Primary congenital and developmental glaucomas.

Glaucoma is the leading cause of irreversible blindness worldwide. Although most glaucoma patients are elderly, congenital glaucoma and glaucomas of childhood are also important causes of visual disability. Primary congenital glaucoma (PCG) is isolated, non-syndromic glaucoma that occurs in the first three years of life and is a major cause of childhood blindness. Other early-onset glaucomas may arise secondary to developmental abnormalities, such as glaucomas that occur with aniridia or as part of Axenfeld-Rieger syndrome. Congenital and childhood glaucomas have strong genetic bases and disease-causing mutations have been discovered in several genes. Mutations in three genes (CYP1B1, LTBP2, TEK) have been reported in PCG patients. Axenfeld-Rieger syndrome is caused by mutations in PITX2 or FOXC1 and aniridia is caused by PAX6 mutations. This review discusses the roles of these genes in primary congenital glaucoma and glaucomas of childhood.

Transgenic TBK1 mice have features of normal tension glaucoma.

Duplication of the TBK1 gene is associated with 1-2% of normal tension glaucoma, a common cause of vision loss and blindness that occurs without grossly abnormal intraocular pressure. We generated a transgenic mouse that has one copy of the human TBK1 gene (native promoter and gene structure) incorporated into the mouse genome (Tg-TBK1). Expression of the TBK1 transgene in the retinae of these mice was demonstrated by real-time PCR. Using immunohistochemistry TBK1 protein was predominantly localized to the ganglion cell layer of the retina, the cell type most affected by glaucoma. More intense TBK1 labelling was detected in the retinal ganglion cells (RGCs) of Tg-TBK1 mice than in wild-type littermates. Tg-TBK1 mice exhibit the cardinal sign of glaucoma, a progressive loss of RGCs. Hemizygous Tg-TBK1 mice (with one TBK1 transgene per genome) had a 13% loss of RGCs by 18 months of age (P = 1.5 × 10-8). Homozygous Tg-TBK1 mice had 7.6% fewer RGCs than hemizygous Tg-TBK1 mice and 20% fewer RGCs than wild-type mice (P = 1.9 × 10-5) at 6 months of age. No difference in intraocular pressures was detected between Tg-TBK1 mice and wild-type littermates as they aged (P > 0.05). Tg-TBK1 mice with extra doses of the TBK1 gene recapitulate the phenotype of normal tension glaucoma in human patients with a TBK1 gene duplication. Together, these studies confirm the pathogenicity of the TBK1 gene duplication in human glaucoma and suggest that excess production of TBK1 kinase may have a role in the pathology of glaucoma.

RetFM-J, an ImageJ-based module for automated counting and quantifying features of nuclei in retinal whole-mounts.

The present article introduces RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of the inner retina from H&E-stained whole-mounted retinas. To illustrate performance, computer-derived outputs were analyzed in inbred C57BL/6J mice. Automated characterization yielded computer-derived outputs that closely matched manual counts. As a method using open-source software that is freely available, inexpensive staining reagents that are robust, and imaging equipment that is routine to most laboratories, RetFM-J could be utilized in a wide variety of experiments benefiting from high-throughput, quantitative, uniform analyses of total cellularity in the inner retina.

Quantitative measurement of retinal ganglion cell populations via histology-based random forest classification.

The inner surface of the retina contains a complex mixture of neurons, glia, and vasculature, including retinal ganglion cells (RGCs), the final output neurons of the retina and primary neurons that are damaged in several blinding diseases. The goal of the current work was two-fold: to assess the feasibility of using computer-assisted detection of nuclei and random forest classification to automate the quantification of RGCs in hematoxylin/eosin (H&E)-stained retinal whole-mounts; and if possible, to use the approach to examine how nuclear size influences disease susceptibility among RGC populations. To achieve this, data from RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of H&E-stained whole-mounted retinas, were used in conjunction with a manually curated set of images to train a random forest classifier. To test performance, computer-derived outputs were compared to previously published features of several well-characterized mouse models of ophthalmic disease and their controls: normal C57BL/6J mice; Jun-sufficient and Jun-deficient mice subjected to controlled optic nerve crush (CONC); and DBA/2J mice with naturally occurring glaucoma. The result of these efforts was development of RetFM-Class, a command-line-based tool that uses data output from RetFM-J to perform random forest classification of cell type. Comparative testing revealed that manual and automated classifications by RetFM-Class correlated well, with 83.2% classification accuracy for RGCs. Automated characterization of C57BL/6J retinas predicted 54,642 RGCs per normal retina, and identified a 48.3% Jun-dependent loss of cells at 35 days post CONC and a 71.2% loss of RGCs among 16-month-old DBA/2J mice with glaucoma. Output from automated analyses was used to compare nuclear area among large numbers of RGCs from DBA/2J mice (n = 127,361). In aged DBA/2J mice with glaucoma, RetFM-Class detected a decrease in median and mean nucleus size of cells classified into the RGC category, as did an independent confirmation study using manual measurements of nuclear area demarcated by BRN3A-immunoreactivity. In conclusion, we have demonstrated that histology-based random forest classification is feasible and can be utilized to study RGCs in a high-throughput fashion. Despite having some limitations, this approach demonstrated a significant association between the size of the RGC nucleus and the DBA/2J form of glaucoma.

Evaluation of sFLT1 protein levels in human eyes with the FLT1 rs9943922 polymorphism.

PURPOSE: Age-related macular degeneration (AMD) is a devastating disease characterized by central vision impairment in individuals with advanced age. Neovascular AMD is a form of end-stage disease in which choroidal vessel outgrowth occurs beneath the retina. While many hypotheses have been raised as to what triggers the formation of pathological choroidal neovascular membranes, the exact mechanism for their initiation remains unresolved. Polymorphisms in the FLT1 gene have previously been associated with neovascular AMD risk, including the rs9943922 single nucleotide polymorphism (SNP). Here, we aimed to determine the association between the high-risk FLT1 genotype and FLT1 protein levels in human retina or retinal pigment epithelium (RPE)/choroid tissue. METHODS: Retina and RPE/choroid tissue from 10 human donor eyes was selected from a collection of eyes genotyped for the rs9943922 SNP. Differences in soluble and membrane bound FLT1 protein levels were assessed for retina versus RPE/choroid donor tissue using ELISA and Western blotting analyses. Genotype-associated changes in FLT1 protein levels were also evaluated. RESULTS: We found soluble FLT1 levels in the RPE/choroid tissue to be approximately three times higher than that of the retina (p < 0.001), while both samples have similar levels of the membrane bound form. When tissue with the rs9943922 SNP was compared with controls, no significant genotypic differences in FLT1 protein levels were observed. CONCLUSIONS: Based on these data, we conclude that the rs9943922 SNP in the FLT1 gene does not result in a large difference in FLT1 protein levels, regardless of whether it is the soluble or the membrane bound form.

Neuroprotection supports signal processing in the hippocampus of Syrian hamsters, a facultative hibernator.

Studies on several species of mammalian seasonal hibernators (those hibernating only in winter) show that their neurons are more tolerant to hypoxia than those in non-hibernating species. Such tolerance has not been studied in facultative hibernators [e.g., Syrian hamsters (Mesocricetus auratus)], which can hibernate at any time of year. We tested the hypotheses that, when exposed to hypoxia, hamster hippocampal pyramidal cells more effectively support signal processing than do rat hippocampal neurons and this protection is enhanced in slices from hibernating versus non-hibernating hamsters and as temperature decreases. Population spike amplitudes (PSAs) were recorded from CA1 pyramidal cells. Slices were perfused in oxygenated artificial cerebral spinal fluid (O(2)ACSF) to establish a baseline. Oxygen was then replaced by nitrogen (N(2)ACSF) for 15 min, followed by a 30-min recovery period in O(2)ACSF. Three minutes after slices were returned to O(2)ACSF, PSAs recovered to 62.4 ± 6.8% of baseline in 15 slices from 8 non-hibernating hamsters but only to 22.7 ± 5.6% in 17 slices from 5 rats. Additionally, PSA recovery was greater in slices from hibernating than non-hibernating hamsters and recovery increased as temperature decreased. These significant differences (P ≤ 0.05) suggest Syrian hamsters are a useful model for studying naturally occurring neuroprotective mechanisms.