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.

Butyrate and type 1 diabetes mellitus: can we fix the intestinal leak?

OBJECTIVES: An intestinal permeability defect precedes type 1 diabetes mellitus and may be a permissive factor in its pathogenesis. Butyrate strengthens the intestinal tight junctions. We hypothesized that enteral administration of sodium butyrate (NaB) in preweaned rats would result in differences in the development of diabetes associated with decreased inflammation and pancreatic ß-cell destruction. MATERIALS AND METHODS: Using biobreeding diabetes-prone rat pups, oral NaB or saline was administered twice per day via micropipette from postnatal days 10 to 23. Rat pups were randomly assigned to 1 of 4 groups for the first experiment (control group, n = 7) and 3 different doses of butyrate groups (n = 8 for each group) and 2 groups for the second and third experiments (control n = 23; NaB at 400 mg · kg(-1) · day(-1), n = 20). Animals were studied into adulthood (up to day 140) for development of diabetes. RESULTS: The results showed that the survival rates were 28% versus 20% (butyrate vs control). No significant differences in survival were seen; however, there was a trend of delaying of onset of diabetes in the butyrate group. There were no differences of pancreatic histology score of islet inflammation between the 2 groups. Cytokine-induced neutrophil chemoattractant-1 was lower in the butyrate group at a dose of 400 mg · kg(-1) · day(-1) in the distal small intestine (P = 0.008) and in the liver (P = 0.01). There were no significant differences in the tracer flux measurements across the distal ileum and colon between the 2 animal groups. CONCLUSIONS: Oral NaB given during the preweaning period did not significantly decrease the subsequent development of death from diabetes in biobreeding diabetes-prone rats.

Live and heat-killed Lactobacillus rhamnosus GG: effects on proinflammatory and anti-inflammatory cytokines/chemokines in gastrostomy-fed infant rats.

Lactobacillus rhamnosus GG (LGG), a probiotics, ameliorates intestinal and other organ inflammation in infant rats. The hypothesis is that live and heat-killed LGG have similar effects on decreasing the inflammatory response induced by E. coli lipopolysaccharide (LPS) in the infant rat. Using a gastrostomy-fed rat model, 7-d-old rat pups were gastrostomy fed with or without live LGG (10(8) or 10(12) cfu x L(-1) x kg(-1) x d(-1)) for 6 d. In a separate experiment, LPS was administered to rat pups with or without live or heat-killed LGG (10(8) cfu x L(-1) x kg(-1) x d(-1)). Cytokine/chemokine proteins were determined by ELISA or multiplex assay. Both live and heat-killed LGG decreased LPS-induced cytokine-induced neutrophil chemoattractant-1 (CINC-1) production in liver and plasma (p < 0.05) and also showed a trend (p = 0.09) in lungs. Live and heat-killed LGG ameliorated LPS-suppressed IL-10 level in lungs (p < 0.05). Both forms of LGG decreased IL-1b production in liver. There was no difference between low and high doses of live LGG in the production of CINC-1, TNF-alpha, and myeloperoxidase (MPO). There was a trend of increase of claudin-1 in both live and heat-killed groups (p = 0.08). In conclusion, both live and heat-killed LGG provided by the enteral route decrease LPS-induced proinflammatory mediators and increase anti-inflammatory mediators.

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.

Postnatal nutrition and adult health programming.

The early postnatal interplay between nutrition, growth patterns, and metabolic and epigenetic phenomena is crucial in determining subsequent health; health that extends through the lifetime of the individual and very likely even into subsequent generations. Recent research in the area of postnatal nutrition and its relationship to adult health, with an emphasis on the appropriate-for-gestational-age infant who is born prematurely and who undergoes growth delays, is presented. Select studies in animals, pertinent to understanding the mechanisms of how early postnatal under- and overnutrition might affect adult health and propagate to subsequent generations, are reviewed. Scientifically based approaches to administering postnatal nutrition designed to improve outcomes and areas where future investigations are needed are presented.

Microbes and the developing gastrointestinal tract.

During the course of mammalian evolution, there has been a close relationship between microbes residing in the gastrointestinal (GI) tract and the mammalian host. Although the host provides the microbes with a warm environment and nutrients, they, in turn, undergo various metabolic processes that aid the host. The host has developed weapons against microbes that are considered foreign, as well as mechanisms to tolerate and live synergistically with most of the microbes in the GI tract. This relationship is proving to be important not only in the neonatal period and during infancy, but it is becoming increasingly evident that microbial colonization in early life may affect the individual's health throughout life. Here we will review this relationship in terms of health and disease, with a focus on the aspects of this relationship during maturation of the host.

Hypoxic-ischemic encephalopathy: a review for the clinician.

IMPORTANCE: Hypoxic-ischemic encephalopathy (HIE) occurs in 1 to 8 per 1000 live births in developed countries. Historically, the clinician has had little to offer neonates with HIE other than systemic supportive care. Recently, the neuroprotective therapy of hypothermia has emerged as the standard of care, and other complementary therapies are rapidly transitioning from the basic science to clinical care. OBJECTIVE: To examine the pathophysiology of HIE and the state of the art for the clinical care of neonates with HIE. EVIDENCE REVIEW: We performed a literature review using the PubMed database. Results focused on reviews and articles published from January 1, 2004, through December 31, 2014. Articles published earlier than 2004 were included when appropriate for historical perspective. Our review emphasized evidence-based management practices for the clinician. FINDINGS: A total of 102 articles for critical review were selected based on their relevance to the incidence of HIE, pathophysiology, neuroimaging, placental pathology, biomarkers, current systemic supportive care, hypothermia, and emerging therapies for HIE and were reviewed by both of us. Seventy-five publications were selected for inclusion in this article based on their relevance to these topics. The publications highlight the emergence of serum-based biomarkers, placental pathology, and magnetic resonance imaging as useful tools to predict long-term outcomes. Hypothermia and systemic supportive care form the cornerstone of therapy for HIE. CONCLUSIONS AND RELEVANCE: The pathophysiology of HIE is now better understood, and treatment with hypothermia has become the foundation of therapy. Several neuroprotective agents offer promise when combined with hypothermia and are entering clinical trials.

UCH-L1 and GFAP Serum Levels in Neonates with Hypoxic-Ischemic Encephalopathy: A Single Center Pilot Study.

OBJECTIVE: We examined two potential biomarkers of brain damage in hypoxic-ischemic encephalopathy (HIE) neonates: glial fibrillary acidic protein (GFAP; a marker of gliosis) and ubiquitin C-terminal hydrolase L1 (UCH-L1; a marker of neuronal injury). We hypothesized that the biomarkers would be measurable in cord blood of healthy neonates and could serve as a normative reference for brain injury in HIE infants. We further hypothesized that higher levels would be detected in serum samples of HIE neonates and would correlate with brain damage on magnetic resonance imaging (MRI) and later developmental outcomes.? STUDY DESIGN: Serum UCH-L1 and GFAP concentrations from HIE neonates (n = 16) were compared to controls (n = 11). The relationship between biomarker concentrations of HIE neonates and brain damage (MRI) and developmental outcomes (Bayley-III) was examined using Pearson correlation coefficients and a mixed model design. RESULT: Both biomarkers were detectable in cord blood from control subjects. UCH-L1 concentrations were higher in HIE neonates (p < 0.001), and associated with cortical injury (p < 0.055) and later motor and cognitive developmental outcomes (p < 0.05). The temporal change in GFAP concentrations during (from birth to 96 h of age) predicted motor developmental outcomes (p < 0.05) and injury to the basal ganglia and white matter. CONCLUSION: Ubiquitin C-terminal hydrolase L1 and GFAP should be explored further as promising serum biomarkers of brain damage and later neurodevelopmental outcomes in neonates with HIE.

Effect of intestinal microbial ecology on the developing brain.

The mammalian gastrointestinal tract harbors a highly diverse microbial population that plays a major role in nutrition, metabolism, protection against pathogens, and development of the immune system. It is estimated that at least 1000 different bacterial species cohabit the human intestinal tract. Most recently, the Human Microbiome Project, using new genomic technologies, has started a catalog of specific microbiome composition and its correlation with health and specific diseases. Herein we provide a brief review of the intestinal microbiome, with a focus on new studies showing that there is an important link between the microbes that inhabit the intestinal tract and the developing brain. With future research, an understanding of this link may help us to treat various neurobehavioral problems such as autism, schizophrenia, and anxiety.

Clinical Implications of Bilirubin-Associated Neuroprotection and Neurotoxicity.

Bilirubin is a primary product of heme catabolism and exhibits both neuroprotective and neurotoxic effects. When present at physiologic concentrations, bilirubin is a potent antioxidant and serves to protect brain tissue from oxidative stress insults. The use of the anesthetic propofol attenuates ischemic injury in rats by exploiting these neuroprotective properties. At pathologic levels, bilirubin has been implicated as a neurotoxic agent, demonstrating the ability to aggregate and adhere to cellular membranes, thereby disrupting normal cellular function. Bilirubin-associated toxicities are amplified by administering drugs such as anesthetics that compete with bilirubin for albumin binding sites, resulting in increased plasma bilirubin concentrations. As such, it is crucial that bilirubin is considered in the critical care management of patients with hemorrhagic stroke, cerebral ischemic damage, and critically ill newborns.

Biomarkers of brain injury in the premature infant.

The term "encephalopathy of prematurity" encompasses not only the acute brain injury [such as intraventricular hemorrhage (IVH)] but also complex disturbance on the infant's subsequent brain development. In premature infants, the most frequent recognized source of brain injury is IVH and periventricular leukomalacia (PVL). Furthermore 20-25% infants with birth weigh less than 1,500 g will have IVH and that proportion increases to 45% if the birth weight is less than 500-750 g. In addition, nearly 60% of very low birth weight newborns will have hypoxic-ischemic injury. Therefore permanent lifetime neurodevelopmental disabilities are frequent in premature infants. Innovative approach to prevent or decrease brain injury in preterm infants requires discovery of biomarkers able to discriminate infants at risk for injury, monitor the progression of the injury, and assess efficacy of neuroprotective clinical trials. In this article, we will review biomarkers studied in premature infants with IVH, Post-hemorrhagic ventricular dilation (PHVD), and PVL including: S100b, Activin A, erythropoietin, chemokine CCL 18, GFAP, and NFL will also be examined. Some of the most promising biomarkers for IVH are S100ß and Activin. The concentrations of TGF-ß1, MMP-9, and PAI-1 in cerebrospinal fluid could be used to discriminate patients that will require shunt after PHVD. Neonatal brain injury is frequent in premature infants admitted to the neonatal intensive care and we hope to contribute to the awareness and interest in clinical validation of established as well as novel neonatal brain injury biomarkers.

Biomarkers of hypoxic-ischemic encephalopathy in newborns.

As neonatal intensive care has evolved, the focus has shifted from improving mortality alone to an effort to improve both mortality and morbidity. The most frequent source of neonatal brain injury occurs as a result of hypoxic-ischemic injury. Hypoxic-ischemic injury occurs in about 2 of 1,000 full-term infants and severe injured infants will have lifetime disabilities and neurodevelopmental delays. Most recently, remarkable efforts toward neuroprotection have been started with the advent of therapeutic hypothermia and a key step in the evolution of neonatal neuroprotection is the discovery of biomarkers that enable the clinician-scientist to screen infants for brain injury, monitor progression of disease, identify injured brain regions, and assess efficacy of neuroprotective clinical trials. Lastly, biomarkers offer great hope identifying when an injury occurred shedding light on the potential pathophysiology and the most effective therapy. In this article, we will review biomarkers of HIE including S100B, neuron specific enolase, umbilical cord IL-6, CK-BB, GFAP, myelin basic protein, UCHL-1, and pNF-H. We hope to contribute to the awareness, validation, and clinical use of established as well as novel neonatal brain injury biomarkers.

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.