Involvement of microglia-neuron interactions in the tumor necrosis factor-alpha release, microglial activation, and neurodegeneration induced by trimethyltin.

Trimethyltin (TMT) is a neurotoxicant known to induce early microglial activation. The present study was undertaken to investigate the role played by these microglial cells in the TMT-induced neurotoxicity. The effects of TMT were investigated in monolayer cultures of isolated microglia or in neuron-enriched cultures and in neuron-microglia and astrocyte-microglia cocultures. The end points used were morphological criteria; evaluation of cell death and cell proliferation; and measurements of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) release in culture supernatant. The results showed that, in cultures of microglia, TMT (10(-6) M) caused, after a 5-day treatment, an increased release of TNF-alpha, without affecting microglial shape or cell viability. When microglia were cocultured with astrocytes, TNF-alpha release was decreased to undetectable levels. In contrast, in neuron-microglia cocultures, TNF-alpha levels were found to increase at lower concentrations of TMT (i.e., 10(-8) M). Moreover, at 10(-6) M of TMT, microglia displayed further morphological activation, as suggested by process retraction and by decrease in cell size. No morphological activation was observed in cultures of isolated microglial cells and in astrocyte-microglia cocultures. With regard to neurons, 10(-6) M of TMT induced about 30% of cell death, when applied to neuron-enriched cultures, whereas close to 100% of neuronal death was observed in neuron-microglia cocultures. In conclusion, whereas astrocytes may rather dampen the microglial activation by decreasing microglial TNF-alpha production, neuronal-microglial interactions lead to enhanced microglial activation. This microglial activation, in turn, exacerbates the neurotoxic effects of TMT. TNF-alpha may play a major role in such cell-cell communications.

Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.

Chromosomal number aberrations and transformation in adult mouse retinal stem cells in vitro.

PURPOSE: The potential of stem cells (SCs) as a source for cell-based therapy on a wide range of degenerative diseases and damaged tissues such as retinal degeneration has been recognized. Generation of a high number of retinal stem cells (RSCs) in vitro would thus be beneficial for transplantation in the retina. However, as cells in prolonged cultivation may be unstable and thus have a risk of transformation, it is important to assess the stability of these cells. METHODS: Chromosomal aberrations were analyzed in mouse RSC lines isolated from adult and from postnatal day (PN)1 mouse retinas. Moreover, selected cell lines were tested for anchorage-dependent proliferation, and SCs were transplanted into immunocompromised mice to assess the possibility of transformation. RESULTS: Marked aneuploidy occurred in all adult cell lines, albeit to different degrees, and neonatal RSCs were the most stable and displayed a normal karyotype until at least passage 9. Of interest, the level of aneuploidy of adult RSCs did not necessarily correlate with cell transformation. Only the adult RSC lines passaged for longer periods and with a higher dilution ratio underwent transformation. Furthermore, we identified several cell cycle proteins that might support the continuous proliferation and transformation of the cells. CONCLUSIONS: Adult RSCs rapidly accumulated severe chromosomal aberrations during cultivation, which led to cell transformation in some cell lines. The culture condition plays an important role in supporting the selection and growth of transformed cells.

IgG and IgM western blot assay for diagnosis of congenital toxoplasmosis

The aim of this study was to evaluate the utility of western blot (WB) analysis as a diagnostic tool for congenital toxoplasmosis in 215 newborn infants. The children were submitted to clinical examinations to assess macular, neurological and hearing signals. The WB results obtained were compared to the persistence of IgG antibodies at the end of 12 months, which is regarded as the "gold standard" diagnosis of congenital toxoplasmosis. Association between the WB results and the clinical signs presented by the infants was also assessed. Of the 215 children, 177 had a confirmed congenital toxoplasmosis diagnosis and 38 were uninfected. IgG-WB showed a sensitivity of 73.5% and a specificity of 97.4%. IgM-WB showed a sensitivity of 54.8% and a specificity of 94.7%. The IgG-WB and IgM-WB combination increased the sensitivity to 86.5%. The IgM-WB-positive children had a 1.4-fold greater risk of presenting active macular lesions than did those that were IgM-WB-negative. This study showed that the WB assay is a useful tool to confirm a diagnosis of congenital toxoplasmosis and that the IgM-WB-positive results can indicate active macular lesions in newborn infants.

Altered expression of beta-galactosidase-1-like protein 3 (Glb1l3) in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout mouse model of Leber's congenital amaurosis

Purpose: In this study, we investigated the expression of the gene encoding beta-galactosidase (Glb)-1-like protein 3 (Glb1l3), a member of the glycosyl hydrolase 35 family, during retinal degeneration in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout (Rpe65(-/-)) mouse model of Leber congenital amaurosis (LCA). Additionally, we assessed the expression of the other members of this protein family, including beta-galactosidase-1 (Glb1), beta-galactosidase-1-like (Glb1l), and beta-galactosidase-1-like protein 2 (Glb1l2).Methods: The structural features of Glb1l3 were assessed using bioinformatic tools. mRNA expression of Glb-related genes was investigated by oligonucleotide microarray, real-time PCR, and reverse transcription (RT) -PCR. The localized expression of Glb1l3 was assessed by combined in situ hybridization and immunohistochemistry.Results: Glb1l3 was the only Glb-related member strongly downregulated in Rpe65(-/-) retinas before the onset and during progression of the disease. Glb1l3 mRNA was only expressed in the retinal layers and the RPE/choroid. The other Glb-related genes were ubiquitously expressed in different ocular tissues, including the cornea and lens. In the healthy retina, expression of Glb1l3 was strongly induced during postnatal retinal development; age-related increased expression persisted during adulthood and aging.Conclusions: These data highlight early-onset downregulation of Glb1l3 in Rpe65-related disease. They further indicate that impaired expression of Glb1l3 is mostly due to the absence of the chromophore 11-cis retinal, suggesting that Rpe65 deficiency may have many metabolic consequences in the underlying neuroretina.

The neuroretina is a novel mineralocorticoid target: aldosterone up-regulates ion and water channels in Müller glial cells.

Glucocorticoids reduce diabetic macular edema, but the mechanisms underlying glucocorticoid effects are imperfectly elucidated. Glucocorticoids may bind to glucocorticoid (GR) and mineralocorticoid (MR) receptors. We hypothesize that MR activation may influence retinal hydration. The effect of the MR agonist aldosterone (24 h) on ion/water channel expression (real-time PCR, Western blot, immunofluorescence) was investigated on cultured retinal Müller glial cells (RMGs, which contribute to fluid homeostasis in the retina), in Lewis rat retinal explants, and in retinas from aldosterone-injected eyes. We evidenced cell-specific expression of MR, GR, and 11-beta-hydroxysteroid dehydrogenase type II. Aldosterone significantly enhances expression of sodium and potassium channels ENaC-alpha (6.5-fold) and Kir4.1 (1.9-fold) through MR and GR occupancy, whereas aquaporin 4 (AQP4, 2.9-fold) up-regulation is MR-selective. Aldosterone intravitreous injection induces retinal swelling (24% increase compared to sham-injected eyes) and activation of RMGs. It promotes additional localization of Kir4.1 and AQP4 toward apical microvilli of RMGs. Our results highlight the mineralocorticoid-sensitivity of the neuroretina and show that aldosterone controls hydration of the healthy retina through regulation of ion/water channels expression in RMGs. These results provide a rationale for future investigations of abnormal MR signaling in the pathological retina.

The effect of standard and high-fluence corneal cross-linking (CXL) on cornea and limbus.

PURPOSE: When treating peripheral ectatic disease-like pellucid marginal degeneration (PMD), corneal cross-linking with UV-A and riboflavin (CXL) must be applied eccentrically to the periphery of the lower cornea, partly irradiating the corneal limbus. Here, we investigated the effect of standard and double-standard fluence corneal cross-linking with riboflavin and UV-A (CXL) on cornea and corneal limbus in the rabbit eye in vivo. METHODS: Epithelium-off CXL was performed in male New Zealand White rabbits with two irradiation diameters (7 mm central cornea, 13 mm cornea and limbus), using standard fluence (5.4 J/cm(2)) and double-standard fluence (10.8 J/cm(2)) settings. Controls were subjected to epithelial removal and riboflavin instillation, but were not irradiated with UV-A. Following CXL, animals were examined daily until complete closure of the epithelium, and at 7, 14, 21, and 28 days. Animals were killed and a corneoscleral button was excised and processed for light microscopy and immunohistochemistry. RESULTS: For both irradiation diameters and fluences tested, no signs of endothelial damage or limbal vessel thrombosis were observed, and time to re-epithelialization was similar to untreated controls. Histological and immunohistochemical analysis revealed no differences in the p63 putative stem cell marker expression pattern. CONCLUSIONS: Even when using fluence twice as high as the one used in current clinical CXL settings, circumferential UV-A irradiation of the corneal limbus does not alter the regenerative capacity of the limbal epithelial cells, and the expression pattern of the putative stem cell marker p63 remains unchanged. This suggests that eccentric CXL may be performed safely in PMD.

Phenotype of two Consanguineous Autosomic Recessive Retinitis Pigmentosa Families Caused by PDE6A and PDE6B Mutations

Purpose: To assess the clinical phenotype in two consanguineous Tunisian families with non syndromic autosomic recessive retinitis Pigmentosa (RP) caused by a PDE6A and PDE6B mutations.Methods: All accessible familiy members were included. Affected members from each family underwent full ophthalmic examination with best corrected Snellen visual acuity, fundus photography, optical coherence tomography and full field electroretinography. Haplotype analyses were used to test linkage in the family to 20 arRP loci, including ABCA4, LRAT, USH2A, RP29, CERKL, CNGA1, CNGB1, CRB1, EYS, RP28, MERTK, NR2E3, PDE6A, PDE6B, RGR, RHO, RLBP1, TULP1. All exons and intron-exon junctions of candidate genes not excluded by haplotype analysis were PCR amplified and directly sequenced.Results: Two family members were clinically affected with arRP in each pedigree. Age range at baseline was 43 to 54 years (mean age at baseline was 48 years). For all affected members, night blindness appeared since early childhood (at 4-5 years old) without nystagmus but with a severe progression and mild to severe loss of central vision at the second decade. Visual acuity at baseline ranged from 20/500 to 20/63. Kinetic visual field was severely constricted for one patient and unrealizable for the others. Funduscopic examination revealed bone spicule-shaped pigment deposits in the mid periphery along with atrophy of the retina, narrowing of the vessels and waxy optic discs. Tomograms showed macular atrophy in both cases of family A, and macular edema in the patients of family B. ERG showed a loss of both rod and cone responses. Haplotype analysis revealed homozygosity for microsatellites markers flanking PDE6A and PDE6B in family A and B, respectively. Sequencing of PDE6A in family A showed a homozygous R102S mutation. In family B, sequencing identified a D600N homozygous mutation. Both mutations cosegregated within each respective pedigree.Conclusions: For these families, affected members developed a severe form of non syndromic arRP. The two reported mutations have already been described. Our data further contribute to our understanding of genotype-phenotype correlations.

Lentiviral Vector Tropism In Degenerating Retinas

Introduction: In normal mice, lentiviral vector (LV) shows a great efficiency to infect the RPE cells, but transduces retinal neurons more efficiently during development. Here, we investigated the tropism of LV in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the interphotoreceptor matrix (IPM). We tested two different LV-pseudotypes using the VSVG and the Mokola envelopes. Methods: Subretinal injections were performed in wild-type (C57/Bl6) and rhodopsin knockout (Rho-/-) mice. We injected LV-VSVG-EFS-GFPII into 3.3-4.9 month old mice and LV-VSVG-Rho-GFP into 1-1.4 month old mice to target the photoreceptors (PR). LV-MOK-CMV-GFP was injected into 2.4-3.3 months old mice. We sacrificed the animals one week post injection, used immunohistochemistry to identify the transduced cells, and investigated the OLM integrity. Results: Using LV-VSVG-EFS-GFPII into 3.3-4.9 months mice, we observed significant retinal and RPE transduction in Rho-/- mice. However, the retinas showed transduction mainly at the injection's site. We mostly observed GFP+ cells having a Müller cell morphology. Using LV-MOK-CMV-GFP into 2.4-3.3 months mice, we evidenced the same pattern of viral infection, but with more Müller cells targeted by the virus. Using LV-VSVG-Rho-GFP into 1-1.4 month old mice, we don't note any difference between Rho-/- and wild-type mice for transduced cells. The IPM stained with ZO1 appears irregular into the 4.9 months old Rho-/- mice; for the youngest mice (Rho-/- and C57/Bl6), there is no modification of the IPM. Conclusion: The degeneration improves retinal cells transduction due to the alteration of the IPM in old Rho-/- mice. Müller cells seem (by morphological evidences) to be the principal cells expressing the transgene. The LV with Mokola envelope can transduce Müller cells in a degenerating retina with an intact IPM. In 1 month old mice, the degeneration doesn't enhance the transduction in rod PR probably because the IPM is not yet altered. The possibility to target photoreceptors at a later stage of the degeneration is under investigation.

Influence of the ankle arthropathies on the 3D kinematics of the lower limbs during gait: analysis with an ambulatory system.

Introduction: Ankle arthropathy is associated with a decreased motion of the ankle-hindfoot during ambulation. Ankle arthrodesis was shown to result in degeneration of the neighbour joints of the foot. Inversely, total ankle arthroplasty conceptually preserves the adjacent joints because of the residual mobility of the ankle but this has not been demonstrated yet in vivo. It has also been reported that degenerative ankle diseases, and even arthrodesis, do not result in alteration of the knee and hip joints. We present the preliminary results of a new approach of this problem based on ambulatory gait analysis. Patients and Methods: Motion analysis of the lower limbs was performed using a Physilog® (BioAGM, CH) system consisting of three-dimensional (3D) accelerometer and gyroscope, coupled to a magnetic system (Liberty©, Polhemus, USA). Both systems have been validated. Three groups of two patients were included into this pilot study and compared to healthy subjects (controls) during level walking: patients with ankle osteoarthritis (group 1), patients treated by ankle arthrodesis (group 2), patients treated by total ankle prosthesis (group 3). Results: Motion patterns of all analyzed joints over more than 20 gait cycles in each subject were highly repeatable. Motion amplitude of the ankle-hindfoot in control patients was similar to recently reported results. Ankle arthrodesis limited the motion of the ankle-hindfoot in the sagittal and horizontal planes. The prosthetic ankle allowed a more physiologic movement in the sagittal plane only. Ankle arthritis and its treatments did not influence the range of motion of the knee and hip joint during stance phase, excepted for a slight decrease of the hip flexion in groups 1 and 2. Conclusion: The reliability of the system was shown by the repeatability of the consecutive measurements. The results of this preliminary study were similar to those obtained through laboratory gait analysis. However, our system has the advantage to allow ambulatory analysis of 3D kinematics of the lower limbs outside of a gait laboratory and in real life conditions. To our knowledge this is a new concept in the analysis of ankle arthropathy and its treatments. Therefore, there is a potential to address specific questions like the difficult comparison of the benefits of ankle arthroplasty versus arthrodesis. The encouraging results of this pilot study offer the perspective to analyze the consequences of ankle arthropathy and its treatments on the biomechanics of the lower limbs ambulatory, in vivo and in daily life conditions.

Astrocytic dysfunction: insights on the role in neurodegeneration.

For decades, astrocytes have been regarded as passive partners of neurons in central nervous system (CNS) function. Studies of the last 20 years, however, challenged this view by demonstrating that astrocytes possess functional receptors for neurotransmitters and respond to their stimulation via release of gliotransmitters, including glutamate. Notably, astrocytes react to synaptically released neurotransmitters with intracellular calcium ([Ca(2+)]) elevations, which result in the release of glutamate via regulated exocytosis and, possibly, other mechanisms. These findings have led to a new concept of neuron-glia intercommunication where astrocytes play an unsuspected dynamic role by integrating neuronal inputs and modulating synaptic activity. The additional observation that glutamate release from astrocytes is controlled by molecules linked to inflammatory reactions, such as the cytokine tumor necrosis factor alpha (TNFalpha) and prostaglandins (PGs), suggests that glia-to-neuron signalling may be sensitive to changes in the production of these mediators occurring in pathological conditions. Indeed, a local, parenchymal brain inflammatory reaction (neuroinflammation) characterized by astrocytic and microglial activation has been reported in several neurodegenerative disorders, including AIDS dementia complex, Alzheimer's disease and amyotrophic lateral sclerosis. This transition may be accompanied by functional de-regulation and even degeneration of the astrocytes with the consequent disruption of the cross-talk normally occurring between these cells and neurons. Incorrect neuron-astrocyte interactions may be involved in neuronal derangement and contribute to disease development. The findings reported in this review suggest that a better comprehension of the glutamatergic interplay between neurons and astrocytes may provide information about normal brain function and also highlight potential molecular targets for therapeutic interventions in pathology.

Claudin-19 mutations and clinical phenotype in Spanish patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsorption in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease.

Neuronal and nonneuronal quantitative BACE immunocytochemical expression in the entorhinohippocampal and frontal regions in Alzheimer's disease.

In this study, we quantitatively investigated the expression of beta-site amyloid precursor protein cleaving enzyme (BACE) in the entorhinohippocampal and frontal cortex of Alzheimer's disease (AD) and old control subjects. The semiquantitative estimation indicated that the intensity of BACE overall immunoreactivity did not differ significantly between AD and controls, but that a significantly stronger staining was observed in the hippocampal regions CA3-4 compared to other regions in both AD patients and controls. The quantitative estimation confirmed that the number of BACE-positive neuronal profiles was not significantly decreased in AD. However, some degeneration of BACE-positive profiles was attested by the colocalization of neurons expressing BACE and exhibiting neurofibrillary tangles (NFT), as well as by a decrease in the surface area of BACE-positive profiles. In addition, BACE immunocytochemical expression was observed in and around senile plaques (SP), as well as in reactive astrocytes. BACE-immunoreactive astrocytes were localized in the vicinity or close to the plaques and their number was significantly increased in AD entorhinal cortex. The higher amount of beta-amyloid SP and NFT in AD was not correlated with an increase in BACE immunoreactivity. Taken together, these data accent that AD progression does not require an increased neuronal BACE protein level, but suggest an active role of BACE in immunoreactive astrocytes. Moreover, the strong expression in controls and regions less vulnerable to AD puts forward the probable existence of alternate BACE functions.

Neuroprotection for optic nerve disorders.

The concept that optic nerve fiber loss might be reduced by neuroprotection arose in the mid 1990s. The subsequent research effort, focused mainly on rodent models, has not yet transformed into a successful clinical trial, but provides mechanistic understanding of retinal ganglion cell death and points to potential therapeutic strategies. This review highlights advances made over the last year. In excitotoxicity and axotomy models retinal ganglion cell death has been shown to result from a complex interaction between retinal neurons and Müller glia, which release toxic molecules including tumor necrosis factor alpha. This counteracts neuroprotection by neurotrophins such as nerve growth factor, which bind to p75NTR receptors on Müller glia stimulating the toxic release. Another negative effect against neurotrophin-mediated protection involves the action of LINGO-1 at trkB brain-derived neurotrophic factor (BDNF) receptors, and BDNF neuroprotection is enhanced by an antagonist to LINGO-1. As an alternative to pharmacotherapy, retinal defences can be stimulated by exposure to infrared radiation. The mechanisms involved in glaucoma and other optic nerve disorders are being clarified in rodent models, focusing on retrograde degeneration following axonal damage, excitotoxicity and inflammatory/autoimmune mechanisms. Neuroprotective strategies are being refined in the light of the mechanistic understanding.

Utilidad de la tomografía de coherencia óptica (OCT) previa a la cirugía de cataratas en el diagnóstico de maculopatías.

Dada la fuerte presión por parte de los pacientes a la hora de conseguir buenos resultados visuales tras la cirugía de cataratas, se considera de gran importancia la correcta evaluación del fondo de ojo para el diagnóstico de posibles maculopatías que puedan ensombrecer el pronóstico postoperatorio. Por ello, con este trabajo se pretende valorar si la tomografía de coherencia óptica macular es un método diagnóstico más fiable y eficiente que la tradicional exploración funduscópica realizada por el propio oftalmólogo, y si debe convertirse en una prueba complementaria imprescindible en las visitas preoperatorias.