Early-onset severe preeclampsia can lead to serious pregnancy complications, including placental abruption and fetal death, which affect maternal and fetal morbidity and mortality. Placental morphology suggests that early-onset preeclampsia is largely a placental disease, whereas late-onset preeclampsia is more a maternal disorder. It is therefore possible that the earlyand late-onset forms should be considered as two different entities when investigating the pathophysiology of preeclampsia.1,2
The present study therefore aimed to investigate the effects of early-onset preeclampsia-like symptoms on feto-placental outcomes and the adverse effects of various factors on placental and fetal growth and development at different gestational stages, using an L-arginine methyl ester (L-NAME) preeclampsia-like mouse model.
Experiments were performed in 72 healthy female C57BL/6 (C57) mice (19-24 g, 8-12 weeks old) purchased from the Department of Laboratory Animal Science of Peking University Health Science Center. The animals were housed individually under controlled conditions of temperature (18°C-28°C), humidity (40%-70%), and lighting (12-hour light/dark cycle), and fed with standard mouse chow with water available ad libitum. Female mice were mated with males and the day of plug detection was designated as day 1 of pregnancy. 3 Pregnant mice were transferred to individual cages during the period of pregnancy. Pregnant mice were divided into three groups: a blank control group (n=12), a control group (n=30) and a preeclampsia group (PE group, n=30). All procedures were approved by the Animal Ethics Committee of the Health Science Center of Peking University.
Establishment of animal model
Preeclampsia was induced using the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester (L-NAME) 50 mg·kg-1 ·d-1, based on previous studies.4 L-NAME was prepared in saline and stored in a freezer at -20°C. 5 Gestational stages were defined as follows: early, days 7-9; middle, days 11-14; and late, days 16-20.6 Mice in the PE group were subdivided into early (n=12), mid (n=12) and late groups (n=6), with L-NAME injections starting at days 7, 11, and 16 of pregnancy, respectively. Gestational age-matched controls were injected with saline at the same time points. The blank control group received no experimental treatment.
The success of the preeclampsia model was determined by measuring the systolic blood pressures of the pregnant mice every 2 days until the mice were anesthetized, using a non-invasive blood pressure measurement system (BP-98A, Softron, Japan). The values were reported as the means of at least three measurements.7 Pregnant C57 mice were placed in individual metabolic cages with free access to food and water, and 24-hour urine samples were collected. Urine was frozen at -80°C until analysis for total protein content using a protein assay kit (Bio-Rad Company, USA), according to the manufacturer's instructions.8
Samples were taken from six mice from each of the earlyand mid-PE groups and their corresponding control groups, and from the blank control group, on day 14 of pregnancy. Samples were taken from the remaining six mice in those groups, and from the mice in the late-PE group and the corresponding control group, on day 18 of pregnancy. Pregnant mice were anesthetized with 10% chloral hydrate (3 ml/kg), and placental and fetal weights were measured. Absorbed and live fetuses were counted to calculate the fetal absorption rate (absorbed fetuses/live fetuses + absorbed fetuses) and fetal survival rate (live fetuses/live fetuses + absorbed fetuses). Placentas were immediately removed from all mice and fixed in 10% formaldehyde. The specimens were dehydrated in a series of graded alcohols and embedded in paraffin. Sections were deparaffinized and stained with hematoxylin-eosin (HE) for histological examination, and 10 of 200× microscopic fields were examined for each placenta using an Olympus microscope (Olympus Corp, Japan). The total number of villi, and the numbers of villi with syncytial knots, interstitial edema, and fibrinoid necrosis respectively were counted and expressed as a percentage.
Data were expressed as mean ± standard error (SE). The measured data were analyzed using analysis of variance (ANOVA) and t-tests, and numerical data were compared using χ2 test. ANOVA and Student-Newman-Keuls tests were used for between-group comparisons. SPSS 11.5 was used for data analysis. P <0.05 was considered statistically significant.
Blood pressure was increased in all PE groups after injection of L-NAME, compared with the respective control groups (P <0.001). There were no significant differences in blood pressure between the PE groups and their controls before injection (P >0.05) (Figures 1 and 2).
Urinary protein contents in the early- and mid-PE groups ((9.56±0.40) μg and (9.35±0.49) μg) were significantly increased at day 14 of pregnancy, compared with the respective control groups ((7.43±0.89) μg and (7.40±0.92) μg) (P <0.01). At day 18 of pregnancy, urinary protein contents in the early-, mid- and late-PE groups ((12.76±1.06) μg, (11.15±1.95) μg and (10.80±1.43) μg) were significantly increased compared with the respective control groups ((9.60±0.62) μg, (8.41±0.64) μg and (8.43±0.79)μg) (P<0.01).
Samples were collected at days 14 and 18 of pregnancy. Live and absorbed fetuses were counted to calculate the fetal survival and absorption rates, and fetal weights were measured.
At day 14 of pregnancy, the fetal survival rate was lower and the fetal absorption rate was higher in the early-PE group compared with its control group (P <0.01), whereas there was no significant difference between the mid-PE group and its control group (P >0.05). At day 18 of pregnancy, the fetal survival rate was lower and the fetal absorption rate was higher in the early-PE group (P <0.001), but there were no significant differences between the mid- and late-PE groups and their corresponding controls (P >0.05) (Tables 1 and 2).
At day 14 of pregnancy, fetal weights in the early- and mid-PE groups ((159.5±13.36) mg and (174.83±13.94) mg) were significantly decreased compared with the respective control groups ((231.33±12.20) mg and (221.66±34.15) mg) (P <0.01), but there were no significant differences between the early- and mid-PE groups (P >0.05). At day 18 of pregnancy, fetal weights in the early- and mid-PE groups ((686.76±59.47) mg and (867.83±33.85) mg) were significantly decreased compared with their control groups ((1198.33±107.04) mg and (1188.66±67.54) mg) (P <0.001), but there was no significant difference between the late-PE group ((1139±13.42) mg) and its control group ((1165±23.91) mg) (P >0.05) (Tables 1 and 2).
At day 14 of pregnancy, placental weights in the early and mid-PE groups ((92.83±8.32) mg and (98.33±11.21) mg) were significantly decreased compared with their control groups ((115.16±6.82) mg and (112.66±11.21) mg) (P<0.01), but there was no significant difference between the early- and the mid-PE groups (P >0.05). At day 18 of pregnancy, placental weights in the early- and mid-PE groups ((102.08±6.08) mg and (117.83±1.60) mg) were significantly decreased compared with their control groups ((134.3±13.51) mg and (142.83±13.16) mg) (P <0.01), but there was no significant difference between the late-PE group ((138.16±6.11) mg) and its control group ((146.66±32.32) mg) (P >0.05). Morphologic changes in the placenta were observed under a light microscope. The mouse placenta contained three different zones: the basal zone, the decidual zone and the labyrinth zone. Villi formed a complicated labyrinth-like structure with maternal blood cells occupying the spaces of the mesh, which was involved in the exchange of material between the maternal and fetal blood. At days 14 and 18 of pregnancy, normal histologic structures were observed in the control groups. However, varying degrees of fibrinoid necrosis and interstitial edema of the villi were observed in the placentas of the early- and mid-PE groups. In addition, no marked pathologic changes were found in the placentas of the late-PE group (Figures 3 and 4).
The total number of villi and the numbers of villi with syncytial knots, interstitial edema, and fibrinoid necrosis were counted. There were significant differences in pathologic changes of the villi between the early- and mid-PE groups and their corresponding control groups at days 14 and 18 of pregnancy, but there was no significant difference between the late-PE group and its control group. Pathologic changes of the villi in the early- and mid-PE groups were more remarkable than those in the late-PE group (P <0.001) (Tables 3 and 4).
Establishment of preeclampsia-like mouse model
Preeclampsia is one of the leading causes of maternal and fetal mortality.9,10 It has been suggested that early- and late-onset preeclampsia have different etiologies and therefore different clinical expressions, but this is still the subject of considerable research.
It is difficult to obtained human placental tissues from women with early-onset severe preeclampsia during the early and middle trimesters of pregnancy. Valid and reliable animal models are thus urgently required to allow a better understanding of the mechanisms underlying preeclampsia. The experimental design of such models has primarily been based on the mechanisms involved in uterine ischemia, impairments in the nitric oxide system, insulin resistance, over activity of the autonomic nervous and/or renin-angiotensin systems, and activation of a systemic inflammatory response.11-13 However, since preeclampsia is a pregnancy-specific disease, none of these approaches represent adequate animal models.
Widespread maternal endothelial dysfunction, generalized vasoconstriction and increased capillary permeability are characteristic of preeclampsia, which is the most serious hypertensive complication of pregnancy. Increased sympathetic activity and disturbances in prostanoid and nitric oxide production may partially explain such changes. Chronic NOS inhibition produces dose- dependent, sustained hypertension in nonpregnant animals. Unlike other models of hypertension, where pregnancy is uniformly antihypertensive, chronic NOS inhibition produces dose-dependent hypertension in pregnant rats that is maintained until term, thus resembling preeclampsia.
We therefore established mouse models using NOS inhibitors such as L-NAME to induce a preeclampsia-like syndrome. Previous preeclampsia-like models have usually been established during the middle or late stages of pregnancy. Early-onset preeclampsia-like mouse models have not been reported.
In this study, early-, mid- and late-onset preeclampsialike mouse models were established using L-NAME. In our study, blood pressure and urinary protein levels in the preeclamptic mice were higher than those in the gestational age-matched controls, which was consistent with previous reports.4,11-13 The successful establishment of these early-, mid- and late-onset models allowed us to obtain placental tissues and fetuses from pregnant mice in the midterm and late trimesters, and to study the effects of preeclampsia-like symptoms onset at different gestational stages on feto-placental outcomes.
Effects of early onset preeclampsia-like symptoms on placenta
Crispi et al14 considered that early- and late-onset preeclampsia were associated with different biochemical and clinical features, and strongly suggested that early-onset preeclampsia was a common placental disorder and a state of endothelial dysfunction, whereas late preeclampsia seemed to be linked more to maternal constitutional factors. Placental morphology suggested that isolated early-onset preeclampsia was associated with abnormal placental morphology, but there were no significant morphologic changes in placentas in late-onset preeclampsia. This confirmed the existence of two subsets of this condition and supported the hypothesis that late-onset preeclampsia is a maternal, rather than a placental disease.15 In our previous study, we found that placental and hepatic damage were the main changes in women with early-onset preeclampsia.16 In the current animal study, marked pathologic changes were observed in placentas from mice with early- and mid-onset preeclampsia. This suggests that an earlier onset and longer duration of preeclampsia-like signs was associated with more severe changes in placental pathology. No significant pathologic changes were observed in the late-PE group. These results are consistent with those from studies of the placenta in humans with preeclampsia, and support the hypothesis that early-onset preeclampsia is a placentopathy that contributes to the maternal systemic inflammatory response.14-16
Effects of early-onset preeclampsia-like symptoms on fetal development
Previous studies found that early pathologic changes associated with preeclampsia could lead to thrombus formation in the placenta, causing placental dysfunction and subsequent intrauterine growth restriction and fetal death.17-19 In this study, we found not only obvious placental pathologic changes in the placenta in early-PE mice, but also a lower fetal survival rate, a higher fetal absorption rate, and more severe fetal growth restriction in the early- and mid-PE groups than in the late-PE group, likely due to impaired early placental development and consequent effects on fetal development. The effects on the placenta and fetus became more pronounced with increasing durations of preeclampsia. Preeclampsia-like symptoms occurring at earlier stages in pregnancy were more likely to be associated with abnormal placental development and pathologic changes, including fibrinoid necrosis and interstitial edema. These changes could increase placental vascular resistance and reduce both placental blood flow and fetal oxygen supply, ultimately resulting in intrauterine growth restriction.20 Although the late-onset preeclampsia-like mice presented with high blood pressure and proteinuria, there were no significant effects on placental morphology or fetal growth, because the placenta was already well developed by this stage. These results support the hypothesis that late-onset preeclampsia is a maternal, rather than a placental disease.
This study showed that preeclampsia-like conditions could be induced by L-NAME in mice at different gestational stages. Preeclampsia-like symptoms occurring on early gestational stages were more likely to affect disease development through the placenta, whereas preeclampsia- like symptoms occurring in late pregnancy had a direct impact on the mother and fetus.
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