aortic occlusion gravid rat model may provide new

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insights into the pathology associated with episodic reductions in uteroplacental blood flow and possibly the pathological pregnancy disease preeclampsia.

ACKNOWLEDGMENTS


This investigation was supported by a Faculty Research Grant and the Department of Biology at The University of Akron. The authors would like to thank the technical expertise of Ms. Emily Njus. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

REFERENCES 1. Lindheimer MD, Roberts JM, Cunningham F. Chesley’s Hypertensive Disorders in Pregnancy. 3rd ed., Stamford, CT: Appleton and Lange, 2009. 2. Broughton Pipkin F, Rubin PC. Pre-eclampsia – the “Disease of Theories”. Br Med Bull 1994; 50:381–396. 3. Khalil RA, Granger JP. Vascular mechanisms of increased arterial pressure in preeclampsia: Lessons from animal models. Am J Physiol Regul Integr Comp Physiol 2002; 283:R29–R45. 4. Roberts JM, Gammill HS. Preeclampsia: Recent insights. Hypertension 2005; 46:1243–1249. 5. Yallampalli C, Garfield RE. Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia. Am J Obstet Gynecol 1993; 169:1316–1320. 6. Maynard SE, Min JY, Merchan J, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003; 111:649–658. 7. Golden JG, Hughes HC, Lang CM. Experimental toxemia in the pregnant guinea pig (Cavia porcellus). Lab Anim Sci 1980; 30:174–179. 8. Makris A, Thornton C, Thompson J, et al. Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1. Kidney Int 2007; 71:977–984. 9. Abitbol MM. Simplified technique to produce toxemia in the rat: Considerations on cause of toxemia. Clin Exp Hypertens B 1982; 1:93–103. 10. Crews JK, Herrington JN, Granger JP, Khalil RA. Decreased endothelium-dependent vascular relaxation during reduction of uterine perfusion pressure in pregnant rat. Hypertension 2000; 35:367–372. 11. Ramirez RJ, Debrah J, Novak J. Increased myogenic responses of mesenteric resistancesized arteries after reduced uterine perfusion pressure in pregnant rats. Hypertension in Pregnancy 2010; in press. 12. Roberts JM, Lain KY. Recent insights into the pathogenesis of pre-eclampsia. Placenta 2002; 23:359–372. 13. Feldman DM. Blood pressure monitoring during pregnancy. Blood Press Monit 2001; 6:1–7. 14. Zahumensky J. Doppler flowmetry in preeclampsia. Bratisl Lek Listy 2009; 110:432–435. 15. Ekici E, Vicdan K, Dayan H, Danisman N, Gokmen O. Reverse end-diastolic uterine artery velocity in a pregnant woman complicated by mild preeclampsia and severe growth retardation. Eur J Obstet Gynecol Reprod Biol 1996; 66:79–82. 16. Lau WL, Lam HS, Leung WC. Reversed diastolic flow in the uterine artery – a new Doppler finding related to placental insufficiency?. Ultrasound Obstet Gynecol 2007; 29:232–235. 17. Boyle DW, Lecklitner S, Liechty EA. Effect of prolonged uterine blood flow reduction on fetal growth in sheep. Am J Physiol 1996; 270:R246–R253. 18. Woods LL. Importance of prostaglandins in hypertension during reduced uteroplacental perfusion pressure. Am J Physiol 1989; 257:R1558–R1561. 19. Woods LL, Brooks VL. Role of the renin-angiotensin system in hypertension during reduced uteroplacental perfusion pressure. Am J Physiol 1989; 257:R204–R209. 20. Woods LL. Role of angiotensin II and prostaglandins in the regulation of uteroplacental blood flow. Am J Physiol 1993; 264:R584–590. 21. Gandley RE, Mclaughlin MK, Koob TJ, Little SA, Mcguffee LJ. Contribution of chondroitindermatan sulfate-containing proteoglycans to the function of rat mesenteric arteries. Am J Physiol 1997; 273:H952–H960.


Aortic Occlusion in Gravid Rat 22. Ramirez RJ, Novak J, Johnston TP, Gandley RE, McLaughlin MK, Hubel CA. Endothelial function and myogenic reactivity in small mesenteric arteries of hyperlipidemic pregnant rats. Am J Physiol Regul Integr Comp Physiol 2001; 281:R1330–R1337. 23. Ramirez RJ, Hubel CA, Novak J, Dicianno JR, Kagan VE, Gandley RE. Moderate ascorbate deficiency increases myogenic tone of arteries from pregnant but not virgin ascorbate-dependent rats. Hypertension 2006; 47:454–460. 24. Alexander BT, Llinas MT, Kruckeberg WC, Granger JP. L-arginine attenuates hypertension in pregnant rats with reduced uterine perfusion pressure. Hypertension 2004; 43:832–836. 25. Nienartowicz A, Link S, Moll W. Adaptation of the uterine arcade in rats to pregnancy. J Dev Physiol 1989; 12:101–108. 26. Anderson CM, Lopez F, Zhang HY, Shirasawa Y, Pavlish K, Benoit JN. Mesenteric vascular responsiveness in a rat model of pregnancy-induced hypertension. Exp Biol Med (Maywood) 2006; 231:1398–1402. 27. Chappell S, Morgan L. Searching for genetic clues to the causes of pre-eclampsia. Clin Sci (Lond) 2006; 110:443–458. 28. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med 1999; 222:222–235. 29. Gupta S, Agarwal A, Sharma RK. The role of placental oxidative stress and lipid peroxidation in preeclampsia. Obstet Gynecol Surv 2005; 60:807–816. 30. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: An endothelial cell disorder. Am J Obstet Gynecol 1989; 161:1200–1204. 31. De Groot CJ, Taylor RN. New insights into the etiology of pre-eclampsia. Ann Med 1993; 25:243–249. 32. Hermida RC, Ayala DE, Mojon A, et al. Differences in circadian blood pressure variability during gestation between healthy and complicated pregnancies. Am J Hypertens 2003; 16:200–208. 33. Okazaki M, Matsuyama T, Kohno T, et al. Induction of epithelial cell apoptosis in the uterus by a mouse uterine ischemia-reperfusion model: Possible involvement of tumor necrosis factor-alpha. Biol Reprod 2005; 72:1282–1288. 34. Thaete LG, Neerhof MG. Endothelin and platelet-activating factor: Significance in the pathophysiology of ischemia/reperfusion-induced fetal growth restriction in the rat. Am J Obstet Gynecol 2006; 194:1377–1383. 35. Harrison N, Larcombe-McDouall JB, Earley L, Wray S. An in vivo study of the effects of ischaemia on uterine contraction, intracellular pH and metabolites in the rat. J Physiol 1994; 476:349–354. 36. Harrison N, Larcombe-McDouall JB, Wray S. A 31P NMR investigation into the effects of repeated vascular occlusion on uterine metabolites, intracellular pH and force, in vivo. NMR Biomed 1995; 8:28–32. 37. Yamazaki K, Endo T, Kitajima Y, et al. Elevation of both cyclooxygenase-2 and prostaglandin E2 receptor EP3 expressions in rat placenta after uterine artery ischemia-reperfusion. Placenta 2006; 27:395–401. 38. Novak J, Danielson LA, Kerchner LJ, Sherwood OD, Ramirez RJ, Moalli PA, Conrad KP. Relaxin is essential for renal vasodilation during pregnancy in conscious rats. J Clin Invest 2001; 107:1469–1475. 39. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol 1997; 272:R441–R463. 40. Barker DJ. Fetal origins of cardiovascular disease. Ann Med 1999; 31(Suppl. 1):3–6. 41. Bell AW, Hay WW JR, Ehrhardt RA. Placental transport of nutrients and its implications for fetal growth. J Reprod Fertil Suppl 1999; 54:401–410.

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