Retinal gene expression changes related to IOP exposure and axonal loss in DBA/2J mice.

PURPOSE: To determine the effects of cumulative IOP exposure and axonal damage on retinal gene expression in DBA/2 mice. METHODS: DBA/2J, DBA/2J(pe) (pearl), and C57BL/6 mice from 3 to 12 months of age were used. IOP was measured with a rebound tonometer, and optic nerve (ON) damage was determined by grading of ON sections. Retinal RNA was subjected to microarray analysis. Comparisons explored the effects of cumulative IOP exposure (cIOPx) as well as ON damage (ONd) in the DBA/2J animals compared with that in the C57BL/6 and pearl mice. RT-PCR was performed to confirm some of the genes and bioinformatic analysis to identify affected gene networks. RESULTS: Microarrays revealed that an increasing number of genes were up- or downregulated in 9- and 12-month DBA/2J mice with various degrees of ONd. A smaller number of genes were expressed differentially between eyes with different cIOPx at the same age, from 6 months on. Expression of 1385 and 1133 genes differed between DBA/2J animals and C57BL/6 or pearl mice, respectively, and some them were confirmed by RT-PCR. Bioinformatics analysis identified functional gene networks, including members of the complement system, that appeared to be related to cIOPx, ONd, or both. CONCLUSIONS: Gene expression changes occur in retinas of DBA/2 mice with various amounts of cIOPx as well as ONd. Genes involved, code for proteins with diverse cellular functions and include among others the complement system. cIOPx and ONd affect common as well as unique gene sets.

Alpha2-adrenergic receptors and their core involvement in the process of axonal growth in retinal explants.

PURPOSE: To determine the patterns of α2-adrenergic receptor (α2-AR) subtype expression in normal and degenerated retinas and to analyze the response of these receptors to the α2-AR agonist brimonidine tartrate (BT). METHODS: The binding characteristics of α2-ARs in the retina were evaluated in experimental and matching sham groups by in vitro quantitative autoradiographic saturation with [(3)H]-clonidine. Retinal explants from juvenile and adult rats with either elevated intraocular pressure or after optic nerve crush (ONC) were cultured with BT over 96 hours in vitro to analyze the effects of BT on axonal growth by videomicroscopy and axon counting. Changes in retinal protein expression by BT were monitored by two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). RESULTS: The total number of α2-ARs in the retina increased significantly after ONC compared with the sham group. BT supported axonal growth in the juvenile, glaucomatous, and injured retinas (P < 0.004) most effectively at a concentration of 0.001 mg/mL, without influencing the axonal growth rate. Immediate supplementation of BT was more effective than delayed supplementation (P < 0.001). Proteomic analysis revealed treatment-specific expression patterns of glial fibrillary acidic protein (GFAP), glucose-related protein (GRP)58, platelet-activating factor (PAF), and laminin-binding protein (LBP). CONCLUSIONS: These data are the first to show differences in α2-AR expression in normal and degenerated retinas. BT supports neuronal growth in cultured retinal pieces, suggesting that α2-ARs play a role in retinal metabolism.

From the protein to the graph: how to quantify immunohistochemistry staining of the skin using digital imaging.

Quantitative immunohistochemistry is needed in order to reliably and accurately assess the expression of cellular proteins in tissue. Skin is a difficult tissue for automated image analysis due to its heterogeneous composition and its architecture. In the present study we used a psoriatic skin model to compare the expression of p53 and bcl-2 before and after treatment with anti-tumor necrosis factor-alpha using digital image analysis. Digital photomicrographs were acquired and analyzed with Scion image software in order to obtain the fraction of p53 and bcl-2 immunoreactive cells' area out of the total area investigated. Statistical analysis with ANOVA revealed a significant increase of p53 expression and a decrease of bcl-2 expression in all 3 epidermal layers during the course of therapy (p<0.001). The results were in line with the conventional histopathological evaluation using an arbitrary scale to grade the extent and intensity of the staining. So, the estimation of volume fraction of immunohistochemically labelled cells in skin tissue can be performed easily and rapidly using commonly available image analysis software and provides reproducible and unbiased numerical estimations of the amount of cell labelling.

Early post-hatching sex differences in cell proliferation and survival in the quail telencephalic ventricular zone and intermediate medial mesopallium.

Previous studies indicated that avian telencephalic areas related to learned behavior, such as song perception and production, are sexually dimorphic. Our study focused on the eventual occurrence of dimorphism in the intermediate medial mesopallium, an area associated with learning in non-singing birds. During early post-hatching life (days 1 and 5) cell proliferation and survival of newborn cells were studied by means of 5-bromo-2-deoxy-uridine immunocytochemistry. Programmed cell death (apoptosis) was investigated at post-hatching day 10. The ventricular zone, intermediate medial part of mesopallium and lateral septal area was analyzed using stereological methods for cell counts. Short-term experiments revealed significantly higher numbers of newborn cells in male ventricular zone of mesopallium compared to female at post-hatching day 1. Long-term survival until post-hatching day 20 showed significantly higher numbers of labeled cells in the male compared to female intermediate medial part of mesopallium, which is the final destination of migrating cells born in the overlying ventricular zone. The vast majority of these early post-hatching newborn cells residing in the intermediate medial part of mesopallium expressed a neuronal phenotype. In addition to neurogenesis, higher numbers of apoptotic figures were found in the male intermediate medial part of mesopallium at post-hatching day 10, suggesting that cell death plays a role in the control of telencephalic regional cell density in males. Our findings indicate that sex-specific mechanisms possibly stimulate increased cell genesis and survival, as well as the counteracting event of increased apoptotic cell death that characterized the male intermediate medial part of mesopallium.