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During analysis of glia in wholemount aged human retinas, frequent projections onto the vitreal surface of the inner limiting membrane (ILM) were noted. The present study characterized these preretinal glial structures. The amount of glial cells on the vitreal side of the ILM was compared between eyes with age-related macular degeneration (AMD) and age-matched control eyes. Retinal wholemounts were stained for markers of retinal astrocytes and activated Müller cells (glial fibrillary acidic protein, GFAP), Müller cells (vimentin, glutamine synthetase) and microglia/hyalocytes (IBA-1). Retinal vessels were labeled with UEA lectin. Images were collected using a Zeiss LSM 710 confocal microscope. Retinas were then cryopreserved. Laminin labeling of cryosections determined the location of glial structures in relation to the ILM. All retinas investigated herein had varied amounts of preretinal glia. These glial structures were classified into three groups based on size: sprouts, blooms, and membranes. The simplest of the glial structures observed were focal sprouts of singular GFAP-positive cells or processes on the vitreal surface of the ILM. The intermediate structures observed, glial blooms, were created by multiple cells/processes exiting from a single point and extending along the vitreoretinal surface. The most extensive structures, glial membranes, consisted of compact networks of cells and processes. Preretinal glia were observed in all areas of the retina but they were most prominent over large vessels. While all glial blooms and membranes contained vimentin and GFAP-positive cells, these proteins did not always co-localize. Many areas had no preretinal GFAP but had numerous vimentin only glial sprouts. In double labeled glial sprouts, vimentin staining extended beyond that of GFAP. Hyalocytes and microglia were detected along with glial sprouts, blooms, and membranes. They did not, however, concentrate in the retina below these structures. Cross sectional analysis identified small breaks in the ILM above large retinal vessels through which glial cells exited the retina. Preretinal glial structures of varied sizes are a common occurrence in aged retinas and, in most cases, are subclinical. While all retinal glia are found in blooms, vimentin labeling suggests that Müller cells form the leading edge. All retinas investigated from eyes with active choroidal neovascularization (CNV) had extensive glial membranes on the vitreal surface of the ILM. Although these structures may be benign, they may exert traction on the retina as they spread along the vitreoretinal interface. In cases with CNV, glial cells in the vitreous could bind intravitreally injected anti-vascular endothelial growth factor. These preretinal glial structures indicate the remodeling of both astrocytes and Müller cells in aged retinas, in particular those with advanced AMD. [ABSTRACT FROM AUTHOR]

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration ( AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 ( LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial ( RPE) cells of Cryba1 conditional knockout ( cKO) mice, but not in control mice. We have recently shown that these mice, which lack βA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine ( C- C motif) ligand 2 ( CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology. [ABSTRACT FROM AUTHOR]

VEGF165b is an anti-angiogenic form of VEGF165 produced by alternative splicing. The localization of pro-angiogenic VEGF165 and anti-angiogenic VEGF165b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF165 immunoreactivity at 7WG and very little VEGF165b. Both forms were elevated at 12WG. VEGF165 then decreased around 17WG when the TVL regresses but VEGF165b remained elevated. In choroid, VEGF165 was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF165b was apical and VEGF165 basal in RPE. Diffuse VEGF165 immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF165b was present in most CXCR4+ progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF165 was present in nerve fibers and VEGF165b in the inner Muller cell process. The localization of VEGF165b was distinctly different from VEGF165 both spatially and temporally and it was often associated with nucleus in progenitors. Developmental Dynamics 241:595-607, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin cd, a basement membrane protein. Despite normal localization of laminin α1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial twohybrid system. Therefore, this mutation could affect interactions between laminin α1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1tm1.1Olf, was generated that exhibits a similar but more severe retinal phenotype than that seen in nmj223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease. [ABSTRACT FROM AUTHOR]

We have previously reported that in the Nuc1 rat, which has a spontaneous mutation in Cryba1 (the gene encoding βA3/A1-crystallin), astrocytes exhibit decreased Notch signaling, leading to reduced promoter activity for glial fibrillary acidic protein (GFAP). Interestingly, in both Nuc1 astrocytes and in wild type astrocytes following knockdown of Cryba1, vascular endothelial growth factor (VEGF) secretion is decreased. This has led us to explore signaling mediators that could be regulated by βA3/A1-crystallin to modulate both GFAP and VEGF. Several studies have shown that the signal transducer and activator of transcription 3 (STAT3) is involved in the co-regulation of GFAP and VEGF. We show that STAT3 and βA3/A1-crystallin may co-regulate each other in astrocytes. Such co-regulation would create a positive feedback circuit; i.e., in the cytosol of astrocytes, βA3/A1-crystallin is necessary for the phosphorylation of STAT3, which then dimerizes and translocates to the nucleus to form DNA-binding complexes, activating transcription of Cryba1. This stoichiometric co-regulation of STAT3 and Cryba1 could potentiate expression of GFAP and secretion of VEGF, both of which are essential for maintaining astrocyte and blood vessel homeostasis in the retina. Consistent with this idea, Cryba1 knockout mice exhibit an abnormal astrocyte pattern and defective remodeling of retinal vessels. [ABSTRACT FROM AUTHOR]

Abstract The choriocapillaris is the source of nutrients and oxygen for photoreceptors, which consume more oxygen per gram of tissue than any other cell in the body. The purpose of this study was to evaluate and compare the ultrastructure of the choriocapillaris and its transport systems in patients with and without age-related macular degeneration (AMD). Ultrastructural changes were also evaluated in subjects that were homozygous for polymorphisms in high risk CFH alleles (Pure 1) only or homozygous only for high risk ARMS2/HTRA1 (Pure 10) alleles. Tissue samples were obtained from the macular region of forty male (n = 24) and female (n = 16) donor eyes and prepared for ultrastructural studies with transmission electron microscopy (TEM). The average age of the aged donors was 74 ± 7.2 (n = 30) and the young donors 31.7 ± 11.25 (n = 10). There was no significant difference in average ages between the adult groups. TEM images of the capillaries in the choriocapillaris (CC) were taken at 4,000X and 25,000X and used to measure the area of endothelial cell somas, the number of fenestrations, and area of caveolae within the endothelial cells per length of Bruchs membrane (BrMb). The Student t-test and Wilcoxon sum rank test were used to determine significant differences. There was no significant difference between young subjects and aged controls in any of the morphological criteria assessed. There was a significant decrease in the number of fenestrations/mm of BrMb in atrophic areas of GA eyes (p = 0.007) when compared with aged control eyes. A significant increase was found in the caveolae area as a percent of the endothelial cell soma of capillaries from GA subjects as compared with the controls (p = 0.03). Loss of capillary segments in choriocapillaris was also evident, especially in areas of geographic atrophy and CNV. In eyes from patients with sequence variations, the capillary endothelial cells often appeared degenerative and exhibited atypical fenestrations and pericytes covering the blood vessels. Subjects that were homozygous for polymorphisms in high risk CFH alleles only had more fenestrations/mm of BrMb than subjects that were homozygous only for high risk ARMS2/HTRA1 alleles (p = 0.04), while the latter had greater caveolae area/endothelial cell area than the former (p = 0.007). This study demonstrated an attenuation of CC and a significant decline in the two major transport systems in CC endothelial cells in AMD. This may contribute to drusen deposition, nutrient transport, and vision loss in AMD subjects. Highlights • Choriocapillaris is the source of nutrients and oxygen for photoreceptors and RPE. • No difference in the TEM criteria evaluated between young and old controls. • Significant reduction in the number of fenestrations in geographic atrophy (GA). • The percentage of endothelial cell area that was caveolae was increased in GA. • There was attenuation of CC and dysfunction in its transport systems in AMD. [ABSTRACT FROM AUTHOR]

Mutations in the Rhodopsin (Rho) gene can lead to autosomal dominant retinitis pigmentosa (RP) in humans. Transgenic mouse models with mutations in Rho have been developed to study the disease. However, it is difficult to know the source of the photoreceptor (PR) degeneration in these transgenic models because overexpression of wild type (WT) Rho alone can lead to PR degeneration. Here, we report two chemically mutagenized mouse models carrying point mutations in Rho (Tvrm1 with an Y102H mutation and Tvrm4 with an 1307N mutation). Both mutants express normal levels of rhodopsin that localize to the PR outer segments and do not exhibit PR degeneration when raised in ambient mouse room lighting; however, severe PR degeneration is observed after short exposures to bright light. Both mutations also cause a delay in recovery following bleaching. This defect might be due to a slower rate of chromophore binding by the mutant opsins compared with the WT form, and an increased rate of transducin activation by the unbound mutant opsins, which leads to a constitutive activation of the phototransduction cascade as revealed by in vitro biochemical assays. The mutant-free opsins produced by the respective mutant Rho genes appear to be more toxic to PRs, as Tvrm1 and Tvrm4 mutants lacking the 11-cis chromophore degenerate faster than mice expressing WT opsin that also lack the chromophore. Because of their phenotypic similarity to humans with Bi Rho mutations, these mutants will be important tools in examining mechanisms underlying Rho-induced RP and for testing therapeutic strategies. [ABSTRACT FROM AUTHOR]

Loss of choriocapillaris (CC) in advanced age-related macular degeneration (AMD) is well documented but changes in early AMD have not been quantified. Postmortem eyes from donors with clinically documented early AMD were examined in choroidal whole mounts to determine the area, pattern, and severity of CC loss. Choroids from postmortem human eyes without AMD (n = 7; mean age = 86.1) and from eyes with a Grade 2 clinical classification of early AMD (n = 7; mean age = 87) were immunolabeled with Ulex europaeus agglutinin (UEA) lectin-FITC to stain blood vessels. Whole mounts were imaged using confocal microscopy and image analysis was performed to determine the area of vascular changes and density of vasculature (percent vascular area, %VA). All areas evaluated had a complete RPE monolayer upon gross examination. In age-matched control eyes, the CC had broad lumens and a homogenous pattern of freely interconnecting capillaries. The mean %VA ± standard deviation in submacula of control subjects was 78.1 ± 3.25%. In eyes with early AMD, there was a significant decrease in mean %VA to 60.1 ± 10.4% (p < 0.0001). The paramacular %VA was not significantly different in eyes with or without AMD. The area of submacular choroid affected by CC dropout was 0.04 ± 0.09 mm2 in control eyes. In eyes with early AMD, the mean area affected by CC dropout was significantly increased (10.4 ± 6.1 mm2; p < 0.001). In some cases, incipient neovascular buds were observed at the border of regions with CC dropout in early AMD choroids. In conclusion, UEA lectin-labeled choroidal whole mounts from donors with clinically documented early AMD has provided a unique opportunity to examine regional changes in vascular pathology associated with choriocapillaris. The study demonstrated attenuation of submacular CC in early AMD subjects but no vascular pathology was observed outside the submacular region. While the affected area in some eyes was quite extensive histologically, these changes may not be detectable clinically using standard in vivo imaging. • There is significant loss of submacular choriocapillaris in subjects with early AMD compared to control subjects. • On the contrary, there was no vascular pathology in paramacular choriocapillaris of the AMD subjects. • Attenuation of choriocapillaris was associated with arteriosclerotic and hypertensive changes in Sattler's layer arterioles. • Some of the areas of choriocapillaris loss had early choroidal neovascularization formations. [ABSTRACT FROM AUTHOR]