Retinopathy of prematurity: Risk factors and variability in Canadian neonatal intensive care units.

OBJECTIVE: To identify predictors of severe retinopathy of prematurity (ROP) in a large population-based cohort and to examine risk-adjusted variations across units. STUDY DESIGN: Retrospective analysis of Canadian Neonatal Network data on neonates with birth weight <1500 g who were screened for ROP between 2003 and 2010. Characteristics of infants with and without ROP were compared and a risk-adjusted model for severe ROP was developed. Rates of severe ROP were compared between sites. RESULTS: 1163 of 9187 (12.7%) infants developed severe ROP. Lower gestational age, male sex, small for gestational age, patent ductus arteriosus, late onset sepsis, more than two blood transfusions, inotrope use, and outborn status were associated with an increased risk of severe ROP. Severe ROP rates varied significantly between units. CONCLUSION: Younger, smaller and sicker male infants had higher adjusted risks of severe ROP and rates varied significantly among sites.

Corticotropin releasing hormone modulates endotoxin-induced inflammatory cytokine expression in human trophoblast cells.

Recent studies have suggested a significant increase in corticotropin releasing hormone (CRH) in maternal plasma and placenta during the course of maternal infection. The aim of this study was to examine the possible role of CRH in lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression using the well-established human extravillous trophoblast cell line HTR-8/SVneo. Exposure of the HTR-8/SVneo cells to LPS resulted in increased secretion of tumour necrosis factor alpha (TNF-alpha) and interleukin (IL)-8. Pre-treatment of the cells with CRH prior to LPS exposure significantly enhanced LPS induced TNF-alpha and IL-8 secretion. This effect was inhibited by the CRH antagonist astressin. Stimulation of the cells with CRH caused a rapid and transient phosphorylation of p38/MAPK while CRH had no effect on ERK1/2 activation. The effect of CRH on p38/MAPK activation was suppressed by astressin and by the p38/MAPK inhibitor SB203580. Exposure of the cells to CRH resulted in increased expression of TLR-4 and this effect was also inhibited by astressin. Taken together, these findings suggest that CRH augments LPS induced cytokine secretion in human trophoblast cells. Modulation of LPS induced immune responses by CRH may be mediated through regulation of TLR-4 and selective activation of the p38/MAPK signalling pathway.

Elevated corticotropin releasing hormone/corticotropin releasing hormone-R1 expression in postmortem brain obtained from children with generalized epilepsy.

The corticotropin releasing hormone (CRH) system has been suggested to initiate seizure activity in the developing brain. However, human data to support this theory is lacking. In this study, we have demonstrated that the expression of CRH, CRH-binding protein, and CRH-R1 (a CRH membrane receptor) were significantly elevated in cortical tissue obtained from 6 children with generalized epilepsy (mean age 8.2+/-1.5 years) relative to age-matched controls (mean age 7.8+/-1.4 years). In contrast, no significant difference in the expression of CRH-R2 was observed. The advent of CRH-R1 receptor antagonists may prove useful as novel anticonvulsants.

The common neurotrophin receptor p75NTR enhances the ability of PC12 cells to resist oxidative stress by a trkA-dependent mechanism.

Functional role(s) for the common neurotrophin receptor p75NTR in nerve growth factor (NGF) signaling have yet to be fully elucidated. Many studies have demonstrated that p75NTR can enhance nerve growth factor-induced survival mediated via the trkA receptor. In addition, newly identified pathways for p75NTR signaling have included distinct p75NTR-specific and trk-independent effects which generally appear to be pro-apoptotic. In the present study, we have examined the influence of p75NTR on NGF-mediated protective effects from hydrogen peroxide (H2O2)-induced apoptotic cell death of PC12 cells. Exposure of PC12 cells to H2O2 resulted in Caspase-3 activation and apoptosis. NGF protected PC12 cells against H2O2-mediated apoptosis in a dose-dependent manner and inhibited Caspase-3 activation. These effects of NGF required activation of both PI 3-kinase and MAP kinase signal pathways. When NGF binding to p75NTR was blocked by treating cells with either BDNF or PD90780, and where p75NTR expression was reduced by treating cells with antisense oligonucleotide to p75NTR, the protective effects of NGF were attenuated. Further, NGF had no effect on cell viability in PC12nn5 cells, which express only p75NTR. When trk-mediated signal transduction was blocked, leaving p75NTR signaling activated, PC12 cells were not more vulnerable to H2O2. These data suggest that p75NTR enhances the ability of PC12 cells to resist oxidative stress by a trkA-dependent mechanism, potentially by allosteric mechanisms. Further, potential trkA-independent and pro-apoptotic signaling of p75NTR does not contribute to apoptotic cell death of PC12 cells in a setting of oxidative insult.

Sublethal hypoxia up-regulates corticotropin releasing factor receptor type 1 in fetal hippocampal neurons.

We analysed the influence of oxygen-glucose deprivation (OGD) on the expression of corticotropin releasing factor (CRF) receptors (CRF-R1 and CRF-R2) in fetal hippocampal neurons in vitro. A 2 h exposure of neurons to OGD resulted in death of 18+/-2.8% cells at 24 h following exposure, which was considered sublethal hypoxia. Expression of both receptors was quantitated by competitive reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Levels of mRNA for CRF-R1 were increased 3.2-fold compared to control neurons, while CRF-R2 mRNA levels remained unchanged. The increase of CRF-R1 mRNA levels was observed at 6 h and peaked at 24 h. CRF-R1 protein levels were also increased by 2.4-fold and 1.7-fold at 24 h and 48 h, respectively. These data suggest that the effects of CRF on neuronal survival are mediated in part through the induction and expression of CRF-R1 following a hypoxic/ischemic insult.

Zinc and copper inhibit nerve growth factor-mediated protection from oxidative stress-induced apoptosis.

We have previously provided evidence that two transition metal cations, Zn2+ and Cu2+, can alter the conformation of nerve growth factor (NGF), rendering it unable to bind to its receptors or to activate signal transduction pathways. In the present study, we have assessed the influence of Zn2+ and Cu2+ on NGF-mediated protection from an oxidative insult. Exposure of rat pheochromocytoma (PC12) cells to hydrogen peroxide resulted in an increase in cell death via apoptosis, which was inhibited by NGF. Zn2+ and Cu2+, when added to cultures at a concentration of 100 microM, prevented NGF-mediated survival-promoting effects. Neither of these ions had an effect on basal cell viability (in the absence of NGF) after an oxidative insult. These results demonstrate that Zn2+ and Cu2+ can selectively inhibit NGF-mediated resistance to an oxidative stress, and have significant implications for neuronal function under both physiological and pathological (e.g. cerebral ischemia) conditions.

Cell biology of astrocyte proteoglycans.

A number of studies have demonstrated that the nervous system produces multiple molecular species of proteoglycans (PGs), and that these PGs on cell surfaces and in the extracellular matrix of the nervous system have the potential to act both as ligands for and receptors on neurons. PGs are now known to play an important role in cell-cell and cell-substrate interactions. This review focuses on a discussion of the expression, characterization and modulation of individual astrocytic PGs and the role that astrocytic PGs play in both the development and pathophysiology of the nervous system.

Quantitative analysis of mRNA expression of neuron-specific growth-associated genes in rat primary neurons by competitive RT-PCR.

The reverse transcriptase-polymerase chain reaction (RT-PCR) application is a sensitive method for detecting gene expression in tissues where the message level is a very small percentage of the total RNA and where only small amounts of sample are available such as in primary cultured hippocampal neurons. Based on a previously developed quantitative competitive RT-PCR strategy, mRNA expression and regulation of the neuron-specific growth-associated genes T alpha1 alpha-tubulin (T alpha1), microtubule-associated protein-2 (MAP-2) and growth-associated protein-43 (GAP-43), all of which have been proposed as putative markers of neurite growth during development and regeneration, were quantitated. This protocol, in combination with morphological evaluation of neurite outgrowth, may provide a useful tool for quantitation of neurite outgrowth during differentiation and regeneration in cultured neurons and may also be applied to detect the expression of other genes where the levels of message are low and in other tissues where small quantities of RNA are available.

Effects of a peptide analogue of the amphiphilic domain of the common neurotrophin receptor on nerve growth factor-mediated motility of human neuroblastoma cells.

Exposure of human neuroblastoma cells (IMR-32) to a peptide mimic of the cytoplasmic amphiphilic domain of the common neurotrophin receptor (p75NTR 367-379) resulted in enhanced nerve growth factor (NGF)-mediated inhibition of cell invasion in vitro. The peptide also enhanced NGF-mediated neurite extension and GAP-43 gene expression but had no effect on NGF-mediated cell survival. These latter functional effects mimicked influences on NGF-mediated neurite growth in other trkA-positive cells as reported previously. NGF-dependent trkA phosphorylation was significantly enhanced by the presence of the peptide, whereas high-affinity binding of 125I-NGF, both NGF receptors mRNA and protein expression, and trkA dimer/monomer ratios were not influenced. The studies suggest that ligand-mediated trkA activation has differential effects on cell motility phenomena and that the amphiphilic domain of p75NTR has a role in this differential signaling.

Collaborative and reciprocal effects of ciliary neurotrophic factor and nerve growth factor on the neuronal phenotype of human neuroblastoma cells.

We have probed the molecular basis of functional effects of ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) on aspects of the neuronal differentiation of LA-N-2 neuroblastoma cells. The influence of CNTF on the cholinergic phenotype can be accounted for by transcriptional/translational effects without implicating posttranslational mechanisms. Although both NGF receptors are expressed constitutively by LA-N-2 cells, CNTF has a marked stimulatory effect on trkA mRNA and protein. The NGF receptors are functional in serum-free conditions where they mitigate CNTF effects on cell adhesion but do not support process extension. Following priming by CNTF, NGF and CNTF have synergistic influences on process formation but not on choline acetyltransferase-specific activity.

Neonatal tracheal obstruction caused by aspiration of decidual tissue.

We report a newborn female born at 29 weeks' gestation who was unable to be resuscitated following a vaginal breech delivery. At autopsy the larynx and trachea contained a 1.2 x 0.2 cm fragment of tissue. Microscopic sections of this tissue revealed it to consist of decidua. This is the first report in the literature of tracheal obstruction secondary to aspiration of decidua. The mechanism by which this tissue may have resulted in completed obstruction of the trachea is discussed.

Congenital erythroleukemia in a neonate with severe hypoxic ischemic encephalopathy.

We report a case of a neonate who presented with hypoxic ischemic encephalopathy, persistent hypoglycemia and hypotension, intractable metabolic acidosis, renal failure and a coagulopathy but who, at autopsy, was found to have massive infiltration of nonhematopoietic tissues with blasts. The diagnosis of congenital erythroleukemia was confirmed by the detection of glycophorin A, a major erythrocyte membrane protein, on the surface of the blasts. The clinical presentation and course of the case described here have not previously been reported for this extremely rare condition.

Effects of neuronal proteoglycans on activity-dependent growth responses of fetal hippocampal neurons.

Excitatory amino-acid (EAA) neurotransmitters act as molecular signals influencing the structure of neurons during development. However, the signal transduction and effector mechanisms responsible for these effects have yet to be fully elucidated. We have previously provided evidence that EAA agonists induce the synthesis and release of proteoglycans (PGs) with neurite-promoting activity from fetal hippocampal neurons. In the present studies exposure of fetal hippocampal neurons to glutamate (100 microM) for 5 min resulted in increases in the neuron-specific growth-associated genes T alpha 1 alpha-tubulin (T alpha 1), microtubule-associated protein-2 (MAP-2) and growth-associated protein-43 (GAP-43). mRNA levels peaked at between 8 and 12 h following exposure as determined by competitive reverse transcription polymerase chain reaction (RT-PCR). Increases in neurite growth as measured by axonal length, the total length of dendrites, the number of branches per axon, the total length of branches per axon and the total neurite length were also observed 48 h after glutamate exposure. The increase in T alpha 1, MAP-2 and GAP-43 mRNA levels following glutamate exposure was mediated via both N-methyl-D-aspartate and metabotropic receptor activation. Heparin, which inhibits the neurite growth-promoting effects of PGs in vitro, and heparitinase, which catalyzes the cleavage of heparan sulphate, also inhibited the glutamate-dependent induction of T alpha 1, MAP-2 and GAP-43 mRNA expression and neurite growth when added to culture medium following glutamate exposure. Chondroitin sulphate and chondroitinase AC had no effects on the mRNA levels tested or on neurite growth. Therefore, these studies suggest that neuronal PGs regulated by activation of EAA receptors mediate neuronal growth responses.

Differential regulation of neuronal proteoglycans by activation of excitatory amino acid receptors.

Cell surface proteoglycans (PGs) have been implicated in neuronal growth, migration and differentiation and can play pivotal roles both in cell-cell and cell-substrate interactions during the development of the nervous system. We have previously shown that glutamate activation of excitatory amino acid receptors induces the synthesis and release of PGs with neurite-promoting activity from hippocampal neurones. In this study, we have investigated the activity-dependent regulation of mRNA expression of two PGs in fetal hippocampal neurones using a competitive reverse transcriptase-polymerase chain reaction and correlated this expression with neuronal growth. Both cerebroglycan (CBG), a glycosylphatidylinositol-anchored heparan sulphate PG, and neurocan, a developmentally regulated chondroitin sulphate PG, are expressed in hippocampal neurones. Exposure of hippocampal neurones to 100 microM glutamate for 5 min resulted in an increase in CBG mRNA levels and an increase in axonal and dendritic length. The increase in CBG mRNA levels following glutamate exposure was mediated via both N-methyl-D-aspartate and metabotropic receptor activation.

Signal transduction mechanisms subserving activity-dependent release of neuronal proteoglycans.

We demonstrate here using dissociated hippocampal neurons that glutamate-induced release of proteoglycans which have been shown to have neurite growth-promoting activity is regulated by serine/threonine kinases of the protein kinase C and calcium/calmodulin type II kinase families, and that the state of phosphorylation of hippocampal neurons is a determinant of the magnitude and duration of the release response. Nitric oxide is also involved in mediating glutamate-induced PG release.

Differentiation effects of ciliary neurotrophic factor on human neuroblastoma cells.

In the human neuroblastoma cell line LA-N-2, recombinant rat ciliary neurotrophic factor (CNTF) induced neurite growth and cholinergic differentiation that were both half-maximally saturated at < 100 pM of the neurokine, but was not required for cell survival in serum-free conditions over a 13-day period. CNTF markedly stimulated choline acetyltransferase activity and acetylcholine synthesis, whereas high-affinity choline transport was only slightly enhanced and acetylcholinesterase activity was unchanged. Leukemia inhibitory factor had effects identical to CNTF on neurite growth and choline acetyltransferase activity, but interleukin 6 had no effect. Radioiodinated CNTF binding and affinity cross-linking studies were consistent with tripartite receptor activation as a mediator of the observed biological effects.

Molecular correlates of spinal cord repair in the embryonic chick: heparan sulfate and chondroitin sulfate proteoglycans.

Proteoglycan (PG) biosynthesis in vivo and PG-associated neurite growth-promoting activity in vitro were examined in the thoracic spinal cord of embryonic chick at times during which functional recovery following axonal damage is permitted, and at later times when such functional recovery is restricted. Over a 10-day period encompassing the permissive and restrictive periods the ratio of newly synthesized heparan sulfate (HS) PG to chondroitin sulfate (CS) PG decreased by more than 50%. Specific PG-associated neurite-promoting activity (NPA) of a PG fraction immobilized on a laminin substrate was 75-fold higher at E9 than at E17. Perturbations of the two families of PGs indicated that all laminin-bound NPA was associated with HSPGs from E9 cord, and that removal of the influence of CSPGs from PG extracts of E17 cord unmasked neurite-promoting activity on a poly-D-lysine substrate of the same magnitude as that observed on a laminin substrate. Neurite-promoting activity associated with HSPGs and high HS to CS ratios of newly synthesized PGs characterize the permissive period for axonal regeneration in the chick embryo spinal cord. In the restrictive period for axonal regeneration neurite promoting activity is masked by the presence of CSPGs which are synthesized at higher levels than HSPGs.

Modulation of neurite promoting proteoglycans by neuronal differentiation.

A human cell line committed to neuronal lineage was used to examine the influence of differentiation on proteoglycan synthesis and function. Where the LA-N-2 cells were stimulated to differentiate towards a phenotype of cholinergic neurons, proteoglycans of the heparan sulphate class increased relative to chondroitin sulphate proteoglycans and displayed more homogeneously shorter glycosaminoglycan chains with increasing degrees of sulphation. The changes were accompanied by increasing potency of the heparan sulphate proteoglycans in neurite growth-promoting activity when immobilized on a laminin substrate. These studies begin to address the role of activity-independent growth and differentiation on the synthesis and release by neurons of neurite growth-promoting proteoglycans. The observations have implications for understanding the role of proteoglycan overexpression and the production of dystrophic neurites in Alzheimer disease.

Activity-dependent regulation of neuronal synthesis and release of neurite-promoting heparan sulfate proteoglycans.

Activity-dependent enduring change in neuronal communication is essential for exquisite connectivity during development of the nervous system and for adaptative responses of the mature nervous system. Here we report that glutamate activation of excitatory amino acid receptors induces the release of proteoglycans (PGs) with neurite growth-promoting activity from fetal hippocampal neurons in dissociated culture. Glutamate exposure produced an increase in the release of PGs of the heparan sulfate class but had no effect on the release of chondroitin sulfate PGs. PG release was mediated via both N-methyl-D-aspartate and metabotropic receptor activation. Conditions producing increased PG release also elicited a sustained increase in neuronal PG synthesis. These studies suggest that a class of substrate-bound growth modulatory glycoconjugates could participate in both the induction and maintenance of activity-dependent synaptic modification.

Axonal transport of proteoglycans in regenerating goldfish optic nerve.

Labeling of goldfish optic nerve and tectum proteoglycans (PGs) was quantified following intraocular injection of 35SO4 and [3H]proline or [3H]glucosamine. Both intact animals and animals which had survived for periods of 10 to 119 days after an optic nerve crush lesion were examined. Regenerating retinal ganglion cell (RGC) axons reached the rostral pole of the tectum by 10 days postcrush and by 21 days had densely innervated the optic synaptic laminae. If the contralateral tectum had been removed, the regenerating RGC axons innervated the remaining ipsilateral tectum with a delay of approximately 14 days. There was a biphasic increase in the synthesis and transport of PGs during optic fiber regeneration which was not affected by the removal of the tectum. More highly sulfated PGs were preferentially off-loaded from the orthograde transport pool proximally in the optic nerve, both in the unoperated animals and during regeneration. These PGs also had longer and/or more glycosaminoglycan (GAG) chains than those off-loaded distally, in the tectum. During early regeneration, the synthesis and transport of chondroitin sulfate PGs (CSPGs) increased more than those of heparan sulfate PGs, and during the period of optic fiber invasion of the synaptic laminae, the PGs retained in the nerve had a higher content of CSPGs than those transported into the tectum. Removal of the contralateral tectum at the time of nerve crush resulted in a decrease in the size and/or numbers of GAGs and overall sulfation of PGs in the nerve by 21 days postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)