James D. Lindsey

Effects of prostaglandins on the aqueous humor outflow pathways.

Topical treatments with certain prostaglandins (PGs), including FP receptor agonists, lower intraocular pressure by increasing uveoscleral outflow. Although the precise mechanism for the increased uveoscleral outflow is not known, there appears to be activation of a molecular transduction cascade and an increase in the biosynthesis of certain metalloproteinases. This leads to reduction of extracellular matrix components within the ciliary muscle, iris root, and sclera. It is possible that this reduction of extracellular matrix present within portions of the uveoscleral pathway may contribute to the mechanism of increased uveoscleral outflow. Additional mechanisms that may contribute to the PG-mediated increase of uveoscleral outflow include relaxation of the ciliary muscle, cell shape changes, cytoskeletal alteration, or compaction of the extracellular matrix within the tissues of the uveoscleral outflow pathway. Future studies should clarify the importance of these various responses that may contribute to increased uveoscleral outflow. At present, there is no compelling evidence for a substantial facility-increasing effect on the trabecular meshwork outflow for any of these compounds.

Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells

PURPOSE The mechanism by which prostaglandin(PG)F2 alpha increases uveoscleral outflow and lowers intraocular pressure in primates is not known. In cultured human ciliary muscle cells, PGF2 alpha induces the expression of the protooncogene c-fos which is known to induce the transcription of genes such as matrix metalloproteinase-1 (MMP-1) and MMP-3 in other cell systems. As these enzymes are initially secreted as proenzymes, the present study was undertaken to determine if PG treatment induces ciliary muscle cells to secrete either proMMP-1 or proMMP-3. METHODS Human ciliary smooth muscle cells were grown to confluence in monolayer cell cultures and then treated with PGF2 alpha, 17-phenyltrinor-PGF2 alpha, or 11-deoxy-PGE1. Medium harvested at various times after treatment was assayed for proMMP-1 and proMMP-3 content using sandwich ELISAs. RESULTS Three days after adding 10 nM PGF2 alpha, proMMP-1 and proMMP-3, concentrations in the culture medium were increased by 254 +/- 33% (mean +/- SE) and 128 +/- 13%, respectively. Compared with vehicle controls, 24 h treatment with 200 nM PGF2 alpha, 17-phenyltrinor-PGF2 alpha, or PGE1, increased proMMP-1 by 116 +/- 29%, 169 +/- 26%, and 273 +/- 16%, respectively. In parallel experiments, proMMP-3 was increased by 99 +/- 18%, 82 +/- 24%, and 214 +/- 16%, respectively. CONCLUSIONS These results suggest that induction of MMPs in situ following topical PG treatment may degrade ciliary muscle extracellular matrix and possibly contribute to increased uveoscleral outflow, as well.

Hippocampal aging in rats: a morphometric study of multiple variables in semithin sections.

Quantitative analyses of pyramidal cells, astrocytes, dark glia (microglia and dark oligodendrocytes), lipofuscin accumulation and astrocyte reactivity were carried out in semithin sections from field CA3 of the hippocampus of rats in 3 age groups (4-7 mo., 13-15 mo., 25-28 mo.). A decrease in neuronal density (approximately 25%) and an increase in dark glia were found in the oldest group. The astrocyte population was stable with age. Lipofuscin increased by 13-15 mo. and increased further by 25-28 mo. Qualitative examples of several kinds of glial reactivity are also described. Our observations indicate that hippocampal changes in aging rats exhibit some similarities to brain changes reported in other mammalian species, and also illustrate the value of semithin sections for examining neuromorphologic correlates of brain aging.

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.

A Selective Inhibitor of Drp1, Mdivi-1, Increases Retinal Ganglion Cell Survival in Acute Ischemic Mouse Retina

PURPOSE To determine whether acute intraocular pressure (IOP) elevation alters dynamin-related protein 1 (Drp1) as well as whether a selective inhibitor of Drp1, mdivi-1, can block apoptotic cell death and subsequently increase retinal ganglion cell (RGC) survival in ischemic mouse retina. METHODS C57BL/6 mice received injections of mdivi-1 (50 mg/kg) or vehicle, and then transient retinal ischemia was induced by acute IOP elevation. RGC survival was measured after FluoroGold labeling. Drp1 and glial fibrillary acidic protein (GFAP) protein expression and distribution were assessed at 12 hours after ischemia-reperfusion by Western blot and immunohistochemistry. Apoptotic cell death was assessed by TUNEL staining. RESULTS Drp1 and GFAP protein expression was significantly increased in the early neurodegenerative events (within 12 hours) of ischemic mouse retina. Mdivi-1 treatment blocked apoptotic cell death in ischemic retina, and significantly increased RGC survival at 2 weeks after ischemia. In the normal mouse retina, Drp1 is expressed in the ganglion cell layer (GCL) as well as the inner plexiform layer, the inner nuclear layer (INL), and the outer plexiform layer (OPL). In the GCL, Drp1 immunoreactivity was strong in RGCs. While Drp1 protein expression was increased in the GCL of vehicle-treated ischemic retina at 12 hours. Mdivi-1 treatment did not change this increase of Drp1 protein expression but significantly decreased GFAP protein expression. CONCLUSIONS These findings suggest that altered Drp1 activity after acute IOP elevation may be an important component of a biochemical cascade leading to RGC death in ischemic retina.

The mechanism of action of prostaglandins on uveoscleral outflow.

It is generally accepted that prostaglandins (PGs) lower intraocular pressure by increasing uveoscleral outflow. The growing use of PGs to lower intraocular pressure has led to increased interest in the uveoscleral outflow. Uveoscleral outflow passes through extracellular spaces within the ciliary muscle and then through the suprachoroidal space to the posterior pole of the eye. Recent studies indicate that this reflects a direct effect of PGs on specific ciliary muscle prostanoid receptors. Activation of these receptors stimulates several linked responses, including cAMP formation and induction of c-Fos and c-Jun expression. These signals lead to increased biosynthesis of matrix metalloproteinases, a family of neutral proteinases that can cleave extracellular matrix molecules. These matrix metalloproteinases may initiate the alteration of collagens in the ciliary muscle to increase spaces among ciliary muscle fibers, thereby reducing hydraulic resistance in the uveoscleral outflow pathway.

Intraocular pressure elevation induces mitochondrial fission and triggers OPA1 release in glaucomatous optic nerve.

PURPOSE To determine whether elevation of intraocular pressure (IOP) triggers mitochondrial fission and ultrastructural changes and alters optic atrophy type 1 (OPA1) expression and distribution in the optic nerve (ON) of glaucomatous DBA/2J mice. METHODS IOP in the eyes of DBA/2J mice was measured, and mitochondrial structural changes were assessed by conventional electron microscopy (EM) and EM tomography. Cytochrome c oxidase IV subunit 1 (COX), OPA1, and Dnm1, a rat homologue of dynamin-related protein-1, mRNA were measured by quantitative (q)PCR. COX and OPA1 protein distribution was assessed by immunocytochemistry and Western blot. RESULTS Excavation of the optic nerve head (ONH), axon loss, and COX reduction were evident in 10-month-old glaucomatous ONHs of eyes with >20 mm Hg IOP elevation. EM analysis showed mitochondrial fission, matrix swelling, substantially reduced cristae volume, and abnormal cristae depletion in 10-month-old glaucomatous ONH axons. The mean length of mitochondrial cross section in these axons decreased from 858.2 +/- 515.3 nm in 3-month-old mice to 583.3 +/- 298.6 nm in 10-month-old glaucomatous mice (P < 0.001). Moderate reductions of COX mRNA were observed in the 10-month-old DBA/2J mices ONHs. Larger reductions of OPA1 immunoreactivity and gene expression were coupled with larger increases of Dnm1 gene expression in 10-month-old glaucomatous ONH. Subcellular fractionation analysis indicates increased release of both OPA1 and cytochrome c from mitochondria in 10-month-old glaucomatous ONs. CONCLUSIONS IOP elevation may directly damage mitochondria in the ONH axons by promoting reduction of COX, mitochondrial fission and cristae depletion, alterations of OPA1 and Dnm1 expression, and induction of OPA1 release. Thus, interventions to preserve mitochondria may be useful for protecting against ON degeneration in glaucoma.

Long-Term In Vivo Imaging and Measurement of Dendritic Shrinkage of Retinal Ganglion Cells

PURPOSE To monitor and measure dendritic shrinkage of retinal ganglion cells (RGCs) in a strain of transgenic mice (Thy-1 YFP) that expresses yellow fluorescent proteins in neurons under the control of a Thy-1 promoter. METHODS A total of 125 RGCs from 16 eyes of Thy-1 YFP transgenic mice were serially imaged with a confocal scanning laser ophthalmoscope for 6 months after optic nerve crush. Quantitative analysis of cell body area, axon diameter, dendritic field, number of terminal branches, total dendritic branch length, branching complexity, symmetry, and distance from the optic disc was used to characterize the morphology of RGCs, describe the patterns of axonal and dendritic degeneration, identify the morphologic predictors for cell survival, and estimate the rate of dendritic shrinkage. RESULTS RGC damage was observed prospectively to begin with progressive dendritic shrinkage, followed by loss of the axon and the cell body. In a small proportion of RGCs, progressive axonal changes including fragmentation, beading, retraction, and bulb formation were also observed. RGCs with a larger dendritic field and a longer total dendritic branch length in general have a better survival probability. The rate of dendritic shrinkage was variable with a slower rate observed in cells having a larger dendritic field, a longer total dendritic branch length, and a greater distance from the optic disc. CONCLUSIONS Estimating the probability of RGC survival and measuring the rate of dendritic shrinkage could become a new paradigm for investigating neuronal degeneration and evaluating the response of neuroprotective treatment.

Memantine blocks mitochondrial OPA1 and cytochrome c release, and subsequent apoptotic cell death in glaucomatous retina

PURPOSE To determine whether intraocular pressure (IOP) elevation alters OPA1 expression and triggers OPA1 release, as well as whether the uncompetitive N-methyl-d-aspartate (NMDA) glutamate receptor antagonist memantine blocks OPA1 release and subsequent apoptotic cell death in glaucomatous DBA/2J mouse retina. METHODS Preglaucomatous DBA/2J mice received memantine (5 mg/kg, intraperitoneal injection, twice daily for 3 months) and IOP in the eyes was measured monthly. RGC loss was counted after FluoroGold labeling. OPA1, Dnm1, Bcl-2, and Bax mRNA were measured by qPCR. OPA1 protein was assessed by immunohistochemistry and Western blot. Apoptotic cell death was assessed by TUNEL staining. RESULTS Memantine treatment significantly increased RGC survival in glaucomatous DBA/2J mice and increased the 75-kDa OPA1 isoform, but did not alter the 80- and 90-kDa isoforms. The isoforms of OPA1 were significantly increased in the cytosol of the vehicle-treated glaucomatous retinas but were significantly decreased in memantine-treated glaucomatous retinas. OPA1 immunoreactivity was decreased in the photoreceptors of both vehicle- and memantine-treated glaucomatous retinas, but was increased in the outer plexiform layer of only the memantine-treated glaucomatous retinas. Memantine blocked apoptotic cell death in the GCL, increased Bcl-2 gene expression, and decreased Bax gene expression. CONCLUSIONS OPA1 release from mitochondria in glaucomatous mouse retina is inhibited by blockade of glutamate receptor activation. Because this OPA1 effect was accompanied by increased Bcl-2 expression, decreased Bax expression, and apoptosis blockade, glutamate receptor activation in the glaucomatous retina may involve a distinct mitochondria-mediated cell death pathway.