Umbilical Cord Blood NOS1 as a Potential Biomarker of Neonatal Encephalopathy

BackgroundThere are no definitive markers to aid in diagnosis of neonatal encephalopathy (NE). The purpose of our study was (1) to identify and evaluate the utility of neuronal nitric oxide synthase (NOS1) in umbilical cord blood as a NE biomarker and (2) to identify the source of NOS1 in umbilical cord blood.MethodsThis was a nested case–control study of neonates >35 weeks of gestation. ELISA for NOS1 in umbilical cord blood was performed. Sources of NOS1 in umbilical cord were investigated by immunohistochemistry, western blot, ELISA, and quantitative PCR. Furthermore, umbilical cords of full-term neonates were subjected to 1% hypoxia ex vivo.ResultsNOS1 was present in umbilical cord blood and increased in NE cases compared with controls. NOS1 was expressed in endothelial cells of the umbilical cord vein, but not in artery or blood cells. In ex vivo experiments, hypoxia was associated with increased levels of NOS1 in venous endothelial cells of the umbilical cord as well as in ex vivo culture medium.ConclusionThis is the first study to investigate an early marker of NE. NOS1 is elevated with hypoxia, and further studies are needed to investigate it as a valuable tool for early diagnosis of neonatal brain injury

Diffusion Tensor Imaging Detects Occult Cerebellar Injury in Severe Neonatal Hypoxic-Ischemic Encephalopathy

BACKGROUND: Despite the benefits of whole-body hypothermia therapy, many infants with hypoxic-ischemic encephalopathy (HIE) die or have significant long-term neurodevelopmental impairment. Prospectively identifying neonates at risk of poor outcome is essential but not straightforward. The cerebellum is not classically considered to be a brain region vulnerable to hypoxic-ischemic insults; recent literature suggests, however, that the cerebellum may be involved in neonatal HIE. In this study, we aimed to assess the microstructural integrity of cerebellar and linked supratentorial structures in neonates with HIE compared to neurologically healthy neonatal controls. METHODS: In this prospective cohort study, we performed a quantitative diffusion tensor imaging (DTI) analysis of the structural pathways of connectivity, which may be affected in neonatal cerebellar injury by measuring fractional anisotropy (FA) and mean diffusivity (MD) within the superior, middle, and inferior cerebellar peduncles, dentate nuclei, and thalami. All magnetic resonance imaging (MRI) studies were grouped into 4 categories of severity based on a qualitative evaluation of conventional and advanced MRI sequences. Multivariable linear regression analysis of cerebellar scalars of patients and controls was performed, controlling for gestational age, age at the time of MRI, and HIE severity. Spearman rank correlation was performed to correlate DTI scalars of the cerebellum and thalami. RESULTS: Fifty-seven (23 females, 40%) neonates with HIE and 12 (6 females, 50%) neonatal controls were included. There were 8 patients (14%) in HIE severity groups 3 and 4 (injury of the basal ganglia/thalamus and/or cortex). Based on a qualitative analysis of conventional and DTI images, no patients had evidence of cerebellar injury. No significant differences between patients and controls were found in the FA and MD scalars. However, FA values of the middle cerebellar peduncles (0.294 vs. 0.380, p < 0.001) and MD values of the superior cerebellar peduncles (0.920 vs. 1.007 × 10-3 mm/s2, p = 0.001) were significantly lower in patients with evidence of moderate or severe injury on MRI (categories 3 and 4) than in controls. In patients, cerebellar DTI scalars correlated positively with DTI scalars within the thalami. CONCLUSION: Our results suggest that infants with moderate-to-severe HIE may have occult injury of cerebellar white-matter tracts, which is not detectable by the qualitative analysis of neuroimaging data alone. Cerebellar DTI scalars correlate with thalamic measures, highlighting that cerebellar injury is unlikely to occur in isolation and may reflect the severity of HIE. The impact of concomitant cerebellar injury in HIE on long-term neurodevelopmental outcome warrants further study

Diffusion tensor imaging detects occult cerebellar injury in severe neonatal hypoxic-ischemic encephalopathy

BACKGROUND: Despite the benefits of whole-body hypothermia therapy, many infants with hypoxic-ischemic encephalopathy (HIE) die or have significant long-term neurodevelopmental impairment. Prospectively identifying neonates at risk of poor outcome is essential but not straightforward. The cerebellum is not classically considered to be a brain region vulnerable to hypoxic-ischemic insults; recent literature suggests, however, that the cerebellum may be involved in neonatal HIE. In this study, we aimed to assess the microstructural integrity of cerebellar and linked supratentorial structures in neonates with HIE compared to neurologically healthy neonatal controls. METHODS: In this prospective cohort study, we performed a quantitative diffusion tensor imaging (DTI) analysis of the structural pathways of connectivity, which may be affected in neonatal cerebellar injury by measuring fractional anisotropy (FA) and mean diffusivity (MD) within the superior, middle, and inferior cerebellar peduncles, dentate nuclei, and thalami. All magnetic resonance imaging (MRI) studies were grouped into 4 categories of severity based on a qualitative evaluation of conventional and advanced MRI sequences. Multivariable linear regression analysis of cerebellar scalars of patients and controls was performed, controlling for gestational age, age at the time of MRI, and HIE severity. Spearman rank correlation was performed to correlate DTI scalars of the cerebellum and thalami. RESULTS: Fifty-seven (23 females, 40%) neonates with HIE and 12 (6 females, 50%) neonatal controls were included. There were 8 patients (14%) in HIE severity groups 3 and 4 (injury of the basal ganglia/thalamus and/or cortex). Based on a qualitative analysis of conventional and DTI images, no patients had evidence of cerebellar injury. No significant differences between patients and controls were found in the FA and MD scalars. However, FA values of the middle cerebellar peduncles (0.294 vs. 0.380, p < 0.001) and MD values of the superior cerebellar peduncles (0.920 vs. 1.007 × 10-3 mm/s2, p = 0.001) were significantly lower in patients with evidence of moderate or severe injury on MRI (categories 3 and 4) than in controls. In patients, cerebellar DTI scalars correlated positively with DTI scalars within the thalami. CONCLUSION: Our results suggest that infants with moderate-to-severe HIE may have occult injury of cerebellar white-matter tracts, which is not detectable by the qualitative analysis of neuroimaging data alone. Cerebellar DTI scalars correlate with thalamic measures, highlighting that cerebellar injury is unlikely to occur in isolation and may reflect the severity of HIE. The impact of concomitant cerebellar injury in HIE on long-term neurodevelopmental outcome warrants further study

Evidence for Sexual Dimorphism in the Response to TLR3 Activation in the Developing Neonatal Mouse Brain: A Pilot Study

Toll-like receptor (TLR)3 activation during the neonatal period produces responses linked to the origins of neuropsychiatric disorders. Although there is sexual dimorphism in neuropsychiatric disorders, it is unknown if brain responses to TLR3 activation are sex-specific. We hypothesized that poly I:C in a post-natal day (P)8 model induces a sexually dimorphic inflammatory responses. C57BL6 mice received intraperitoneal injection of poly I:C (10 mg/kg) or vehicle [normal saline (NS)] at P8. Pups were killed at 6 or 14 h for caspase 3 and 8 activity assays, NFkB ELISA, IRF3, AP1, and GFAP western blotting and cytokines/chemokines gene expression real time qRT-PCR (4–6/group). A second group of pups were killed at 24 h (P9) or 7 days (P15) after poly I:C to assess astrocytic (GFAP) and microglia (Iba1) activation in the hippocampus, thalamus and cortex using immunohistochemistry, and gene and protein expression of cytokines/chemokines using real time RT-PCR and MSD, respectively (4–6/group). Non-parametric analysis was applied. Six hours after poly I:C, caspase-3 and -8 activities in cytosolic fractions were 1.6 and 2.8-fold higher in poly I:C-treated than in NS-treated female mice, respectively, while gene expressions of pro-inflammatory cytokines were upregulated in both sexes. After poly I:C, IRF3 nuclear translocation occurred earlier (6 h) in female mice and later (14 h) in male mice. At 14 h after poly I:C, only male mice also had increased nuclear NFκB levels (88%, p &lt; 0.001) and GFAP expression coinciding with persistent IL-6 and FAS gene upregulation (110 and 77%, respectively; p &lt; 0.001) and IL-10 gene downregulation (-42%, p &lt; 0.05). At 24 h after poly I:C, IL-1β, CXCL-10, TNF-α, and MCP-1 were similarly increased in both sexes but at 7 days after exposure, CXCL-10 and INFγ were increased and IL-10 was decreased only in female mice. Accordingly, microglial activation persisted at 7 days after poly I:C in the hippocampus, thalamus and cortex of female mice. This preliminary study suggests that TLR3 activation may produce in the developing neonatal mouse brain a sexually dimorphic response with early activation of caspase-dependent pathways in female mice, activation of inflammatory cascades in both sexes, which then persists in female mice. Further well-powered studies are essential to confirm these sex-specific findings

Basal forebrain magnocellular cholinergic systems are damaged in mice following neonatal hypoxia-ischemia

Neonatal hypoxic-ischemic encephalopathy (HIE) causes lifelong neurologic disability. Despite the use of therapeutic hypothermia, memory deficits and executive functions remain severely affected. Cholinergic neurotransmission from the basal forebrain to neocortex and hippocampus is central to higher cortical functions. We examined the basal forebrain by light microscopy and reported loss of choline acetyltransferase-positive (ChAT)+ neurons, at postnatal day (P) 40, in the ipsilateral medial septal nucleus (MSN) after neonatal hypoxia-ischemia (HI) in mice. There was no loss of ChAT+ neurons in the ipsilateral nucleus basalis of Meynert (nbM) and striatum. Ipsilateral striatal and nbM ChAT+ neurons were abnormal with altered immunoreactivity for ChAT, shrunken and crenated somas, and dysmorphic appearing dendrites. Using confocal images with 3D reconstruction, nbM ChAT+ dendrites in HI mice were shorter than sham (p = .0001). Loss of ChAT+ neurons in the MSN directly correlated with loss of ipsilateral hippocampal area. In the nbM and striatum, percentage of abnormal ChAT+ neurons correlated with loss of ipsilateral cerebral cortical and striatal area, respectively. Acetylcholinesterase (AChE) activity increased in adjacent ipsilateral cerebral cortex and hippocampus and the increase was linearly related to loss of cortical and hippocampal area. Numbers and size of cathepsin D+ lysosomes increased in large neurons in the ipsilateral nbM. After neonatal HI, abnormalities were found throughout the major cholinergic systems in relationship to amount of forebrain area loss. There was also an upregulation of cathepsin D+ particles within the nbM. Cholinergic neuropathology may underlie the permanent dysfunction in learning, memory, and executive function after neonatal brain injury

Clinical and neuroimaging features as diagnostic guides in neonatal neurology diseases with cerebellar involvement

Cerebellar abnormalities are encountered in a high number of neurological diseases that present in the neonatal period. These disorders can be categorized broadly as inherited (e.g. malformations, inborn errors of metabolism) or acquired (e.g. hemorrhages, infections, stroke). In some disorders such as Dandy-Walker malformation or Joubert syndrome, the main abnormalities are located within the cerebellum and brainstem. In other disorders such as Krabbe disease or sulfite oxidase deficiency, the main abnormalities are found within the supratentorial brain, but the cerebellar involvement may be helpful for diagnostic purposes. In In this article, we review neurological disorders with onset in the neonatal period and cerebellar involvement with a focus on how characterization of cerebellar involvement can facilitate accurate diagnosis and improved accuracy of neuro-functional prognosis