Candace Rossignol

A Pilot Study of Novel Biomarkers in Neonates With Hypoxic-Ischemic Encephalopathy

Severe hypoxic-ischemic encephalopathy (HIE) is a devastating condition that can lead to mortality and long-term disabilities in term newborns. No rapid and reliable laboratory test exists to assess the degree of neuronal injury in these patients. We propose two possible biomarkers: 1) phosphorylated axonal neurofilament heavy chain (pNF-H) protein, one of the major subunits of neurofilaments, found only in axonal cytoskeleton of neurons and 2) Ubiquitin C-terminal hydrolase 1 (UCHL1 protein) that is heavily and specifically concentrated in neuronal perikarya and dendrites. High-serum pNF-H and UCHL1 levels are reported in subarachnoid hemorrhage and traumatic brain injury, suggesting that they are released into blood following neuronal injury. We hypothesized that serum pNF-H and UCHL1 were higher in neonates with moderate-to-severe HIE than in healthy neonates. A time-limited enrollment of 14 consecutive patients with HIE and 14 healthy controls was performed. UCHL1 and pNF-H were correlated with clinical data and brain MRI. UCHL1 and pNF-H serum levels were higher in HIE versus controls. UCHL1 showed correlation with the 10-min Apgar score, and pNF-H showed correlation with abnormal brain MRI. Our findings suggest that serum UCHL1 and pNF-H could be explored as diagnostic and prognostic tools in neonatal HIE.

Transplantation of multipotent astrocytic stem cells into a rat model of neonatal hypoxic-ischemic encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) in neonates results in long-term disabilities. Stem cell therapy may offer an attractive treatment for HIE. Multipotent astrocytic stem cells (MASCs) from mice transplanted into a rat model of hypoxia-ischemia (HI) survived the transplantation and showed signs of migration towards the injured cortex. Some MASCs around the injured cortex differentiated into neuronal and astrocytic phenotypes. MASCs transplanted into non-ischemic pups survived but retained their astrocytic phenotype. These data suggest that transplanted MASCs can survive and differentiate into neurons and astrocytes in the post-injury milieu of the neonatal brain injured by HI.

Ontogeny of the neutral amino acid transporter SAT1/ATA1 in rat brain

The glutamine-glutamate/GABA cycle is critical for the developing brain as glutamatergic neurotransmission is important for neuronal survival and drives synaptogenesis and activity-dependent synaptic plasticity. GABAergic transmission may be essential for the formation of neural circuits. Recently a cDNA encoding a brain-enriched System A transporter (SAT1/ATA1), has been identified which may provide glutamine to neurons for the biosynthesis of neurotransmitters glutamate and gamma-aminobutyric acid (GABA). In this study, we have examined the developmental expression pattern of SAT1/ATA1 protein in rat brain by immunohistochemistry. We find that SAT1/ATA1 was present in the developing rat brain at all gestational ages examined including prenatal days 17 and 19 and postnatal days 2, 10, 14, and adult. SAT1/ATA1 immunoreactivity was seen in the neocortex, hippocampus, and neuroepithelium at the earliest time point examined, prenatal day 17. SAT1/ATA1 was prominent in the striatum, the hippocampus and the cortex in the postnatal animals. In adults, SAT1/ATA1 was limited to the cell body region while in developing animals SAT1/ATA1 protein was found in neuronal processes. These results contribute to our understanding of the relationship between the cycling of glutamate and glutamine between astrocytes and glia and the pathophysiological conditions that occur in hypoxic ischemic encephalopathy.

Neurotrophin-Induced Migration and Neuronal Differentiation of Multipotent Astrocytic Stem Cells In Vitro

Hypoxic ischemic encephalopathy (HIE) affects 2–3 per 1000 full-term neonates. Up to 75% of newborns with severe HIE die or have severe neurological handicaps. Stem cell therapy offers the potential to replace HIE-damaged cells and enhances the autoregeneration process. Our laboratory implanted Multipotent Astrocytic Stem Cells (MASCs) into a neonatal rat model of hypoxia-ischemia (HI) and demonstrated that MASCs move to areas of injury in the cortex and hippocampus. However, only a small proportion of the implanted MASCs differentiated into neurons. MASCs injected into control pups did not move into the cortex or differentiate into neurons. We do not know the mechanism by which the MASCs moved from the site of injection to the injured cortex. We found neurotrophins present after the hypoxic-ischemic milieu and hypothesized that neurotrophins could enhance the migration and differentiation of MASCs. Using a Boyden chamber device, we demonstrated that neurotrophins potentiate the in vitro migration of stem cells. NGF, GDNF, BDNF and NT-3 increased stem cell migration when compared to a chemokinesis control. Also, MASCs had increased differentiation toward neuronal phenotypes when these neurotrophins were added to MASC culture tissue. Due to this finding, we believed neurotrophins could guide migration and differentiation of stem cell transplants after brain injury.

The Effects of Hypoxia-Ischemia on Neutral Amino Acid Transporters in the Developing Rat Brain

The neutral amino acid transporters SNAT1–3 and ASCT1 play critical roles in the recycling of glutamine, and subsequently glutamate, via the glutamine-glutamate cycle. Hypoxia-ischemia was induced in rat pups using the Rice-Vannucci model. Brains were harvested at 1 h, 24 h and 7 days after ischemia. The expression of NAATs was evaluated using immunoblotting, real-time PCR, and immunohistochemistry. Results were compared with age-matched controls and shams. SNAT1 mRNA decreased at 1 h after injury in both hemispheres when compared with the control animals and correlated with a decrease in protein expression at 24 h in the hippocampus and cortex. SNAT1 protein expression increased globally at 7 days after injury and specifically in the hippocampus. Finally, SNAT2 and 3 demonstrated subtle changes in various brain regions after injury. These data suggest that neutral amino acid transporters remain largely intact after hypoxia-ischemia.

Gelatinized copper-capillary alginate gel functions as an injectable tissue scaffolding system for stem cell transplants.

In severe hypoxic–ischemic brain injury, cellular components such as neurons and astrocytes are injured or destroyed along with the supporting extracellular matrix. This presents a challenge to the field of regenerative medicine since the lack of extracellular matrix and supporting structures makes the transplant milieu inhospitable to the transplanted cells. A potential solution to this problem is the use of a biomaterial to provide the extracellular components needed to keep cells localized in cystic brain regions, allowing the cells to form connections and repair lost brain tissue. Ideally, this biomaterial would be combined with stem cells, which have been proven to have therapeutic potentials, and could be delivered via an injection. To study this approach, we derived a hydrogel biomaterial tissue scaffold from oligomeric gelatin and copper–capillary alginate gel (GCCAG). We then demonstrated that our multipotent astrocytic stem cells (MASCs) could be maintained in GCCAG scaffolds for up to 2 weeks in vitro and that the cells retained their multipotency. We next performed a pilot transplant study in which GCCAG was mixed with MASCs and injected into the brain of a neonatal rat pup. After a week in vivo, our results showed that: the GCCAG biomaterial did not cause a significant reactive gliosis; viable cells were retained within the injected scaffolds; and some delivered cells migrated into the surrounding brain tissue. Therefore, GCCAG tissue scaffolds are a promising, novel injectable system for transplantation of stem cells to the brain.

Ontogeny of the neutral amino acid transporter SNAT1 in the developing rat

SummarySystem A is a highly regulated, Na+-dependent transporter that accepts neutral amino acids containing short, polar side chains. System A plays an important role during rat development as decreased pup weights are observed in dams infused during gestation with a non-metabolizable System A substrate. Given the potential importance of SNAT1 during development in the rat brain, we examined whether SNAT1 would be present at an earlier gestation during organogenesis in multiple organs by immunohistochemistry and immunoblotting. SNAT1 protein was observed in the developing lungs, intestines, kidneys, heart, pancreas, and skeletal muscle of rats at prenatal days 14, 17, 19, 21, and postnatal day 2 rats. SNAT1 protein expression decreased in the liver and intestine shortly after birth and as the rat matured. SNAT1 expression was constant throughout development in the lungs and kidney and increased in the heart from prenatal day 19 to postnatal day 2. Highest levels of expression in older animals were seen in organs undergoing rapid cell division.

A pH-dependent increase in neuronal glutamate efflux in vitro: Possible involvement of ASCT1

Efflux of glutamate from intracellular pools during hypoxia-ischemia has been postulated to be mediated by amino acid transporters and can lead to excitotoxicity. In addition, a decrease in pH seen during global hypoxia-ischemia may influence which transporter is responsible for this glutamate efflux. For example, the neutral amino acid transporter ASCT1 is an effective transporter of glutamate at low pH. We have examined the effects of pH, pH and temperature, and hypoxia on glutamate efflux in a rat primary neuronal cell culture model. We observed a marked increase of glutamate efflux as pH was decreased from 7.4 to 5.5. This pH-dependent efflux is likely due to a transporter-mediated process because it was seen in the presence of tetrodotoxin and was blunted by decreasing the temperature to either 35 degrees C or 33 degrees C. In addition, no increase in LDH was seen at pH 5.5 suggesting that increased glutamate levels were not due to cellular death. No change in glutamate levels was seen when the oxygen tension of the medium was lowered from 150 mm Hg to either 30 or 15 mm Hg. Given that EAAT transporters are inhibited by low pH, other transporters, such as ASCT1, may be responsible for this pH-dependent efflux of glutamate.

Enteral Feeding as an Adjunct to Hypothermia in Neonates with Hypoxic-Ischemic Encephalopathy

Background: Withholding enteral feedings during hypothermia lacks supporting evidence. Objectives: We aimed to determine if minimal enteral nutrition (MEN) during hypothermia in patients with hypoxic-ischemic encephalopathy was associated with a reduced duration of parenteral nutrition, time to full oral feeds, and length of stay, but would not be associated with increased systemic inflammation or feeding complications. Methods: We performed a pilot, retrospective, matched case-control study within the Florida Neonatal Neurologic Network from December 2012 to May 2016 of patients who received MEN during hypothermia (n = 17) versus those who were not fed (n = 17). Length of stay, feeding-related outcomes, and brain injury identified by MRI were compared. Serum inflammatory mediators were measured at 0–6, 24, and 96 h of life by multiplex assay. MRI were scored using the Barkovich system. Results: MEN subjects had a reduced length of hospital stay (mean 15 ± 11 vs. 24 ± 19 days, p < 0.05), days receiving parenteral nutrition (7 ± 2 vs. 11 ± 6, p < 0.05), and time to full oral feeds (8 ± 5 vs. 18 ± 18, p < 0.05). MEN was associated with a significantly reduced serum IL-12p70 at 24 and 96 h (p < 0.05). Brain MRI scores were not significantly different between groups. Conclusion: MEN during hypothermia was associated with a reduced length of stay and time to full feeds, but did not increase feeding complications or systemic inflammation.

A Pilot Study of Inhaled CO Therapy in Neonatal Hypoxia-Ischemia: Carboxyhemoglobin Concentrations and Brain Volumes

Objective: The objective of this pilot study was to start evaluating the efficacy and the safety (i.e., carboxyhemoglobin concentration of carbon monoxide (CO)) as a putative neuroprotective therapy in neonates. Study Design: Neonatal C57BL/6 mice were exposed to CO at a concentration of either 200 or 250 ppm for a period of 1 h. The pups were then sacrificed at 0, 10, 20, 60, 120, 180, and 240 min after exposure to either concentration of CO, and blood was collected for analysis of carboxyhemoglobin. Following the safety study, 7-day-old pups underwent a unilateral carotid ligation. After recovery, the pups were exposed to a humidified gas mixture of 8% oxygen and 92% nitrogen for 20 min in a hypoxia chamber. One hour after the hypoxia exposure, the pups were randomized to one of two groups: air (HI+A) or carbon monoxide (HI+CO). An inhaled dose of 250 ppm of CO was administered to the pups for 1 h per day for a period of 3 days. At 7 days post-injury, the pups were sacrificed and the brains analyzed for cortical and hippocampal volumes. Results: CO exposure at 200 and 250 ppm produced a peak carboxyhemoglobin concentration of 21.52 ± 1.18% and 27.55 ± 3.58%, respectively. The carboxyhemoglobin concentrations decreased rapidly, reaching control concentrations by 60 min post exposure. At 14 days of age (7 days post injury), the HI+CO (treated with 1 h per day of 250 ppm of CO for 3 days post injury) had significant preservation of the ratio of ipsilateral to contralateral cortex (median 1.07, 25% 0.97, 75% 1.23, n = 10) compared the HI+A group (p < 0.05). Conclusion: CO exposure of 250 ppm did not reach carboxyhemoglobin concentrations which would induce acute neurologic abnormalities and was effective in preserving cortical volumes following hypoxic-ischemic injury.