Hypoxia inducible factor-1α (HIF-1α) and some HIF-1 target genes are elevated in experimental glaucoma.

Low levels of hypoxia have been suggested to be a mechanism of retinal damage in glaucoma. To test the hypothesis that the activation of the hypoxia-responsive transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) is involved in the pathophysiology of glaucoma, we used a rat model of glaucoma to study (1) HIF-1alpha retinal protein levels by immunoblot analysis, (2) cellular localization of HIF-1alpha in the retina by immunohistochemistry, and (3) expression of retinal HIF-1 gene targets by quantitative real-time polymerase chain reaction. Glaucoma was unilaterally induced in rats by injecting hypertonic saline in episcleral veins. We find that HIF-1alpha protein was increased in the retina following elevation of intraocular pressure, specifically in Müller glia and astrocytes but not in activated microglia. Eight established HIF-1 target genes were measured in experimental glaucoma. Retinal Epo, Flt-1, Hsp-27, Pai-1, and Vegfa mRNA levels were increased and Et-1, Igf2, and Tgfbeta3 levels were decreased in the glaucomatous retinas. Thus, the increase in HIF-1alpha levels in Müller glia and astrocytes is accompanied by a marked up regulation of some, but not all, HIF-1 transcriptional targets. These data support the hypothesis that HIF-1alpha becomes transcriptionally active in astrocytes and Müller cells but not microglia or neurons in glaucoma, arguing against a global hypoxia stimulus to the retina.

Efficient estimation of retinal ganglion cell number: a stereological approach.

Retinal ganglion cells (RGCs) are the only output neurons of the retina, and their degeneration after damage to the optic nerve or in glaucoma is a well established system for studying apoptosis in the central nervous system. Frequently used procedures for assessing RGC number in retinal flat mounts suffer from two problems: RGC densities are not uniform across retinal flat mounts, and density measures may therefore not reflect total number, and flat mounts do not allow efficient use of tissue. To overcome these problems we developed a stereological method for efficiently assessing RGC number in cryostat sections of the retina. We empirically demonstrate that only approximately 1:20 sections need be assessed to accurately estimate the total number of RGCs in the rat retina, providing ample tissue for additional studies in the same retina and saving considerably on more exhaustive sampling strategies. Using this method, we estimate that there are 86,282+/-4759 RGCs in the normal Brown Norway rat retina. These counts match well with estimates of axon counts in optic nerve. In a pilot study of experimental glaucoma, we determined a reduction of RGCs to 53,862+/-4272 (p<0.05). The current technique should prove advantageous to assess neuroprotective strategies in these experimental models.

Expression of mRNA for glutamate receptor subunits distinguishes the major classes of retinal neurons, but is less specific for individual cell types.

PURPOSE: To investigate the expression of ionotropic glutamate receptor subunits by retinal neurons, to assess the extent to which different functional types of retinal neurons are characterized by the expression of the receptor subtypes. METHODS: Rod photoreceptor cells and bipolar cells were identified in retina dissociates. Amacrine cells were identified in dissociates from transgenic mice or by staining with an antibody against the extracellular carbohydrate epitope CD15. Ganglion cells were identified by retrograde axonal transport of FITC-dextran or by green fluorescent protein (GFP) fluorescence in a transgenic strain. We examined the receptors simultaneously using non-quantitative single-cell reverse transcriptase polymerase chain reaction for GluR1-R4 (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors), GluR5-R7, and KA1 and 2 (kainate receptors), delta1 and delta2 subunits, and the N-methyl-D-aspartate (NMDA) receptor subunits NR1, 2a-d, and 3a. RESULTS: The expression of glutamate receptors on bipolar cells and rod photoreceptors was limited: Neither expressed functional NMDA receptors, and rods were also negative for AMPA receptors. The sample of ganglion cells included examples of many ganglion cell types; these were distinguished morphologically using quantitative parameters defined in a previous cluster analysis. All types of ionotropic glutamate receptors were found to be expressed on ganglion cells. The iGluR subunits GluR4, KA2, delta1, and NR1 were expressed on almost all ganglion cells examined. CONCLUSIONS: Despite the heterogeneity of ganglion cell types, differences among them in this PCR-based method were minor. Thus, retinal interneurons are characterized by expression of distinctive glutamate receptor types, but functional differences among ganglion cells seem to be reflected instead in the amounts as well as spatial distributions of a widely expressed group of receptors.

Organotypic culture of physiologically functional adult mammalian retinas.

BACKGROUND: The adult mammalian retina is an important model in research on the central nervous system. Many experiments require the combined use of genetic manipulation, imaging, and electrophysiological recording, which make it desirable to use an in vitro preparation. Unfortunately, the tissue culture of the adult mammalian retina is difficult, mainly because of the high energy consumption of photoreceptors. METHODS AND FINDINGS: We describe an interphase culture system for adult mammalian retina that allows for the expression of genes delivered to retinal neurons by particle-mediated transfer. The retinas retain their morphology and function for up to six days- long enough for the expression of many genes of interest-so that effects upon responses to light and receptive fields could be measured by patch recording or multielectrode array recording. We show that a variety of genes encoding pre- and post-synaptic marker proteins are localized correctly in ganglion and amacrine cells. CONCLUSIONS: In this system the effects on neuronal function of one or several introduced exogenous genes can be studied within intact neural circuitry of adult mammalian retina. This system is flexible enough to be compatible with genetic manipulation, imaging, cell transfection, pharmacological assay, and electrophysiological recordings.

Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice.

Using a variety of double and triple labeling techniques, we have reevaluated the death of retinal neurons in a mouse model of hereditary glaucoma. Cell-specific markers and total neuron counts revealed no cell loss in any retinal neurons other than the ganglion cells. Within the limits of our ability to define cell types, no group of ganglion cells was especially vulnerable or resistant to degeneration. Retrograde labeling and neurofilament staining showed that axonal atrophy, dendritic remodeling, and somal shrinkage (at least of the largest cell types) precedes ganglion cell death in this glaucoma model. Regions of cell death or survival radiated from the optic nerve head in fan-shaped sectors. Collectively, the data suggest axon damage at the optic nerve head as an early lesion, and damage to axon bundles would cause this pattern of degeneration. However, the architecture of the mouse eye seems to preclude a commonly postulated source of mechanical damage within the nerve head.

CD15 immunoreactive amacrine cells in the mouse retina.

The mouse retina has become an important model in vision research, mainly because of the wide availability of transgenic animals. In order to study cell function and connectivity in the inner retina, antibodies that differentially stain one cell type, or a small number of cell types, are helpful as markers. Here we characterize the CD15 (3[alpha1-3]-fucosyl-N-acetyl-lactosamine)-positive cells in the mouse retina using immunofluorescence confocal microscopy and reverse-transcription polymerase chain reaction. CD15 immunoreactivity was observed in two distinct types of amacrine cells and, faintly, in some cone bipolar cells. Type I CD15+ amacrine cells are GABAergic wide-field cells that stratify in lamina 3 and 4/5 of the inner plexiform layer. Type II CD15+ amacrine cells are also GABAergic and costratify with the dopaminergic tyrosine hydroxylase-positive cells in lamina 1 of the inner plexiform layer. The densities of types I and II CD15+ amacrine cells in mid-periphery were 258 cells/mm(2) and 274 cells/mm(2). Double labeling with several other markers for amacrine cell types showed that neither type belongs to another previously identified subpopulation of amacrine cells. Single-cell RT-PCR showed that CD15+ amacrine cells coexpress several AMPA receptors - GluR1, GluR2, and GluR4 being the most common combination.

Comparison of hedgehog and patched-1 protein expression in the cranial sutures of craniosynostotic and wild-type rabbits.

Craniosynostosis is characterized by premature fusion of the cranial sutures. At the molecular level, mutations in homeobox genes, transcription factors, and growth factor receptors have been implicated in the pathogenesis of this disorder, but the specific etiologic pathways have not yet been elucidated. To further study the molecular biology behind craniosynostosis, perisutural tissues in a unique rabbit model with congenital delayed-onset coronal craniosynostosis were examined for the presence of the hedgehog family of growth factors and their receptor, patched-1. Expression of desert hedgehog, Indian hedgehog, sonic hedgehog, and patched-1 was evaluated in four areas: suture, endosteum, periosteum, and osteocytes, using immuno-histochemistry (n = 8). Protein levels in affected animals were compared with protein levels in wild-type control rabbits (n = 8). Overall, sonic hedgehog, Indian hedgehog, and patched-1 protein levels were greater in affected animals. Specifically, areas of increased staining were seen along the bony interface of the endosteum and periosteum and in the osteocytes of the synostotic rabbits. Interestingly, in the suture, increased levels of Indian hedgehog and sonic hedgehog, but not patched-1, were seen. There was minimal expression of desert hedgehog in both rabbit types. The increased overall presence of hedgehog and patched-1 proteins in synostotic rabbits may be a reactive change to the disorder or part of the pathogenic process. Although the specific cause cannot be determined from the data, it is clear that the molecular milieu of the cranial sutures in synostotic rabbits is markedly different from that of wild-type rabbits.

Changes in the protein expression of hedgehog and patched-1 in perisutural tissues induced by cranial distraction.

With the modern emphasis on minimally invasive therapies, the concept of distraction is being applied in the treatment of craniosynostosis. Although specific genetic mutations have been identified in craniosynostotic patients, changes in the gene expression induced by cranial distraction have not yet been explored. The effects of cranial distraction on hedgehog and patched-1 expression were evaluated in a rabbit model for craniosynostosis. Rabbits (n = 8) were divided into four groups: affected rabbits, wild-type rabbits, affected rabbits subject to cranial distraction, and wild-type rabbits subject to distraction. Perisutural tissue was examined using immunohistochemistry in four areas: suture, endosteum, periosteum, and osteocytes, for the expression of Indian hedgehog, sonic hedgehog, and desert hedgehog and their receptor, patched-1. Two experimental groups were compared: (1) wild-type before distraction to wild-type after distraction, and (2) synostotic before distraction to synostotic after distraction. Distraction produced several variable and interesting changes in hedgehog protein presence. In wild-type rabbits, the predominant effect was a mild decrease in Indian hedgehog levels. Sonic and desert hedgehog and patched-1 protein levels were unchanged. In synostotic rabbits, the predominant effect of distraction was to decrease Indian hedgehog, sonic hedgehog, and patched-1 protein levels. This was especially true in the periosteum and endosteum. Cranial distraction of normal and affected rabbits differentially changed both the expression levels and patterns of the hedgehog and patched-1 proteins in the cranial tissues examined. These results suggest that molecular and genetic parameters of distraction and bone response may be different in craniosynostotic individuals, which may influence treatment protocols in these patients.