Morphologic evidence of photoreceptor differentiation of pinealocytes in the neonatal rat.

The pineal body and the retina of the neonatal Sprague-Dawley rat were studied by light and electron microscopy, and the morphologic differentiation of the parenchymal cells of the pineal body was compared with that of the developing photoreceptor cells of the retina. Between the ages of 4 and 12 days after birth, some of the developing pinealocytes were observed to become elongated and polarized, with their nuclei located at one pole. "Synaptic" ribbons were observed within the cell body. At the opposite pole the cells developed elongated cell processes that initially contained microtubules and ribosomes. These cell processes projected into luminal spaces and were attached by structures resembling zonulae adherentes to the adjacent cells. Extending from the tips of the cell processes, cilia with a 9 + 0 arrangement were observed. Lamellated and vesicular membranes were noted at the tips of the cilia. Such morphologic differentiation, however, could be observed only in rats younger than 17 days. Comparison of the morphologic features of the neonatal pinealocytes with those of the developing retinal photoreceptor cells showed much similarity. It is suggested that the pinealocytes of the neonatal rat undergo "photoreceptor-like" differentiation during a transient neonatal period. Such morphologic differentiation may provide an explanation for light-induced biochemical changes described in neonatal rats whose eyes had been enucleated.

Chronic lead administration in neonatal rats: electron microscopy of the retina.

The morphologic effects on the retina resulting from chronic lead exposure were assessed in neonatal rats. Newborn rats nursed from dams were given a low (0.115%) or a high (4.5%) concentration of lead in their diet. At day 21 the pups were weaned to the mother's diet. The retinas of the pups were studied by electron microscopy at various ages up to day 60. High and low lead concentrations produced necrosis of photoreceptor cells and cells of the inner nuclear layer. The high lead concentration, in addition, was associated with swelling of endothelial cells of the retinal vessels and narrowing of the lumen. Increased permeability of the retinal vessels and pigment epithelium to horseradish peroxidase was also observed under the high-dose condition. The authors conclude that lead can produce direct neuronal damage and, at high doses, produces retinal vascular lesions and alteration of the blood-retinal barrier.

Repair and late degeneration of the primate foveola after injury by argon laser.

Each foveola of nine rhesus monkey eyes was subjected to a single, mild, calibrated exposure from an argon laser (1 to 1.4 mW for 10 to 20 min). Observations from ophthalmoscopy, fundus photography, and fluorescein angiography were correlated with light and electron microscopic studies. The ophthalmoscopic changes consisted of initial whitening and subsequent but persistent depigmentation of the foveola. Fluorescein angiography showed a pattern consistent with "window defect" of the retinal pigment epithelium (RPE). In the first 6 months after injury, the pathologic changes consisted of initial vacuolar changes of the RPE, followed by persistent hypopigmentation and slow development of membranous bodies or lipoidal degeneration in these cells. Initial alteration and subsequent incomplete reformation of cone outer segments were also noted. In animals sacrificed 3 to 4 years after injury, separation of RPE from Bruch's membrane, with production of various abnormal basement membranes and intercellular cystoid changes in the overlying retina, was observed. The morphologic observations suggest that although the mildly injured epithelial cells may recover, they may develop functional incompetence at a later date, resulting in serous detachment of RPE and foveal (macular) edema.

Experimental macular edema after lens extraction.

Macular edema has been observed frequently in man after cataract extraction, but pathogenic mechanisms remain unclear. Seven eyes of four young adult rhesus monkeys underwent lens extraction. The retinas and maculae of these eyes were examined by ophthalmoscopy fundus photography, fluorescein angiography, light and electron microscopy, and the horseradish peroxidase tracer technique. In the macular region, the blood-retinal barrier at the retinal vasculature was disrupted in three of the seven eyes. All three eyes had had vitreous loss during lens extraction. Horseradish peroxidase was observed both intracellularly and extracellularly in the maculae. In contrast, the blood-retinal barrier at both the retinal pigment epithelium and the retinal vasculature of the peripheral retina in most eyes was intact. We conclude that macular edema secondary to lens extraction is due to disruption of the blood-retinal barrier at the levels of the retinal vasculature and the retinal pigment epithelium.

Vitreous fluorophotometry in patients with senile macular degeneration.

Twenty-two phakic eyes of 16 patients with varying stages of senile macular degeneration (SMD) underwent vitreous fluorophotometry. The upper tolerance limit of the penetration ratio in 17 eyes of 17 age-matched controls was 6.33 X 10(-6) min-1. Ten of the 22 eyes with SMD had values exceeding this, indicating abnormal blood-retinal barrier function. When the fluorescence recorded in the vitreous but not related to the local intravitreal dye was evaluated, it was greater in those eyes with more severe forms of the disease. All ten eyes with drusen alone and no SMD had normal fluorophotometry values. Vitreous fluorophotometry may be helpful in the diagnosis and classification of SMD.

c-Fos protein in photoreceptor cell death after photic injury in rats.

PURPOSE: To examine the involvement of c-Fos protein in light-induced photoreceptor cell death in rats. METHODS: Thirty-two Lewis albino rats were exposed to green fluorescent light (480-520 nm) of 300 to 320 foot-candles (3228-3443.2 lux) for 3 hours, allowed to recover in the dark, and euthanatized at 0, 1, 3, 6, 12, 24, or 96 hours after light exposure. c-Fos was detected immunohistochemically and nicked DNA by in situ TdT-dUTP terminal nick-end labeling (TUNEL). Double labeling of c-Fos and DNA nicks was also performed. RESULTS: There was a time-dependent change in the number of c-Fos-positive photoreceptor nuclei after light injury, which paralleled the change in the number of TUNEL-positive nuclei. The temporal and spatial appearance of these nuclei also matched the appearance of pyknotic nuclei of the outer nuclear layer. Double-labeling study revealed that some c-Fos-positive nuclei were also TUNEL-positive nuclei. CONCLUSIONS: There was an acute accumulation of c-Fos protein in photoreceptors associated with cell death. This study further supports other studies showing that c-Fos is linked to apoptotic photoreceptor cell death.

Immunoreactivity against tau, amyloid precursor protein, and beta-amyloid in the human retina.

PURPOSE: Increased immunoreactivity (IR) of beta-amyloid and the amyloid-associated proteins tau and amyloid precursor protein (APP) in the brain have been linked to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. However, the expression of these proteins has not been investigated in the normal or diseased human retina. METHODS: Using immunohistochemical techniques, we examined the distribution and age-related changes of anti-tau-1, anti-tau-2, anti-APP, and anti-beta-amyloid IR in the human retina at various ages (n = 24), in retinitis pigmentosa (RP, n = 6), and in age-related macular degeneration (ARMD, n = 10). RESULTS: Tau-1 immunoreactivity was intense in the inner retinal layers and did not change with age or in RP. Eyes with ARMD showed less intense staining but exhibited a similar distribution. Tau-2 IR was faint and did not change with age but was mildly increased in the retinal pigment epithelium (RPE) of eyes with RP and in the retina of eyes with ARMD. APP IR was most prominent in the ganglion cell and nerve fiber layer, and it appeared to increase in ganglion cells of older persons and in RPE cells of eyes with RP and ARMD. Beta-amyloid IR was only detected focally in sub-RPE deposits in eyes from older persons. CONCLUSIONS: The proteins investigated in this study are present in the human retina. The staining pattern of tau is different from the brain, but it shows no age-related changes. The increased immunoreactivity of APP in retinal ganglion cells of older eyes and in RPE cells of eyes with RP and ARMD, as well as the patchy staining of beta-amyloid within sub-RPE deposits, might indicate a relationship of these proteins to retinal aging and possibly to retinal degeneration in RP.

Tau-2 immunoreactivity of corpora amylacea in the human retina and optic nerve.

PURPOSE: To characterize the constituents of corpora amylacea in the human retina and optic nerve. METHODS: Immunohistochemistry was performed on sections of retina, optic nerve, and brain tissue using antibodies against tau 1, tau-2, and amyloid precursor protein. RESULTS: Consistent anti-tau-2 immunoreactivity was noted in the corpora amylacea in the retina, optic nerve, and brain tissue, albeit with variations in pattern and intensity of staining. No immunoreactivity was observed with antibodies anti-tau 1 and anti-amyloid precursor protein. CONCLUSION: Our findings suggest the accumulation of possibly abnormal tau-2 within the corpora amylacea, which may be either astrocytic or axonal in origin.

Effects of basic fibroblast growth factor in retinal ischemia.

PURPOSE: Basic fibroblast growth factor (bFGF), a 17- to 24-kDa protein known to be essential for the survival of neurons, induced fiber outgrowth of ganglion cells in cultures of rat retina and rescued photoreceptor cell loss in the retina of Royal College of Surgeon rats. The authors evaluated the efficacy of bFGF in rescuing the neuronal loss in rat retina after retinal ischemia. METHODS: Retinal ischemia was induced in 29 eyes of 17 albino Lewis rats by increasing the intraocular pressure to 110 mm Hg for 45 minutes via an intracameral catheter. A total of 800 ng of bFGF was delivered into the anterior chamber at the time of induction of ischemia. Sixteen eyes of nine rats received bFGF, and 13 eyes of eight rats received heparin in phosphate-buffered saline as vehicle control. The animals were euthanized 7 or 14 days after reperfusion. RESULTS: Morphologic examination of the retinas at both time points showed that necrosis of the retinal ganglion cells (RGCs) and thinning of the inner plexiform and inner nuclear layers were less severe in the bFGF-treated eyes than in the vehicle-treated eyes. On morphometric examination, 7 days after reperfusion, the mean thickness of the inner retinal layers and the RGC counts on flat preparations of retina in both the posterior and the peripheral portions of the retina were significantly higher in the bFGF-treated eyes than in the vehicle-treated eyes (P < 0.02). At 14 days, similar beneficial effects were noted in all morphometric parameters, except RGC counts in the posterior pole. CONCLUSIONS: These results demonstrate that bFGF partially protects the RGCs and other inner retinal elements from ischemic injury.

Reduced and oxidized ascorbates in guinea pig retina under normal and light-exposed conditions.

Both reduced and oxidized ascorbates were measured in aqueous, neural retina, and pigment epithelium-choroid complex (PE-C) of pigmented guinea pigs. Normal values for total ascorbate of 16 mg/dl in aqueous, 22 mg/dl in neural retina, and 7 mg/dl in PE-C were found. After mild photic damage caused by varying lengths of exposure of 10,000 to 20,000 lux of fluorescent lighting, reduced ascorbate concentrations generally decreased in the neural retina, while oxidized ascorbate generally increased in PE-C. In both normal and light-exposed retinas, reduced ascorbate was predominant in the neural retina, and oxidized ascorbate was predominant in the PE-C. Histochemical localization of reduced ascorbate occurred in the Müller cell fibers and at the apices of the retinal pigment epithelium.

An inherited retinopathy in collies. A light and electron microscopic study.

A recessively inherited retinopathy in collies aged 8 to 189 days was studied by light and electron microscopy. The disease is produced when the outer segments of rods and cones fail to develop normally. Retinal pigment epithelial changes found in several litters appeared to form a separate disease entity. We compared the collie retinopathy with other canine models and the collie ectasia syndrome.

Experimental allergic optic neuritis in guinea pigs: preliminary report.

An experimental model for acute allergic optic neuritis was produced in adult strain 13 guinea pigs by sensitization with isogenic spinal cord emulsion in complete Freund's adjuvant. These animals exhibited two distinct clinical patterns: (1) "retrobulbar optic neuritis," with a diminished pupillary response to light despite a normal fundus, and (2) "neuroretinitis," with a diminished pupillary response associated with hyperemia and swelling to the disc and juxtapapillary retinal edema. Histopathologic study of those animals with "retrobulbar neuritis" revealed that some had no abnormalities in the optic nerve or chiasm, but showed foci of mononuclear cell infiltration in the brain. Others had a mononuclear cell infiltration localized to the retrobulbar portion of the optic nerve and chiasm with multiple foci of axial and periaxial demyelination. Similar pathologic changes were present in the animals with "neuroretintis", but the lesions were located just behind the lamina scleralis. These animals also exhibited marked swelling of the axons at the lamina retinalis. On examination by light microscopy, the alterations in the region of optic nerve head appeared characteristic of papilledema.

Pathologic studies of the blood--retinal barrier in the spontaneously diabetic BB rat.

The BB rat spontaneously develops a diabetic state that closely resembles human type I diabetes. The authors studied the pathologic changes of the retina and retinal pigment epithelium of four normal and nine diabetic BB rats using (1) light and electron microscopy with the horseradish peroxidase tracer technique, and (2) trypsin digest preparations of the retinal vessels. They observed a retinal pigment epitheliopathy characterized by (1) derangement of the plasmalemma infoldings; (2) patchy organelle degeneration leading to focal necrosis; (3) increased permeability to horseradish peroxidase; and (4) repair of the pigment epithelium. Focal thickening of the retinal vascular basement membrane was seen occasionally, but the trypsin digest preparations were unremarkable. These studies suggest that diabetic retinal pigment epitheliopathy may be one of the early changes of diabetic retinopathy and may provide a pathogenetic mechanism for early disruption of the blood-retinal barrier.

Apoptosis and caspases after ischemia-reperfusion injury in rat retina.

PURPOSE: Extensive cell loss in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) was noted in a rat model of retinal ischemia-reperfusion injury by transient elevated intraocular pressure (IOP). The possible involvement of apoptosis and caspases was examined in this model of neuronal loss. METHODS: Transient elevated IOP was induced in albino Lewis rats through the insertion of a needle into the anterior chamber connected to a saline column. Elevated IOP at 110 mm Hg was maintained for 60 minutes. Groups of animals were euthanatized at various times after reperfusion, and their retinas were evaluated by morphology, agarose gel electrophoresis of DNA, in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL), immunohistochemistry of caspases II (ICH1) and III (CPP32), and morphometry. YVAD.CMK, a tetrapeptide inhibitor of caspases, was used to examine the involvement of caspases. RESULTS: A marked ladder pattern in retinal DNA gel analysis, typical of internucleosomal DNA fragmentation and characteristic of apoptosis, was present 12 and 18 hours after reperfusion. Labeling of nuclei in the RGCL and the inner nuclear layer (INL) by TUNEL was noted between 8 and 18 hours after reperfusion. Histologic and ultrastructural features typical of apoptosis were also observed in the inner retina after ischemia. YVAD.CMK administered during the ischemic period inhibited apoptotic fragmentation of retinal DNA and ameliorated the tissue damage. When administered intravitreally 0, 2, or 4 hours after reperfusion, YVAD.CMK was also effective in preserving the inner retina but had no significant effect when administered 6 or 8 hours after reperfusion. The inner retina showed transient elevated immunoreactivity of caspases II and III 4 and 8 hours after reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retinal ganglion cell layer and the INL. Caspases may have a pivotal role in the early events of the apoptotic pathway(s). Rescue by using anti-apoptotic agents after ischemia-reperfusion is feasible.

A comparative study of methods of photoreceptor morphometry.

A comparative study was performed to compare methods of quantitative evaluation of damage to photoreceptor cells in rat eyes exposed to light. Using a manual count of individual photoreceptor nuclei as the standard, the following measurements were compared: (1) outer nuclear layer column count, (2) area, (3) thickness, and (4) the number of nuclei of the outer nuclear layer of the retina using a newly developed computer program that counts a digitized image. Linear-regression analysis of the data showed that the computer method was the most accurate with a Pearson correlation coefficient of 0.9911. The other methods tested also correlated well with the manual count, with coefficients ranging from 0.9367-0.9532. All these methods were reasonably accurate measurements of photoreceptor layer integrity, with the computer count most closely approximating the values obtained by a manual count. However, the outer nuclear layer thickness measurement appeared to be the fastest and most convenient method to evaluate photoreceptor cell damage quantitatively. Computer counting could be reserved for situations in which a high degree of accuracy is required.

'On' response defect in paraneoplastic night blindness with cutaneous malignant melanoma.

Response properties of rod and cone systems were assessed in a patient with an acquired form of night blindness associated with a metastatic cutaneous malignant melanoma. The night blindness, a sensation of shimmering lights, and selective reductions in the amplitudes of both rod and cone electroretinographic (ERG) b-waves were present before and after chemotherapy, confirming that this disorder was a paraneoplastic consequence of the melanoma rather than a response to chemotherapy. During ERG testing with flashes of extended duration, the cone b-wave abnormality was found to be a predominant loss of the cone ERG "on" response with relative preservation of the "off" response, similar to that observed in patients with congenital stationary night blindness. An impairment in signal transmission specific for retinal "on" pathways may be a primary defect in both of these forms of night blindness.

Tissue transglutaminase in apoptosis of photoreceptor cells in rat retina.

PURPOSE: The possible involvement of tissue transglutaminase (tTG) in apoptosis during photoreceptor degeneration was examined in retinal photic injury in rats and in retinal dystrophy of Royal College of Surgeons (RCS) rats. METHODS: Retinal photic injury was induced in 48 male Lewis albino rats by exposure to green fluorescent light of 300 to 320 foot-candles. The retinal tTG was examined by enzyme assay, immunohistochemistry, and Western blot analysis after 9, 12, or 24 hours of exposure or at 6 or 24 hours of dark adaptation after 24 hours of light exposure. Retinas from RCS rats at various stages of degeneration also were examined with similar methods. RESULTS: There was a progressive increase in retinal tTG activity after 300 to 320 ft-c of light exposure, reaching a peak after 24 hours of light exposure. In the RCS rats, tTG activity increased with age. Western blot analysis revealed an immunoreactive band at 80 kDa, which increased in accordance with the transglutaminase activity in both models. In normal rat retinas, tTG immunolabeling was present only in the outer segments. There was an increased number of immunolabeled photoreceptor nuclei from 12 hours of light exposure to 24 hours of light exposure. In the RCS rat, increasing numbers of immunopositive photoreceptor nuclei from 20 to 50 days of age were noted. CONCLUSIONS: The data associated increased retinal tTG activity and enzyme levels with photoreceptor cells undergoing apoptosis. The tTG-dependent irreversible cross-linking of intracellular protein may play an important role in causing the structural changes in cells undergoing apoptosis in the retina.

N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina.

PURPOSE: The involvement of apoptosis in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in adult rat retinas was examined. METHODS: Excitotoxic loss of inner retinal elements was induced by intravitreal injections of various concentrations of neutralized NMDA in adult albino Lewis rats. Tissue responses were quantified by measuring the inner retinal thickness (IRT) in plastic sections of the retinas and cell counts in the retinal ganglion cell layer in flatmount preparations of the whole retinas. Internucleosomal DNA fragmentation, a hallmark of apoptosis, was assayed with agarose DNA gel electrophoresis. The in situ TdT-mediated biotin-dUTP nick end labeling (TUNEL) method was used to locate nicked DNA in paraffin sections of the retinas. Ultrastructural changes of the degenerating cells were examined by electron microscopy. The efficacy of Ac-Tyr-Val-Ala-Asp-CMK (YVAD-CMK), a peptidyl caspase inhibitor, and 3-aminobenzamide (ABA), an inhibitor of poly(ADP-ribose) polymerase (PARP), in ameliorating the loss of inner retinal elements was evaluated using morphometry to examine the apoptotic pathways. RESULTS: Intravitreal injection of NMDA induced a dose-dependent loss of inner retinal elements as evidenced by the measurements of IRT and RGCCs. There were time- and dose-related appearances of internucleosomal fragmentation of retinal DNA and a time-related appearance of TUNEL-positive nuclei in the inner retinas after intravitreal NMDA injection. Ultrastructural features consistent with classic apoptotic changes were noted in degenerating cells in the retinal ganglion cell layer and the inner nuclear layer. Control retinas given vehicle, N-methyl-L-aspartate (the L-isomer of NMDA), or NMDA plus MK-801, a specific antagonist, did not show these changes. Simultaneous administration of NMDA and YVAD-CMK or ABA abolished or attenuated the loss of RGCCs in the posterior retinas. CONCLUSIONS: NMDA-induced excitotoxicity involved apoptosis and caspases and PARP may play important roles in the pathways.

Iron-induced apoptosis in the photoreceptor cells of rats.

PURPOSE: To determine whether apoptosis is involved in retinal degeneration induced by intravitreal implantation of 5 iron particles in rat eyes. METHODS: Autoclaved iron particles were implanted in the vitreous cavities of the experimental eyes. Glass chips were implanted in the control eyes. The experimental eyes were enucleated at various time intervals from days 1 to 15. Retinal degeneration was examined using the TdT-mediated, dUTP-biotin nick-end labeling (TUNEL) method. Electrophoresis on agarose gel was used to detect internucleosomal DNA fragmentation. RESULTS: TUNEL-positive nuclei were observed only in the outer nuclear layer beginning on day 2. The nuclei spread throughout the outer nuclear layer by the end of day 3. No TUNEL-positive nuclei were observed in other layers throughout the experimental period. Analysis of DNA, extracted from the retinas by electrophoresis on agarose gel, revealed a typical ladder pattern of internucleosomal DNA cleavage in the experimental eyes. CONCLUSIONS: Apoptosis occurred during photoreceptor cell death at the early phase of iron-induced retinopathy in these rats. Like photic injury, iron-induced apoptosis was limited to the outer nuclear layer.