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Objective: The purpose of this study was to evaluate the prognostic role of placental ultrasound imaging at 19-23 weeks of gestation in clinically high-risk women with abnormal uterine artery Doppler (UTAD)., Study Design: Placentas of 60 women with abnormal UTAD were examined at 19-23 weeks of gestation for shape and texture abnormalities. Findings were correlated with clinical outcomes (preterm delivery at <32 weeks of gestation; birth weight <10th percentile [small for gestational age]; preeclampsia/hemolysis, elevated liver enzymes, low platelets; early-onset intrauterine growth restriction with abnormal umbilical artery Doppler; and intrauterine fetal death) and maternal serum screening data. Placental disease was reviewed by 2 perinatal pathologists., Results: Women with abnormal placental shape at 19-23 weeks of gestation (n = 28) had higher odds of intrauterine fetal death (odds ratio, 4.5; 95% CI, 1.3-15.6), delivery at <32 weeks of gestation (odds ratio, 4.7; 95% CI, 1.6-14.1]), and intrauterine growth restriction (odds ratio, 4.7; 95% CI, 1.4-15.1]) than did the women with a normal placental shape. Thirty-two of 41 placentas (74%) weighed <10th percentile, and 36 of 43 placentas (83%) had ischemic-thrombotic pathologic condition. There was no association between abnormal placental shape at 19-23 weeks of gestation and placental weight, but 5 of 6 placentas that were <10 cm long were <10th percentile for weight at delivery. There was a poor correlation between measures of ultrasound texture at 19-23 weeks of gestation and the presence of specific lesions at delivery., Conclusion: Combined abnormal UTAD and placental dysmorphologic condition before fetal viability identifies a subset of women who are at risk of adverse outcomes. Placental size is critical in the determination of the outcome in this situation because of the very high prevalence of destructive lesions, although present methods of placental imaging have significant limitations.

Meloxicam (MEL), a cyclooxygenase (COX)-2 inhibitor, decreases prostaglandin production and blocks preterm labor in sheep. The objective of this study is to investigate MEL dosage regimens on COX-1, COX-2, and prostaglandin dehydrogenase (PGDH) expression in ovine intrauterine and fetal tissues. Animals in preterm labor received maternal infusions of saline or MEL at maintained high or graded doses (study 1) or acute graded doses (study 2). MEL blocked preterm labor. In study 1, MEL decreased COX-2 expression in the endometrium, myometrium, and amnion but not placenta or fetal tissues. In study 2, COX-2 expression was unchanged. COX-1/PGDH expression was unaffected. While MEL is an effective tocolytic, reductions in COX-2 protein occurred only with maintained MEL exposure. MEL effects are tissue specific and do not affect COX-1 or PGDH expression. Maternal MEL does not affect fetal COX expression in the sheep, possibly contributing to its lack of fetal side effects.

We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we previously reported in fibrinogen-like protein 2 (Fgl2) knockout (KO) mice. We therefore used ultrasound biomicroscopy to assess left ventricular (LV) cardiac structure and function during development in Fgl2 KO and wild-type (WT) mice. The only deaths observed between embryonic day (E)8.5 (onset of heart beating) and postnatal day (P)28 (weaning) were within 3 days after birth, when 33% of Fgl2 KO pups died. Histopathology and Doppler assessments suggested that death was due to acute congestive cardiac failure without evidence of valvular or other obvious cardiac structural abnormalities. Heart rates in Fgl2 KO embryos were significantly reduced at E8.5 and E17.5, and irregular heart rhythms were significantly more common in Fgl2 KO (21/26) than WT (2/21) embryos at E13.5. Indexes of systolic and/or diastolic cardiac function were also abnormal in KO mice at E13.5 and E17.5, in postnatal mice studied at P1, and in KO mice surviving to P28. M-mode analysis showed no difference in LV diastolic chamber dimension, although posterior wall thickness was thinner at P7 and P28 in Fgl2 KO mice. We conclude that Fgl2 deficiency is not associated with obvious structural cardiac defects but is associated with a high incidence of neonatal death as well as contractile dysfunction and rhythm abnormalities during embryonic and postnatal development in mice.

Mouse models of human disease are increasingly used to study the nature of cardiovascular diseases such as atherosclerosis. The pulse wave velocity (PWV) provides an indirect measure of arterial stiffness and can be useful for characterizing disease progression. In this study, the PWV was measured noninvasively in the left common carotid artery of seven young mice using two image-guided approaches: a regional transit-time (TT) method and a local flow-area (QA) method. The QA approach measures the cross-sectional area and volume flow through the vessel using high frame-rate retrospective colour flow imaging. The QA method was found to correlate well with the TT method (r2=0.80, p<0.001). The mean difference between methods was 0.05+/-0.21 m/s. This study demonstrates the feasibility of measuring both regional and local PWV in mice using image-based high-frequency ultrasound methodologies.

This study evaluates microcomputed tomography (micro-CT) as a method to obtain quantitative three-dimensional (3D) information on the arterial and venous vasculature of the mouse placenta. Surface renderings at embryonic days (E) 13.5, 15.5, and 18.5 (full term) revealed that the arterial and venous vasculature branched within the chorionic plate whereas only the arterial vasculature deeply penetrated the placenta. Umbilical vessel diameters measured by micro-CT did not significantly differ from those measured non-invasively in vivo by ultrasound biomicroscopy. Variability in umbilical diameters, and surface area and volume measurements of arterial and venous vascular trees due to experimental error was low relative to biological variability, and significant inter-litter differences within gestational ages were detected. Furthermore, umbilical vessel diameter increased significantly and incrementally to an arterial diameter of 0.631+/-0.009 mm and a venous diameter of 0.690+/-0.018 mm at E18.5. Umbilical vein diameter was 3-9% greater than the artery, and both were significantly correlated with embryonic body weight (R> or =0.96). Surface area and volume were determined for vessels greater than the minimum resolvable diameter of 0.03 mm which therefore excluded capillaries. Arterial surface area and volume were unchanged from E13.5-15.5 but then more than doubled at E18.5 (to 170+/-13 mm(2) and 7.2+/-0.8mm(3), respectively). Venous surface areas and volumes changed similarly with development although surface areas were lower than their arterial counterparts. We conclude that micro-CT has sufficient accuracy and precision to quantify late gestational changes in the 3D structure of the arterial and venous vasculature of the mouse placenta.

The objective of this study was to characterize offspring responses to maternal dietary restriction (DR) in two phylogenetically distant strains of mice: A/J and C57BL/6J (B6). Pregnant mice were fed 100% or 70% of ad libitum between 6.5 and 17.5 days (d) gestation. Offspring were fed 100% ad libitum postweaning. All comparisons were made to strain and sex matched controls. Male DR-B6 offspring initially grew slower than controls; however, by 77 d and 182 d they were significantly heavier (P<0.05). Further, they had an increase percentage fat mass (+70%, P<0.01) by 182 d and were glucose intolerant at both 80 d (P<0.001) and 186 d (P<0.05). In contrast, weight, %Fat mass and glucose tolerance in DR-A/J males during postnatal life were not different from controls. Female DR-B6 mice showed catch-up growth during the first 77 d of life; however, their weight, %Fat mass and glucose tolerance were not different from controls at 80 d and 186 d. Although female DR-A/J were heavier than controls at 182 d (P<0.05), their %Fat mass and glucose tolerance were not different from controls at 182 d and 186 d. The observed strain and sex differences offer a unique opportunity to begin to define gene-environment interactions that contribute to developmental origins of health and disease.

In human pregnancy, abnormal placental hemodynamics likely contribute to the etiology of early-onset preeclampsia and fetal intrauterine growth restriction. The mouse is increasingly being deployed to study normal and abnormal mammalian placental development, yet the placental hemodynamics in normal pregnancy in mice is currently unknown. We used ultrasound biomicroscopy to noninvasively image and record Doppler blood velocity waveforms from the maternal and embryonic placental circulations in mice throughout gestation. In the uterine artery, peak systolic velocity (PSV) increased significantly from 23+/-2 (SE) to 59+/-3 cm/s, and end-diastolic velocity (EDV) increased from 7+/-1 to 28+/-2 cm/s in nonpregnant versus full-term females so that the uterine arterial resistance index (RI) decreased from 0.70+/-0.02 to 0.53+/-0.02. Velocities in the maternal arterial canal in the placenta were low and nearly steady and increased from 0.9+/-0.03 cm/s at embryonic day 10.5 (E10.5) to 2.4+/-0.07 cm/s at E18.5. PSV in the umbilical artery increased steadily from 0.8+/-0.1 cm/s at E8.5 to 15+/-0.6 cm/s at E18.5, whereas PSV in the vitelline artery increased from 0.6+/-0.1 cm/s at E8.5 to 4+/-0.2 cm/s at E13.5 and then remained stable to term. In the umbilical artery, the EDV detection rate was 0% at

Objective: Preterm birth occurs in 5% to 10% of all pregnancies and is associated with considerable neonatal mortality and morbidity. Effective and safe drugs to prevent preterm labor are not currently available. We have hypothesized that the nonsteroidal anti-inflammatory drug meloxicam, a more selective cyclooxygenase-2 inhibitor will successfully inhibit labor but avoid the complications associated with inhibition of cyclooxygenase-1., Study Design: Preterm labor was induced in chronically catheterized sheep by RU486 administration. Animals were then randomized to receive maternal infusions of saline (n = 5) or meloxicam (n = 4) for 48 hours or until delivery when the animals were killed and tissues and blood samples collected., Results: Maternal infusion of meloxicam inhibited uterine contractions, increasing contraction duration, and attenuating frequency and amplitude. Saline-treated animals progressed to delivery. Administration of meloxicam was not associated with any change in fetal or maternal blood gas status, osmolality, arterial pressure, heart rate, or fetal blood flows., Conclusion: Meloxicam may represent a potentially safe and effective tocolytic agent.

In humans, the increased cardiovascular demands of pregnancy are met by increases in cardiac output (CO), stroke volume (SV), plasma volume (PV), and cardiac and aortic inner dimensions and a concurrent decrease in arterial pressure that indicates a fall in total peripheral vascular resistance. The mechanisms responsible for these changes are incompletely understood, but NO synthase (NOS) is believed to play a central role. We assessed whether C57Bl/6J (B6) mice show similar changes and whether these changes are altered in mice lacking the gene for endothelial NOS (eNOS). The CO of B6 mice increased 28% by day 9.5 of gestation because of a 25% increase in SV, and increased 48% by day 17.5 because of a 41% increase in SV. The increase in SV at day 17.5 was associated with a 27% increase in PV, a 15% decrease in arterial pressure, and 10% to 15% increases in aortic and left-ventricular inner dimensions. In the absence of eNOS, CO increased 22% by day 9.5 because of increases in SV (14%) and heart rate (9%), but increased no further by day 17.5. SV near term was lower than B6 mice despite similar 26% increases in PV and 14% decreases in arterial pressure in association with blunted left-ventricular chamber enlargement. All reported changes are P<0.05. We conclude that cardiovascular changes during pregnancy are similar in B6 mice and humans. eNOS plays a critical role in increasing stroke volume in late gestation by promoting cardiac remodeling.

Background: In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5-E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure., Results: In 40 MHz ultrasound images at E6.5, the ectoplacental cone region and proamniotic cavity could be visualized. The ectoplacental cone region was successfully targeted with 13.8 nL of a fluorescent bead suspension with few or no beads off-target in 51% of concepti microinjected at E6.5 (28/55 injected). Seventy eight percent of the embryos survived 2 to 12 days post injection (93/119), 73% (41/56) survived to term of which 68% (38/56) survived and appeared normal one week after birth. At E7.5, the amniotic and exocoelomic cavities, and ectoplacental cone region were discernable. Our success at targeting with few or no beads off-target was 90% (36/40) for the ectoplacental cone region and 81% (35/43) for the exocoelomic cavity but tended to be less, 68% (34/50), for the smaller amniotic cavity. At E11.5, beads microinjected at E7.5 into the ectoplacental cone region were found in the placental spongiotrophoblast layer, those injected into the exocoelomic cavity were found on the surface or within the placental labyrinth, and those injected into the amniotic cavity were found on the surface or within the embryo. Following microinjection at E7.5, survival one week after birth was 60% (26/43) when the amniotic cavity was the target and 66% (19/29) when the target was the ectoplacental cone region. The survival rate was similar in sham experiments, 54% (33/61), for which procedures were identical but no microinjection was performed, suggesting that surgery and manipulation of the uterus were the main causes of embryonic death., Conclusion: Ultrasound-guided microinjection into the ectoplacental cone region at E6.5 or E7.5 and the amniotic cavity at E7.5 was achieved with a 7 day postnatal survival of >/=60%. Target accuracy of these sites and of the exocoelomic cavity at E7.5 was > or =51%. We suggest that this approach may be useful for exploring gene function during early placental and embryonic development.

Since genetically modified mice have become more common in biomedical research as models of human disease, a need has also grown for efficient and quantitative methods to assess mouse phenotype. One powerful means of phenotyping is characterization of anatomy in mutant vs. normal populations. Anatomical phenotyping requires visualization of structures in situ, quantification of complex shape differences between mouse populations, and detection of subtle or diffuse abnormalities during high-throughput survey work. These aims can be achieved with imaging techniques adapted from clinical radiology, such as magnetic resonance imaging and computed tomography. These imaging technologies provide an excellent nondestructive method for visualization of anatomy in live individuals or specimens. The computer-based analysis of these images then allows thorough anatomical characterizations. We present an automated method for analyzing multiple-image data sets. This method uses image registration to identify corresponding anatomy between control and mutant groups. Within- and between-group shape differences are used to map regions of significantly differing anatomy. These regions are highlighted and represented quantitatively by displacements and volume changes. This methodology is demonstrated for a partially characterized mouse mutation generated by N-ethyl-N-nitrosourea mutagenesis that is a putative model of the human syndrome oculodentodigital dysplasia, caused by point mutations in the gene encoding connexin 43.

Considerable progress has been made in adapting existing and developing new technologies to enable increasingly detailed phenotypic information to be obtained in embryonic and newborn mice. Sophisticated methods for imaging mouse embryos and newborns are available and include ultrasound and magnetic resonance imaging (MRI) for in vivo imaging, and MRI, vascular corrosion casts, micro-computed tomography, and optical projection tomography (OPT) for postmortem imaging. In addition, Doppler and M-mode ultrasound are useful noninvasive tools to monitor cardiac and vascular hemodynamics in vivo in embryos and newborns. The developmental stage of the animals being phenotyped is an important consideration when selecting the appropriate technique for anesthesia or euthanasia and for labeling animals in longitudinal studies. Study design also needs to control for possible differences between inter- and intralitter variability, and for possible long-term developmental effects caused by anesthesia and/or procedures. Noninvasive or minimally invasive intravenous or intracardiac injections or blood sampling, and arterial pressure and electrocardiography (ECG) measurements are feasible in newborns. Whereas microinjection techniques are available for embryos as young as 6.5 days of gestation, further advances are required to enable minimally invasive fluid or tissue samples, or blood pressure or ECG measurements, to be obtained from mouse embryos in utero. The growing repertoire of techniques available for phenotyping mouse embryos and newborns promises to accelerate knowledge gained from studies using genetically engineered mice to understand molecular regulation of morphogenesis and the etiology of congenital diseases.

Background: We investigated the locus of flow regulation of vascular tone in carotid arteries of C57 Bl/6 and eNOS(-/-) mice., Methods: Arterial segments (2-3 mm) were mounted in a perfusion myograph and preconstricted with 1 muM phenylephrine before monitoring flow-induced changes in lumen diameter., Results: Both control and eNOS(-/-) mice demonstrated flow-dependent relaxation. This response was not attenuated by the NO synthase antagonist L-NAME, the cyclooxygenase inhibitor indomethacin, the selective guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), the adenylate cyclase inhibitor Rp-8-Br-cAMPs, integrin-binding RGD peptides, or by removal of the endothelium. Hypoxia, a physiological stimulus known to alter endothelium-dependent flow regulation of vascular tone, also failed to attenuate the observed flow-mediated dilation., Conclusions: These findings indicate the existence of a previously unidentified endothelium-independent mechanism of flow-induced dilation in the carotid artery. Further investigations to identify the mechanisms that underlie this response may provide novel therapeutic directions in the treatment of disorders characterized by abnormal flow regulation of vascular tone.

This chapter describes methods for making vascular corrosion casts of the uteroplacental and fetoplacental vasculature of the mouse placenta. A catheter placed in the ascending thoracic aorta of a pregnant mouse permits the introduction of a methyl methacrylate casting compound into the lower body vasculature, including the uterus and placenta. A fine-tipped glass cannula attached to a double-lumen catheter is used to instill the same casting compound in the fetoplacental vessels of mouse placentas. Following polymerization of the casting compound, tissue is digested off of the placental casts using 20% KOH. The washed and dried casts are then available for light or scanning electron microscopy. The methods described have been used to cast the mouse uteroplacental vasculature from 5.5-18.5 d gestation and the fetoplacental vasculature from 12.5 d gestation to term.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.

Alopecia is a common dermatological condition in humans and other mammals. Here, we present two similar but histologically distinct mouse models of scarring alopecia. Both mutant lines were generated using random genome-wide N-ethyl-N-nitrosourea mutagenesis, and both harbor dominant mutations on chromosome 11. In both mutants, there is an early onset of alopecia that progresses to nearly complete pelage hair loss in both males and females by 20 weeks of age. Histologically, there is an increased dermal cellularity due to inflammatory cell infiltration at 7-10 days of age. By 3 weeks of age, the epidermis is acanthotic and the dermis is approximately twice as thick as in control mice due to a substantial, mostly mononuclear, inflammatory cell infiltrate. This infiltrate becomes more perifollicular by 4-5 weeks of age but is localized differently in the two mutants. In alopecia 1 (Alo-1), the perifollicular infiltrate is confined to the portion of the follicle within the dermis, whereas in Alo-2, the infiltrate extends the full length of the follicle. Expression of major histocompatibility complex (MHC) class I on the follicular epithelium in the two mutants is much greater than that in non-mutants. Furthermore, MHC class I expression is localized differently in the two mutant lines and mirrors the pattern of the inflammatory infiltrate. Despite these differences, the clinical progression of alopecia is identical in both mutants. The early onset of the disease, predictable progression, and differences in inflammatory cell localization between the two mutants make these mice particularly useful models for inflammatory hair loss and autoimmune diseases in general.

High resolution ultrasound imaging of the mouse placenta during development revealed highly echogenic foci localized near the materno-placental interface in early gestation and, near term, in the placental labyrinth (the exchange region of the placenta). Echogenic foci and calcium deposits identified in histological sections using Alizarin red staining showed similar localization and changes with gestation. Calcium deposits caused the echogenic foci because incubating uteri in a decalcifying solution eliminated both the deposits and echogenic foci. Transmission electron microscopy, X-ray microanalysis, and electron diffraction were used to show that deposits were calcium hydroxyapatite crystals. Calcium deposits were extensive and densely packed at days 7.5-9.5 of gestation at the border between the maternal decidua and the fetal trophoblast giant cells of ectoplacental cone. After the formation of the chorio-allantoic placenta (approximately day 10.5), calcification deposits appeared larger and more rarefied but were still localized at the border between the maternal decidua and the fetal trophoblast giant cells of the placenta. Calcification deposits were not observed in the labyrinthine region of the mouse placenta until > or = day 15.5 (day 18.5 is full term). We conclude that deposits of calcium hydroxyapatite crystals in the mouse placenta are detectable by high resolution ultrasound imaging. These deposits provide an ultrasound detectable marker of the maternal-placental interface that is particularly prominent during the establishment of the chorio-allantoic placenta between days 7.5 and 9.5 of gestation., ((c) Elsevier Ltd. All rights reserved.)

Tissue-specific transcription factors regulate several important aspects of embryonic development. They must function in the context of DNA assembled into the higher-order structure of chromatin. Enzymatic complexes such as the Swi/Snf-like BAF complexes remodel chromatin to allow the transcriptional machinery access to gene regulatory elements. Here we show that Smarcd3, encoding Baf60c, a subunit of the BAF complexes, is expressed specifically in the heart and somites in the early mouse embryo. Smarcd3 silencing by RNA interference in mouse embryos derived from embryonic stem cells causes defects in heart morphogenesis that reflect impaired expansion of the anterior/secondary heart field, and also results in abnormal cardiac and skeletal muscle differentiation. An intermediate reduction in Smarcd3 expression leads to defects in outflow tract remodelling reminiscent of human congenital heart defects. Baf60c overexpressed in cell culture can mediate interactions between cardiac transcription factors and the BAF complex ATPase Brg1, thereby potentiating the activation of target genes. These results reveal tissue-specific and dose-dependent roles for Baf60c in recruiting BAF chromatin remodelling complexes to heart-specific enhancers, providing a novel mechanism to ensure transcriptional regulation during organogenesis.

Mouse blood imaged using high-frequency ultrasound (US) is more echogenic in embryos than in adults. Studying changes in blood echogenicity in embryos may be of fundamental interest in studies on the genetic regulation of normal and abnormal blood development in mutant mice. Embryonic red blood cells (RBCs) are large and nucleated in midgestation but decrease in size and become enucleated as they mature. We therefore hypothesised that these structural alterations are responsible for variations in echogenicity of embryonic blood with gestational age and development. The objective of the current study was to quantify these structural changes in echogenicity (echo brightness) and apparent integrated backscatter (AIB) from embryonic blood at high US frequencies in vivo in mice. Results from anaesthetised pregnant mice studied using transcutaneous US showed that echogenicity of embryonic blood in the heart, aorta and umbilical cord and AIB within the heart chambers peaked at embryonic day (ED) 13.5 and then decreased progressively toward term. Between EDs 13.5 and 17.5 (near term), RBC mean cell volume decreased from 133 to 109 fL, haematocrit increased from 12 to 34%, and the percentage of nucleated RBCs decreased from 59 to 2%. Relative to younger ages, RBC nuclei at ED 13.5 were small and dense (pyknotic) which may have contributed to the peak in echogenicity and AIB at this age. To calculate the AIB, radiofrequency (RF) signals with centre frequencies of 28 MHz and 35 MHz were integrated over the 16- to 35-MHz and 21- to 42-MHz frequency range, respectively. At 28 MHz, mean apparent integrated backscatter of blood in the embryonic heart increased significantly from 0.0023 +/- 0.0004 Sr.cm(-1) (mean +/- SEM) at ED 12.5 to peak at 0.0037 +/- 0.0005 Sr.cm(-1) at ED 13.5. The mean AIB then decreased progressively with advancing gestation to 0.0002 +/- 0.0001 Sr.cm(-1) at ED 17.5. At 35 MHz, the mean AIB changed similarly with gestational age, except that values were lower than at 28 MHz at all ages. Higher attenuation of US at 35 MHz than at 28 MHz in tissue likely accounted for the lower AIB of blood insonified at 35 MHz. We speculate that developmental changes in red cell morphology are responsible for the observed changes in echogenicity and AIB of embryonic blood with gestational age in mice.

Objective: Our aim was to study the hemodynamic effects of betamethasone on fetuses with intrauterine growth restriction (IUGR) with absent or reversed end-diastolic (ARED) umbilical artery flow., Study Design: Color/pulsed Doppler waveforms were obtained before and after intramuscular injections of betamethasone in 19 consecutive fetuses with IUGR/ARED and 6 control fetuses. Peak velocities and pulsatility index (PI) values were obtained from the umbilical (UA) and middle cerebral (MCA) arteries and intrahepatic umbilical vein (UV)., Results: Ten ARED fetuses developed transient positive umbilical end-diastolic flow after steroids, whereas nine fetuses showed persistent ARED. The persistent ARED subgroup demonstrated increased UA and UV peak velocities after steroids, which may indicate fetal hypertension. Fetal death (n=2) and severe acidosis (n=2) were confined to the subgroup with persistent ARED., Conclusion: Preterm IUGR/ARED fetuses exhibit divergent cardiovascular responses to prenatal steroids. Intensive Doppler-based fetal monitoring may identify a subset of fetuses prone to decompensation after maternal steroid administration.

Developmental changes in left and right ventricular diastolic filling patterns were determined noninvasively in isoflurane-anesthetized outbred ICR mice. Blood velocities in the mitral and tricuspid orifices were recorded in 16 embryos at days 14.5 (E14.5) and 17.5 of gestation (E17.5) using an ultrasound biomicroscope and also serially in three groups of postnatal mice aged 1-7 days (n = 23), 1-4 wk (n = 18), and 4-12 wk (n = 27) using 20-MHz pulsed Doppler. Postnatal body weight increased rapidly to 8 wk. Heart rate increased rapidly from approximately 180 beats/min at E14.5 to approximately 380 beats/min at 1 wk after birth and then more gradually to plateau at approximately 450 beats/min after 4 wk. Ventricular filling was quantified using the ratio of peak velocity of early ventricular filling due to active relaxation (E wave) to that of the late ventricular filling caused by atrial contraction (A wave) (peak E/A ratio) and the ratio of the peak E velocity to total time-velocity integral of E and A waves (peak E/total TVI ratio). Both ventricles had similar diastolic filling patterns in embryos (peak E/A ratio of 0.28 +/- 0.02 for mitral flow and 0.27 +/- 0.02 for tricuspid flow at E14.5). After birth, mitral peak E/A increased to >1 between the third and fifth day, continued to increase to 2.25 +/- 0.25 at approximately 3 wk, and then remained stable. The tricuspid peak E/A ratio increased much less but stabilized at the same age (increased to 0.79 +/- 0.03 at 3 wk). The peak E/total TVI ratio showed similar left-right differences and changes with development. Age-related changes were largely due to increases in peak E velocity. The results suggest that diastolic function matures approximately 3 wk postnatally, presumably in association with maturation of ventricular recoil and relaxation mechanisms.

Mammalian embryos have an intimate relationship with their mothers, particularly with the placental vasculature from which embryos obtain nutrients essential for growth. It is an interesting vascular bed because maternal vessel number and diameter change dramatically during gestation and, in rodents and primates, the terminal blood space becomes lined by placental trophoblast cells rather than endothelial cells. Molecular genetic studies in mice aimed at identifying potential regulators of these processes have been hampered by lack of understanding of the anatomy of the vascular spaces in the placenta and the general nature of maternal-fetal vascular interactions. To address this problem, we examined the anatomy of the mouse placenta by preparing plastic vascular casts and serial histological sections of implantation sites from embryonic day (E) 10.5 to term. We found that each radial artery carrying maternal blood into the uterus branched into 5-10 dilated spiral arteries located within the metrial triangle, populated by uterine natural killer (uNK) cells, and the decidua basalis. The endothelial-lined spiral arteries converged together at the trophoblast giant cell layer and emptied into a few straight, trophoblast-lined "canals" that carried maternal blood to the base of the placenta. Maternal blood then percolated back through the intervillous space of the labyrinth toward the maternal side of the placenta in a direction that is countercurrent to the direction of the fetal capillary blood flow. Trophoblast cells were found invading the uterus in two patterns. Large cells that expressed the trophoblast giant cell-specific gene Plf (encoding Proliferin) invaded during the early postimplantation period in a pattern tightly associated with spiral arteries. These peri/endovascular trophoblast were detected only approximately 150-300 microm upstream of the main giant cell layer. A second type of widespread interstitial invasion in the decidua basalis by glycogen trophoblast cells was detected after E12.5. These cells did not express Plf, but rather expressed the spongiotrophoblast-specific gene Tpbp. Dilation of the spiral arteries was obvious between E10.5 and E14.5 and was associated with a lack of elastic lamina and smooth muscle cells. These features were apparent even in the metrial triangle, a site far away from the invading trophoblast cells. By contrast, the transition from endothelium-lined artery to trophoblast-lined (hemochorial) blood space was associated with trophoblast giant cells. Moreover, the shaping of the maternal blood spaces within the labyrinth was dependent on chorioallantoic morphogenesis and therefore disrupted in Gcm1 mutants. These studies provide important insights into how the fetoplacental unit interacts with the maternal intrauterine vascular system during pregnancy in mice.

We report here on the design and evaluation of the first high-frequency ultrasound (US) imaging system specifically designed for microimaging of the mouse. High-frequency US or US biomicroscopy (UBM) has the advantage of low cost, rapid imaging speed, portability and high resolution. In combination with the ability to provide functional information on blood flow, UBM provides a powerful method for the investigation of development and disease models. The new UBM imaging system is demonstrated for mouse development from day 5.5 of embryogenesis through to the adult mouse. At a frequency of 40 MHz, the resolution voxel of the new mouse scanner measures 57 microm x 57 microm x 40 microm. Duplex Doppler provides blood velocity sensitivity to the mm per s range, consistent with flow in the microcirculation, and can readily detect blood flow in the embryonic mouse heart, aorta, liver and placenta. Noninvasive UBM assessment of development shows striking similarity to invasive atlases of mouse anatomy. The most detailed noninvasive in vivo images of mouse embryonic development achieved using any imaging method are presented., (Copyright 2002 World Federation for Ultrasound in Medicine & Biology)

A new multifrequency (19-55 MHz) ultrasound biomicroscope with two-dimensional imaging and integrated Doppler ultrasound was evaluated using phantoms and isoflurane-anesthetized mice. Phantoms revealed the biomicroscope's lateral resolution was between 50 and 100 microm, whereas that of a conventional 13 MHz ultrasound system was 200-500 microm. This difference was apparent in the markedly higher resolution images achieved using the biomicroscope in vivo. Transcutaneous images of embryos in pregnant mice from approximately 2 days after implantation (7 days gestation) to near term (17.5 days) were obtained using frequencies from 25 to 40 MHz. The ectoplacental cone and early embryonic cavities were visible as were the placenta and embryonic organs throughout development to term. We also evaluated the ability of the biomicroscope to detect important features of heart development by examining embryos from 8.5 to 17.5 day gestation in exteriorized uteri using 55 MHz ultrasound. Cardiac looping, division of the outflow tract, and ventricular septation were visible. In postnatal imaging, we observed the heart and kidney of neonatal mice at 55 MHz, the carotid artery in juveniles (approximately 8 g body wt) and adults (approximately 25 g body wt) at 40 MHz, and the adult heart, aorta, and kidney at 19 MHz. The coefficient of variation of carotid and aortic diameter measurements was 1-3%. In addition, blisters in GRIP1 -/- embryos and aortic valvular stenosis in two adults were readily visualized. Using image-guided Doppler function, low blood velocities in vessels as small as 100 microm in diameter including the primitive heart tube at day 8.5 were measurable, but high blood velocities (>37.5 cm/s) such as in the heart and large arteries in late gestation and postnatal life were off-scale. Accurate cardiac dimension measurements were impeded by poor temporal resolution (4 frames/s). In summary, the multifrequency ultrasound biomicroscope is a versatile tool well suited to detailed study of the morphology of various organ systems throughout development in mice and for hemodynamic measurements in the low velocity range.

Members of the TGF-beta family have been shown to play an important role in numerous tissues during development. In the present study we have investigated the spatial and temporal expression of TGF-beta 3, in human umbilical cord development. Total TGF-beta 3 protein content, assessed by immunoblotting, increased with advancing gestation as did immunostaining and mRNA in Wharton's jelly fibroblasts. Immunohistochemical analysis revealed that TGF-beta 3 was present in all cell types. Temporal changes in TGF-beta 3 expression were observed in the vascular smooth muscle cells, such that with advancing gestation TGF-beta 3 protein expression and became mostly restricted to the extracellular compartment of the vascular media. This was associated with a decrease in TGF-beta 3 mRNA expression in umbilical vascular smooth muscle cells. Of clinical significance, umbilical cords from pregnancies complicated by pre-eclampsia, showed a significant reduction in total TGF-beta 3 protein expression when compared to those of age-matched patients. Both TGF-beta 3 mRNA and protein expression were downregulated in the endothelium and smooth muscle layers of the umbilical arteries, as well as in the Wharton jelly fibroblasts. Our data demonstrate that during umbilical cord development TGF-beta 3 expression is spatially and temporally regulated and that TGF-beta 3 expression is altered in umbilical cords of pregnancies complicated by pre-eclampsia. We speculate that the downregulation of TGF-beta 3 expression found in pre-eclamptic umbilical cord may contribute to the abnormal structure and mechanical properties seen in these pathological umbilical cords., (Copyright 2002 Elsevier Science Ltd.)

Genetically altered mice may provide useful models for exploring cardiovascular regulation during pregnancy and postpartum if changes in mice mimic humans. We found in awake ICR (CD-1) mice at 17.5 days gestation that hematocrit was reduced 18%, and the pressor response to intravenous angiotensin II was reduced ~33%. Arterial pressure in awake mice was 12% lower in early pregnancy (3.5 days) than late pregnancy (17.5 days) and postpartum (3 and 17 days after delivery), whereas heart rate was 10-20% higher in the peripartum period (17.5 days gestation and 3 days postpartum). In late pregnancy, cardiac output under isoflurane anesthesia was 64% higher than in nonpregnant mice, due to a 37% increase in stroke volume and a 17% increase in heart rate. All changes P < 0.05. We conclude that, as in humans, mice exhibit hypotension in early pregnancy, and a blunted pressor response to angiotensin II, a decrease in hematocrit, and a marked increase in cardiac output in late pregnancy.

Vasa vasorum are important sources of oxygen and nutrients to vascular tissues and their proliferation influences the pathogenesis of arterial disease; however, the regulation of their growth is poorly understood partly because of a lack of appropriate experimental models. We cuffed common carotid arteries of rabbits with segments of the contralateral carotid artery, a procedure that resulted in rapid and extensive elaboration of adventitial vasa vasorum and connective tissue. Endothelium-lined microvessels were observed at 1 week but vessels as large as 300 microm with an organizing media were common by 3 weeks. These vasa vasorum arose primarily from the vascular supply to contiguous tissues, but also from the carotid artery. This angiogenesis was accompanied by increased expression of the angiogenic factor, vascular endothelial growth factor (VEGF), in the invading connective tissue cells and increased expression of the transcriptional regulator of VEGF, hypoxia-inducible factor-1alpha (HIF-1alpha), in these connective tissues and in the cuffing artery. These findings are consistent with the hypothesis that upregulation of HIF-1alpha and VEGF expression drives angiogenesis of vasa vasorum in this model. This simple model may be amenable to the study of the development and elaboration of vasa vasorum, especially in the context of vascular pathologies.

One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. In rodents and primates, new blood vessels form through angiogenesis, dilate and then become modified such that the blood enters into trophoblast cell-lined sinuses (hemochorial). Evidence from gene knockout mice suggests that factors from the placenta regulate the uterine vasculature. Consistent with this, trophoblast giant cells produce a number of angiogenic and vasoactive substances that may mediate these effects. Teratocarcinomas containing large numbers of trophoblast giant cells (derived from Parp1 gene-deficient ES cells) show similar 'hemochorial' host blood flow, implying that the effects are not specific to the uterine vascular bed. As in primates, murine trophoblast cells also invade into the uterine arteries of the mother. However, in normal pregnancy, dilation of the uterine arteries may be largely mediated by the effect of uterine natural killer cells.

We have previously shown in the sheep fetus at 0.7 and 0.9 gestation that the choroid plexus, unlike brain parenchyma, catabolizes prostaglandins (PGs). Peculiarly, in the choroid plexus, PGE(2) catabolism persists throughout the neonatal period to abate in the adult, while PGF(2alpha) catabolism abates shortly after birth. To explain this differential behavior and elucidate the function of catabolic enzymes, we examined the cellular location and activity of the rate-limiting enzyme for PGE(2) and PGF(2alpha) catabolism, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Immunofluorescence histochemistry and immunogold electronmicroscopy revealed abundant 15-PGDH expression in the epithelial cytosol close to the brush-border membrane at 0.7 and 0.9 gestation. In contrast, at 5 and 15 days postnatal, 15-PGDH was found throughout the cytosol of stromal fibroblasts. No staining was observed at either location in pregnant adults. PGF(2alpha) catabolism was minimal in the total homogenate and 100000xg supernatant of the fetal choroid plexus at 0.7 and 0.9 gestation, while PGE(2) catabolism was evident at 0.7 gestation only. In contrast, both PGs were catabolized in minced specimens at either age. In conclusion, our study shows immunoreactive 15-PGDH in the choroid plexus from fetal and neonatal, but not pregnant adult, sheep. Results suggest that PGE(2) catabolism is not as critically dependent as that of PGF(2alpha) on tissue integrity and 15-PGDH location. Given the key role being assigned to the choroid plexus in PG removal from brain, we speculate that persistence of PGE(2) catabolism into the early postnatal period protects against central respiratory depression caused by the compound during this susceptible stage of development.

PGE(2) has centrally mediated respiratory, febrile, and cardiovascular effects that markedly differ between fetal and adult life. We hypothesized that the transition from fetal to adult responses to PGE(2) occurs in the newborn period. Thus effects of an intracarotid infusion of PGE(2) (3 microg/min for 60 min) were determined in unanesthetized newborn lambs at 5, 10, and 15 days after birth. At 5 days, PGE(2) reduced central CO(2) sensitivity, reduced lung ventilation due to a decrease in breathing frequency, and induced hypercapnia. By 15 days, these effects of PGE(2) had waned significantly. In contrast, phasic (expiratory) thyroarytenoid muscle electromyogram activity, number of short apneas, and incidence of Biot periodic breathing were similarly increased at all three ages. PGE(2) induced a sustained fever at 10 and 15 days. Heart rate and mean arterial blood pressure were unchanged in contrast to marked increases observed by others in adults. Results showed that the transition from fetal to adult respiratory and febrile responses to PGE(2) occurs in early postnatal life, whereas adult cardiovascular responses develop later in life in sheep.

We hypothesized that placental resistance was elevated and transfer reduced in cotyledons from intrauterine growth-restricted (IUGR) fetuses. We perfused 10 cotyledons from term, normally grown fetuses, six from preterm, normally grown fetuses with normal umbilical arterial end-diastolic velocities (EDV), and six from preterm IUGR fetuses (<3rd centile) with absent or reversed umbilical arterial EDV. Perfused cotyledons were pressure-fixed, and villi were observed by scanning electron microscopy. The groups did not differ in fetoplacental resistance at baseline; neither did they differ in the change in resistance that followed the administration of nitroglycerin or angiotensin II. The increase in resistance during hypoxia was similar in the two preterm groups but greater in the term than in the preterm normally grown group (p < 0.05). Groups did not differ in net maternofetal transfer of oxygen or glucose, or in clearance of aminoisobutyric acid or antipyrine. However, glucose consumption was doubled in cotyledons of preterm IUGR versus preterm normally grown fetuses (p < 0.05). Terminal villi of perfused cotyledons from preterm IUGR fetuses displayed less terminal villous branching and budding than preterm controls, as anticipated from previous work. IUGR fetuses with absent or reversed umbilical arterial EDV in vivo may have high placental resistance due to a vasoconstrictive rather than anatomic abnormality and an elevated placental glucose consumption that may impair glucose transfer.

The early postnatal decrease in prostaglandin (PG)E(2) levels in cerebrospinal fluid (CSF) likely contributes to the establishment of continuous breathing. To elucidate mechanisms underlying this event, choroid plexuses from lateral (L-CP) and third/fourth (III/IV-CP) ventricles were incubated with [3H]-PGE(2) and label uptake (tissue-to-medium ratio for radioactivity, T/M) and catabolism (%radioactivity associated with metabolites, PGM) were measured. [3H]-PGF(2alpha) was a reference. Uptake of [3H]-PGE(2) was lower than [3H]-PGF(2alpha) in the term fetus (L-CP: 5.9+/-0.5 vs. 9.6+/-0. 9, n=11; III/IV-CP: 2.7+/-0.4 vs. 7.7+/-1.0, n=5) and 17 d lamb (L-CP: 5.3+/-0.8 vs. 11.0+/-1.2, n=7; III/IV-CP: 3.1+/-0.2 vs. 11. 6+/-2.8, n=3 and 4, respectively). This difference was not significant in the pregnant adult. Release of the two compounds was similar and did not change with age. [3H]-PGE(2) uptake was reduced by probenecid (1 mM) and excess PG (60 microM PGE(2) or PGF(2alpha)). Excess PG also reduced catabolism in the fetus, which was extensive for [3H]-PGE(2) and [3H]-PGF(2alpha)60%). In the lamb, catabolism remained high for [3H]-PGE(2) (L-CP: 64+/-4%, n=7; III/IV-CP: 41+/-4%, n=3), but not [3H]-PGF(2alpha) (L-CP: 26+/-4%, n=7; III/IV-CP: 4+/-1%, n=4). In the pregnant adult, catabolism was above background only for [3H]-PGE(2) in the L-CP (26+/-5%, n=11). Unlike the perinatal animal, this catabolism was reduced by probenecid. In conclusion, PGE(2) uptake and catabolism operate independently in the choroid plexus from perinatal sheep. Differences between PGE(2) and PGF(2alpha) are developmentally-regulated for both mechanisms. While neither process explains the postnatal decrease in CSF PGE(2), both may help keep CSF levels low during early postnatal development.

We previously reported changes in mechanical properties and collagen cross-linking of the ovine thoracic aorta during perinatal development and postnatal maturation, and we now report changes in biochemical composition (elastin, collagen, and DNA contents per mg wet wt) over the same developmental intervals. A comparison of results from the present and previous studies has yielded novel and important observations concerning the relationship between aortic mechanics and composition during maturation. Developmental changes in aortic incremental elastic modulus at low tensile stress (E(low)) closely followed changes in relative elastin content (i.e., per mg wet wt). An 89% increase in E(low) during the perinatal period was associated with a 69% increase in relative elastin content, whereas neither variable changed during postnatal life. Incremental elastic modulus at high tensile stress (E(high)) did not change during the perinatal period but increased 88% during postnatal life. This pattern closely paralleled changes in collagen cross-linking index, which did not change perinatally but almost doubled postnatally. In contrast, relative collagen content (per mg wet wt) increased only slightly from fetal to adult life, a trend that was unrelated to aortic mechanics. Substantial, progressive decreases in measures of wall viscosity (pressure wave attenuation coefficient and viscoelastic phase angle) from fetal to adult life followed the pattern observed for relative DNA (smooth muscle cell) content (per mg wet wt). Our findings suggest that accumulation of elastin per milligram wet weight contributes most to developmental changes in E(low), change in collagen cross-linking is the primary determinant of developmental changes in E(high), and cell accumulation contributes most to developmental changes in wall viscosity.

Increased arterial wall tension stimulates growth and remodeling of arteries, but little is known about the effects of decreased wall tension, despite its developmental and pathological significance. Consequently, we cuffed 1 carotid artery in rabbits with a portion of the contralateral artery to off-load circumferential wall tension. The model produced rapid and extensive atrophy of the cuffed artery that yielded decreases in the DNA content of the cuffed artery (a measure of cell number) from 8.0+/-0.5 microgram/cm of in situ vessel length to 5.6+/-0.5 microgram/cm at 21 days postoperatively. The elastin content of the cuffed artery was also significantly reduced, from 399+/-17 to 283+/-17 microgram/cm, and collagen content was reduced from 468.0+/-59.0 to 154+/-24 microgram/cm (P<0.05) at 21 days postoperatively. Detection of DNA oligonucleosomes by gel electrophoresis implicated apoptotic cell death in remodeling due to cuffing. Upregulation of matrix metalloproteinases (MMPs), including MMP-2, MMP-9, and unidentified gelatinases, indicated that these enzymes may also be involved in remodeling. No further changes in wall structure were seen between 3 weeks and 6 months, and the excised artery that was used as a cuff exhibited normal medial morphology for at least 6 months postoperatively. We infer from these experiments that off-loading of arterial wall tension induces rapid and extensive atrophy of the arterial media.

The flow pulsatility index, the ratio of flow pulse amplitude to mean flow over the cardiac cycle, has been used to quantify pulsatility of blood flow in the umbilical artery. In experiments with fetal sheep, we showed that the flow pulsatility index in the umbilical artery is accurately estimated by the ratio of total umbilico-placental vascular resistance (mean arterial pressure divided by mean umbilical flow) divided by fundamental impedance (umbilical vascular impedance at the heart rate frequency) times the pulsatility index (pulse/mean) of the arterial pressure that drives flow through this bed. The pulsatility index of arterial pressure is primarily determined by upstream factors (e.g. heart rate) whereas fundamental impedance depends primarily on the radius and viscoelastic wall properties of the umbilical artery. An increase in resistance in the microcirculation and/or veins causes proportional changes in the flow pulsatility index because these sites have little influence on fundamental impedance. However, an increase in resistance in the highly vasoactive umbilical arteries has offsetting effects on impedance and resistance; consequently, flow pulsatility changes little even when arterial vasoconstriction markedly reduces mean flow. We conclude that when arterial pressure pulsatility is stable, a change in the flow pulsatility index provides a useful indication of a change in resistance in the microcirculation and/or veins but will not reliably detect a resistance change in the artery.

The human placenta contains two types of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). The exclusive oxidase 11beta-HSD2 has been suggested to protect the fetus from high levels of maternal glucocorticoids by converting cortisol to inactive cortisone. Perfused term human placenta was used to examine the activity of the oxoreductase 11beta-HSD1 and to determine the regulation of cortisol effects on placental vascular tone and corticotropin-releasing hormone (CRH) output by 11beta-HSD. Radioimmunoassay showed that there was substantial cortisol (295+/-57 nM) detected in the fetal vein upon perfusion of cortisol (2 microM; perfusion rate, 12 ml/min) into the maternal intervillous space. Output of cortisol increased to 559+/-22 nM on the fetal side (P<0.05) with concurrent perfusion of carbenoxolone (CBX; 1 microM), a non-specific 11beta-HSD inhibitor. Cortisol formation increased in a dose-dependent manner with infusion of cortisone (0.1-2 microM) into the maternal intervillous space reaching 15 and 23 nM in fetal and maternal venous outflows respectively at 2 microM cortisone perfusion. There was no significant effect of cortisol either alone or in combination with CBX on the fetal arterial perfusion pressure, but cortisol perfusion increased CRH output into the fetal vein. It is concluded that activities of both 11beta-HSD1 and -2 are demonstrable in perfused human placenta in vitro, and these enzymes affect transplacental glucocorticoid transfer. These activities may provide a precise mechanism to control the passage of maternal glucocorticoids to the fetal circulation, and to regulate glucocorticoid effects within the placenta.

We have used hydrothermal isometric tension (HIT) techniques in a sheep model to assess collagen crosslink stability and its contribution to the mechanical properties of the ovine thoracic aorta during perinatal and postnatal development. Aortic tissue was studied from fetal sheep, lambs, and adult sheep. Strips of tissue were loaded under isometric tension and heated to a 90 degrees C isotherm which was sustained for 3 hours. The decrease in load at this temperature is associated with collagen peptide bond hydrolysis and chain slippage, and the rate of this decrease is an inverse indicator of collagen crosslinking. The half-time of load decay (t1/2) was computed before and after tissue was treated with NaBH4 which stabilizes immature, reducible crosslinks. We observed a two-fold increase in t1/2 of untreated tissue from the lamb to the adult, indicating that aortic collagen crosslinking increased during postnatal development. Furthermore, the t1/2 of NaBH4-stabilized lamb tissue was similar to that of the untreated adult tissue, suggesting that much of the immature crosslinking in the lamb is stabilized during postnatal development. These observations suggest (a) increased crosslinking occurs during postnatal development and (b) that this increase is largely due to a conversion of immature crosslinks into their mature heat stable form.

Expression in sheep brain of EP transcripts and their ontogeny in the brainstem was determined using RT-PCR. Sheep EP subtypes showed high homology (>80%) with other species. EP1, EP2, EP3 and EP4 were expressed in spinal cord, medulla, pons, diencephalon, hippocampus, pituitary, cortex and cerebellum of the fetal, newborn and adult sheep brain. Expression of all four peaked in the newborn, whereas fetal and adult expression were similar. A similar ontogeny suggests a common regulation., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

The cardiovascular effects of repeated administration of the nitric oxide (NO) synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) were assessed daily for 3 days in fetal sheep near term (124-126 days gestation) beginning 4 days after surgery (n = 7). In the first hour on day 1, fetal infusion of L-NAME (30 mg bolus, 6 mg/min infusion iv for 3 h) significantly increased fetal arterial pressure from 41 +/- 2 to 58 +/- 3 mmHg, decreased heart rate from 173 +/- 5 to 134 +/- 3 beats/min, increased umbilicoplacental resistance from 0.16 +/- 0.02 to 0.28 +/- 0.07 mmHg.ml-1.min, and inhibited the hypotensive response to acetylcholine (ACh; 2 micrograms iv bolus). All changes were sustained except for arterial pressure, which decreased significantly to 50 +/- 3 mmHg in the third hour. Within 17 h, all cardiovascular variables returned to control. L-NAME readministered on days 2 and 3 had no effect on cardiovascular variables. L-NAME did not potentiate the pressor response to angiotensin II on day 2 and caused a surprising attenuation of the pressor response to endothelin-1 on day 3. We conclude that, whereas NO normally contributes to low arterial pressure, high heart rate, and low umbilicoplacental vascular resistance in fetal sheep near term, the role of NO in these functions is replaced by an alternate mechanism within 17 h after NO synthesis inhibition with L-NAME.

The mammalian aorta undergoes rapid remodeling during the perinatal period and more gradual remodeling during subsequent development, but the implications of this remodeling for arterial mechanics are poorly understood. In this study in vivo and in vitro techniques were used to determine the static and viscoelastic properties of the thoracic aortas of 119-day-gestation fetal sheep (full term = 145 days), 21-day-old lambs, and adult sheep at control distending pressures and after 70% increases or 30% decreases in pressure. In the weeks surrounding birth, aortic wall tissue became substantially stiffer (static elastic modulus in vitro increased by 28%, and pressure wave velocity in vivo increased by 61%) but less viscous (pressure wave attenuation in vivo decreased by 46%, and viscoelastic phase angle in vitro decreased by 15%), whereas the wall thickness-to-radius ratio was unchanged. By contrast, modest changes in tissue viscoelasticity from neonatal to adult life were accompanied by a halving of the wall thickness-to-radius ratio from 0.19 +/- 0.01 to 0.10 +/- 0.01. The relative thinning of the vessel wall, combined with a doubling of blood pressure after birth, resulted in a 265% increase in aortic wall tensile stress over the period of study. We concluded that rapid remodeling in the perinatal period primarily alters the viscoelastic properties of aortic wall tissues, whereas more gradual postnatal remodeling largely affects vessel geometry.

The role of nitric oxide in hypoxic fetoplacental vasoconstriction (HFPV) was investigated using dually perfused human placental cotyledons. Standard medium (Earle's salt solution with added dextran and L-arginine) was equilibrated with 95 per cent O2 and 5 per cent CO2 (maternal side) and 94 per cent N2 and 6 per cent CO2 (fetal side). Part 1 consisted of perfusion for 1 h, then maternal perfusate equilibrated with a 95 per cent N2 and 5 per cent CO2 for 20 min (hypoxia), and then the original perfusion conditions resumed for 40 min. In part 2, this sequence was repeated with standard medium alone (n = 6), or with added N-nitro-L-arginine methyl ester (L-NAME) (n = 6), or L-NAME and nitroglycerin (n = 6). When standard medium was used throughout, basal fetal perfusion pressure (30 +/- 2 mmHg) and the hypoxia-induced increase in perfusion pressure (18 +/- 1 mmHg) did not change significantly between parts 1 and 2. L-NAME increased basal perfusion pressure from 33 +/- 3 to 56 +/- 2 mmHg whereas perfusion pressure remained unchanged with L-NAME and nitroglycerin or nitroglycerin alone. The hypoxic vasoconstriction observed during part 1 in the L-NAME (14 +/- 3 mmHg) and the L-NAME with nitroglycerin groups (18 +/- 2 mmHg) was abolished during part 2 (to - 4 +/- 1 and 0.4 +/- 0.5 mmHg, respectively) whereas nitroglycerin alone significantly blunted the response (21 +/- 3 to 6 +/- 1 mmHg). Results suggest that a reduction in basal NO release mediates hypoxic fetoplacental vasoconstriction in the perfused human placental cotyledon in vitro.

We have previously reported that prostaglandin(PG) E2 levels in sheep cerebrospinal fluid (CSF) are high prenatally and abate rapidly after birth. This event may contribute to the establishment of continuous breathing. To explain this change, we have examined PG disposal mechanisms in the perinatal and adult (pregnant and non-pregnant animal) sheep brain by measuring the capacity of the isolated choroid plexus to concentrate [3H]PGF2alpha and [3H]PGE2. At 0.9 gestation, [3H]PGF2alpha uptake (expressed as the tissue-to-medium ratio, T/M) attained a steady-state by 15 min and was maintained thereafter (T/M at 60 min, 5.6 +/- 0.6; n = 16). Likewise, [3H]PGE2 was taken up by the tissue, but the actual accumulation was smaller (T/M at 60 min, 2.6 +/- 0.2; n = 8). Thin-layer radiochromatographic analysis of the tissue following incubation with [3H]PGF2alpha showed that 55 +/- 4% (n = 10) of radioactivity migrated as the 15-keto-13,14-dihydro metabolite. [3H]PGF2alpha uptake decreased upon treatment with probenecid (1 mM) (T/M, 2.5 +/- 0.2; n = 10) or after adding unlabelled PGF2alpha to the medium (1-60 microM) (T/M at 60 microM, 1.8 +/- 0.1; n = 13). The yield of labelled metabolite was also lower when using excess PGF2alpha (14% of control at 60 microM; n = 13), while it was not affected by probenecid. Uptake of both PGs did not change through development, from 0.7 gestation to day 18 postnatal, and attained higher values in the pregnant adult. Conversely, PGF2alpha catabolism decreased postnatally and became negligible by adult age. We conclude that during the perinatal period PGs can be removed from CSF by two distinct processes in the choroid plexus, active transport and catabolism. Neither process, however, can account for the birth-related change in CSF PGE2.

Indomethacin, a prostaglandin synthesis inhibitor, transforms normally intermittent fetal breathing into nearly continuous breathing. If indomethacin acts in brain, we hypothesized that indomethacin would stimulate breathing at a lower dose when given via the lateral cerebral ventricle (i.c.v) than via the fetal jugular vein (i.v.). Chronically catheterized fetal sheep were studied at 0.88 of gestation (128 days). A 2-h control period was followed by an indomethacin infusion at 7 micrograms.kg-1 fetal body weight over the first 10 min of each hour for 3 h then at 28 micrograms.kg-1 over the first 10 min of each hour for the next 3 h, either i.c.v. (n = 7) or i.v. (n = 6). Plasma prostaglandin E2 concentrations decreased similarly by both routes, although the decrease was only significant for the i.c.v. route. Indomethacin at 7 micrograms.kg-1.h-1 did not change fetal breathing activity by either route within 3 h. During the 2nd and 3rd h at 28 micrograms.kg-1.h-1, breathing incidence increased (from approximately 35 to approximately 80% time), breath amplitude and rate increased, and arterial O2 content decreased slightly (all changes significant). ANOVA revealed no significant differences in responses evoked by the two routes. Fetal arterial pressure, heart rate, Pco2, and pH were unchanged (both doses, both routes). The indomethacin dose (105 micrograms.kg-1 over 6 h) was > or = 10-fold lower than that used in previous studies on breathing in fetal sheep but similar to that used clinically to treat patent ductus arteriosus. We conclude that site(s) mediating effects of indomethacin on breathing respond to a low dose and are equally accessible by i.c.v. and i.v. routes.

Prostaglandin (PG) E2 acts on the brain stem to modulate breathing activity in the ovine fetus. The source of this PGE2 is unknown and we hypothesized that it is produced locally in the developing brain and functions in a paracrine and/or autocrine manner. The purpose of the present study was to establish whether prostaglandin-H synthase-1 (PGHS-1), a crucial enzyme in de novo prostaglandin synthesis, is present and its gene expressed in the ovine fetal brain. Immunohistochemical and molecular hybridization techniques were used to identify sites of PGHS-1 immunoreactivity and PGHS-1 mRNA expression respectively in the brain of the ovine fetus in late gestation (approximately 126 days gestation, term 145 days). PGHS-1 immunoreactivity was localized to specific regions of the fetal brain, including the cortex, hypothalamus, hippocampal formation, superior colliculus of the midbrain, parabrachial nucleus of the pons, and the reticular formation, raphe, nucleus of the solitary tract, and gracile and cuneate nuclei of the medulla. The relative abundance of PGHS-1 mRNA in selected brain regions, as determined by Northern blot analysis, correlated qualitatively with the number of PGHS-1 immunoreactive neurons identified in each region. In situ hybridization demonstrated PGHS-1 mRNA to be localized in the same neurons or nuclei as PGHS-1 immunoreactivity. These results indicate that PGHS-1 synthesized de novo in many brain regions including two that are important in respiratory control: the pneumotaxic center (parabrachial nucleus) and the dorsal respiratory group (nucleus tractus solitarius) suggesting that prostaglandins that modulate fetal respiratory activity are synthesized endogenously.

We investigated the hypothesis that the precipitous decrease in prostaglandin E2 (PGE2), a potent inhibitor of fetal breathing, from high plasma concentrations during labor causes a rebound stimulation of breathing without newborn concentrations falling below prelabor fetal values. Fetal plasma PGE2 concentration was gradually increased from 384 +/- 82 (SE) pg/ml in 2-h steps [0 (baseline), 1.5, 3, and 6 micrograms/min] to labor levels (1,230 +/- 381 pg/ml at 6 micrograms/min) and then was maintained for 24 h (n = 9). PGE2 at 1.5 micrograms/min significantly decreased breathing incidence [from 42 +/- 4 (baseline) to 14 +/- 4%] and breath amplitude (from 2.1 +/- 0.5 to 1.5 +/- 0.2 arbitrary units) and increased breath-to-breath interval (from 1.16 +/- 0.07 to 1.56 +/- 0.06 s). No further dose-related changes were observed. During the first 2 h after PGE2 infusion was stopped, PGE2 concentration returned to basal (352 +/- 64 pg/ml) but breathing incidence and amplitude were significantly higher (74 +/- 8% and 2.4 +/- 0.3 arbitrary units, respectively) and breath-to-breath interval was significantly lower (0.95 +/- 0.10 s) than were basal levels. Changes arose within approximately 15 min and were maintained for at least 4 h. Breathing did not change significantly in the saline-treated group (n = 7). Results suggest that the rapid decrease in plasma PGE2 concentration at birth promotes the onset of breathing.

Endothelin-1 produced by umbilicoplacental tissues may regulate fetal placental perfusion. To investigate its site of action, we measured segmental resistance in this bed in unanesthetized fetal sheep near term during fetal endothelin-1 infusion. A 15-min intravenous infusion of endothelin-1 at 1 micrograms/min significantly increased fetal blood pressure in the aorta (+33%), cotyledon artery and vein, and inferior vena cava, and endothelin-1 decreased fetal heart rate (-40%). Vascular resistance in the placental microcirculation increased significantly (+332%), but smaller increases in resistance of the umbilical artery and vein were not significant. Nevertheless, the stiffness of the umbilical arterial wall appeared to increase because vascular input impedance increased significantly both at the heart rate frequency (+85%) and when averaged > 2 Hz (characteristic impedance; +138%). Mean blood flow in the umbilical artery decreased by 64%, and the flow pulsatility index increased 137% (P < 0.05 for both). Despite the large decrease in placental perfusion, there was no significant change in descending aortic oxygen tension or oxygen content, because fetal oxygen consumption was reduced by 40%. We conclude that endothelin-1 is a potent constrictor of the placental microcirculation in sheep. Endothelin-1 also decreases fetal oxygen consumption by an unknown mechanism.

An electrical analog model of the umbilicoplacental circulation that was based on hemodynamic measurements in fetal sheep was used to predict the mean blood velocity waveform in the umbilical artery. A 300% increase in placental resistance reduced the normally forward diastolic blood velocity in the umbilical artery nearly to zero. When resistance was normal, variations in umbilical arterial wall thickness (h) and elastic modulus (E) had little effect on the velocity waveform, whereas a 20% increase in radius (a) halved the ratio of the diastolic-to-systolic blood velocity (D/S). When placental resistance was high, abnormal umbilical arterial wall properties (a large, or h or E small) further reduced D/S. We conclude that high placental resistance, low umbilical arterial resistance, or a combination of high placental resistance with abnormal umbilical arterial wall properties are the primary downstream determinants of elevated pulsatility in umbilical arterial blood velocity.

We investigated whether the weak febrile response to pyrogens in newborns is due to a diminished activation of the putative pyrogen mediator, prostaglandin (PG)E2. Indwelling cannulas in the third ventricle of lambs (age, 5-31 days) and adult ewes were used to collect cerebrospinal fluid (CSF) for radioimmunoassay of PGE2. Intravenous (i.v.) endotoxin caused a smaller increase in body temperature but a larger increase in CSF PGE2 in lambs compared to adults. PGE2 by intracarotid infusion raised body temperature in 5 of 7 trials in 3 lambs and in 4 of 4 trials in 1 adult. Endotoxin given intracerebroventricularly (i.c.v.) induced a rise in temperature and CSF PGE2 in the lamb but, in the adult, these responses were delayed and smaller. Interleukin-1 i.c.v. and PGE2 i.c.v. were weak pyretic agents at both ages. We conclude that the lamb's diminished febrile response to endotoxin i.v. is not caused by a lesser rise in CSF PGE2, rather it may be due, at least in part, to reduced responsiveness to this putative mediator. Regardless of age, the sheep differs from other species in that pyrogen/PGE2 coupling occurs primarily at a site in brain that is better accessible from blood than CSF.