ABSTRACT
PURPOSE: To characterize a long-term elevated intraocular pressure (IOP) glaucoma model in the rat with respect to electroretinographic (ERG) changes and the pattern and mechanism of retinal ganglion cell (RGC) death. METHODS; An approximate doubling of IOP was induced in one eye (G) of female Wistar rats (150-180 g) by cautery of 3 episcleral/limbal veins. At intervals over 3 to 4 months, measurements of IOP and ERG changes (contact-lens electrode) were made in both the G and contralateral normal (N) eyes. At the end of 3 to 4 months of elevated IOP, RGCs were fluorescently labeled with Fluorogold (retrogradely from the superior colliculus), or retinas were labeled by intravitreal injection of a mitochondrial potential indicator dye and stained for apoptotic nuclei with a DNA dye. Flatmounts of fixed, dye-labeled retinas were examined by epifluorescence, confocal, or interference contrast microscopy. RESULTS: Elevated IOP was consistently maintained for up to 4 months in G eyes, but ERG a- and b-waves showed a statistically significant decline, of 30% to 40% in amplitude, after 3 months. Loss of RGCs in G retinas was primarily focal with no statistically significant loss demonstrable outside of the focal areas when assessed by an area sampling method for counting RGCs, which totaled 2% to 3% of the entire retinal area. Mitochondrial membrane potential of cells in the RGC layer was reduced by 17.5% (P: < 0.05) in regions surrounding areas of focal loss compared with comparable locations in control N eyes. After 3.5 months' elevated IOP the G retinas showed cell nuclei at various stages of apoptosis, from initial DNA condensation to fragmentation. CONCLUSIONS: The three-vein episcleral/limbal vein occlusion model for inducing glaucomatous pathology in the rat eye gives a consistent long-term elevation of IOP. After 3 to 4 months of approximately 100% increased IOP, the ERG responses begin to decline, there is a variable focal loss of RGCs, and some of the remaining RGCs show characteristics of stress and apoptosis. These changes seem consistent with retinal damage in human glaucoma (focal field defects), and this rat model appears to mimic some features of primary open-angle glaucoma.
Subject(s)
Glaucoma, Open-Angle/complications , Intraocular Pressure , Retinal Diseases/etiology , Retinal Ganglion Cells/pathology , Stilbamidines , Animals , Cell Death , Cell Nucleus/pathology , DNA Fragmentation , Disease Models, Animal , Electroretinography , Female , Fluorescent Dyes , Glaucoma, Open-Angle/physiopathology , Membrane Potentials/physiology , Microscopy, Confocal , Microscopy, Fluorescence , Mitochondria/physiology , N-Methylaspartate/administration & dosage , Rats , Rats, Wistar , Retinal Diseases/pathology , Retinal Diseases/physiopathology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/physiology , Time FactorsABSTRACT
Cultured testes and spermatocytes from the frog Xenopus laevis have been incubated (40-42 h) with adriamycin or colcemid followed by quantitation of chromosome aberrations in secondary spermatocytes and quantitation of micronuclei in secondary spermatocytes, early round spermatids, and round spermatids with acrosomal vacuoles (AV) at 18-162 h of culture. Micronucleus frequencies were consistently higher in secondary spermatocytes relative to round spermatids after exposure to either adriamycin or colcemid due to a higher rate of micronucleus formation during meiosis I compared to meiosis II. Also, some of the micronuclei formed during meiosis I did not survive meiosis II to form micronucleated spermatids. Micronucleus formation occurred in 3-7% of secondary spermatocytes with detectable chromosome aberrations, depending upon drug treatment. Thus, the ratio of micronuclei to total chromosome aberrations in secondary spermatocytes was always higher in colcemid-treated cells compared to adriamycin-treated cells following 18- and 42-h treatment periods. Adriamycin induced significant increases in micronuclei in both secondary spermatocytes and spermatids after 162 h of culture, the time for initial pachytene stages to develop into secondary spermatocytes and spermatids. The data show that cultured testes and spermatocytes from Xenopus may be used to quantify specific meiotic chromosome aberrations induced by both clastogens and spindle poisons using either a rapid secondary spermatocyte micronucleus assay or meiotic chromosome analysis.
Subject(s)
Chromosome Aberrations , Demecolcine/toxicity , Doxorubicin/toxicity , Micronuclei, Chromosome-Defective/drug effects , Spermatids/drug effects , Spermatocytes/drug effects , Animals , Cells, Cultured , Culture Techniques , Male , Meiosis , Micronucleus Tests , Spermatids/cytology , Spermatocytes/cytology , Xenopus laevisABSTRACT
Methods are described for the attachment of isolated spermatocytes to glass slides and the subsequent hypotonic swelling and gradual fixation of the metaphase I and metaphase II cells. The methods minimize cell loss and cell disruption and meiotic metaphase chromosomes become spread within residual cytoplasm thus reducing artefactual chromosome loss. Metaphase II complements from mouse, rat and frog spermatocytes prepared by these procedures had relatively low frequencies of hypoploidy (0.5-1.6%). Bivalent loss was not detected in 916 metaphase I complements. Injection of 0.1 mg/kg demecolcine into mice increased the incidence of metaphase II hypoploidy 8-fold. The hypoploid and hyperploid frequencies here increased equally. The results suggest that the methods described may be useful for the analysis of mechanisms of meiotic aneuploidy including aneuploidy resulting from chromosome loss during meiosis I.
