ß-Hydroxybutyrate Detection with Proton MR Spectroscopy in Children with Drug-Resistant Epilepsy on the Ketogenic Diet.

BACKGROUND AND PURPOSE: The ketogenic diet, including both classic and modified forms, is an alternative to antiepileptic medications used in the treatment of drug-resistant epilepsy. We sought to evaluate the utility of proton MR spectroscopy for the detection of ß-hydroxybutyrate in a cohort of children with epilepsy treated with the ketogenic diet and to correlate brain parenchymal metabolite ratios obtained from spectroscopy with ß-hydroxybutyrate serum concentrations. MATERIALS AND METHODS: Twenty-three spectroscopic datasets acquired at a TE of 288 ms in children on the ketogenic diet were analyzed with LCModel using a modified basis set that included a simulated ß-hydroxybutyrate resonance. Brain parenchymal metabolite ratios were calculated. Metabolite ratios were compared with serum ß-hydroxybutyrate concentrations, and partial correlation coefficients were calculated using patient age as a covariate. RESULTS: ß-hydroxybutyrate blood levels were highly correlated to brain ß-hydroxybutyrate levels, referenced as either choline, creatine, or N-acetylaspartate. They were inversely but more weakly associated with N-acetylaspartate, regardless of the ratio denominator. No strong concordance with lactate was demonstrated. CONCLUSIONS: Clinical MR spectroscopy in pediatric patients on the ketogenic diet demonstrated measurable ß-hydroxybutyrate, with a strong correlation to ß-hydroxybutyrate blood levels. These findings may serve as an effective tool for noninvasive monitoring of ketosis in this population. An inverse correlation between serum ß-hydroxybutyrate levels and brain tissue N-acetylaspartate suggests that altered amino acid handling contributes to the antiepileptogenic effect of the ketogenic diet.

Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line.

Catalase, superoxide dismutase, and dimethylsulfoxide were tested for their ability to prevent the cytotoxic effect of 6-hydroxydopamine (6-OHDA) on the human neuroblastoma line SY5Y. Viability was measured at two time points after 6-OHDA treatment: at 3 hr by means of amino acid incorporation and at 24 hr by trypan blue dye exclusion. Survival of cells treated concomitantly with catalase (50 microgram/ml) and 6-OHDA was at least 90 per cent that of untreated controls. Cells receiving 6-OHDA alone showed less than 30 per cent survival relative to untreated controls. Superoxide dismutase (50 microgram/ml) temporarily protected cells from a high concentration of 60-OHDA. Dimethylsulfoxide treatment increased survival from the control level 24 hr after treatment with 6-OHDA. Two other cell lines (A1B1 human glial cells and CHO fibroblasts) had intermediate and high resistance to the drug, respectively, compared to the low resistance of SY5Y cells. CHO and SY5Y cells had similar responses to 6-OHDA and to H2O2 when tested at twice the molarity of 6-OHDA. Specific activities of three enzymes known to detoxify H2O2 or H2O2-generated organic hydroperoxides (catalase, glutathione S-transferase, and glutathione peroxidase) were compared in the three cell lines. Catalase activity was 2.5 times as high as in A1B1 and CHO cells as in SY5Y cells when expressed as units/mg protein and 7 times as high in units/culture dish. Other enzyme activities showed no correlation to 6-OHDA resistance.

Atypical manifestations of two cases of trisomy 9 syndrome: rethinking development delay.

Trisomy 9 syndrome is characterized by "bulbous" nose, microphthalmia, dislocated limbs, and other anomalies of skeletal, cardiac, genitourinary, and central nervous systems. With the exception of one reported case study, all surviving infants have had severe mental impairment. The prospect of severe mental retardation often overwhelms parents who are faced with prenatal diagnosis of trisomy 9. We report on two new cases of mosaic trisomy 9, both of whom are only mildly developmentally delayed. One patient presented with the distinctive facial appearance, large fontanels, and joint abnormalities. The other had none of the typical congenital abnormalities. However, the patient was found to have a congenital heart defect and hypoplastic left heart syndrome, which to our knowledge has not been reported previously in the trisomy 9 syndrome. When these two patients are added to the published patients with this syndrome, there appears to be a range of manifestations, especially with respect to mental status, which has not fully been recognized.

Expression of glial fibrillary acidic protein by differentiated astrocytes is regulated by serum antagonistic factors.

We report here that, in culture, the expression of glial fibrillary acidic protein (GFAP) by astrocytes, as well as their shape (flat-polygonal vs. stellate) can be regulated by 4 serum antagonistic factors. Three of these factors are stimulatory, while the fourth exerts an inhibitory effect upon these astrocytic properties. As suggested by temperature and trypsin treatments, the inhibitory factor is a polypeptide or a protein of 15-35 kDa. The stimulatory factors are smaller: two of them have a mol. wt. between 0.2 and 5 kDa; the third is smaller than 0.2 kDa. Treatments with chloroform/methanol, ammonium sulfate, neuraminidase, and papain, indicate that at least one glycolipid and one glycoprotein are involved. We speculate that, during development, cells from the astrocytic line could be susceptible selectively to one or another of these factors, which would explain their great plasticity.

Expression of beta-nerve growth factor in cultured cells derived from the hypothalamus and cerebral cortex.

Although the synthesis of nerve growth factor (NGF) in brain regions innervated by magnocellular cholinergic neurons of the basal forebrain is well documented, the cell type(s) able to produce NGF in the central nervous system (CNS) remain only partially characterized. Moreover, little is known regarding the ability of brain areas not innervated by magnocellular cholinergic neurons to express NGF protein. The hypothalamus, which controls the endocrine system, is one of such regions. Primary culture of mixed populations of cells from the fetal hypothalamus were used to identify the presence of NGF in this brain area. Immunocytochemistry revealed that hypothalamic oligodendrocytes and a subpopulation of neurons expressed the NGF protein. In contrast, astrocytes were either immunonegative or equivocally stained. To define whether synthesis of NGF is restricted to a particular cell type, cultures of purified astrocytes, oligodendrocyte progenitor (oligoP) cells and neurons were utilized. They were obtained from the neonatal cerebral cortex to ensure an adequate yield of glial cells. Virtually the entire population of cerebral oligoP cells were found to express NGF protein. In contrast, and similar to hypothalamic astrocytes, cerebral type I astrocytes isolated at the same time as oligoP cells exhibited little or no NGF staining. When type I astrocytes were induced to differentiate in the presence of a serumless, chemically defined medium, a subpopulation of the culture became more robustly positive for the NGF protein. Contrasting with these differences in NGF immunoreactivity, Northern analysis of RNA isolated from purified cerebral type I astrocytes, oligoP cells and neurons demonstrated that NGF mRNA was expressed in each of these cell types at approximately the same levels. The results indicate that: (a) when placed in culture, each of the major cell types within the CNS has the capability of transcribing the NGF gene, and (b) despite similar NGF mRNA levels the cellular content of NGF protein is greater in a subpopulation of neurons and in oligodendrocytes than in astrocytes, suggesting differences in NGF post-transcriptional regulation between these cell types. In addition, the presence of NGF in hypothalamic cells suggests that NGF may be involved in the regulation of specific hypothalamic neuronal systems.

Myelination of axons within cytosine arabinoside treated mouse cerebellar explants by cultured rat oligodendrocytes.

Cell suspensions of cultured purified rat oligodendrocytes prepared by the differential substrate adhesion method were applied to neonatal mouse cerebellar explant cultures in which myelination and oligodendrocyte maturation had been irreversibly inhibited by exposure to cytosine arabinoside. Myelination of Purkinje cell axons within 92% of the host explants was observed 2-5 days after oligodendrocyte application. Ultrastructurally, mature oligodendrocytes and axons surrounded by compact myelin, as well as spherules of compact myelin membranes without axons, were present within the cerebellar explants. It is evident that cultured dissociated purified oligodendrocytes retain the ability to myelinate appropriate axons. Such oligodendrocytes may be hyperreactive with regard to myelin membrane formation, as suggested by the presence of spheres of compact myelin without axons.

The control of glial populations in brain: changes in astrocyte mitogenic and morphogenic factors in response to injury.

Injury to rat brain induces a 3-10-fold increase in the activity of factors capable of stimulating astrocyte DNA synthesis and cell division in vitro. Maximum mitogenic activity was reached 10-15 days post-lesion in both the tissue surrounding the wound and in the gelfoam filling the wound cavity. Factors capable of transforming the astrocyte morphology from polygonal-flat to fibrous-like (morphogens) could also be observed in brain tissue and showed increased activity beginning at 10 days postlesion. On the other hand, morphogenic activity was very low or absent in gelfoam extracts until 15 days postlesion. Both mitogenic and morphogenic factors were nondiffusible and were partly temperature and trypsin sensitive, i.e. they had the properties of protein-like substances, but seemed different from both epidermal and fibroblast growth factors. As judged by their filtration behavior on Amicon membranes, the molecular weight of mitogens and morphogens ranged from lower than 30,000 to greater than 100,000. Inhibitors of both mitogenic and morphogenic activities with molecular weight lower than 30,000 seemed to be also present in the brain extracts. The factors described here can account for the processes of astrocytosis and astrogliosis observed in vivo in response to CNS injury.

Epilepsy surgery in infancy.

Surgery for intractable epilepsy is being offered at progressively younger ages, including infancy. The most common causes of catastrophic epilepsy in very young surgical candidates are focal malformations of cortical development and low-grade tumors. Additional causes include Sturge-Weber syndrome, epidermal nevus syndrome, hemimegalencephaly, and prenatal or perinatal infarction. Many infants manifest with focal seizures, whereas some patients have infantile spasms in the setting of a focal epileptogenic lesion. Video electroencepholography, magnetic resonance imaging, and positron emission tomography are critical investigations to explore surgical options. In small series, the percentage of infants free of seizures after surgery was in the range of 60%. This is similar to that seen after epilepsy surgery in older children, adolescents, and adults. However, larger series with long-term follow up will be important. Furthermore, the extensive procedures required in infants for removal of the epileptogenic developmental lesions entail some risk, and should not be offered in the absence of severe epilepsy. Most infant candidates for epilepsy surgery have significant developmental delay. Few data are available, but anecdotal experience suggests that surgical relief of catastrophic epilepsy may result in resumption of developmental progression. For each infant, the timing of surgery must be carefully considered based on full assessment of the relative risks and benefits, derived from a detailed presurgical evaluation.

Acute neurotoxicity of meperidine in an infant.

We report a 6-week-old boy with meperidine neurotoxicity. What distinguished our patient from those previously reported was his minimal exposure to therapeutic doses of meperidine in the setting of normal renal function, and no history of sickle cell anemia, cancer, hepatitis, or cirrhosis. In addition, our patient had no abnormal changes in the electroencephalogram during the event. After only 2 doses of meperidine, he exhibited acute orofacial dyskinesias consisting of tongue thrusting, lip pursing, and facial grimacing combined with prominent flexion of the arms and stiffening of his legs. However, a normal sucking response remained. His symptoms resolved over the next 36 hours and did not respond to naloxone. We believe that this unique presentation of meperidine-induced neurotoxicity may be due to changes in the basal ganglia resulting from perinatal hypoxemia.

When might hemispheric favouring of epileptiform discharges begin?

PURPOSE: EEG studies based on adult populations report interictal epileptiform discharges (EDS) favour the left hemisphere. It is not clear when favouring becomes apparent as similar paediatric studies have not been performed. METHODS: The authors reviewed 1,579 paediatric EEG interpretations for evidence of hemispheric favouring of focal epileptiform discharges. Analysis focused on first-time EEG results. RESULTS: Right hemispheric favouring of interictal epileptiform discharges occurs in childhood, it remits around 5 years of age whereupon left-sided favouring occurs more frequently (P=0.004, Fisher's Exact). CONCLUSION: Hemispheric vulnerabilities to interictal focal epileptiform activity may display discrete age-related favouring. These findings are discussed in context of normal hemispheric maturation.

The information highway for the pediatrician: an understanding of the Internet for the clinician.

The author details the information highway available to the computer-literate pediatrician: the Internet, Electronic mail (E-mail), the World Wide Web, and explains how to access the information.

Tract-based spatial statistical analysis of diffusion tensor imaging in pediatric patients with mitochondrial disease: widespread reduction in fractional anisotropy of white matter tracts.

BACKGROUND AND PURPOSE: Often diagnosed at birth or in early childhood, mitochondrial disease presents with a variety of clinical symptoms, particularly in organs and tissues that require high energetic demand such as brain, heart, liver, and skeletal muscles. In a group of pediatric patients identified as having complex I or I/III deficits on muscle biopsy but with white matter tissue appearing qualitatively normal for age, we hypothesized that quantitative DTI analyses might unmask disturbance in microstructural integrity. MATERIALS AND METHODS: In a retrospective study, DTI and structural MR brain imaging data from 10 pediatric patients with confirmed mitochondrial disease and 10 clinical control subjects were matched for age, sex, scanning parameters, and date of examination. Paired TBSS was performed to evaluate differences in FA, MD, and the separate diffusion direction terms (λr and λa). RESULTS: In patients with mitochondrial disease, significant widespread reductions in FA values were shown in white matter tracts. Mean diffusivity values were significantly increased in patients, having a sparser distribution of affected regions compared with FA. Separate diffusion maps showed significant increase in λr and no significant changes in λa. CONCLUSIONS: Despite qualitatively normal-appearing white matter tissues, patients with complex I or I/III deficiency have widespread microstructural changes measurable with quantitative DTI.

Characterization of cultured rat oligodendrocytes proliferating in a serum-free, chemically defined medium.

A serumless, chemically defined medium has been developed for the culture of oligodendrocytes isolated from primary neonatal rat cerebral cultures. Combined together, insulin, transferrin, and fibroblast growth factor synergistically induced an essentially homogenous population (95-98%) of cells expressing glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) activity to undergo cell division. Proliferating cels were characterized by several criteria: (i) ultrastructural analysis by transmission electron microscopy identified the cell type as an oligodendrocyte; (ii) biochemical assays showed expression of three oligodendrocyte biochemical markers, induction of both glycerol phosphate dehydrogenase and lactate dehydrogenase (EC 1.1.1.27), and presence of 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37); and (iii) immunocytochemical staining showed cultures to be 95-98% positive for glycerol phosphate dehydrogenase, 90% for myelin basic protein, 60-70% for galactocerebroside, and 70% for A2B5. Few cells (less than 5%) stained positive for glial fibrillary acidic protein, and none were detected positive for fibronectin.

Effect of mitogens in various organs and cell culture conditioned media on rat oligodendrocytes.

Extracts prepared from embryonic, neonatal and adult rat brain were examined for the presence of oligodendroglial mitogens. Brain-derived mitogenic activities were found in all developmental stages with specific activity increasing during neonatal development. Extracts from postnatal day 7 contained the highest specific activity. Upon fractionation by molecular weight, each developmental stage expressed a peak of mitogenic activity corresponding to 67,000 daltons. This fraction was able to induce proliferation in cultures grown in serum, serumless chemically defined medium or serum-free medium alone. Neonatal and adult brain extracts had an additional peak of activity at 14,000 daltons. This latter activity was expressed only under serum-supplemented culture conditions. Mitogenic activity was also found in conditioned media from the clonal glioma cell line C6 and primary astrocytes and in extracts derived from neonatal rat liver. These data indicate that a limited range of brain-derived mitogens for oligodendrocytes exist during development and adulthood.

Traumatic intracerebral venous thrombosis associated with an abnormal golf swing.

OBJECTIVES: To describe the occurrence of cerebral venous thrombosis in a 40-year-old man whose cerebral event was induced by a poor golf swing, to review the literature on possible mechanisms producing venous thrombosis, and to compare this case with the literature. BACKGROUND: Headache is the most frequent symptom in patients with cerebral venous thrombosis. However, patients presenting with a headache due to cerebral venous thrombosis are uncommon. The known risk factors for thrombosis include both acquired and genetic factors. When the interaction of these two groups occurs, the magnitude of this interaction is thought to produce a dynamic state that can favor thrombosis. Our case report illustrates that moderate levels of anticardiolipin antibodies together with the mild trauma of a golf swing can induce a cerebral venous thrombosis. This case also suggests that although headache is rarely due to cerebral venous thrombosis, it should be excluded by good medical acumen and testing. RESULTS: Minor trauma induced by a poor golf swing was chronologically related to the development of a progressive cerebral venous thrombosis. The patient had none of the risk factors associated with a predisposition to venous thrombosis: hypercoagulable state, concurrent infection, pregnancy/puerperium, collagen vascular disorder, malignancy, migraine, false-positive VDRL, previous deep vein thrombosis, renal disease, factor V Leiden, or a hematological disorder. There was no anatomical abnormality that would predispose the patient to a cerebral venous thrombosis. The only laboratory abnormality was a moderate anticardiolipin antibody level (25 GPL). The patient was placed on warfarin sodium therapy and is currently without clinical sequela from the venous thrombotic event. CONCLUSIONS: Under certain circumstances, minor trauma can induce cerebral venous thrombosis. A review of the literature indicates that cerebral venous thrombosis in the presence of anticardiolipin antibodies and in the absence of systemic lupus erythematosus is a rare event. Previously, only major traumatic events have been reported to be associated with cerebral venous thromboses. The chronological development of cerebral venous thrombosis after a faulty golf swing strongly indicates that given a background of moderate levels of anticardiolipin antibodies, even minor trauma can induce a venous thrombotic event.

Interleukin-2 inhibition of oligodendrocyte progenitor cell proliferation depends on expression of the TAC receptor.

Interleukin-2 (IL-2) has been shown to inhibit oligodendrocyte progenitor cell proliferation. Within the immune system, IL-2 biological action is dependent strictly on the expression of the IL-2 receptor. The antibody TAC, which specifically binds the lymphocyte IL-2 receptor, has been shown to also bind oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium. The expression of the TAC antigen was found necessary for IL-2 inhibition of oligodendrocyte progenitor cell proliferation. After IL-2 induced down-regulation of the TAC antigen, the progenitor cell was unresponsive to IL-2, even 72 hr after IL-2 withdrawal. During this unresponsive period, the oligodendrocyte progenitor cell was immunocytochemically negative for the TAC antigen. Thus, in contrast to IL-2 receptors on T-cells, IL-2 does not up-regulate its receptor on oligodendrocyte progenitor cells. However, upon interleukin 1 (IL-1) addition both IL-2 responsiveness and TAC immunocytochemical staining reappeared. These data suggest that IL-2 inhibition of progenitor cell proliferation depends on the expression of the TAC antigen, which can be regulated by IL-1.

Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver.

Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.

Developmental expression of rat brain mitogens for cultured astrocytes.

Extracts prepared from prenatal, neonatal, and adult rat brain were examined for the presence of astrocyte mitogens. Extracts were characterized by molecular weight separation on Sephadex G-100. Two major peaks of mitogenic activity, at 80K and 23K daltons, were identified in the adult brain extract. Extract prepared from adult tissue also possessed the lowest specific activity. Extract prepared from neonatal rat brain exhibited four general peaks of activity at approximately 80K, 45K, 23K, and 17K daltons. This extract possessed the highest specific activity. Extract prepared from prenatal rat brain contained six general peaks of activity with unique peaks at 67K, 14K, and less than 10K daltons. In addition, this extract possessed a specific activity intermediate between the adult and postnatal extracts. Differences were also noted among the extracts in their requirement for "permissive" factors found in serum necessary for mitogenic activity. The data have implications for the normal development of astrocytes, as well as for the pathological process of gliosis.