calcimycin and Glaucoma

This paper seeks to investigate differences between the neonatal and adult retinal ganglion cell populations to apoptotic death stimuli. DESIGN AND SAMPLES: In vitro and ex vivo paradigms involving P6 and P60 Sprague-Dawley rat retinal explants and retinal ganglion cells were employed..  Postnatal day 6 (P6) and 60 (P60) Sprague-Dawley retinal ganglion cells and retinal explants were either serum starved or subjected to excitotoxicity using calcium ionophore A23187.. Apoptosis was detected in both models using terminal dUTP nick end labelling. Expression of Apaf-1, active caspases-3 and 9 in P6 and P60 retinas, and in the ganglion cell layer was examined using Western blotting.. In both the dissociated retinal ganglion cell and retinal explant models, P60 retinal ganglion cells were significantly less susceptible to excitoxicity and serum starvation than their P6 counterparts. Western blotting indicated that active caspase-3 and Apaf-1 are downregulated in the Sprague-Dawley rat retina at P60 compared with P6.. We demonstrate that neonatal Sprague-Dawley retinal ganglion cells are more susceptible to glaucoma-related death stimuli than their adult counterparts in dissociated retinal ganglion cells and axotomized retinal explant models. It is apparent that these different retinal ganglion cell populations are inherently designed to react differently to death stimuli. Thus caution should be exercised when noting the high susceptibility of neonatal retinal ganglion cells to glaucomatous death stimuli.

Topics: Aging; Animals; Animals, Newborn; Apoptosis; Apoptotic Protease-Activating Factor 1; Blotting, Western; Calcimycin; Caspase 3; Cells, Cultured; Disease Susceptibility; Fluorescent Antibody Technique, Indirect; Glaucoma; In Situ Nick-End Labeling; Nerve Growth Factors; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells

The purpose of this study was to examine the importance of calpains in retinal ganglion cell (RGC) apoptosis and the protection afforded by calpain inhibitors against cell death.. Two different models of RGC apoptosis were used, namely the RGC-5 cell line after either intracellular calcium influx or serum withdrawal and retinal explant culture involving optic nerve axotomy. Flow cytometry analysis with Annexin V/PI staining was used to identify RGC-5 cells undergoing apoptosis after treatment. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis in retinal explant sections under various conditions. Serial sectioning was used to isolate the cell population of the ganglion cell layer (GCL). Western blotting was used to demonstrate calpain cleavage and activity by detecting cleaved substrates.. In the RGC-5 cell line, the authors reported the activation of mu-calpain and m-calpain after serum starvation and calcium ionophore treatment, with concurrent cleavage of known calpain substrates. They found that the inhibition of calpains leads to the protection of cells from apoptosis. In the second model, after a serial sectioning method to isolate the cells of the ganglion cell layer (GCL) on a retinal explant paradigm, protein analysis indicated the activation of calpains after axotomy, with concomitant cleavage of calpain substrates. The authors found that inhibition of calpains significantly protected cells in the GCL from cell death.. These results suggest that calpains are crucial for apoptosis in RGCs after calcium influx, serum starvation, and optic nerve injury.

Topics: Animals; Annexin A5; Apoptosis; Axotomy; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Flow Cytometry; Glaucoma; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Optic Nerve; Organ Culture Techniques; Propidium; Retina; Retinal Ganglion Cells

The ionophores A23187 and X537A, which increase the permeability of cell membranes to calcium and other divalent cations, produced significant elevation of intraocular pressure in rabbits. Topical instillation of these ionophores in concentrations of 1.0 and 0.1 per cent were effective. Aqueous humor protein and facility of outflow were similar in ionophore-treated and control eyes. Pretreatment with indomethacin did not block the intraocular pressure rise induced by A23187. Alterations of intracellular calcium might control cellular processes within the eye as in many other biological systems.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Aqueous Humor; Calcimycin; Dose-Response Relationship, Drug; Eye Proteins; Glaucoma; Indomethacin; Intraocular Pressure; Ionophores; Lasalocid; Prednisolone; Premedication; Rabbits