Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures.

The intravenous, short-acting general anesthetic propofol was applied to three-dimensional (aggregating) cell cultures of fetal rat telencephalon. Both the clinically used formulation (Disoprivan, ICI Pharmaceuticals, Cheshire, England) and the pure form (2,6-diisopropylphenol) were tested at two different periods of brain development: immature brain cell cultures prior to synaptogenesis and at the time of intense synapses and myelin formation. At both time periods and for clinically relevant concentrations and time of exposure (i.e., concentrations > or = 2.0 micrograms/ml for 8 hr), propofol caused a significant decrease of glutamic acid decarboxylase activity. This effect persisted after removal of the drug, suggesting irreversible structural changes in GABAergic neurons. The gamma-aminobutyric acid type A (GABAA) blocking agents bicuculline and picrotoxin partially attenuated the neurotoxic effect of propofol in cultures treated at the more mature phase of development. This protective effect was not observed in the immature brain cells. The present data suggest that propofol may cause irreversible lesions to GABAergic neurons when given at a critical phase of brain development. In contrast, glial cells and myelin appeared resistant even to high doses of propofol.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

A comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ1-42, okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Examining the Agreement of Structural Loss Measured With Heidelberg Retinal Tomograhy and Functional Loss With Standard Automated Perimetry (SAP; Octopus), Pulsar Perimetry (PP), and Moorfields Motion Displacement Test (MDT) Perimetry

Purpose: To examine the relationship of functional measurements with structural measures. Methods: 146 eyes of 83 test subjects underwent Heidelberg Retinal Tomography (HRTIII) (disc area<2.43, mphsd<40), and perimetry testing with Octopus (SAP; Dynamic), Pulsar (PP; TOP) and Moorfields MDT (ESTA). Glaucoma was defined as progressive structural or functional loss (20 eyes). Perimetry test points were grouped into 6 sectors based on the estimated optic nerve head angle into which the associated nerve fiber bundle enters (Garway-Heath map). Perimetry summary measures (PSM) (MD SAP/ MD PP/ PTD MDT) were calculated from the average total deviation of each measured threshold from the normal for each sector. We calculated the 95% significance level of the sectorial PSM from the respective normative data. We calculated the percentage agreement with group1 (G1), healthy on HRT and within normal perimetric limits, and group 2 (G2), abnormal on HRT and outside normal perimetric limits. We also examined the relationship of PSM and rim area (RA) in those sectors classified as abnormal by MRA (Moorfields Regression Analysis) of HRT. Results: The mean age was 65 (range= [37, 89]). The global sensitivity versus specificity of each instrument in detecting glaucomatous eyes was: MDT 80% vs. 88%, SAP 80% vs. 80%, PP 70% vs. 89% and HRT 80% vs. 79%. Highest percentage agreement of HRT (respectively G1, G2, sector) with PSM were MDT (89%, 57%, nasal superior), SAP (83%, 74%, temporal superior), PP (74%, 63%, nasal superior). Globally percentage agreement (respectively G1, G2) was MDT (92%, 28%), SAP (87%, 40%) and PP (77%, 49%). Linear regression showed there was no significant trend globally associating RA and PSM. However, sectorally the supero-nasal sector had a statistically significant (p<0.001) trend with each instrument, the associated r2 coefficients are (MDT 0.38 SAP 0.56 and PP 0.39). Conclusions: There were no significant differences in global sensitivity or specificity between instruments. Structure-function relationships varied significantly between instruments and were consistently strongest supero-nasally. Further studies are required to investigate these relationships in detail.

IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies-A Price Comparison with Sanger Sequencing.

The molecular diagnosis of retinal dystrophies (RD) is difficult because of genetic and clinical heterogeneity. Previously, the molecular screening of genes was done one by one, sometimes in a scheme based on the frequency of sequence variants and the number of exons/length of the candidate genes. Payment for these procedures was complicated and the sequential billing of several genes created endless paperwork. We therefore evaluated the costs of generating and sequencing a hybridization-based DNA library enriched for the 64 most frequently mutated genes in RD, called IROme, and compared them to the costs of amplifying and sequencing these genes by the Sanger method. The production cost generated by the high-throughput (HT) sequencing of IROme was established at CHF 2,875.75 per case. Sanger sequencing of the same exons cost CHF 69,399.02. Turnaround time of the analysis was 3 days for IROme. For Sanger sequencing, it could only be estimated, as we never sequenced all 64 genes in one single patient. Sale cost for IROme calculated on the basis of the sale cost of one exon by Sanger sequencing is CHF 8,445.88, which corresponds to the sale price of 40 exons. In conclusion, IROme is cheaper and faster than Sanger sequencing and therefore represents a sound approach for the diagnosis of RD, both scientifically and economically. As a drop in the costs of HT sequencing is anticipated, target resequencing might become the new gold standard in the molecular diagnosis of RD.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

The sand rat, Psammomys obesus, develops type 2 diabetic retinopathy similar to humans.

PURPOSE: Diabetic retinopathy (DR) is a leading cause of blindness, yet pertinent animal models are uncommon. The sand rat (Psammomys obesus), exhibiting diet-induced metabolic syndrome, might constitute a relevant model. METHODS: Adult P. obesus (n = 39) were maintained in captivity for 4 to 7 months and fed either vegetation-based diets (n = 13) or standard rat chow (n = 26). Although plant-fed animals exhibited uniform body weight and blood glucose levels over time, nearly 60% of rat chow-raised animals developed diabetes-like symptoms (test group). Animals were killed, and their eyes and vitreous were processed for immunochemistry. RESULTS: Compared with plant-fed animals, diabetic animals showed many abnormal vascular features, including vasodilation, tortuosity, and pericyte loss within the blood vessels, hyperproteinemia and elevated ratios of proangiogenic and antiangiogenic growth factors in the vitreous, and blood-retinal barrier breakdown. Furthermore, there were statistically significant decreases in retinal cell layer thicknesses and densities, accompanied by profound alterations in glia (downregulation of glutamine synthetase, glutamate-aspartate transporter, upregulation of glial fibrillar acidic protein) and many neurons (reduced expression of protein kinase Cα and Cξ in bipolar cells, axonal degeneration in ganglion cells). Cone photoreceptors were particularly affected, with reduced expression of short- and mid-/long-wavelength opsins. Hypercaloric diet nondiabetic animals showed intermediate values. CONCLUSIONS: Simple dietary modulation of P. obesus induces a rapid and severe phenotype closely resembling human type 2 DR. This species presents a valuable novel experimental model for probing the neural (especially cone photoreceptor) pathogenic modifications that are difficult to study in humans and for screening therapeutic strategies.

Retinal thickening in HLA-B27 associated acute anterior uveitis: evolution with time and association with severity of inflammatory activity.

Purpose: To describe the evolution of retinal thickness in eyes affected with acute anterior uveitis (AAU) in the course of follow-up and to assess its correlation with severity of inflammatory activity in the anterior chamber. Methods: Design: Prospective, cohort study Setting: Institutional study Patient population: 72 eyes (affected and fellow eyes) of 36 patients Observation procedure: Patients were followed daily until beginning of resolution of inflammatory activity and weekly thereafter. Optical coherence tomography and laser flare photometry were performed at each visit. Treatment consisted of topical corticosteroids Main outcome measures: Retinal thickness of affected eyes, difference in retinal thickness between affected and fellow eyes and their evolution in time, association between maximal retinal thickness and initial laser flare photometry. Results: Difference in retinal thickness between affected and fellow eyes became significant on average seven days from baseline and remained so through-out follow-up (p<0.001). There was a steep increase in retinal thickness of affected eyes followed by a progressive decrease after reaching a peak value. Maximal difference in retinal thickness between affected and fellow eyes was observed between 17 and 25 days from baseline and exhibited a strong, positive correlation with initial laser flare photometry values (p=0.015). Conclusions: Retinal thickness in eyes affected with AAU presents a steep increase over 3 to 4 weeks and then gradually decreases. Severity of inflammation at baseline predicts the amount of retinal thickening in affected eyes. A characteristic pattern of temporal response of retinal anatomy to inflammatory stimuli seems to arise.

Retinal Dystrophy In The Oculo-auricular Syndrome Due to HMX1 Mutation.

Purpose: To report on the clinical and electrophysiological findings in a patient with oculo-auricular syndrome due to HMX1 mutation, with a follow-up of 12 years. Background: Oculo-auricular syndrome (MIM: 612109) is a rare developmental recessive condition affecting the eye and external ear that results from a mutation in the HMX1 gene. Previously described ocular abnormalities include bilateral microcornea, posterior synechiae, cataract, chorioretinal colobomas and rod-cone dystrophy. Methods: Retrospective chart review of an affected boy followed over a period of 12 years who had serial complete ophthalmologic examinations, fundus photographs, Goldmann perimetry and full-field electroretinograms (ERG). Results: Initial ERG tracings revealed generalized rod more than cone dysfunction. Thereafter, a rapid deterioration in rod and cone function was detected on follow up ERGs. Conclusion: The retinal degeneration in the recessively inherited oculo-auricular syndrome is a progressive rod-cone dystrophy. Visual prognosis is guarded considering the progressive nature of the retinal dystrophy in early infancy.

Mitochondrial DNA (mt DNA) A3243G mutation associated with an annular perimacular retinal atrophy

Résumé:Background:La mutation 3243 de 1'ADN mitochondrial est associee avec le syndrome l\/HDD (surdite, diabète transmis par la mère) et le syndrome MELAS (Myopathie, Encéphalopathie, acidose Lactique et attaques cérébrales). Elle est aussi associe à des troubles cardiaques, digestifs, endo- et exocrines. Nous rapportons deux cas de maculopathie associée à cette mutation.Histoire et symptomes: pCas l: il s'agit d'une femme de 60 ans soufrant d'un diabète et d'une surdité sans plainte visuelle lors de la présentation. Son acuité visuelle était de 10/ l0 des deux yeux.Cas 2: il s'agit d'une femme de 54 ans souffrant d'une surdité et d'un diabète qui se plaint d'une baisse de vision principalement de l'oeil gauche. Son acuité visuelle était de 6/10 pour l'oeil droit et de 0.5/l0 pour l'oeil gauche.Les deux patientes présentaient une atrophie choriorétinienne aréolaire centrale. La patiente 1 a été suivie durant plus de 15 ans. Une évolution lente et progressive de la maculopathie a été observée. Lors de la dernière visite, l'acuité visuelle était de 6/ l0 dans les deux yeux. Elle présentait un handicap marqué des suites du scotome annulaire.Thérapie et pronostic:AucunConclusion:Les deux patientes présentaient une atrophie rétinienne annulaire périmaculaire. Les patients atteints d'une mutation 3243 de l'ADN mitochondrial devraient bénéficier d'un examen du fond d'oeil à la recherche d'une maculopahtie, même s'ils sont asymptomatiques.Inversement, la découverte d'une telle maculopathie géographique devrait suggérer la possibilité d'une mutation au locus 3243 de l'ADN mitochondrial, surtout en présence d'un diabète et/ou d'une surdité.

Periphere okklusive Vaskulopathie bei einer Patientin mit kombinierter Präeklampsie und Alpha-Thalassämie minor [Peripheral occlusive retinal vasculopathy in a woman with combined preeclampsia and alpha thalassemia minor].

Normalerweise eine Störung der ersten Schwangerschaft, ist die Präeklampsie charakterisiert durch eine arterielle Hypertonie (> 140 mmHg systolisch oder > 90 mmHg diastolisch), die in der Regel nach der 20. Schwangerschaftswoche auftritt und von einer Proteinurie begleitet wird [1]. Die Präeklampsie wird als ,,schwer" bezeichnet, wenn sie mit einer wesentlichen Erhöhung des Blutdrucks (> 160 mmHg systolisch oder > 110 mmHg diastolisch), schwerer Proteinurie, Oligurie, Lungenödem, abdominalen Schmerzen, Leberfunktionsstörungen, Thrombozytopenie und visuellen oder zerebralen Symptomen einhergeht. Eine Eklampsie wiederum ist durch die Entwicklung von tonisch-klonischen Anfällen bei einer präeklamptischen Patientin charakterisiert. Bei der Alpha-Thalassämie tritt ein Defekt von 2 oder mehr der 4 Alpha-Globin-Gene auf. Von einer Alpha-Thalassämie minor spricht man, wenn 2 Alpha-Ketten-Gene deletiert sind. Sie tritt häufig bei Menschen aus Afrika, Südostasien, dem westindischen und mediterranen Raum auf. Die Alpha-Thalassämie minor verursacht eine milde bis moderate mikrozytäre Anämie. Wir berichten über eine Patientin mit peripherer okklusiver Vaskulopathie im Rahmen einer kombinierten Präeklampsie und Alpha-Thalassämie minor.

Décollement de rétine rhegmatogène et hypertonie: penser au syndrome de Schwartz-Matsuo [Rhegmatogenous retinal detachment and hypertension: Schwartz-Matsuo syndrome]

PURPOSE: To assess the usefulness of electron microscopy of the aqueous cells when confronted with the clinical association of rhegmatogenous retinal detachment after trauma, high intraocular pressure (IOP) and aqueous cells. METHOD: We report a clinical history of a 50-years-old man who had ocular trauma with perforation in 1944, intraocular lens for traumatic cataract in 1988, Yag capsulotomy in 1993 and retinal detachment with oral dialysis, high IOP and aqueous cells in anterior chamber in 1995. During the surgical therapy we performed an anterior chamber puncture to analyse the aqueous cells. An electron microscopic study was performed on 0.2 ml of aqueous humor mixed in the same volume of 2.5% glutaraldehyde and fixed with 1% osmium acid. RESULTS: Electron microscopic ultrastructural study of the aqueous cells showed numerous photoreceptor outer segments, some of them appearing degenerated. CONCLUSION: The combination of rhegmatogenous retinal detachment with tears near the ora serrata, high IOP and aqueous cells in the anterior chamber should lead the physician to do an anterior chamber puncture and analyse the aqueous cells structure. The combination of those three clinical signs associated with the photoreceptor outer segments in the anterior chamber allowed to diagnose the Schwartz-Matsuo syndrome.