Neocortical layer formation of human developing brains and lissencephalies: consideration of layer-specific marker expression.

To investigate layer-specific molecule expression in human developing neocortices, we performed immunohistochemistry of the layer-specific markers (TBR1, FOXP1, SATB2, OTX1, CUTL1, and CTIP2), using frontal neocortices of the dorsolateral precentral gyri of 16 normal controls, aged 19 gestational weeks to 1 year old, lissencephalies of 3 Miller-Dieker syndrome (MDS) cases, 2 X-linked lissencephaly with abnormal genitalia (XLAG) cases, and 4 Fukuyama-type congenital muscular dystrophy (FCMD) cases. In the fetal period, we observed SATB2+ cells in layers II-IV, CUTL1+ cells in layers II-V, FOXP1+ cells in layer V, OTX1+ cells in layers II or V, and CTIP2+ and TBR1+ cells in layers V and VI. SATB2+ and CUTL1+ cells appeared until 3 months of age, but the other markers disappeared after birth. Neocortices of MDS and XLAG infants revealed SATB2+, CUTL1+, FOXP1+, and TBR1+ cells diffusely located in the upper layers. In fetal FCMD neocortex, neurons labeled with the layer-specific markers located over the glia limitans. The present study provided new knowledge indicating that the expression pattern of these markers in the developing human neocortex was similar to those in mice. Various lissencephalies revealed abnormal layer formation by random migration.

Magnetic resonance imaging volumetry and clinical analysis of epilepsy patients with unilateral hippocampal abnormality.

BACKGROUND: In order to clarify the correlation between morphological characteristics and clinical features in epilepsy patients with unilateral hippocampal abnormality, morphological and morphometric magnetic resonance imaging studies were performed. METHODS: We selected a series of childhood-onset epilepsy patients with unilateral hippocampal abnormality. The volume of hippocampal formation and anterior temporal lobe were measured, and the hippocampal morphology was compared with their clinical features. The morphological characteristics of the hippocampal formation were classified into three groups: group I, diffuse and severe volume reduction of the hippocampal formation and anterior temporal lobe with abnormal signal; group II, focal atrophy or focal abnormal signal in the hippocampal formation; and group III, no significant volume reduction but an enlargement of the temporal horn. RESULTS: All of the patients in group I had a history of status epilepticus in infancy. Temporal lobe epilepsy (TLE) was found in three of four patients. Group II contained TLE in three and frontal lobe epilepsy in one. One patient with intractable TLE had a history of status epilepticus in infancy. Group III contained miscellaneous epilepsies, including benign partial epilepsy with centro-temporal spikes in three of seven patients. Five patients in group III showed some characteristic features of hippocampal malrotation, which refers to incomplete hippocampal infolding. CONCLUSIONS: Diffuse and severe volume reduction of the hippocampal formation and anterior temporal lobe with unilateral hippocampal sclerosis was strongly associated with status epilepticus in infancy. Both hippocampal sclerosis and hippocampal malrotation suggest significant roles in the pathogenesis of epilepsy.

Near-infrared light scattering and water diffusion in newborn brains.

OBJECTIVE: MRI provides useful information regarding brain maturation and injury in newborn infants. However, MRI studies are generally restricted during acute phase, resulting in uncertainty around upstream clinical events responsible for subtle cerebral injuries. Time-resolved near-infrared spectroscopy non-invasively provides the reduced scattering coefficient ( µ s ' ), which theoretically reflects tissue structural complexity. This study aimed to test whether µ s ' values of the newborn head reflected MRI findings. METHODS: Between June 2009 and January 2015, 77 hospitalised newborn infants (31.7 ± 3.8 weeks gestation) were assessed at 38.8 ± 1.3 weeks post-conceptional age. Associations of µ s ' values with MRI scores, mean diffusivity and fractional anisotropy were assessed. RESULTS: Univariable analysis showed that µ s ' values were associated with gestational week (p = 0.035; regression coefficient [B], 0.065; 95% confidence interval [CI], 0.005-0.125), fractional anisotropy in the cortical grey matter (p = 0.020; B, -5.994; 95%CI, -11.032 to -0.957), average diffusivity in the cortical grey matter (p < 0.001; B, -4.728; 95%CI, -7.063 to -2.394) and subcortical white matter (p = 0.001; B, -2.071; 95%CI, -3.311 to -0.832), subarachnoid space (p < 0.001; B, -0.289; 95%CI, -0.376 to -0.201) and absence of brain abnormality (p = 0.042; B, -0.422; 95%CI, -0.829 to -0.015). The multivariable model to explain µ s ' values comprised average diffusivity in the subcortical white matter (p < 0.001; B, -2.066; 95%CI, -3.200 to -0.932), subarachnoid space (p < 0.001; B, -0.314; 95%CI, -0.412 to -0.216) and absence of brain abnormality (p = 0.021; B, -0.400; 95%CI, -0.739 to -0.061). INTERPRETATION: Light scattering was associated with brain structure indicated by MRI-assessed brain abnormality and diffusion-tensor-imaging-assessed water diffusivity. When serially assessed in a larger population, µ s ' values might help identify covert clinical events responsible for subtle cerebral injury.

Foetal growth, birth transition, enteral nutrition and brain light scattering.

If the brain structure is assessed at neonatal intensive care units, covert clinical events related with subtle brain injury might be identified. The reduced scattering coefficient of near-infrared light (µS') obtained using time-resolved near-infrared spectroscopy from the forehead of infants is associated with gestational age, body weight and Apgar scores, presumably reflecting subtle changes of the brain related to foetal growth and birth transition. One hundred twenty-eight preterm and term infants were studied to test whether µS' obtained from the head at term-equivalent age is associated with foetal growth, birth transition and nutritional status after birth, which are key independent variables of developmental outcomes. As potential independent variables of µS', birth weight, Apgar scores, age at full enteral feeding and post-conceptional age at the study were assessed to represent foetal growth, birth transition and nutritional status after birth. Subsequently, higher µS' values were associated with higher Apgar scores (p = 0.003) and earlier establishment of enteral feeding (p < 0.001). The scattering property of near-infrared light within the neonatal brain might reflect changes associated with birth transition and nutritional status thereafter, which might be used as a non-invasive biomarker to identify covert independent variables of brain injury in preterm infants.

[Brain imaging and pathology in children with neurological diseases].

Morphological and functional imaging of the brain is rapidly developing, while knowledge of brain pathology is also progressing at the levels of protein and RNA along with molecular biology. In this review, brain imaging and pathology were compared developmentally. Brain imaging reveals the early stages and progression of brain damage, and the pathology confirms the characteristics of the lesions. However, some pathological lesions such as pontosubicular neuron necrosis (death) cannot be diagnosed by brain imaging. Further accumulation of brain images and pathological tissues may be helpful to clarify the pathogenesis and effective preventive strategies.

[Bench to bedside, tertiary centres to developing world --"tailoring" best available neuroprotection in high and low resource settings].

Neuroprotection following brain injury has been a top priority in neuroscience since the Hippocratic era. However, of hundreds of "experimentally-neuroprotective treatments", currently only hypothermia is clinically available. Interpretation of bench-work may have to account for the variation in developmental stages and types of injury. For example GABAergic stimulators have been used to reduce cerebral energy consumption; recent studies suggested the paradoxical excitation of premature neurons following GABAergic stimuli because of the high intracellular chloride concentration during the perinatal period. Even for hypothermia, many questions remain unanswered:selective-head cooling following perinatal hypoxia-ischemia appeared rather deleterious for infants with the most severe encephalopathy or low birth-weight. In a piglet model, different cooling levels provided differential regional brain protection:35 degrees C was optimal for deep-grey-matter whereas the cortical-grey-matter was better protected at 33 degrees C, suggesting that a single cooling level may not be equally optimal for the entire brain. Another experimental study used magnetic-resonance spectroscopy to demonstrate that the more severe the acute event was, the shorter the therapeutic time window was, explaining the lower effectiveness of neuroprotection following the most severe hypoxia-ischemia. Another suggestion from our translational research was the dependence of cerebral temperatures on body weight under selective-head cooling:the smaller the body weight, the lower the brain temperature, presumably because of the larger surface area to brain volume ratio in small subjects. Thus translational research is essential to achieve "tailored" treatments, that address for individual and regional cerebral conditions. Although therapeutic options have become available in the tertiary centre settings, neuroprotection should not remain an expensive treatment only available in rich countries. We have recently developed a cooling system that can maintain stable hypothermia over 24 hours without an electric supply. Experimental studies to optimise high-tech cooling need to run in parallel with studies to increase the distribution of these treatments worldwide.

Sodium phenylbutyrate improved the clinical state in an adult patient with arginase 1 deficiency.

An adult female patient was diagnosed with arginase 1 deficiency (ARG1-D) at 4 years of age, and had been managed with protein restriction combined with sodium benzoate therapy. Though the treatment was successful in ameliorating hyperammonemia, hyperargininemia persisted. After being under control with a strict restriction of dietary protein, severe fall of serum albumin levels appeared and her condition became strikingly worsened. However, after sodium phenylbutyrate (NaPB) therapy was initiated, the clinical condition and metabolic stability was greatly improved. Current management of ARG1-D is aimed at lowering plasma arginine levels. The nitrogen scavengers, such as NaPB can excrete the waste nitrogen not through the urea cycle but via the alternative pathway. The removal of nitrogen via alternative pathway lowers the flux of arginine in the urea cycle. Thereby, the clinical complications due to insufficient amount of protein intake can be prevented. Thus, NaPB therapy can be expected as a useful therapeutic option, particularly in patients with ARG1-D.

Phosphatidyl ethanolamine with increased polyunsaturated fatty acids in compensation for plasmalogen defect in the Zellweger syndrome brain.

To elucidate the neuropathological mechanism of Zellweger syndrome (ZS), we studied changes in the molecular species of glycerophospholipids in the cerebral tissue by thin-layer chromatography (TLC) and fast atom bombardment mass spectrometry (FABMS). First, we estimated the amount of plasmalogens by TLC. Plasmalogen-type phosphatidyl ethanolamine (PE) accounted for 30% of the total PE in the control brain, but was absent in the ZS brain. Plasmalogen-type phosphatidyl choline (PC) was undetectable in both control and ZS brains. Next, we analyzed plasmalogen-type PE by FABMS. Oleic (18:1), arachidonic (20:4) and docosapentanoic (22:5) acids were present in the control gray matter, but not in the ZS gray matter. In compensation for the defect of plasmalogen, the level of diacyl PE with polyunsaturated fatty acids, 20:4, 22:4, 22:5 and 22:6, was higher in the ZS brain than that in the control brain. These results indicate an alteration in the molecular species of PE, which may cause abnormal neural membrane fluidity and excessive vulnerability to oxygen stress.

Body Temperature, Heart Rate, and Short-Term Outcome of Cooled Infants.

Therapeutic hypothermia following neonatal encephalopathy is neuroprotective. However, approximately one in two cooled infants still die or develop permanent neurological impairments. Further understanding of variables associated with the effectiveness of cooling is important to improve the therapeutic regimen. To identify clinical factors associated with short-term outcomes of cooled infants, clinical data of 509 cooled infants registered to the Baby Cooling Registry of Japan between 2012 and 2014 were evaluated. Independent variables of death during the initial hospitalization and survival discharge from the cooling hospital at ≤28 days of life were assessed. Death was associated with higher Thompson scores at admission (p < 0.001); higher heart rates after 3-72 hours of cooling (p < 0.001); and higher body temperature after 24 hours of cooling (p = 0.002). Survival discharge was associated with higher 10 minutes Apgar scores (p < 0.001); higher blood pH and base excess (both p < 0.001); lower Thompson scores (at admission and after 24 hours of cooling; both p < 0.001); lower heart rates at initiating cooling (p = 0.003) and after 24 hours of cooling (p < 0.001) and lower average values after 3-72 hours of cooling (p < 0.001); higher body temperature at admission (p < 0.001); and lower body temperature after 24 hours and lower mean values after 3-72 hours of cooling (both p < 0.001). Survival discharge was best explained by higher blood pH (p < 0.05), higher body temperature at admission (p < 0.01), and lower body temperature and heart rate after 24 hours of cooling (p < 0.01 and <0.001, respectively). Lower heart rate, higher body temperature at admission, and lower body temperature during cooling were associated with favorable short-term outcomes.

Developmental changes in KCNQ2 and KCNQ3 expression in human brain: possible contribution to the age-dependent etiology of benign familial neonatal convulsions.

Several mutations of KCNQ2 and KCNQ3 are considered to be associated with benign familial neonatal convulsions (BFNC). BFNC is characterized by seizures starting within several days of life and spontaneous remission within weeks to months. KCNQ channel is a heteromeric voltage-dependent potassium channel consisting of KCNQ2 and KCNQ3 subunits. To clarify the age-dependent etiology of BFNC, we examined the developmental changes in KCNQ2 and KCNQ3 expression in human hippocampus, temporal lobe, cerebellum and medulla oblongata obtained from 23 subjects who died at 22 gestation weeks to adulthood. Formalin-fixed and paraffin-embedded specimens were used for immunohistochemistry. Unique developmental changes in KCNQ2 and KCNQ3 were found in each region. A high expression of KCNQ2 was identified in the hippocampus, temporal cortex, cerebellar cortex and medulla oblongata in fetal life, but such expression decreased after birth. The expression of KCNQ3 increased in late fetal life to infancy. Simultaneous and high expressions of KCNQ2 and KCNQ3 were observed in each region from late fetal life to early infancy, coinciding with the time when BFNC occurs. Such coexpression may contribute to the pathogenesis of BFNC.

Estimation of elevated intracranial pressure in infants with hydroce-phalus by using transcranial Doppler velocimetry with fontanel compression.

For infants with acute progressive hydrocephalus, invasive drainage of cerebrospinal fluid (CSF) is performed until a ventriculo-peritoneal shunt can be inserted. Surrogate markers of intracranial pressure (ICP) may help optimise the timing of invasive procedures. To assess whether RI with/without fontanel compression helps distinguish between infants with normal (<5 cmH2O), mild (5-11 cmH2O), and moderate (>11 cmH2O) ICP elevation, 74 ICP measures before/after CSF removal and 148 related Doppler measures of the middle cerebral artery were assessed. Higher RI was associated with fontanel compression, elevated ICP, and their interaction (all p < 0.001). Without compression, differences in RI were observed between normal and moderate (p < 0.001) and between mild and moderate ICP elevation (p = 0.033). With compression, differences in RI were observed for all pairwise comparisons among normal, mild, and moderate ICP elevation (all p < 0.001). Without compression, areas under the receiver-operating characteristic curve for prediction of mild and moderate ICP elevation were 0.664 (95% confidence interval (CI), 0.538-0.791; p = 0.020) and 0.727 (95% CI, 0.582-0.872; p = 0.004), respectively, which improved to 0.806 (95% CI, 0.703-0.910; p < 0.001) and 0.814 (95% CI, 0.707-0.921; p < 0.001), respectively, with compression. RI with fontanel compression provides improved discrimination of infants with absent, mild, and moderate ICP elevation.

Methyl CpG-binding protein 2 (a mutation of which causes Rett syndrome) directly regulates insulin-like growth factor binding protein 3 in mouse and human brains.

Rett syndrome (RTT) is a major neurodevelopmental disorder, characterized by mental retardation and autistic behavior. Mutation of the MeCP2 gene, encoding methyl CpG-binding protein 2, causes the disease. The pathomechanism by which MeCP2 dysfunction leads to the RTT phenotype has not been elucidated. We found that MeCP2 directly regulates expression of insulin-like growth factor binding protein 3 (IGFBP3) gene in human and mouse brains. A chromatin immunoprecipitation assay showed that the IGFBP3 promoter contained an MeCP2 binding site. IGFBP3 overexpression was observed in the brains of mecp2-null mice and human RTT patients using real-time quantitative polymerase chain reaction and Western blot analyses. Moreover, mecp2-null mice showed a widely distributed and increased number of IGFBP3-positive cells in the cerebral cortex, whereas wild-type mice at the same age showed fewer IGFBP3-positive cells. These results suggest that IGFBP3 is a downstream gene regulated by MeCP2 and that the previously reported BDNF and DLX5 genes and MeCP2 may contribute directly to the transcriptional expression of IGFBP3 in the brain. Interestingly, the pathologic features of mecp2-null mice have some similarities to those of IGFBP3-transgenic mice, which show a reduction of early postnatal growth. IGFBP3 overexpression due to lack of MeCP2 may lead to delayed brain maturation.

Increase of ceramide monohexoside and dipalmitoyl glycerophospholipids in the brain of Zellweger syndrome.

The lipid composition and molecular species of phospholipids were examined in the brain of a patient with Zellweger syndrome (ZS), and were compared with those of control infants. In the cerebral gray matter of the ZS patient, the amounts of ceramide monohexoside and cholesterol ester were larger than those of controls. By contrast, the amount of ceramide monohexoside in the white matter was smaller in the ZS patient than that in the age-matched control. Although the amount of phosphatidylcholine (PC) plus phosphatidylserine (PS) was the same, dipalmitoyl PC and PS were increased in both the gray and white matter of the ZS cerebrum. These alterations in the molecular species of brain lipids may play crucial roles in the pathogenesis of ZS.

A case of partial 14q- with facial features of holoprosencephaly and hydranencephaly.

We report a rare case of facial features of holoprosencephaly associated with hydranencephaly, with a de novo proximal interstitial deletion of the long arm of chromosome 14, specifically, del(14)(q13q21). She was born at 37 weeks of gestation and transferred to our institution at 3 years of age. The patient had midline facial anomalies consisting of cleft palate, defective nasal septum, and hypotelorism, together with endocrine abnormalities such as diabetes insipidus and hypothyroidism. Cranial computed tomography revealed the near-total loss of all cerebral tissue, with a frontal part of the cerebral falx lacking. None of the few reports of holoprosencephaly with 14q- chromosomal abnormality describe holoprosencephaly in association with hydranencephaly. The partial deletion of chromosome 14, del(14)(q13q21), may underlie the association of facial features of holoprosencephaly and hydranencephaly.

Altered nestin expression in the cerebrum with periventricular leukomalacia.

Nestin is a cytoskeletal protein expressed by neural stem cells, and by immature neurons and glial cells. In an effort to explore the potential of the infant brain for repair and plasticity, we immunohistochemically studied nestin expression in the human cerebral cortex of control subjects and of patients with periventricular leukomalacia. During normal development, nestin immunoreactivity of the cortical gray and white matter was detectable throughout the fetal period, and disappeared around birth. In brain with periventricular leukomalacia, nestin expression was altered in a time- and space-dependent manner. In the cortical gray matter, neuronal immunoreactivity was often reduced in the subacute stage, but was increased in chronic and remote stages. In the white matter near a lesion of periventricular leukomalacia, glial immunoreactivity was increased in all stages. In many cases, neurons and axons far from a lesion also showed an altered expression of nestin. These findings indicate that in brain with periventricular leukomalacia, neurons and glial cells may recapitulate nestin expression in response to ischemic brain injury, suggesting functional relevance in repair and plasticity.

[A questionnaire survey on daily medical care problems at special schools for severely neurologically impaired students in southern Fukuoka prefecture].

With increasing numbers of children having severe motor and intellectual disabilities, there is growing demand for daily medical care such as tube feeding and sputum suctioning in special schools. We devised a questionnaire survey for special school teachers investigating the need for daily medical care in four schools in southern Fukuoka prefecture. One-hundred fifty-two out of 633 (24%) students needed medical care; specifically, 38 of 152 (25%) needed tube feeding and 41 of 152 (27%) required sputum suctioning. Such patients lived in their houses, hospitals or medical centers for the severely disabled. Mothers whose children commuted from home had to go to school themselves to provide medical care for their children, such as sputum suctioning. School teachers were worried about their students' feeding difficulties and respiratory disorders. More support for teachers to care for students is needed and provision of medical care under supervision of school nurses and doctors should be promoted.

Neuroimaging and neuropathological characteristics of cerebellar injury in extremely low birth weight infants.

OBJECTIVE: To determine the morphological characteristics and pathogenic factors of cerebellar injury in extremely low birth weight infants (ELBWI). SUBJECTS AND METHODS: Neuroimaging examination was performed on 17 eligible surviving ELBWI. Their MR images were assessed and classified its pattern of cerebellar injuries. Brain pathology was examined on 15 patients, who isolated this neuroimaging subjects. The trend of brain pathologies was revealed. RESULTS: Four types of morphological pattern were recognized: (i) the absence of major portions in the cerebellum (6/17 cases); (ii) focal cerebellar tissue loss (2/17); (iii) unilateral cerebellar atrophy/hypoplasia (3/17); (iv) small cerebellum with entrapped fourth ventricle (6/17). In cerebellar pathology, the most common findings were focal or widespread cerebellar subarachnoid hemorrhage (12/15) and olivocerebellar degeneration (12/15). In addition, one-third of the cases indicated remote cerebellar parenchymal hemorrhage. CONCLUSION: In MRI-defined lesions, the absence of major portions or focal tissue loss was associated with cerebellar parenchymal hemorrhage and/or hemorrhagic infarction, that is destructive lesion. On the other hand, small cerebellum or unilateral atrophy/hypoplasia, that is impaired development, may be related to the cerebellar neuron loss due to hemosiderin deposits in the surface of the cerebellum. The cerebellar injury in ELBWI is probably caused by not only environmental factors such as hemorrhage, hypoxia-ischemia, or other deleterious effect, but also immaturity of the rapidly growing cerebellum in particular gestational age.

Evaluation of Cognitive Function When Hearing One's Own Name in Patients With Brain Injuries in Early Developmental Stages.

PURPOSE: The level of residual cognitive function in patients with early brain injury is a key factor limiting rehabilitation and the quality of life. Although understanding residual function is necessary for appropriate rehabilitation, the extent of its effects on cognitive improvement remains unknown. This study evaluated cognitive function in patients with severe motor and intellectual disabilities after early brain injuries due to cerebral hemorrhage or periventricular leukomalacia. We focused on neural responses to hearing the subject's own name (SON). According to previous studies, differences in response to SON are associated with several types of cognitive dysfunction. METHODS: We examined healthy subjects (aged 21.4 ± 1.10 years; control) and patients with a previous brain injury (aged 13-27 years at the time of our analysis) resulting in periventricular leukomalacia or a cerebral hemorrhage during the perinatal period or childhood. We recorded EEG responses to the SON and to other Japanese words, obtaining EEG-evoked potentials with wavelet transformations. RESULTS: Compared with healthy controls, beta power (not alpha power) revealed differences in response to SON by patients with brain injury, especially those with cerebral hemorrhage. CONCLUSIONS: We suggest that alpha and beta power differences reflect different cognitive functions and that the SON response reveals more than one process. Beta powers may reflect the intellectual disability of cognitive function in response to self-relevant stimuli, especially in patients with cerebral hemorrhage. Meanwhile, alpha powers did not differ from those of the healthy controls, suggesting that the patients perhaps paid attention to their own names.

Association of impaired neuronal migration with cognitive deficits in extremely preterm infants.

Many extremely preterm infants (born before 28 gestational weeks [GWs]) develop cognitive impairment in later life, although the underlying pathogenesis is not yet completely understood. Our examinations of the developing human neocortex confirmed that neuronal migration continues beyond 23 GWs, the gestational week at which extremely preterm infants have live births. We observed larger numbers of ectopic neurons in the white matter of the neocortex in human extremely preterm infants with brain injury and hypothesized that altered neuronal migration may be associated with cognitive impairment in later life. To confirm whether preterm brain injury affects neuronal migration, we produced brain damage in mouse embryos by occluding the maternal uterine arteries. The mice showed delayed neuronal migration, ectopic neurons in the white matter, altered neuronal alignment, and abnormal corticocortical axonal wiring. Similar to human extremely preterm infants with brain injury, the surviving mice exhibited cognitive deficits. Activation of the affected medial prefrontal cortices of the surviving mice improved working memory deficits, indicating that decreased neuronal activity caused the cognitive deficits. These findings suggest that altered neuronal migration altered by brain injury might contribute to the subsequent development of cognitive impairment in extremely preterm infants.