White and gray matter development in human fetal, newborn and pediatric brains

Brain anatomy is characterized by dramatic growth from the end of the second trimester through the neonatal stage. The characterization of normal axonal growth of the white matter tracts has not been well-documented to date and could provide important clues to understanding the extensive inhomogeneity of white matter injuries in cerebral palsy (CP) patients. However, anatomical studies of human brain development during this period are surprisingly scarce and histology-based atlases have become available only recently. Diffusion tensor magnetic resonance imaging (DTMRI) can reveal detailed anatomy of white matter. We acquired diffusion tensor images (DTI) of postmortem fetal brain samples and in vivo neonates and children. Neural structures were annotated in two-dimensional (2D) slices, segmented, measured, and reconstructed three-dimensionally (3D). The growth status of various white matter tracts was evaluated on cross-sections at 19-20 gestational weeks, and compared with 0-month-old neonates and 5- to 6-year-old children. Limbic, commissural, association, and projection white matter tracts and gray matter structures were illustrated in 3D and quantitatively characterized to assess their dynamic changes. The overall pattern of the time courses for the development of different white matter is that limbic fibers develop first and association fibers last and commissural and projection fibers are forming from anterior to posterior part of the brain. The resultant DTNIRI-based 3D human brain data will be a valuable resource for human brain developmental study and will provide reference standards for diagnostic radiology of premature newborns. (c) 2006 Elsevier Inc. All rights reserved.

How many glomerular profiles must be measured to obtain reliable estimates of mean glomerular areas in human renal biopsies?

The objective of this study was to investigate the number of glomerular profiles that are required for accurate estimates of mean profile area in a renal biopsy series. Slides from 384 renal biopsies from one center were reviewed. They contained a median of seven glomerular profiles or of four profiles without sclerosis. Profile areas were measured using stereologic point counting. The true individual mean for each biopsy was calculated and the true population mean for groups of biopsies derived. Individual and population random sample means then were calculated from a random sampling of profiles in each biopsy and were compared with true means for the same biopsies. The effect on the true population means of the entire group of biopsies was also assessed, as the minimum number of glomerular profiles that were required for inclusion was changed. In a single biopsy, random sampling of >= 10 profiles without exclusions and of eight profiles or more without sclerosis reliably estimated the true mean areas. In a group of 30 biopsies, random sampling of five or more glomeruli per biopsy reliably estimated the true population mean. In the aggregate series, inclusion of all 384 biopsies produced the most robust true population mean; the reliability of the estimates decreased as the numbers of eligible biopsies diminished with increasing requisite minimum numbers of profiles per biopsy. We conclude that, while >= 10 profiles might be needed for reliable area estimates in a single biopsy, far fewer profiles per biopsy can suffice when groups of biopsies are studied. In analyses of groups of biopsies, all available biopsies should be used without consideration of the number of glomerular profiles in each. Stipulation of a specific minimum number of glomeruli in each biopsy for inclusion reduces the power of analyses because fewer biopsies are available for evaluation.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 mu l) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 -> 255.3). The assay was linear from 0.2 to 25 mu g/l (r(2) > 0.997, n = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 mu g/l) were 95.8-103.2 and < 5.5%, respectively. At the lower limit of quantification (0.2 mu g/l) the interand intra-day analytical recovery was 99.0-102.8% with imprecision of < 7.6% (n = 5). The assay had a mean relative recovery of 100.5 +/- 5.8% (n = 15). Extracted samples were stable for 16 h. IFTY720 quality control samples were stable at room temperature for 16 h at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans. (c) 2006 Elsevier B.V. All rights reserved.

Comorbidity and genotype modify receptor expression in human alcoholics

Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic ætiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelæ to the liver damage.

Detection of elevated protein modification in human cerebellum in alcoholic cerebellar degeneration

Modification of proteins by reactive ethanol metabolites has been known for some time to occur in the liver, the main site of ethanol metabolism. In more recent studies of laboratory animals, similar modifications have been detected in organs with lesser ability to metabolize ethanol, such as skeletal and cardiac muscle and brain. Such modification may alter protein function or form a neoantigen, making it a target for immune attack. We now report an analysis of protein modification derived from ethanol metabolites in human brain tissue by ELISA using adduct-specific antibodies. We obtained autopsy cerebellum samples from 10 alcoholic cerebellar degeneration cases and 10 matched controls under informed written consent from the next of kin and clearance from the UQ Human Ethics Committee. Elevated levels of protein modifications derived from acetaldehyde (unreduced-acetaldehyde and acetaldehyde-advanced glycation end-product adducts), from malondialdehyde (malondialdehyde adducts) and from combined adducts (malondialdehydeacetaldehyde (MAA) adducts) were detected in alcoholic cerebellar degeneration samples when compared to controls. Other adduct types found in liver samples, such as reduced-acetaldehyde and those derived from hydroxyethyl radicals, were not detected in brain samples. This may reflect the different routes of ethanol metabolism in the two tissues. This is the first report of elevated protein modification in alcoholic cerebellar degeneration, and suggests that such modification may play a role in the pathogenesis of brain injury. Supported by NIAAA under grant NIH AA12404 and the NHMRC (Australia) under grant #981723.

Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis