Newborn Rabbits Research Articles

Reconstruction of rabbit corneal stroma with skin fibroblasts

To explore whether skin fibroblasts could be used as a cell source for reconstruction of the corneal stroma. It was an experimental study. Skin fibroblast cells were isolated from newborn rabbits, cultured and expanded in vitro. Cells were labeled with green fluorescence protein (GFP) gene by retro-viral infection. Fibroblasts at passage 3 were seeded on polyglycolic acid (PGA) non-woven fibers to form a cell-scaffold construct. Constructs were then implanted into the adult rabbit corneal stroma layer after being cultured in vitro for 1 week. Engineered stroma were observed continuously and harvested after 8 weeks of transplantation for gross, histological evaluation and Keratocan examination. PGA alone was used as control. The engineered tissue in the cornea became transparent gradually over a period of 8 weeks. Histological analysis showed that engineered stromal lamellar was relatively regular and the orientation of fibers was parallel to the surface of cornea, which is similar to normal cornea. The implanted cells were confirmed by GFP expression under fluorescent microscope, which also express Keratocan. By transmission electron microscopy examination, no significant difference in the diameter of collagen fiber was observed between engineered stroma (33.08 + or - 2.47) nm and normal stroma (t = 1.80, P = 0.0771). Skin fibroblast cells could be used as seed cells for reconstruction of the corneal stroma.

Physical contact while handling is not necessary to reduce fearfulness in the rabbit

Rabbits handled around nursing time during the first week after birth show reduced fear response toward humans. Our earlier attempt to reduce the duration of daily treatment necessary to achieve this effect showed that even a minimal human contact, characteristic of animal caretaking in intensive rabbitries, results in reduced fearfulness. Being descendants of a nocturnal mammal species, olfactory cues are of central importance in rabbits, especially just after parturition, when the other sensory organs are undeveloped. In the present experiment, we investigated whether exposing newborn rabbit pups to human smell at nursing time is sufficient to reduce fear of humans in rabbits. For this, we exposed rabbit pups to one of the following handling treatments in the first week of life: (1) full handling, within 0.5 h after nursing, which consisted of removing the pups from the nest and weighing them (about 5 min/litter), (2) exposing rabbit pups to the smell of humans for about 5 min/litter, without touching them, (3) untreated controls. At 28 days of age, the timidity of the pups was measured in a 5 min approach test. Pups that were either handled or exposed to human smell appeared to be equally less fearful as they approached the experimenter's hand with a lower latency and more frequently than untreated controls. This indicates that olfactory exposure during handling results in imprinting even without a human contact in rabbits.

In VivoCartilage Engineering with Collagen Hydrogel and Allogenous Chondrocytes After Diffusion Chamber Implantation in Immunocompetent Host

In vivo cartilage reconstruction at an ectopic site was not successful in immunocompetent animals, possibly because of immunoreaction and the failure of material design. A diffusion chamber, which has been predominantly adopted to study cell differentiation, was effective in preventing host immune rejection, host cell invasion, and vascular invasion. In this study, we proposed to regenerate ectopic cartilage tissue in rabbits by implanting a diffusion-chamber system subcutaneously for 8 weeks. Inside the chamber, biomimetic scaffolds loaded with allogenous chondrocytes from newborn rabbits were enclosed. Tissue with characteristics of cartilage was formed inside the chamber with collagen gel as a scaffold, which was demonstrated using histological, immunohistochemical, and reverse transcriptase polymerase chain reaction assays. In contrast, for implant without diffusion chamber, vascular invasion was observed and results showed much less expression of cartilage extracellular matrix (ECM). Collagen type I hydrogel and sponge were compared as scaffolds. No cartilage tissue was found in the collagen sponge inside the chamber, presumably because of the different cell-seeding characteristics of gel. In addition, allogenous chondrocytes were adopted as a cell resource and were proved viable for the regeneration of cartilage tissue in this model. The results revealed that the diffusion chamber and scaffold design are both important in providing a more favorable biomimetic microenvironment for the formation of cartilage in vivo at an ectopic site, even with allogenous cells. Moreover, preliminary repair of a cartilage defect using the engineered tissue for 4 weeks showed the growth of new cartilage, obtaining a satisfactory interface with the original cartilage inside the defect. The model of engineering cartilage in vivo was proven to be useful. This study is the preliminary exploration for the reconstruction of ectopic cartilage in an immunocompetent host to be applied for cartilage repair. It may provide a valuable reference for the clinical application of cartilage repair.

Elemental and configural processing of odour mixtures in the newborn rabbit

The processing of odour mixtures by young organisms is poorly understood. Recently, the perception of an AB mixture, known to engage configural perception in adult humans, was suggested also to be partially configural in newborn rabbits. In particular, pups did not respond to AB after they had learned A or B. However, two alternative hypotheses might be suggested to explain this result: the presence in the mixture of a novel odorant that inhibits the response to the learned stimulus, and the unevenness of the sensory and cognitive processes engaged during the conditioning and the behavioural testing. We conducted four experiments to explore these alternative hypotheses. In experiment 1, the learning of A or B ended in responses to mixtures including a novel odorant (AC or BC). Experiment 2 pointed to the absence of overshadowing. Therefore, a novelty effect cannot explain the non-response to AB after the learning of A or B. In experiment 3, pups having learned A or B in AC or BC did not respond to AB. However, they generalized odour information acquired in AB to AC or BC in experiment 4. Thus, the balancing of the perceptual tasks between the conditioning and retention test does not enhance the response to the AB mixture. To sum up, the present experiments give concrete support to the partially configural perception of specific odour mixtures by newborn rabbits.

Pheromone-induced olfactory memory in newborn rabbits: Involvement of consolidation and reconsolidation processes

Mammary pheromone (MP)-induced odor memory is a new model of appetitive memory functioning early in a mammal, the newborn rabbit. Some properties of this associative memory are analyzed by the use of anisomycin as an amnesic agent. Long-term memory (LTM) was impaired by anisomycin delivered immediately, but not 4 h after either acquisition or reactivation. Thus, the results suggest that this form of neonatal memory requires both consolidation and reconsolidation. By extending these notions to appetitive memory, the results reveal that consolidation and reconsolidation processes are characteristics of associative memories of positive events not only in the adult, but also in the newborn.

Hypoxia–ischemia causes persistent movement deficits in a perinatal rabbit model of cerebral palsy: assessed by a new swim test

The relationship of movement between different muscle groups has not been quantified before in the newborn period. Cerebral palsy (CP), which often occurs as a result of perinatal hypoxia–ischemia (H–I), is categorized depending on clinical presentation, brain region involvement and extent of involvement. In order to test different brain region involvement, this study investigates individual and multi-joint involvement in a rabbit model of CP. Pregnant rabbits at 70% gestation were subjected to 40-min uterine ischemia. Newborn rabbit kits were subjected to a swim test at 5 time points over the first 11 days of life. H–I kits were divided into hypertonic and non-hypertonic groups based on muscle tone at birth. The ranges and velocity of angular movement of the forelimb and hind limb joints (wrist, elbow, shoulder, ankle, knee and hip) during supported swimming were determined. Severely impaired (hypertonic) animals have significantly reduced range and angular velocity of joint motion, which do not improve over time. The non-hypertonic group showed deficits in wrist and hind limb movements that were not evident on prolonged observation. Preventive treatment with an inhibitor of neuronal nitric oxide synthase decreased the incidence of severely impaired kits; the non-hypertonic kits showed a different pattern of swimming. Supported swimming allows quantification of limb and joint motion in the principal plane of movement in the absence of weight bearing and decreases the need for balance control. Identification and quantification of milder deficits allows mechanistic studies in the causation of H–I injury as well as estimation of recovery with therapeutic agents.

Beneficial effect of insulin-like growth factor-1 on hypoxemic renal dysfunction in the newborn rabbit

Acute normocapnic hypoxemia can cause functional renal insufficiency by increasing renal vascular resistance (RVR), leading to renal hypoperfusion and decreased glomerular filtration rate (GFR). Insulin-like growth factor 1 (IGF-1) activity is low in fetuses and newborns and further decreases during hypoxia. IGF-1 administration to humans and adult animals induces pre- and postglomerular vasodilation, thereby increasing GFR and renal blood flow (RBF). A potential protective effect of IGF-1 on renal function was evaluated in newborn rabbits with hypoxemia-induced renal insufficiency. Renal function and hemodynamic parameters were assessed in 17 anesthetized and mechanically ventilated newborn rabbits. After hypoxemia stabilization, saline solution (time control) or IGF-1 (1 mg/kg) was given as an intravenous (i.v.) bolus, and renal function was determined for six 30-min periods. Normocapnic hypoxemia significantly increased RVR (+16%), leading to decreased GFR (-14%), RBF (-19%) and diuresis (-12%), with an increased filtration fraction (FF). Saline solution resulted in a worsening of parameters affected by hypoxemia. Contrarily, although mean blood pressure decreased slightly but significantly, IGF-1 prevented a further increase in RVR, with subsequent improvement of GFR, RBF and diuresis. FF indicated relative postglomerular vasodilation. Although hypoxemia-induced acute renal failure was not completely prevented, IGF-1 elicited efferent vasodilation, thereby precluding a further decline in renal function.

The developing mammalian retina is partially protected from gentamicin toxicity

Gentamicin retinal toxicity was tested in numerous studies on adult animal models, but not in the developing retina. Since differentiating cells and developing tissues may exhibit different degrees of sensitivity to toxic drugs, we aimed here to test the susceptibility of the developing mammalian retina to the toxic action of gentamicin. Gentamicin was injected in the right eye of newborn rabbits, aged 11–38 days. The left eye of each rabbit was injected with saline. Eight weeks after injection, electroretinographic (ERG) responses were recorded in the dark- and light-adapted states. The rabbits were then sacrificed, and the retinas were prepared for morphological examination and immunostaining for Glial Fibrillary Acidic Protein (GFAP) and Protein Kinase C (PKC). The ERG responses demonstrated practically no functional damage to the retina of rabbits injected at postnatal age of 11 and 13 days, and a gradually increasing severity of functional damage with increasing postnatal age of intravitreal gentamicin injection. The histopathological studies yielded similar results. GFAP immunoreactivity showed staining in Müller cells only in retinas that exhibited functional and structural damage. PKC immunoreactivity indicated lesser damage to the rod-bipolar cells compared to the photoreceptors. The ERG data and morphological observations suggest that processes involved in development of the rabbit retina, such as outer segment elongation may provide partial or even complete protection to the retina from toxic insults such as a single dose of gentamicin.

Heart Failure in Children

Why is heart failure in children important? If we just consider the number of individuals affected, adult heart failure is clearly a more compelling public health problem. However, the relatively small numbers belie the overall economic and social impact of pediatric heart failure. When a child is admitted to the hospital for heart failure, the costs are considerably higher for children than adults because of the frequent need for surgical or catheter-based intervention. The demands of medical care can fray the family structure and adversely affect parental economic productivity. When a child dies of heart failure, the economic impact is magnified enormously because of the number of potentially productive years lost per death. For these and other reasons, heart failure in children is a serious public health concern. In addition, growing numbers of children with heart failure are reaching adulthood because of the successful application of medical and surgical heart failure therapies and the improved outcomes of congenital heart surgery. A greater understanding of the pathophysiology of heart failure in childhood may help inform therapeutic strategies once these children become adults. Furthermore, given the recent explosion of research in the impact of cardiac development and cardiogenetics on pediatric cardiovascular disease, it is not outside the realm of possibility for a pediatric discovery to be made that will also benefit adults with heart failure. We may not yet be able to agree on a definition of heart failure, but the cardinal symptoms, dyspnea, anasarca (“dropsy”), and cachexia, were well recognized in antiquity.1 These symptoms were not specific for cardiac pathology, and it was not until the 17th century, when William Harvey definitively identified the heart as an organ that pumped blood rather than generating heat, that the heart could begin to be understood as a source of dyspnea, edema, and …

POLYETHYLENE GLYCOL ADDITION DOES NOT IMPROVE EXOGENOUS SURFACTANT FUNCTION IN AN EXPERIMENTAL MODEL OF MECONIUM ASPIRATION SYNDROME

Meconium (MEC) is a potent inactivator of pulmonary surfactant. The authors studied the effects of polyethylene glycol addition to the exogenous surfactant over the lung mechanics and volumes. Human meconium was administrated to newborn rabbits. Animals were ventilated for 20 minutes and dynamic compliance, ventilatory pressure, and tidal volume were recorded. Animals were randomized into 3 study groups: MEC group (without surfactant therapy); S100 group (100 mg/kg surfactant); and PEG group (100 mg/kg porcine surfactant plus 5% PEG). After ventilation, a pulmonary pressure-volume curve was built. Histological analysis was carried out to calculate the mean alveolar size (Lm) and the distortion index (DI). Both groups treated with surfactant showed higher values of dynamic pulmonary compliance and lower ventilatory pressure, compared with the MEC group (P <.05). S100 group had a larger maximum lung volume, V30, compared with the MEC group (P <.05). Lm and DI values were smaller in the groups treated with surfactant than in the MEC group (P <.05). No differences were observed between the S100 and PEG groups. Animals treated with surfactant showed significant improvement in pulmonary function as compared to nontreated animals. PEG added to exogenous surfactant did not improve lung mechanics or volumes.

Meconium-induced release of nitric oxide in rabbit alveolar cells

Previous studies have shown meconium-induced lung injury occurs throughout release of inflammatory cytokines. The exact mechanism of cytokine-induced apoptosis is not known. In this study we hypothesized that meconium-induced apoptosis in the lungs is mediated through the production of inducible nitric oxide (NO). We studied two groups of newborn rabbit pups: one group was instilled with meconium and other with normal saline. We measured precursors of NO in lung lavage from both groups of rabbits and NO levels were calculated accordingly. The levels of NO and NO-derivatives increased significantly in both groups. However NO expression in meconium group 2 h after meconium instillation was significantly higher than in saline-instilled group suggesting NO production plays a role in meconium-induced inflammation.

Age-related changes in selected haematology parameters in rabbits

Even though there is an abundance of information on the reference values of haematological parameters in adult rabbits, a little is known about the changes in haematology in newborn rabbits or during their postnatal development. Therefore, the aim of our study was to investigate changes in red blood cells (RBC), white blood cells (WBC) and differential leukocyte counts in SPF New Zealand White rabbits from the age of one day to 20 weeks. Significant age-related changes during the first four weeks of life were detected. These included an increase of RBC and WBC, reversal of the neutrophil/lymphocyte ratio and increase of total counts of eosinophils and basophils. From the age of six weeks of life, all of the studied haematological parameters were comparable to those of adult rabbits.

Endogenous clock gene expression in the suprachiasmatic nuclei of previsual newborn rabbits is entrained by nursing

The rabbit is particularly suitable for investigating the development of mammalian circadian function. Blind at birth, the pups are only visited by the mother to be nursed once every 24 h for about 3 min and so can be studied largely without maternal interference. They anticipate the mother's visit with increased behavioral arousal and with a rise in body temperature, both of which represent endogenous circadian rhythms. We now report that in newborn pups the suprachiasmatic nuclei of the hypothalamus (SCN; the main circadian pacemaker in mammals) show endogenous 24-h rhythmicity in the expression of the clock genes Per1, Per2, and Bmal1. Pups nursed from postnatal days 1 to 7 and fasted to day 9 showed the same rhythms of clock gene expression as normally nursed controls. We also report that these rhythms are entrained by nursing. Pups killed on postnatal days 3-4 showed the same rhythms in gene expression as pups in the previous experiment, whereas littermates subsequently nursed from postnatal days 4 to 7 with nursing delayed 6 h showed a corresponding shift in the diurnal pattern of clock gene expression. Consistent with this, two groups of pups implanted with telemetric thermal sensors and nursed 6 h apart had daily patterns in body temperature synchronized with the two different nursing times. We conclude that the expression of clock genes associated with the newborn rabbit's circadian system is entrained by nonphotic cues accompanying nursing, the exact nature of which now needs to be clarified.

Abstract 2609: Mitochondrial Oxidative Stress Promotes Failure of the Pressure-Loaded Infant Right Ventricle

Background: Right ventricular hypertrophy (RVH) and failure induced by chronic RV pressure overload is a major problem in congenital heart disease. We have previously shown that mitochondrial (Mito) damage accompanied load-induced infant RV failure. Here we assessed Mito electron transport chain(ETC) dysfunction and Mito oxidative stress in the transition of infant RVH to failure. Methods : Newborn rabbits underwent pulmonary artery banding (or sham) causing stable RV pressure load (~40 mmHg) and RVH by 2 weeks; RV contractile function (echocardiography) was preserved for ~4 weeks, followed by failure onset at ~8 weeks. Thus, RV free wall was assayed at 3, 4 and 8 weeks for ETC enzyme activities, high energy phosphates, Mito GSH/GSSH ratio (index of Mito ROS scavenging) , Mito 8-hydroxy-2-deoxyguanosine (8-OHdG, oxidative Mito DNA damage), Mito protein carbonyls (oxidative Mito protein damage) and Mito ultrastructure (electron microscopy). Results: By 4 weeks (RVH, preserved function), Mito 8-OHdG and protein carbonyl markedly increased, accompanied by decreased complex I and III activity. At 8 weeks (early failure), there were further increases in Mito 8-OHdG and protein carbonyls, significantly decreased GSH/GSSG ratio, further loss of complex I and III, and decreased activities of complexes IV and V; ATP content and ATP/ADP ratio were also significantly reduced. In parallel the number of abnormal disrupted RV mitochondria increased markedly. Conclusion: Pressure-load of the infant RV rapidly induces Mito oxidative stress. Due to the limited Mito scavenging and repair systems, this results in a cycle of Mito DNA and protein damage, progressive loss of ETC enzyme activity and further oxidative stress, as well as loss of functional mitochondria. This load-induced oxidative stress may be a therapeutic target to preserve RV function and oxidative metabolism and prevent the transition of RVH to RV failure. Changes in banded and sham-operated hearts after surgery

Intrauterine administration of endotoxin leads to motor deficits in a rabbit model: a link between prenatal infection and cerebral palsy

This study was undertaken to determine whether maternal intrauterine endotoxin administration leads to neurobehavioral deficits in newborn rabbits. Pregnant New Zealand white rabbits were injected with 1 mL saline solution (n = 8) or 20 microg/kg of lipopolysaccharide in saline solution (n = 8) into the uterine wall on day 28/31 of gestation. On postnatal day 1, kits (saline solution [n = 30] and lipolysaccharide in saline solution [n = 18] from 4 consecutive litters) underwent neurobehavioral testing. Neonatal brains were stained for microglial cells and myelin. Kits in the lipopolysaccharide in saline solution group were hypertonic and demonstrated significant impairment in posture, righting reflex, locomotion, and feeding, along with neuroinflammation indicated by activated microglia and hypomyelination in the periventricular regions. A greater mortality was noted in the lipopolysaccharide in saline solution group (16 stillbirths from 3 litters vs 3 from 1 litter). Maternal intrauterine endotoxin administration leads to white matter injury and motor deficits in the newborn rabbit, resulting in a phenotype that resembles those found in periventricular leukomalacia and cerebral palsy.

Differential development of body equilibrium among littermates in the newborn rabbit

Interest is growing among psychobiologists and behavioral ecologists in the role of sibling relations in shaping individual development and life histories. In litters of domestic rabbits Oryctolagus cuniculus the heaviest pups at birth are more likely to survive the critical first postnatal week, they compete more effectively with littermates for milk and well-insulated positions in the litter huddle, and are the heaviest at weaning. Here we report that high birth weight pups are also better able to maintain body equilibrium. Testing pups' ability to maintain equilibrium when placed on a 15 degrees ramp for 2 min each day during the first postnatal week, we found that pups showed a continual daily improvement in their ability to maintain balance while moving on the ramp, rarely lost balance by postnatal day 8, and that heavier pups could maintain balance better and earlier than their lighter littermates. Better ability to maintain body equilibrium, however achieved, may help explain heavier pups' advantage in competing for vital resources such as milk and in gaining access to better-insulated positions in the litter huddle. It also provides further support for the usefulness of birth weight, not only as an absolute measure but also relative to the weight of other littermates, as a predictor of different developmental trajectories, behavioral and physiological, among same-age siblings in this mammal.

Cloning rabbits from fetal fibroblasts

Live rabbits were generated through nuclear transfer using adult cells as nuclear donors. We showed that, in addition to adult cell nuclei, cultured fetal rabbit fibroblasts could support full-term embryonic development following nuclear transfer to enucleated oocytes. Following nuclear transfer, 24.4% (21/86) of resulting embryos developed to the blastocyst stage, and 289 embryos were transferred to oviducts of 11 recipient mothers, resulting in 6 pregnancies. Three mothers carried the pregnancy to term, and three newborn rabbits were subsequently delivered by caesarian, one of which survived for more than 4 months. DNA analyses confirmed that all 3 rabbits were genetically identical to fetal donor cells. This study demonstrated that rabbits could be cloned from fetal fibroblasts, although at a lower frequency than when using adult somatic cells as nuclear donors.

Simultaneous acquisition of dual analyser-based phase contrast X-ray images for small animal imaging

Analyser-based phase contrast X-ray imaging can provide high-contrast images of biological tissues with exquisite sensitivity to the boundaries between tissues. The phase and absorption information can be extracted by processing multiple images acquired at different analyser orientations. Recording both the transmitted and diffracted beams from a thin Laue analyser crystal can make phase retrieval possible for dynamic systems by allowing full field imaging. This technique was used to image the thorax of a mechanically ventilated newborn rabbit pup using a 25 keV beam from the SPring-8 synchrotron radiation facility. The diffracted image was produced from the (1 1 1) planes of a 50 mm × 40 mm, 100 μm thick Si analyser crystal in the Laue geometry. The beam and analyser were large enough to image the entire chest, making it possible to observe changes in anatomy with high contrast and spatial resolution.

Perception of odor blending mixtures in the newborn rabbit

In adult mammals, the processing of complex odor mixtures is elemental or configural. Here, we challenged these processes in newborn rabbits, in evaluating their perception of a binary odor mixture for which perceptual blending occurs in humans. This model of newborn animal was interesting since general questions remain on how odor cues are processed in immature organisms, and since rabbit pups present abilities of rapid odor learning. In the present study, we first demonstrated (Exp. 1) that rabbit pups rapidly acquired the odor of the binary mixture through associative conditioning (when the mammary pheromone was used as unconditioned stimulus). Then, we compared how they responded to the mixture, its components and the mammary pheromone, after they had learned the mixture or one of its constituents. After they had learned the odor of the mixture, they responded to its odor and the odor of its constituents (Exp. 2). However, after they had learned one constituent's odor, they responded to this odor but not to the mixture's odor (Exp. 3). The response to the mixture appeared nevertheless when pups successively acquired the odor of the two components (Exp. 4). Therefore, both elemental and configural processing of the mixture seem to be displayed by rabbit pups, suggesting that neonatal perception of a simple odor mixture may involve more than the perception of its constituents.

A Novel Rabbit Model for Studying RPE Transplantation

The goal of this project was to develop a model of retinal pigment epithelium (RPE) transplantation that permits extensive and reliable analysis of the transplants. Cultures of newborn rabbit RPE were evaluated by morphology, electrophysiology, and the expression of zonula occludens-1, cytokeratin, and the melanocyte marker S-100. Cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) were transplanted into the subretinal space of rabbits with a 30-gauge needle without making a conjunctival flap or sclerotomy. The transplants were examined by fundus photography, confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), and angiography. At 2 months, the retina was examined histochemically. A 1-minute incubation at 37 degrees C with 20 muM CFDA-SE did not affect morphology or the expression of marker proteins. In coculture, the labeled cells integrated into monolayers that developed a normal transepithelial electrical resistance of 400 to 450 Omega . cm(-2). Dye was not transferred from labeled to nonlabeled RPE cells. Transplanted RPE was detectable for at least 2 months. Angiography demonstrated an intact blood-retinal barrier. The normal morphology of the retina and lack of debris in the subretinal space suggested that the transplanted RPE was functional. Primary cultures of newborn rabbit RPE were highly differentiated, even when labeled with CFDA-SE. Labeled cells were observed long-term in vitro and in vivo. This model can be used to examine how culture and transplantation protocols affect the reformation of a functional RPE monolayer. The similar size of rabbit and human eyes will facilitate the translation of these protocols to the bedside.