Newborn Animal Models Research Articles

PPAR Gamma Receptor: A Novel Target to Improve Morbidity in Preterm Babies.

Worldwide, three-quarters of a million babies are born extremely preterm (<28 weeks gestation) with devastating outcomes: 20% die in the newborn period, a further 35% develop bronchopulmonary dysplasia (BPD), and 10% suffer from cerebral palsy. Pioglitazone, a Peroxisome Proliferator Activated Receptor Gamma (PPARγ) agonist, may reduce the incidence of BPD and improve neurodevelopment in extreme preterm babies. Pioglitazone exerts an anti-inflammatory action mediated through Nuclear Factor-kappa B repression. PPARγ signalling is underactive in preterm babies as adiponectin remains low during the neonatal period. In newborn animal models, pioglitazone has been shown to be protective against BPD, necrotising enterocolitis, and lipopolysaccharide-induced brain injury. Single Nucleotide Polymorphisms of PPARγ are associated with inhibited preterm brain development and impaired neurodevelopment. Pioglitazone was well tolerated by the foetus in reproductive toxicology experiments. Bladder cancer, bone fractures, and macular oedema, seen rarely in adults, may be avoided with a short treatment course. The other effects of pioglitazone, including improved glycaemic control and lipid metabolism, may provide added benefit in the context of prematurity. Currently, there is no formulation of pioglitazone suitable for administration to preterm babies. A liquid formulation of pioglitazone needs to be developed before clinical trials. The potential benefits are likely to outweigh any anticipated safety concerns.

A New Technique for Collection of Cerebrospinal Fluid in Rat Pups.

BACKGROUNDNeuroprotective strategies to prevent or decrease brain injury in hypoxic ischemic newborns are one of the main research lines in neonatology. Animal models have been used to assess the efficiency of new therapeutic strategies. Brain damage biomarkers in cerebrospinal fluid (CSF) are frequently used to evaluate the outcome at the bedside. Despite the importance of this approach in clinical practice, there are many difficulties in using it in small animals. The aim of this paper was to describe a new technique for collecting CSF in rat pups. Furthermore the reference values of S100β protein levels, commonly used in common clinical practice, were analyzed in animals between 7 to 12 days.METHODS42 Wistar rat pups aged 7 to 12 days were used. CSF was obtained by direct puncture of the cisterna magna with a 24-gauge needle. S100β protein levels were determined with enzyme-linked immunosorbent assay (ELISA).RESULTSCSF was successfully obtained in 96% of the cases, with an average amount of 21.28 μl (5–40 μl). Normal values for S100β were described. HI animals presented higher S100β values than controls.CONCLUSIONSA simple, reproducible technique for CSF collection in rat pups has been described. This new method will allow study of brain injury biomarkers in newborn hypoxic ischemic animal models.

Animal models of bronchopulmonary dysplasia. The preterm and term rabbit models

Bronchopulmonary dysplasia (BPD) is an important lung developmental pathophysiology that affects many premature infants each year. Newborn animal models employing both premature and term animals have been used over the years to study various components of BPD. This review describes some of the neonatal rabbit studies that have contributed to the understanding of BPD, including those using term newborn hyperoxia exposure models, premature hyperoxia models, and a term newborn hyperoxia model with recovery in moderate hyperoxia, all designed to emulate aspects of BPD in human infants. Some investigators perturbed these models to include exposure to neonatal infection/inflammation or postnatal malnutrition. The similarities to lung injury in human premature infants include an acute inflammatory response with the production of cytokines, chemokines, and growth factors that have been implicated in human disease, abnormal pulmonary function, disordered lung architecture, and alveolar simplification, development of fibrosis, and abnormal vascular growth factor expression. Neonatal rabbit models have the drawback of limited access to reagents as well as the lack of readily available transgenic models but, unlike smaller rodent models, are able to be manipulated easily and are significantly less expensive than larger animal models.

Combined Treatment of Xenon and Hypothermia in Newborn Rats - Additive or Synergistic Effect?

BackgroundBreathing the inert gas Xenon (Xe) enhances hypothermic (HT) neuroprotection after hypoxia-ischemia (HI) in small and large newborn animal models. The underlying mechanism of the enhancement is not yet fully understood, but the combined effect of Xe and HT could either be synergistic (larger than the two effects added) or simply additive. A previously published study, using unilateral carotid ligation followed by hypoxia in seven day old (P7) rats, showed that the combination of mild HT (35°C) and low Xe concentration (20%), both not being neuroprotective alone, had a synergistic effect and was neuroprotective when both were started with a 4 h delay after a moderate HI insult. To examine whether another laboratory could confirm this finding, we repeated key aspects of the study.Design/MethodsAfter the HI-insult 120 pups were exposed to different post-insult treatments: three temperatures (normothermia (NT) NT37°C, HT35°C, HT32°C) or Xe concentrations (0%, 20% or 50%) starting either immediately or with a 4 h delay. To assess the synergistic potency of Xe-HT, a second set (n = 101) of P7 pups were exposed to either HT35°C+Xe0%, NT+Xe20% or a combination of HT35°C+Xe20% starting with a 4 h delay after the insult. Brain damage was analyzed using relative hemispheric (ligated side/unligated side) brain tissue area loss after seven day survival.ResultsImmediate HT32°C (p = 0.042), but not HT35°C significantly reduced brain injury compared to NT37°C. As previously shown, adding immediate Xe50% to HT32°C increased protection. Neither 4 h-delayed Xe20%, nor Xe50% at 37°C significantly reduced brain injury (p>0.050). In addition, neither 4 h-delayed HT35°C alone, nor HT35°C+Xe20% reduced brain injury. We found no synergistic effect of the combined treatments in this experimental model.ConclusionsCombining two treatments that individually were ineffective (delayed HT35°C and delayed Xe20%) did not exert neuroprotection when combined, and therefore did not show a synergistic treatment effect.

PO-0396 The Sigma-1 Receptor Agonist Pre-084 Protects Against Glutamate Induced Neurotoxicity In Primary Hippocampal Neurons

Background Prematurity is a major determinant of neonatal mortality and morbidity. The number of preterm birth is still on the rise. Recently we and others could demonstrate neuroprotective effects of sigma-1 receptor ligands in adult and newborn animal models of brain injury. Since sigma-1 receptor agonists are already undergoing clinical trials in adult neurological disease, they might be considered a promising therapeutic option also in preterm brain injury. We have previously shown that the selective sigma-1 receptor agonist PRE-084 (2-(4-morpholinethyl)1-phenylcyclohexane-carboxylate) protects against neonatal excitotoxic brain injury in vivo . The aim of the present study was to investigate whether PRE-084 is able to prevent neurotoxicity following glutamate exposure in vitro . Methods Cultured primary hippocampal neurons (day in vitro 10) were pre-treated with PRE-084 before glutamate was applied. Subsequently cell death was quantified by means of PI/calcein – AM staining using live confocal microscopy. Neurons were randomly assigned to one of the following groups: i) control, ii) glutamate or iii) glutamate+PRE-084. PRE-084 was applied in two dosages (10 and 100 µM) prior to glutamate. Results The application of PRE-084 significantly reduced the percentage of dead cells (PRE-084 10 µM: 22.09 (20.50;28.84) and 100 µM: 25.87 (18.77;33.40)) compared to the untreated glutamate control group 43.56 (39,86;46.02). Conclusion Our data show that administration of PRE-084 protects against glutamate induced cell death in primary hippocampal neurons. PRE-084 shows considerable promise as a therapeutic strategy in preterm brain injury and might provide an adequate means of combating this major cause of neurological disability in infancy.

Urine metabolomic profiling in neonatal nephrology

Metabolomics, the latest “omics” technology aims to study the complete set of low molecular weight metabolites that may change according to the physiological or the pathological state of the organism. Clinical studies dealing with metabolomics in neonatal and pediatric nephrology are very few. In this paper we present the experimental studies in newborn animal models, together with the available data on human newborns. Finally the urine metabolomic profiling of 3 newborns who suffered from severe perinatal asphyxia and were treated with hypothermia. They are located in a different part of the multivariate space, the reason of the differences being the basal metabolic profile (resilience) of each neonate: 1 died and 2 survived (one of them developed an acute kidney injury). The main metabolites responsible for the different metabolic profile among the 3 newborns are presented. In the future each neonatologist and nephrologist should become skilled in the metabolomic field.

Aspects fondamentaux de la toxicite éventuelle des drogues anesthésiques

Brain development is a complex phenomenon in which several stages of production, maturation, and organization of neural cells in a network succeed each other. Various environmental factors can disrupt these stages. During the last decade, numerous in vitro and in vivo experimental studies in newborn animal models have established the neurotoxic effects of most anesthetic and sedative drugs used in pediatrics. These effects are essentially responsible for neuronal apoptosis and have been associated with learning disorders in adulthood. This neurotoxicity is time-varying: there is a vulnerability period during synaptogenesis. These toxic effects were attributed to agonist properties on GABA receptors or antagonist properties on NMDA receptors, which are characteristics of all implicated anesthetics. Excessive activation of the GABA pathway and/or excessive inhibition of the NMDA pathway activate cellular mechanisms leading to apoptosis. The intensity of neurotoxic effects is dose- and time-exposure-dependent. These numerous experimental data must be interpreted with caution with regard to their validity in humans, mainly because of interspecies differences as well as differences between experimental conditions and clinical practice. Today, these data are insufficient to change our practices, taking into account the indisputable benefits of the use of anesthetics and sedative drugs. However, progress in experimental research will help us identify the safest therapeutic strategies and neuroprotective treatments.

PERINATAL ASPHYXIA MAY CAUSE REDUCTION IN CSF DOPAMINE METABOLITE CONCENTRATIONS

Perinatal asphyxia (PA) is a major determinant of neurologic disability. In the immature brain, dopaminergic neurons are very sensitive to asphyxia, and abnormal dopamine levels can lead to functional and morphologic disturbances. The early dopaminergic input from the midbrain may play an important role in the development of the basal ganglia and the cerebral cortex.1 In newborn animal models, an excessive increase in extracellular dopamine is seen shortly following PA.2 But PA also produces long-lasting abnormalities in the dopaminergic pathways (mainly in the mesostriatal/mesolimbic dopamine system).2 Monoamine changes following PA have seldom been reported in human newborns and always restricted to the first hours of life, showing higher CSF dopamine metabolites in moderate-to-severe PA than in control or mild cases.3 Within the CNS, the end product of dopamine is homovanillic acid (HVA) and of serotonin, 5-hydroxyindoleacetic acid (HIAA); both can be measured in CSF. We describe chronological changes in cerebral monoamine neurotransmitters in newborns following a moderate to severe PA. ### Methods. We recruited seven term infants …

The Seek of Neuroprotection: Introducing Cannabinoids

The cannabinoid system is constituted by some endogenous ligands (endocannabinoids), usually arachydonic acid derivatives, and their specific receptors. The endogenous cannabinoid system (ECS) is involved in the control of synaptic transmission, modulating memory, motivation, movement, nociception, appetite and thermoregulation. ECS also exert extraneural effects, mainly immunomodulation and vasodilation. Two cannabinoid receptors have been cloned so far: CB(1) receptors are expressed in the central nervous system (CNS) but can also be found in glial cells and in peripheral tissues; CB(1) receptors are Gi/o protein coupled receptors that modulate the activity of several plasma membrane proteins and intracellular signaling pathways. CB(2) receptors are also Gi/o protein-coupled receptors; although it is accepted that CB(2) receptors are not expressed in forebrain neurons, they have been described in activated glia. Some of the cannabinoids activate other receptors, for instance vanilloid receptors (TRPV1). Lately, the ECS is emerging as a natural system of neuroprotection. This consideration is based on some properties of cannabinoids as their vasodilatory effect, the inhibition of the release of excitotoxic amino acids and cytokines, and the modulation of oxidative stress and toxic production of nitric oxide. Such effects have been demonstrated in adult and newborn animal models of acute and chronic neurodegenerative conditions, and postulate cannabinoids as valuable neuroprotective agents. Patents related to cannabinoid receptors are also discussed.

Spatial and temporal expression of surfactant proteins in hyperoxia-induced neonatal rat lung injury

BackgroundBronchopulmonary dysplasia, a complex chronic lung disease in premature children in which oxidative stress and surfactant deficiency play a crucial role, is characterized by arrested alveolar and vascular development of the immature lung. The spatial and temporal patterns of expression of surfactant proteins are not yet fully established in newborn infants and animal models suffering from BPD.MethodsWe studied the mRNA expression of surfactant proteins (SP) A, -B, -C and -D and Clara cell secretory protein (CC10) with RT-PCR and in situ hybridization and protein expression of CC10, SP-A and -D with immunohistochemistry in the lungs of a preterm rat model, in which experimental BPD was induced by prolonged oxidative stress.ResultsGene expression of all surfactant proteins (SP-A, -B, -C and -D) was high at birth and initially declined during neonatal development, but SP-A, -B, and -D mRNA levels increased during exposure to hyperoxia compared to room-air controls. Peak levels were observed in adult lungs for SP-A, SP-C and CC10. Except for SP-A, the cellular distribution of SP-B, -C, -D and CC10, studied with in situ hybridization and/or immunohistochemistry, did not change in room air nor in hyperoxia. Exposure to normoxia was associated with high levels of SP-A mRNA and protein in alveolar type 2 cells and low levels in bronchial Clara cells, whereas hyperoxia induced high levels of SP-A expression in bronchial Clara cells.ConclusionThe increased expression of SP-A mRNA under hyperoxia can be attributed, at least in part, to an induction of mRNA and protein expression in bronchial Clara cells. The expanded role of Clara cells in the defence against hyperoxic injury suggests that they support alveolar type 2 cell function and may play an important role in the supply of surfactant proteins to the lower airways.

Perinatal corticosteroids: A review of research. Part I: Antenatal administration.

The premature infant may receive therapeutic glucocorticoid drugs while in utero or in the postnatal period. This article (part I of a two-part series) discusses the benefits and risks of in utero, or antenatal, corticosteroids (ACS) for the premature infant. Part II addresses the benefits and risks of postnatal corticosteroid (PCS) use. There are numerous clinical studies on the therapeutic use of these steroids for the prevention of respiratory distress syndrome and chronic lung disease in the premature infant, although research results on the efficacy of repeated steroid exposure among premature infants vary. Premature infants who are exposed to repeated courses of ACS and/or high-cumulative-dose PCS may show no neurologic side effects until later in life. Research in newborn animal models focused on the timing, duration, and amounts of ACS and PCS. Current clinical research includes examination of the neurodevelopment of infants who are therapeutically exposed to perinatal corticosteroids, to identify safer minimal dose protocols. Over the past 30 years, corticosteroids have been increasingly prescribed before and after birth. Understanding the potential treatment benefits and risks to human fetuses and neonates is vital to clinical practice. This review presents historic and pharmacokinetic information about prenatal use of corticosteroids. It also offers scientific evidence of the benefits and risks identified in animal models and clinical trials, to stimulate thought that gtiides neonatal clinical practice.

Hypoxic preconditioning reduces apoptosis in a rat model of immature brain hypoxia-ischaemia

Hypoxic events are common in newborns but their consequences on brain development have not been demonstrated. It has been reported that in newborn animal models of cerebral hypoxic-ischaemic insult, short-term hypoxia before the insult completely prevented brain damage. The mechanisms of this brain tolerance have not been fully elucidated. Using a rat model of hypoxic preconditioning at day 6 followed by carotid ligation and hypoxic insult at day 7, we found a decrease in the number of apoptotic cells 24 and 48 h after the insult in the striatum, hippocampus and cortex. We demonstrated here that regulation of apoptotic cell death is one of the mechanisms involved in tolerance to hypoxia-ischaemia induced by hypoxic preconditioning.

Perinatal asphyxia: a clinical review, including research with brain hypothermia.

Perinatal asphyxia may occur in utero, during labor and delivery, or in the postnatal period. There are numerous causes, and the clinical manifestations vary. Infants who experience mild asphyxia may show no neurologic injury. Severe asphyxia may be fatal in utero, or immediately after birth, with survivors showing extensive neurologic sequelae, with or without cognitive deficits. Mild brain hypothermia appears promising in the prevention of further neurologic damage in encephalopathic infants following asphyxia. Recent research on newborn animal models has focused on the timing, duration, and depth of hypothermia. Promising new research is now under way in nurseries in the U.S. in an attempt to establish clinical protocols for use of hypothermia in human neonates.

Cooling the asphyxiated brain - ready for clinical trials?

Recent research using newborn animal models of hypoxia-ischaemia has shown that cooling the subject from 3 to 6 degrees C starting after the insult and lasting up to 72 h offers long-lasting neuroprotection. One concern is that additional stress during hypothermia seems to reduce the beneficial effect. Phase 1 studies on cooling severely asphyxiated infants have not yet revealed any systemic adverse effects. The evidence is strongly in favor of hypothermia reducing brain damage after asphyxia and the time has come for undertaking randomized trials.

Motonuclear changes after cranial nerve injury and regeneration.

Little is known about the mechanisms at play in nerve regeneration after nerve injury. Personal studies are reported regarding motonuclear changes after regeneration of injured cranial nerves, in particular of the facial and oculomotor nerves, as well as the influence that the natural molecule acetyl-L-carnitine (ALC) has on post-axotomy cranial nerve motoneuron degeneration after facial and vagus nerve lesions. Adult and newborn animal models were used. Massive motoneuron response after nerve section and reconstruction was observed in the motonuclei of all nerves studied. ALC showed to have significant neuroprotective effects on the degeneration of axotomized motoneurons. Complex quantitative, morphological and somatotopic nuclear changes occurred that sustain new hypotheses regarding the capacities of motoneurons to regenerate and the possibilities of new neuron proliferation. The particularities of such observations are described and discussed.

EFFECT OF INHIBITORS OF PROTEIN KINASE C(PKC) AND PROTEIN TYROSINE KINASE(PTK) ON THE CEREBROVASCULAR RESPONSE TO ADENOSINE 2276

Adenosine is an important factor in cerebrovascular regulation in the newborn especially under conditions low cerebral oxygen availability. Post receptor signaling associated with adenosine receptor(s) in the newborn cerebral circulation are poorly understood. Recent studies suggest that certain adenosine receptors are coupled to PKC-and/or PTK-dependent signal transduction pathways. We evaluated the hypothesis that activation of PKC modulates the cerebrovascular response to adenosine in vivo by examining the effect of staurosporine, an inhibitor of PKC, using the closed cranial window preparation in twelve newborn piglets. In that paradigm, staurosporine (9 nM) treatment augmented by 4-fold the increase pial arteriolar diameter induced by 1 μM exogenous adenosine (Δdiameter control = 4±2%[79±4 μm to 82±3 μm]; Δdiameter staurosporine = 17±3%[56±3 μm to 101±5 μm]). In addition, the adenosine-induced vasodilation was prolonged in staurosporine-treated preparations. Recent experiments show a similar effect, i.e., enhanced the dose-dependent relaxation response to adenosine in isolated, newborn lamb middle cerebral artery (MCA) preparations in tissue bath. An inhibitor of PTK (genistein, 20 μM) showed similar trend but was not statistically significant. The data suggest that activation of staurosporine-sensitive signal transduction pathways such as PKC may play a role in the ultimate expression of contractile properties of cerebral arteries from newborn animal models. We propose that certain adenosine receptors activate PKC which modulate the contraction/relaxation response and/or modify adenosine uptake and metabolism. (Supported by the American Heart Association national council, #95009270)

Impairment of Cerebral Blood Flow Autoregulation in the Newborn Lamb by Hypoxia

Autoregulation of cerebral blood flow has been demonstrated in both fetal and newborn animal models under normoxic conditions. In the present experiments we have attempted to define the minimal hypoxic insult which impairs autoregulation in the newborn lamb and to assess the time to recovery. We measured cerebral blood flow by the intracarotid 133Xe method in fifteen 4- to 9-day-old lambs and tested autoregulation of cerebral blood flow by increasing blood pressure 20-30% with phenylephrine. Autoregulation was tested in the control state and at successive time intervals after an hypoxic stress (PaO2 of 30 mm Hg) of 10 or 20 min. We found that cerebral autoregulation was abolished after 20 min of hypoxia and recovered within 7 h. Since this model identifies the minimal hypoxic insult to abolish autoregulation it might be used to study means to protect autoregulation or to hasten its recovery after hypoxia.