Abnormalities in expression of human retinal mRNA in retinitis pigmentosa.

The causative genetic defects of retinitis pigmentosa (RP) might be expected to affect the expression of messenger RNA in the retina. Total cellular RNA from retinas of normal and dystrophic individuals was therefore subjected to (i) in vitro translation and analysis by two-dimensional SDS polyacrylamide gel electrophoresis and (ii) Northern blot analysis using probes specific for ?-tubulin, interphotoreceptor retinoid-binding protein (IRBP) and glial fibrillary acidic protein (GFAP) mRNAs. Both methods revealed quantitative abnormalities in specific mRNA levels in RP. These differences appeared to reflect (i) the loss of photoreceptors in RP (decreased ?-tubulin and IRBP mRNA, and a reduced translation product comigrating with rhodopsin) and (ii) the proliferation of glial elements in the RP retinas (increased GFAP mRNA and its translation product). In addition, translation products of 21 and 39 kDa were consistently increased in RP retinas. These have not been previously detected in gliosis and their characterization may provide insight into the mechanisms of disease in RP.

Light damage in rod outer segments: the effects of fixation on ultrastructural alterations.

PURPOSE: In this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina. METHODS: The alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures. RESULTS: Light exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar. CONCLUSIONS: We conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation.

Necrosis and apoptosis in neuroretina and pigment epithelium after diffuse photodynamic action in rats: a light and electron microscopic study.

Cell death in the rat retina has been shown to occur mainly along two distinct pathways after pressure-induced ischemia. The authors conducted a qualitative investigation to determine whether similar types of cell death could be identified after diffuse photodynamic injury and photothrombosis. After i.v. injection of rose bengal (40 mg/kg), the eyes of 21 albino rats were exposed to light for durations ranging from 2 to 15 minutes, at a light intensity of 15-17,000 ft-cd, with interposition of a Kodak Wratten gelatin filter #15. Survival times ranged from 15 minutes to 6 days. The specimens were studied by light and electron microscopy. We identified two types of retinal cell death in the neuroretina and in the retinal pigment epithelium (RPE). Type I cell death was consistent with necrosis, and type-II cell death showed many features of apoptosis. These observations may not be exhaustive. The patterns of retinal cell death in this model appeared to be identical with those found after pressure-induced ischemia. The morphology of apoptosis of the RPE is hitherto undescribed. This study corroborates the concept of a limited number of types of cell death in the retina after exposure to noxious stimuli of various kinds.

[Histopathologic studies of the rabbit retina after use of perfluorodecalin]. / Badania histopatologiczne siatkówki królika po zastosowaniu perfluorodekaliny.

PURPOSE: To investigate the retinal toxicity of a vitreous substitute perfluorodecalin (PFD) in the rabbit eye up to 2 weeks after injection. MATERIAL AND METHODS: A space was created in the vitreous cavity by injecting 0.4 cc of perfluoropropane gas. After 3 days gas-fluid exchange was performed. Experimental eyes were injected with 1 cc of PFD and control eyes with 1 cc of Ringer solution. The eyes were enucleated 1, 3, 6 and 14 days after the procedure and histological studies were conducted. RESULTS: Control samples showed almost normal histology. One day after PFD injection photoreceptor nuclei dropout and migration below the outer limiting membrane, pyknosis and occasional densification of cell bodies were observed. After 3 days glial cell hypertrophy and accumulation of macrophages above the inner limiting membrane was also noted. After 6 days necrosis was observed in the outer and inner retinal layers. Foci of cell death in the retina were also observed 14 days after PFD injection. CONCLUSION: PFD induced degenerative changes in the rabbit retina within the first 24 hours after administration. Structural alterations in the inner and outer retina persisted within the two weeks of observation. Our results support the removal of PFD at the end of the primary surgical procedure.

Retinitis pigmentosa and the question of photoreceptor connecting cilium defects.

BACKGROUND: A generalized structural defect of the cilia in various tissues, including photoreceptor connecting cilium, has been postulated as occurring in some forms of retinitis pigmentosa (RP). However, the literature on ciliary abnormalities in RP contains contradictory findings. METHODS: In this study the fine structure of photoreceptors from 17 RP donors including X-linked RP, X-linked RP carrier state, autosomal dominant RP and autosomal recessive RP was examined by electron microscopy. RESULTS: Photoreceptor preservation was commonly observed even in the most advanced cases of the disease, especially in the perimacular area, in the proximity of the optic nerve and in the periphery. Primary ciliary defects, expressed as additional or missing microtubules, were found in none of the samples. Comparison of photoreceptors in normal and RP retinae showed thinner cilia in RP cells but no defect in the microtubule arrangements within the connecting cilium. CONCLUSION: Additional or missing microtubules in ciliated cells are not uncommon and have been reported in the literature and recorded in some studies of RP tissue. Such defects, however, are believed to be acquired rather than inherited abnormalities of cilia and were not observed in the photoreceptor connecting cilia of RP patients examined in this study. Thinning of the cilium may also be a secondary effect related to cell shrinkage early during apoptosis, which is postulated to be a common pathway in photoreceptor degeneration.

Light damage in the rat retina: glial fibrillary acidic protein accumulates in Müller cells in correlation with photoreceptor damage.

Low intensity diffuse white fluorescent light (1,000 lx for 2 h) exclusively induced photoreceptor damage in the inferior retina of albino rats; the temporal retina showed extensive damage, whereas the nasal retina revealed threshold lesions prior to recovery. To expand our morphological data, further experiments were undertaken to determine if glial fibrillary acidic protein (GFAP) expression was associated with the regions of photoreceptor damage. To follow the time course of GFAP expression, immunoblot analysis was carried out on retinal homogenates of dark-adapted (control) rats and light-exposed rats returned to cyclic light for 0 h, 1, 2, 3 and 6 days. A significant twofold increase in GFAP immunoreactivity over controls was observed in the retinas of light-exposed rats returned to cyclic light for 6 days. Using an indirect immunohistochemical method, retinal sections of the control and light-exposed rats allowed to recover for 6 days were stained for GFAP. GFAP immunoreactivity was localised to the astrocytes and Müller cells. Moreover, GFAP staining in Müller cells in the retinas of control animals was uniformly restricted to rare end-feet. In contrast, a gradient of GFAP immunoreactivity was observed in experimental rats, rising from the superior retina to the inferior temporal quadrant; the GFAP staining in the inferior nasal quadrant was intermediate. Thus, GFAP immunoreactivity was proportional to photoreceptor damage. Interestingly, no GFAP induction could be demonstrated in the pineal glands of light-exposed rats.

Haematoporphyrin photosensitisation treatment of experimental choroidal melanoma.

Greene amelanotic melanoma transplanted to pigmented rabbit choroid provided the experimental model for studying photosensitisation treatment of choroidal melanoma. Administration of haematoporphyrin derivative (HPD) and subsequent photoradiation with green laser light destroyed much of the melanoma with minimal side effects to normal uvea. The results of treatment were documented by fundus photography and fluorescein angiography. By 24 hours, the irradiated melanoma and surrounding retina appeared whitened and necrotic, with complete non-perfusion of the area. Over the course of one week, the clinical appearance did not alter. Histopathology at 24 hours and 8 days confirmed massive central tumour necrosis, but in all cases this was subtotal. Viable cells were evident at the periphery and also along the base of the tumour. Experimental evaluation of treatment parameters is required before this technique can be recommended for human choroidal melanoma.