Identification of early metabolic markers of various degrees of ischemia in the mouse brain by localized H-1 magnetic resonance spectroscopy

Objectives: Magnetic resonance (MR) imaging and spectroscopy (MRS) allow the establishment of the anatomical evolution and neurochemical profiles of ischemic lesions. The aim of the present study was to identify markers of reversible and irreversible damage by comparing the effects of 10-mins middle cerebral artery occlusion (MCAO), mimicking a transient ischemic attack, with the effects of 30-mins MCAO, inducing a striatal lesion. Methods: ICR-CD1 mice were subjected to 10-mins (n = 11) or 30-mins (n = 9) endoluminal MCAO by filament technique at 0 h. The regional cerebral blood flow (CBF) was monitored in all animals by laser- Doppler flowmetry with a flexible probe fixed on the skull with < 20% of baseline CBF during ischemia and > 70% during reperfusion. All MR studies were carried out in a horizontal 14.1T magnet. Fast spin echo images with T2-weighted parameters were acquired to localize the volume of interest and evaluate the lesion size. Immediately after adjustment of field inhomogeneities, localized 1H MRS was applied to obtain the neurochemical profile from the striatum (6 to 8 microliters). Six animals (sham group) underwent nearly identical procedures without MCAO. Results: The 10-mins MCAO induced no MR- or histologically detectable lesion in most of the mice and a small lesion in some of them. We thus had two groups with the same duration of ischemia but a different outcome, which could be compared to sham-operated mice and more severe ischemic mice (30-mins MCAO). Lactate increase, a hallmark of ischemic insult, was only detected significantly after 30-mins MCAO, whereas at 3 h post ischemia, glutamine was increased in all ischemic mice independently of duration and outcome. In contrast, glutamate, and even more so, N-acetyl-aspartate, decreased only in those mice exhibiting visible lesions on T2-weighted images at 24 h. Conclusions: These results suggest that an increased glutamine/glutamate ratio is a sensitive marker indicating the presence of an excitotoxic insult. Glutamate and NAA, on the other hand, appear to predict permanent neuronal damage. In conclusion, as early as 3 h post ischemia, it is possible to identify early metabolic markers manifesting the presence of a mild ischemic insult as well as the lesion outcome at 24 h.

Development of place navigation in rats from weaning to puberty.

Young hooded rats were trained to escape onto a hidden platform after swimming in a pool of opaque water. Subjects 21, 28, 35, 42, and 64 days of age on the first training day were given 28 trials on 5 consecutive days. Half of the rats were required to localize the platform in relation to external room cues only ("place only" condition) and the other half were helped by the presence of a visible cue on the platform ("cue + place" condition). A deficiency in place navigation was observed in the 21- and 28-day groups; they showed slow escape and took circuitous routes more often than older rats. This deficiency was related to a poor spatial bias toward the training position when the subjects were allowed to swim for 30 s in the absence of the platform, at the end of the 28-trial training period (probe trial). The 35-day group showed adult-like learning ability in both training conditions, but failed to show searching behavior during the probe trial after having been trained in the presence of the proximal cue. Only rats older than 40 days showed typical adult behavior such as swimming directly toward the platform from any starting position and localized searching around the absent platform's position during the probe trial, no matter what the training conditions were. These results suggest that central nervous system structures responsible for place learning in the rat are functional from around 32 days of age, but fail to trigger searching behavior following cued training before the sixth week.

Corneal Opacities in Trisomy 8 Mosaic Syndrome : Clinical, Histologic and Genetic Features

Purpose: To describe the clinical, histologic and genetic findings of corneal opacities in the trisomy 8 mosaic syndrome. Methods: 3 children aged 8 years (Patients A), 6 years (Patients B) and 1 month (Patients C) respectively, were referred with corneal opacities for ophthalmologic evaluation. The 2 older patients had been previously diagnosed with trisomy 8 mosaicism, while the third was diagnosed after the ocular examination. Automated lamellar keratoplasty (ALTK) was performed on the most amblyopic eye. Histopathologic analysis with immunohistochemical markers and cytogenetic studies by FISH using haploid probes for chromosome 8 and chromosome 16 (control) were performed on the excised corneal lesion. Results: All patients presented vascularized corneal opacities involving the superficial stroma, and amblyopia with a bilateral involvement in two of them (Patients A and B). Post-operative follow-up (range 6-20 months) was satisfactory, with the graft remaining clear and improved visual acuity, allowing iso-acuity and stereoscopy in the one month old child (Patients C). The clinically observed corneal opacities corresponded histopathologically to the replacement of the normal anterior corneal stroma by a choristomatous loose richly vascularized connective tissue containing mucopolysacharides. Bowman's membrane was absent. There were no adnexal structures. The overlaying epithelium expressed keratin 3 in all three cases. Keratin 19 was found in the suprabasal epithelial cells in one case but was absent in the other cases. There were no expression of keratin 7 and 1 as well as MUC5AC in the epithelial cells. FISH analysis from 100 interphase cells of the affected tissue and normal conjontival probe revealed normal diploid cells. Conclusions: In this series, the corneal opacities associated with trisomy 8 mosaic syndrome share a common clinical, histopathological and genetic features. ALTK should be considered at diagnosis to prevent amblyopia in these children.

mitoKATP channel activation in the postanoxic developing heart protects E-C coupling via NO-, ROS-, and PKC-dependent pathways.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Multiple adenosine 3',5'-cyclic [corrected] monophosphate response element DNA-binding proteins generated by gene diversification and alternative exon splicing.

The protein sequence deduced from the open reading frame of a human placental cDNA encoding a cAMP-responsive enhancer (CRE)-binding protein (CREB-327) has structural features characteristic of several other transcriptional transactivator proteins including jun, fos, C/EBP, myc, and CRE-BP1. Results of Southwestern analysis of nuclear extracts from several different cell lines show that there are multiple CRE-binding proteins, which vary in size in cell lines derived from different tissues and animal species. To examine the molecular diversity of CREB-327 and related proteins at the nucleic acid level, we used labeled cDNAs from human placenta that encode two different CRE-binding proteins (CREB-327 and CRE-BP1) to probe Northern and Southern blots. Both probes hybridized to multiple fragments on Southern blots of genomic DNA from various species. Alternatively, when a human placental c-jun probe was hybridized to the same blot, a single fragment was detected in most cases, consistent with the intronless nature of the human c-jun gene. The CREB-327 probe hybridized to multiple mRNAs, derived from human placenta, ranging in size from 2-9 kilobases. In contrast, the CRE-BP1 probe identified a single 4-kilobase mRNA. Sequence analyses of several overlapping human genomic cosmid clones containing CREB-327 sequences in conjunction with polymerase chain reaction indicates that the CREB-327/341 cDNAs are composed of at least eight or nine exons, and analyses of human placental cDNAs provide direct evidence for at least one alternatively spliced exon. Analyses of mouse/hamster-human hybridoma DNAs by Southern blotting and polymerase chain reaction localizes the CREB-327/341 gene to human chromosome 2. The results indicate that there is a dichotomy of CREB-like proteins, those that are related by overall structure and DNA-binding specificity as well as those that are related by close similarities of primary sequences.

Micro-Structural Brain Alterations in Aviremic HIV+ Patients with Minor Neurocognitive Disorders: A Multi-Contrast Study at High Field.

OBJECTIVE: Mild neurocognitive disorders (MND) affect a subset of HIV+ patients under effective combination antiretroviral therapy (cART). In this study, we used an innovative multi-contrast magnetic resonance imaging (MRI) approach at high-field to assess the presence of micro-structural brain alterations in MND+ patients. METHODS: We enrolled 17 MND+ and 19 MND- patients with undetectable HIV-1 RNA and 19 healthy controls (HC). MRI acquisitions at 3T included: MP2RAGE for T1 relaxation times, Magnetization Transfer (MT), T2* and Susceptibility Weighted Imaging (SWI) to probe micro-structural integrity and iron deposition in the brain. Statistical analysis used permutation-based tests and correction for family-wise error rate. Multiple regression analysis was performed between MRI data and (i) neuropsychological results (ii) HIV infection characteristics. A linear discriminant analysis (LDA) based on MRI data was performed between MND+ and MND- patients and cross-validated with a leave-one-out test. RESULTS: Our data revealed loss of structural integrity and micro-oedema in MND+ compared to HC in the global white and cortical gray matter, as well as in the thalamus and basal ganglia. Multiple regression analysis showed a significant influence of sub-cortical nuclei alterations on the executive index of MND+ patients (p = 0.04 he and R(2) = 95.2). The LDA distinguished MND+ and MND- patients with a classification quality of 73% after cross-validation. CONCLUSION: Our study shows micro-structural brain tissue alterations in MND+ patients under effective therapy and suggests that multi-contrast MRI at high field is a powerful approach to discriminate between HIV+ patients on cART with and without mild neurocognitive deficits.

In vivo quantification of neuro-glial metabolism and glial glutamate concentration using 1H-[13C] MRS at 14.1T.

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo (13) C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-(13) C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using (1) H-[(13) C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg ) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT ) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC ) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vx (g) and Vx (n) ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations. In this (13) C-acetate labeling study, we propose a refined two-compartment analysis of brain energy metabolism based on (13) C turnover curves of acetate, glutamate and glutamine measured with state of the art in vivo dynamic MRS at high magnetic field in rats, enabling a deeper understanding of the specific role of glial cells in brain oxidative metabolism. In addition, the robustness of the metabolic fluxes determination relative to MRS data quality was carefully studied.

Pragmatic approach for interpreting antiretroviral drug concentrations based on a systematic review of population pharmacokinetic studies

Objectives: The study objective was to derive reference pharmacokinetic curves of antiretroviral drugs (ART) based on available population pharmacokinetic (Pop-PK) studies that can be used to optimize therapeutic drug monitoring guided dosage adjustment.¦Methods: A systematic search of Pop-PK studies of 8 ART in adults was performed in PubMed. To simulate reference PK curves, a summary of the PK parameters was obtained for each drug based on meta-analysis approach. Most models used one-compartment model, thus chosen as reference model. Models using bi-exponential disposition were simplified to one-compartment, since the first distribution phase was rapid and not determinant for the description of the terminal elimination phase, mostly relevant for this project. Different absorption were standardized for first-order absorption processes.¦Apparent clearance (CL), apparent volume of distribution of the terminal phase (Vz) and absorption rate constant (ka) and inter-individual variability were pooled into summary mean value, weighted by number of plasma levels; intra-individual variability was weighted by number of individuals in each study.¦Simulations based on summary PK parameters served to construct concentration PK percentiles (NONMEM®).¦Concordance between individual and summary parameters was assessed graphically using Forest-plots. To test robustness, difference in simulated curves based on published and summary parameters was calculated using efavirenz as probe drug.¦Results: CL was readily accessible from all studies. For studies with one-compartment, Vz was central volume of distribution; for two-compartment, Vz was CL/λz. ka was directly used or derived based on the mean absorption time (MAT) for more complicated absorption models, assuming MAT=1/ka.¦The value of CL for each drug was in excellent agreement throughout all Pop-PK models, suggesting that minimal concentration derived from summary models was adequately characterized. The comparison of the concentration vs. time profile for efavirenz between published and summary PK parameters revealed not more than 20% difference. Although our approach appears adequate for estimation of elimination phase, the simplification of absorption phase might lead to small bias shortly after drug intake.¦Conclusions: Simulated reference percentile curves based on such an approach represent a useful tool for interpretating drug concentrations. This Pop-PK meta-analysis approach should be further validated and could be extended to elaborate more sophisticated computerized tool for the Bayesian TDM of ART.

Effects of antibacterial agents and drugs monitored by atomic force microscopy.

Originally invented for topographic imaging, atomic force microscopy (AFM) has evolved into a multifunctional biological toolkit, enabling to measure structural and functional details of cells and molecules. Its versatility and the large scope of information it can yield make it an invaluable tool in any biologically oriented laboratory, where researchers need to perform characterizations of living samples as well as single molecules in quasi-physiological conditions and with nanoscale resolution. In the last 20 years, AFM has revolutionized the characterization of microbial cells by allowing a better understanding of their cell wall and of the mechanism of action of drugs and by becoming itself a powerful diagnostic tool to study bacteria. Indeed, AFM is much more than a high-resolution microscopy technique. It can reconstruct force maps that can be used to explore the nanomechanical properties of microorganisms and probe at the same time the morphological and mechanical modifications induced by external stimuli. Furthermore it can be used to map chemical species or specific receptors with nanometric resolution directly on the membranes of living organisms. In summary, AFM offers new capabilities and a more in-depth insight in the structure and mechanics of biological specimens with an unrivaled spatial and force resolution. Its application to the study of bacteria is extremely significant since it has already delivered important information on the metabolism of these small microorganisms and, through new and exciting technical developments, will shed more light on the real-time interaction of antimicrobial agents and bacteria.

13q deletion syndrome and retinoblastoma in identical dichorionic diamniotic monozygotic twins.

PURPOSE: To report the case of identical dichorionic diamniotic female twins with unilateral retinoblastoma in 13q deletion syndrome. METHODS: Clinical and ophthalmoscopic evaluation, combination of multiple ligation-dependent probe amplification, array-comparative genomic hybridization analyses, and magnetic resonance imaging were performed. RESULTS: Peculiar facial features, marked hypotonia, gastroesophageal reflux, interatrial septal defect with left to right shunt and light dilatation of right chambers, 5th finger hypoplasia, 3rd-5th toes clinodactyly, 2nd toe overlapped to 3rd toe, and cutis marmorata were found. Ophthalmoscopic evaluation revealed unilateral retinoblastoma in both girls. Magnetic resonance imaging detected corpus callosum hypoplasia in both twins. A 34.4-Mb deletion involving bands 13q13.2-q21.33 and including the RB1 gene was identified in both twins. The deletion was not present in the DNA of their parents and older brother. CONCLUSIONS: Dysmorphic features in children must be always suspicious of 13q deletion syndrome and a short ophthalmoscopic follow-up is necessary to detect the presence of a retinoblastoma.