Fetal Rabbit Research Articles

Distinguishing Laterality in Brain Injury in Rabbit Fetal Magnetic Resonance Imaging Using Novel Volume Rendering Techniques

Introduction: Our laboratory has been exploring the MRI detection of fetal brain injury, which previously provided a prognostic biomarker for newborn hypertonia in an animal model of cerebral palsy (CP). The biomarker relies on distinct patterns of diffusion-weighted imaging-defined apparent diffusion coefficient (ADC) in fetal brains during uterine hypoxia-ischemia (H-I). Despite the challenges posed by small brains and tissue acquisition, our objective was to differentiate between left and right brain ADC changes. Methods: A novel aspect involved utilizing three-dimensional rendering techniques to refine ADC measurements within spheroids encompassing fetal brain tissue. 25-day gestation age of rabbit fetuses underwent global hypoxia due to maternal uterine ischemia. Results: Successful differentiation of left and right brain regions was achieved in 28% of the fetal brains. Ordinal analysis revealed predominantly higher ADC on the left side compared to the right at baseline and across the entire time series. During H-I and reperfusion-reoxygenation, the right side exhibited a favored percentage change. Among these fetal brains, 73% exhibited the ADC pattern predictive of hypertonia. No significant differences between left and right sides were observed in patterns predicting hypertonia, except for one timepoint during H-I. This study also highlights a balance between left-sided and right-sided alterations within the population. Conclusion: This study emphasizes the importance of investigating laterality and asymmetric hemispheric lesions for early diagnosis of brain injury, leading to CP. The technological limitations in obtaining a clear picture of the entire fetal brain for every fetus mirror the challenges encountered in human studies.

Prenatal developmental sequences of the esophageal epithelium in the New Zealand white rabbits: Light and electron microscopic analysis.

Several morphogenetic sequences occur during esophageal development and birth defects occur due to defects in foregut morphogenesis. This work aimed to record the cellular events in the morphogenesis of rabbits' esophageal epithelium. On the 16th day of gestation, the esophageal epithelium varied from stratified ciliated columnar to stratified squamous type. The surface epithelium presented mucous cells with mucigen granules of various sizes occupying their supranuclear cytoplasm. Cytoplasmic vacuolation was evident in all layers of the esophageal epithelium at this age. On the 18th gestational day, some light cells could be detected in the middle portion of the epithelium, while others occupied the whole epithelial length. On the 21st day, mucous cells are more frequently observed at the apical esophageal part as well as at the surface epithelium. Numerous elongated dark cells could be distinguished embedded between the basal cells. On the 24th gestational day the number of the mucous cells reached its peak. Reaching the 30th gestational day, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased. Collectively, detection of surface mucous and dark cells together with the non-cornified surface in some regions of the rabbit esophageal epithelium at the end of gestation ensure a postnatal development to reach the adult epithelium essential to sustain the passage of the harsh raw food. Future immunohistochemical studies are recommended to investigate the components of secretions in mucous cells and functional studies to highlight the dark cells significance. RESEARCH HIGHLIGHTS: Esophageal epithelium of fetal rabbit was analyzed by light and transmission microscopy. Surface epithelium presented mucous cells with mucigen granules of various sizes. They reached their maximum number on 24th day then decreased. On the 16th day, cytoplasmic vacuolation was evident in all epithelial layers. On the 21st day, numerous elongated dark cells could be distinguished embedded between the basal cells. Before birth, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased.

Long-term pulmonary and neurodevelopmental impairment in a fetal growth restriction rabbit model.

Fetal growth restriction (FGR) remains one of the main obstetrical problems worldwide, with consequences beyond perinatal life. Animal models with developmental and structural similarities to the human are essential to understand FGR long-term consequences and design novel therapeutic strategies aimed at preventing or ameliorating them. Herein, we described the long-term consequences of FGR in pulmonary function, structure, and gene expression, and characterized neurodevelopmental sequelae up to preadolescence in a rabbit model. FGR was induced at gestational day 25 by surgically reducing placental blood supply in one uterine horn, leaving the contralateral horn as internal control. Neonatal rabbits born near term were assigned to foster care in mixed groups until postnatal day (PND) 21. At that time, one group underwent pulmonary biomechanical testing followed by lung morphometry and gene expression analysis. A second group underwent longitudinal neurobehavioral assessment until PND 60 followed by brain harvesting for multiregional oligodendrocyte and microglia quantification. FGR was associated with impaired pulmonary function and lung development at PND 21. FGR rabbits had higher respiratory resistance and altered parenchymal biomechanical properties in the lungs. FGR lungs presented thicker alveolar septal walls and reduced alveolar space. Furthermore, the airway smooth muscle content was increased, and the tunica media of the intra-acinar pulmonary arteries was thicker. In addition, FGR was associated with anxiety-like behavior, impaired memory and attention, and lower oligodendrocyte proportion in the frontal cortex and white matter. In conclusion, we documented and characterized the detrimental pulmonary function and structural changes after FGR, independent of prematurity, and beyond the neonatal period for the first time in the rabbit model, and describe the oligodendrocyte alteration in pre-adolescent rabbit brains. This characterization will allow researchers to develop and test therapies to treat FGR and prevent its sequelae.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1−/− rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1−/− ovaries did not undergo meiosis and folliculogenesis. XX DMRT1−/− adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

Pregnancy Complications and Feto-Maternal Monitoring in Rabbits.

Rabbit production holds significant relevance in modern agriculture due to its potential as a sustainable source of high-quality protein and efficient feed conversion, contributing to food security and economic diversification. Nevertheless, studies incorporating feto-maternal monitoring in this species are uncommon. This review gathers research on the monitoring and evaluation of factors affecting rabbit gestation, providing a better understanding of the causes of prenatal development abnormalities. These include studies regarding how chronic maternal hypertension, gestational diabetes, maternal stress, ectopic gestation, maternal uterine ischemia and fetal hypoxia, intrauterine growth restriction, superfetation, maternal age, maternal nutritional status, maternal physical condition, maternal and embryonic genotype, and the intrauterine location of rabbit fetuses can potentially impact rabbits' reproduction and maternal and fetal health. Among other monitoring techniques, ultrasonography, considered one of the best tools for diagnosing pregnancy and conducting follow-up, is also reviewed. Details on measurable fetal-development parameters in rabbits and precautions to be considered before and during the examination are also provided. Additional studies are required to understand why some events occur and their consequences throughout gestation, allowing the determination of new biomarkers or cut-offs that can be helpful for early diagnosis and improve reproductive efficiency.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

OC16.02: The study of ultrasound guidance injection of bone marrow mesenchymal stem cells to treat fetal rabbit bronchopulmonary dysplasia

OC16.02: The study of ultrasound guidance injection of bone marrow mesenchymal stem cells to treat fetal rabbit bronchopulmonary dysplasia

Regeneration of vital organs of mammals in fetal period

BACKGROUND: This study is relevant owing to the importance of fetal regeneration of pathologically altered organs in mammals for fundamental science and practical medicine.
 AIM: To elucidate the regenerative potential of the cardiac, brain, and liver tissues in fetuses during fetal development following mechanical trauma.
 MATERIALS AND METHODS: As experimental animals, 12 mature female chinchilla rabbits weighing 39004350 g and 30 outbred female rats weighing 265280 g were examined. Mechanical trauma to the heart and brain was inflicted on rabbit fetuses (n=50) and to the liver of rat fetuses. The operated fetuses (n=84) were withdrawn from the experiment 1, 3, 5, 7, 9, 11, 15, and 30 days after the operation. Histological, morphometric, electron microscopic, and immunohistochemical methods were employed.
 RESULTS: Following the healing of mechanical trauma in the heart of rabbit fetuses, the necrotic area was completely restored. In rate fetuses, the liver did not fully regenerate, and the fibrotic area at the site of the dead tissue only decreased. The brain of rabbit fetuses did not fully recover, and a glial scar appeared in the place of the dead tissue. The difference in the healing outcomes of organ damage in fetuses is associated with the absence of an exudative reaction to injury in the heart of rabbit fetuses, whereas in rat fetuses, this reaction occurred during injury. In the nervous tissue, the activation of the protein synthetic function of neurocytes was recorded, which can be regarded as a sign of intracellular regeneration.
 CONCLUSIONS: The results suggest that the vital organs of mammalian fetuses have high regenerative potencies.

Synthetic surfactant with a combined SP-B and SP-C analogue is efficient in rabbit models of adult and neonatal respiratory distress syndrome

Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace. Here, we develop a combined surfactant protein B (SP-B) and SP-C peptide analogue (Combo) that can be efficiently expressed and purified from Escherichia coli without any solubility or purification tag. NMR spectroscopy shows that Combo peptide forms α-helices both in organic solvents and in lipid micelles, which coincide with the helical regions described for the isolated SP-B and SP-C parts. Artificial Combo surfactant composed of synthetic dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol, 1:1, mixed with 3 weights % relative to total phospholipids of Combo peptide efficiently improves tidal volumes and lung gas volumes at end-expiration in a premature rabbit fetus model of RDS. Combo surfactant also improves oxygenation and respiratory parameters and lowers cytokine release in an acid instillation-induced ARDS adult rabbit model. Combo surfactant is markedly more resistant to inhibition by albumin and fibrinogen than a natural-derived surfactant in clinical use for the treatment of RDS. These features of Combo surfactant make it attractive for the development of novel therapies against human ARDS.

Concordance between alizarin red stained skeleton and micro-CT skeleton evaluation methods: A case study in New Zealand White rabbits.

The objective of this study was to examine the fetal skeletons using both alizarin red stain and micro-computed tomography (CT) images; investigate differences, and to determine if the conclusions of the study were the same regardless of the examination method. A candidate drug was given orally by gavage to pregnant New Zealand White rabbits on gestation day (GD) 7 to GD 19 (mating = GD 0) at doses of 0 (control), 0.02, 0.5, 5, and 15 mg/kg/day. Maternal toxicity was evident at ≥0.02 mg/kg/day. The 199 fetal skeletons (totaling 50,546 skeletal elements) obtained at cesarean delivery on GD29 were first stained with Alizarin Red S, then imaged by a Siemens Inveon micro-CT scanner. All fetal skeletons were examined by both methods, without knowledge of dose group, and the results were compared. In total, 33 types of skeletal abnormalities were identified. There was 99.8% concordance of results comparing stain to micro-CT. Ossification of the middle phalanx of the forepaw digit 5 showed the greatest difference between the two methods. Overall, micro-CT imaging is a realistic, and robustalternative to skeletal staining to examine fetal rabbit skeletons in developmental toxicity studies.

Congenital diaphragmatic hernia increases the sensitivity of pulmonary arteries to nitric oxide

Left congenital diaphragmatic hernia (CDH) can lead to pulmonary arteries abnormalities in the contralateral and ipsilateral sides of the diaphragm. Nitric oxide (NO) is the main therapy used to attenuate the vascular effects of CDH, but it is not always effective. We hypothesized that the left and right pulmonary arteries do not respond similarly to NO donors during CDH. Therefore, vasorelaxant responses of the left and right pulmonary arteries to sodium nitroprusside (SNP, a NO donor) were determined in a rabbit experimental model of left CDH. CDH was surgically induced in the fetuses of rabbits on the 25th day of pregnancy. On the 30th day of pregnancy, a midline laparotomy was performed to access the fetuses. The fetuses’ left and right pulmonary arteries were isolated and mounted in myograph chambers. Vasodilation was evaluated by cumulative concentration-effect curves to SNP. Protein expression of guanylate cyclase isoforms (GCα, GCβ) and the α isoform of cGMP-dependent protein kinase 1 (PKG1α), and the concentration of NO and cGMP were determined in the pulmonary arteries. The left and right pulmonary arteries of newborns with CDH exhibited increased vasorelaxant responses to SNP (i.e. the potency of SNP was increased) compared to the control group. GCα, GCβ, and PKG1α expression were decreased, while NO and cGMP concentrations were increased in the pulmonary arteries of newborns with CDH compared to the control group. The increased cGMP mobilization may be responsible for the increased vasorelaxant responses to the SNP in the pulmonary arteries during left CDH.

Histological observation on cardiogenesis during second trimester of domestic rabbit's fetuses (Oryctolagus Cuniculus)

The present work was designed to investigate the histological developmental characteristics of heart in the local rabbit fetuses. It which included the detection of timing of heart beat onset and studied morphohistogenesis of heart components. The study was performed on 35 rabbit fetuses distributed at seven groups according to the following ages: (9, 10, 12, 14, 16, 18, 20) days gestational age. The results of the current study showed that differentiation and formation of the heart began between 9th and 10th day of pregnancy in which the smooth-walled heart loop had an outer myocardial mantle, cardiac jelly and present of endocardium by histological study for serial section in each stage. The histological structures for each age showed that the heart passed through different sequential morphological stages in embryonic life represented by heart tube, looping heart, chamber formation and cardiac septation. The purpose of our study was to provide a more complete histological description of the stages of heart formation in rabbits during prenatal development.

In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae)

BackgroundAnisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host’s gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature.MethodsTesting two commercially available media (RPMI 1640, Schneider’s Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii.ResultsSchneider’s Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages.ConclusionsClosing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug–target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3.Graphical

Brain development is altered in rabbit fetuses with congenital diaphragmatic hernia.

Children with congenital diaphragmatic hernia (CDH) are at risk for neurodevelopmental delay. Some changes are already present prenatally. Herein,we further examined how the brain develops in fetal rabbits with surgically created DH. Two fetuses underwent surgical DH creation on day 23 (term=d31). DH pups and littermate controls were harvested at term. Ten DH pups and 11 controls underwent transcardial perfusion for brain fixation and measurement of brain volume, brain folding, neuron and synaptic density, pre-oligodendrocyte count, proliferation, and vascularization. Twelve other DH and 11 controls had echocardiographic assessment of cardiac output and aortic and cerebral blood flow, magnetic resonance imaging (9.4T) for cerebral volumetry, and molecular assessment of vascularization markers. DH pups had lower lung-to-body weight ratio (1.3±0.3 vs. 2.4±0.3%; p<0.0001) and lower heart-to-body weight ratio (0.007±0.001 vs. 0.009±0.001; p=0.0006) but comparable body weight and brain-to-body weight ratio. DH pups had a lower left ventricular ejection fraction, aortic and cerebral blood flow (39±8 vs. 54±15mm/beat; p=0.03) as compared to controls but similar left cardiac ventricular morphology. Fetal DH-brains were similar in volume but the cerebellum was less folded (perimeter/surface area: 25.5±1.5 vs. 26.8±1.2; p=0.049). Furthermore, DH brains had a thinner cortex (143±9 vs. 156±13μm; p=0.02). Neuron densities in the white matter were higher in DH fetuses (124±18 vs. 104±14; p=0.01) with comparable proliferation rates. Pre-oligodendrocyte count was lower, coinciding with the lower endothelial cell count. Rabbits with DH had altered brain development compared to controls prenatally, indicating that brain development is already altered prenatally in CDH.

Local host response of commercially available dural patches for fetal repair of spina bifida aperta in rabbit model.

Fetal surgery for spina bifida aperta (SBA) by open hysterotomy typically repairs anatomical native tissue in layers. Increasingly, fetoscopic repair is performed using a dural patch followed by skin closure. We studied the host response to selected commercially available patches currently being used in a fetal rabbit model for spina bifida repair. SBA was surgically induced at 23-24days of gestation (term=31days). Fetal rabbits were assigned to unrepaired (SBA group), or immediate repair with Duragen™ or Durepair™. Non-operated littermates served as normal controls. At term, spinal cords underwent immunohistochemical staining including Nissl and glial fibrillary acidic protein. We hypothesized that spinal cord coverage with a dural patch and skin closure would preserve motor neuron density within the non-inferiority limit of 201.65cells/mm2 and reduce inflammation compared to unrepaired SBA fetuses. Motor neuron density assessed by Nissl staining was conserved both by Duragen (n=6, 89.5; 95% CI -158.3 to -20.6) and Durepair (n=6, 37.0; 95% CI -132.6 to -58.5), whereas density of GFAP-positive cells to quantify inflammation was lower than in unrepaired SBA-fetuses (SBA 2366.0±669.7cells/mm2 vs. Duragen 1274.0±157.2cells/mm2 ; p=0.0002, Durepair 1069.0±270.7cells/mm2 ; p<0.0001). Covering the rabbit spinal cord with either Duragen or Durepair followed by skin closure preserves motor neuron density and reduces the inflammatory response.

Effect of prenatal diaphragmatic hernia on pulmonary arterial morphology.

Congenital diaphragmatic hernia (CDH) is a major cause of severe lung hypoplasia and pulmonary hypertension in the newborn. While the pulmonary hypertension is thought to result from abnormal vascular development and arterial vasoreactivity, the anatomical changes in vascular development are unclear. We have examined the 3D structure of the pulmonary arterial tree in rabbits with a surgically induced diaphragmatic hernia (DH). Fetal rabbits (n= 6) had a left-sided DH created at gestational day 23 (GD23), delivered at GD30, and briefly ventilated; sham-operated litter mates (n= 5) acted as controls. At postmortem the pulmonary arteries were filled with a radio-opaque resin before the lungs were scanned using computed tomography (CT). The 3D reconstructed images were analyzed based on vascular branching hierarchy using the software Avizo 2020.2. DH significantly reduced median number of arteries (2,579 (8440) versus 576 (442), p= .017), artery numbers per arterial generation, mean total arterial volume (43.5± 8.4 vs. 19.9± 3.1μl, p= .020) and mean total arterial cross-sectional area (82.5± 2.3 vs. 28.2± 6.2 mm2 , p=.036). Mean arterial radius was increased in DH kittens between the eighth and sixth branching generation and mean arterial length between the sixth and 28th branching generation. A DH in kittens resulted in threefold reduction in pulmonary arterial cross-sectional area, primarily due to reduced arterial branching. Thus, the reduction in arterial cross-sectional area could be a major contributor to pulmonary hypertension infants with CDH.

The metabolic and lipidomic profiling of the effects of tracheal occlusion in a rabbit model of congenital diaphragmatic hernia

PurposeFetal tracheal occlusion (TO) reverses the pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH), but its mechanism of action remains poorly understood. ‘Omic’ readouts capture metabolic and lipid processing function, which aid in understanding CDH and TO metabolic mechanisms. MethodsCDH was created in fetal rabbits at 23 days, TO at 28 days and lung collection at 31 days (Term ∼32 days). Lung-body weight ratio (LBWR) and mean terminal bronchiole density (MTBD) were determined. In a cohort, left and right lungs were collected, weighed, and samples homogenized, and extracts collected for non-targeted metabolomic and lipidomic profiling via LC–MS and LC–MS/MS, respectively. ResultsLBWR was significantly lower in CDH while CDH + TO was similar to controls (p = 0.003). MTBD was significantly higher in CDH fetuses and restored to control and sham levels in CDH + TO (p < 0.001). CDH and CDH + TO resulted in significant differences in metabolome and lipidome profiles compared to sham controls. A significant number of altered metabolites and lipids between the controls and CDH groups and the CDH and CDH + TO fetuses were identified. Significant changes in the ubiquinone and other terpenoid-quinone biosynthesis pathway and the tyrosine metabolism pathway were observed in CDH + TO. ConclusionCDH + TO reverses pulmonary hypoplasia in the CDH rabbit, in association with a specific metabolic and lipid signature. A synergistic untargeted ‘omics’ approach provides a global signature for CDH and CDH + TO, highlighting cellular mechanisms among lipids and other metabolites, enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology and recovery. Type of StudyBasic Science, Prospective. Level of EvidenceII.

Histogenesis of the rabbit liver (pars hepatica) with particular reference to the portal area

Rabbits are small mammals used as standard lab animals in biomedical research. The liver is the largest internal organ, providing essential metabolic, exocrine, and endocrine functions. The present study was performed on rabbit embryos and fetuses at different gestational periods and neonatal kittens to find out the histological structure of the liver. Histogenesis of the liver was performed by using different histological stains, Harris's H and E and Masson's trichrome. The liver bud was developed around the 10th day of gestation from the caudo-ventral part of the foregut. In comparison, the hepatoblasts developed at the 12th day of gestation. Then, the liver increased in size, and cellular differentiation occurred throughout the entire length of the gestation period. The hepatic parenchymal cells, central vein, and hematopoietic cells were studied for their developmental sequences. The portal area differentiation was the focus of our study. This study clarified that this area was essential for detecting mesenchymal stem cell markers through immunohistochemistry.

The myocardial capillary network is altered in congenital diaphragmatic hernia in the fetal rabbit model

Congenital diaphragmatic hernia (CDH) is associated with thoracic compression of the lungs and heart caused by the herniated abdominal content, leading to cardiac modifications including pressure and vascular changes. Our aim was to investigate the experimental immunoexpression of the capillary proliferation, activation, and density of Ki-67, VEGFR2, and lectin in the myocardium after surgical creation of a diaphragmatic defect. Pregnant New Zealand rabbits were operated on the 25th gestational day in order to create left-sided CDH (LCDH, n=9), right-sided CDH (RCDH, n=9), and Control (n=9), for a total of 27 fetuses in 19 pregnant rabbits. Five days after the procedure, animals were sacrificed, and histology and immunohistochemistry studies of the harvested hearts were performed. Total body weight and heart weight were not significantly different among groups (P=0.702 and 0.165, respectively). VEGFR2 expression was increased in both ventricles in the RCDH group (P<0.0001), and Ki-67 immunoexpression was increased in the left ventricle in the LCDH group compared to Control and RCDH groups (P<0.0001). In contrast, capillary density was reduced in the left ventricle in the LCDH compared to the Control and RCDH groups (P=0.002). Left and right ventricles responded differently to CDH in this model depending on the laterality of the diaphragmatic defect. This surgical model of diaphragmatic hernia was associated with different expression patterns of capillary proliferation, activation, and density in the myocardium of the ventricles of newborn rabbits.

Abstract 11961: Ductus Arteriosus Oxygen Response is Mediated by Mitochondrial Subunit NDUFS2

Introduction: The ductus arteriosus (DA) is a fetal vessel connecting the pulmonary artery (PA) and aorta, diverting oxygenated blood away from the developing lungs to the systemic circulation. The DA rapidly constricts in response to the increase in arterial oxygen (O 2 ) following the first breath. Failure of the DA to close in response to O 2 results in persistent ductus arteriosus, one of the most common complications of preterm birth. Mitochondria are known to play an important role in the O 2 response of DA smooth muscle cells (DASMC), however the mechanism is not fully understood. A subunit of mitochondrial Complex I, NDUFS2 (NADH:ubiquinone oxidoreductase core subunit 2) mediates the O 2 response of other specialized O 2 sensing tissues (PA and carotid body); we assess whether NDUFS2 also plays a role in DA O 2 sensing. Methods: DASMC were isolated from male term (29 days gestation) fetal New Zealand white rabbits. Cell lines were purified via fluorescence activated cell sorting, excluding CD90 + and CD31 + cells (fibroblasts and endothelial cells, respectively). Smooth muscle cell purity was confirmed using flow cytometry for α-smooth muscle actin (α-SMA). Primary cultures were 97.65% ± 0.65% CD90 - CD31 - , and were 100% α-SMA + post-sorting. We measured the O 2 responsiveness of DASMC using confocal microscopy, measuring changes in intracellular calcium with Cal-520AM. Cells were cultured in hypoxia (2.5% O 2 , pO 2 = 41 mmHg), with O 2 -induced increase in calcium relative to hypoxic baseline serving as a surrogate for constriction. DASMC were treated for 48 hours with 30 pmol siRNA and 90 pmol Lipofectamine® RNAiMAX to knockdown gene expression of NDUFS2 or a negative control. Knockdown was confirmed using qPCR. Results &amp; Conclusions: There was a 14.7% (±2.6%, n=33, p&lt;0.0001) increase in intracellular calcium relative to hypoxic baseline in siNC-treated cells. O 2 response was obliterated when treated with siNDUFS2 (0.7% ± 2.1%, n=29, p=0.798), with a 90.9% ± 1.89% knockdown of NDUFS2 mRNA expression. These data suggest a critical role for the mitochondrial electron transport chain, specifically NDUFS2, in DA oxygen responsiveness. While further functional studies will be required, our findings suggest a novel potential therapeutic target to modulate DA patency.