carbocyanines and Glaucoma

To determine whether cerebrospinal fluid (CSF) entry into the optic nerve is altered in glaucoma.. Fluorescent 10-kDa dextran tracer was injected into the CSF of 2-month-old (n = 9) and 10-month-old DBA/2J glaucoma mice (n = 8) and age-matched controls (C57Bl/6; n = 8 each group). Intraocular pressure (IOP) was measured in all mice before tracer injection into CSF. Tracer distribution was assessed using confocal microscopy of optic nerve cross-sections of mice killed 1 hour after injection. Paravascular tracer distribution in the optic nerve was studied in relation to isolectin-stained blood vessels. Tracer intensity and cross-sectional area in the laminar optic nerve were quantitatively assessed in all four groups and statistically compared. Aquaporin 4 (AQP4) and retinal ganglion cell axonal phosphorylated neurofilament (pNF) were evaluated using immunofluorescence and confocal microscopy.. IOP was elevated in 10-month-old glaucoma mice compared with age-matched controls. One hour after tracer injection, controls showed abundant CSF tracer in the optic nerve subarachnoid space and within the nerve in paravascular spaces surrounding isolectin-labeled blood vessels. CSF tracer intensity and signal distribution in the optic nerve were significantly decreased in 10-month-old glaucoma mice compared with age-matched controls (P = 0.0008 and P = 0.0033, respectively). AQP4 immunoreactivity was similar in 10-month-old DBA and age-matched control mice. Half of the 10-month-old DBA mice (n = 4/8) showed a decrease in pNF immunoreactivity compared to controls. Altered pNF staining was seen only in DBA mice lacking CSF tracer at the laminar optic nerve (n = 4/5).. This study provides the first evidence that CSF entry into the optic nerve is impaired in glaucoma. This finding points to a novel CSF-related mechanism that may help to understand optic nerve damage in glaucoma.

Topics: Animals; Aquaporin 4; Axons; Carbocyanines; Cerebrospinal Fluid; Female; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Glaucoma; Intraocular Pressure; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Microscopy, Confocal; Neurofilament Proteins; Optic Nerve Diseases; Phosphorylation; Retinal Ganglion Cells

Glaucoma is a major cause of vision impairment, which arises from the sustained and progressive apoptosis of retinal ganglion cells (RGC), with ocular hypertension being a major risk or co-morbidity factor. Because RGC death often continues after normalization of ocular hypertension, growth factor-mediated protection of compromised neurons may be useful. However, the therapeutic use of nerve growth factor (NGF) has not proven effective at delaying RGC death in glaucoma. We postulated that one cause for the failure of NGF may be related to its binding to two receptors, TrkA and p75. These receptors have distinct cellular distribution in the retina and in neurons they induce complex and sometimes opposing activities. Here, we show in an in vivo therapeutic model of glaucoma that a selective agonist of the pro-survival TrkA receptor was effective at preventing RGC death. RGC loss was fully prevented by combining the selective agonist of TrkA with intraocular pressure-lowering drugs. In contrast, neither NGF nor an antagonist of the pro-apoptotic p75 receptor protected RGCs. These results further a neurotrophic rationale for glaucoma.

Topics: Animals; Carbocyanines; Cell Death; Cell Survival; Female; Glaucoma; Intraocular Pressure; Nerve Degeneration; Nerve Growth Factor; Neuroprotective Agents; Peptides; Rats; Rats, Wistar; Receptor, Nerve Growth Factor; Receptor, trkA; Retinal Degeneration; Retinal Ganglion Cells; Stilbamidines

Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the betaGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice.. Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of betaGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal.. A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma.

Topics: Age Factors; Animals; beta-Glucosidase; Carbocyanines; Cell Count; Chi-Square Distribution; Disease Models, Animal; Disease Progression; DNA-Binding Proteins; Glaucoma; Mice; Mice, Inbred DBA; Mice, Transgenic; Optic Nerve Diseases; Retinal Degeneration; Retinal Ganglion Cells; Time Factors; Transcription Factors; Ubiquitin-Protein Ligase Complexes

The present study was conducted to examine whether the morphology of the retinal ganglion cells is altered in advanced glaucoma. Perikaryal, axonal, and dendritic alterations were monitored in glaucoma-resistant retinal ganglion cells by postvitam application of the fluorescent dye DiI.. The retinas of four amaurotic glaucomatous eyes and four normal eyes enucleated after death were used in this study. The retinas were freed from surrounding tissue, prepared as flatmounts on a nitrocellulose filter, and fixed overnight in 4% paraformaldehyde. The retinal ganglion cells were labeled by introducing crystals of the fluorescent carbocyanine dye DiI, into the optic fiber layer. This dye diffuses along membranes of ganglion cell axons, completely labeling them and their cell bodies and dendrites. Further characterization of the retinas and optic nerves included hematoxylin-eosin and van Gieson histochemical staining as well as immunohistochemistry against glial fibrillary acidic protein.. Because of the advanced stage of the disease, the retinas were almost completely depleted of ganglion cells, which had degenerated and therefore could not be stained. The few remaining ganglion cells were considered to be resistant to glaucoma. They showed drastic morphologic alterations, such as abnormal axonal beading, the cell bodies were normal in size but had irregular silhouettes or swellings, and there were fewer dendritic bifurcations. The size of the dendritic trees was smaller, implicating pruning of smaller dendritic branches. Glial cells were also detected immunocytochemically indicating their involvement in the pathologic course of glaucoma.. The data suggest that the few ganglion cells that survive the elevated intraocular pressure associated with loss of visual function display morphologic changes that are manifested both on the cell body and on their intraretinal processes, including axons and dendrites.

Topics: Adult; Axons; Blindness; Carbocyanines; Cell Survival; Dendrites; Eye Enucleation; Fluorescent Dyes; Glaucoma; Glial Fibrillary Acidic Protein; Humans; Intraocular Pressure; Microscopy, Fluorescence; Middle Aged; Nerve Fibers; Optic Nerve Diseases; Retinal Diseases; Retinal Ganglion Cells

In this study we investigated changes to horizontal cells in human retinae affected by glaucoma. Glaucoma is characterized by raised intraocular pressure and is responsible for retinal ganglion cell and, possibly, photoreceptor degeneration. It was therefore assumed that horizontal cells might also be affected. The carbocyanine dye DiI was placed at discrete points on fixed, whole-mounted retinae obtained from normal and glaucomatous patients. After allowing 6-24 weeks for intramembranous diffusion within the lipid layers of the nerve cells and, therefore, fluorescent labeling, we measured horizontal cell soma and dendritic field sizes. Selected cells were then embedded in Araldite and cut at 4 microns. Horizontal cells in glaucomatous eyes appeared larger and had a granulated outline as compared with cells from normal retinae. Analysis of the mean cell soma size indicated that cells were 26% larger in the glaucomatous retinae and that this increase was significantly different from that seen in normal retinae (P < 0.05). The dendritic field size was unaffected (P > 0.05). As seen in cross section there was a clear loss of photoreceptor outer segments, and shrunken silhouettes of photoreceptor inner segments with pyknotic nuclei were observed. It is proposed that the increase in some size is indicative of horizontal cell responses that are likely to culminate in degeneration as a result of heightened intraocular pressure. In addition, this paper provides further evidence that photoreceptors are affected by advanced glaucoma.

Topics: Carbocyanines; Cell Size; Dendrites; Fluorescent Dyes; Glaucoma; Humans; Neurons; Photoreceptor Cells; Retinal Degeneration