Methodological approaches to quantitative evaluation of microcirculation in tissues with dynamic magnetic resonance tomography

We observed the process of disappearance of the choriocapillaris after loss of the retinal pigment epithelium (RPE) induced by intravitreal injection or ornithine. Three hours after administration of ornithine, the RPE cells swelled remarkably in the posterior pole, but, the endothelial cells of the choriocapillaris remained intact. At 3 days, the RPE cells became necrotic, but the choriocapillaris still preserved its in normal appearance. At 7 days, RPE disappeared completely in the posterior pole and the choriocapillaris displayed evidence of atrophy; the swollen lumen of the choriocapillaris became narrow the cytoplasm of the endothelium was swollen, and the number of fenestrae was reduced. On the other hand, these changes were not seen where the RPE remained. At 14 days, in the posterior pole, the lumen of the choriocapillaris occluded by the swollen endothelial cells. At 28 days, the choriocapillaris completely disappeared and the large choroidal vessel was directly in contact with Bruch's membrane. These results showed that the RPE is correlated with the presence of the choriocapillaris.     

Polarity and the development of the outer blood-retinal barrier

The retinal pigment epithelium (RPE) is a monolayer that separates the outer surface of the neural retina from the choriocapillaris. Because the choriocapillaris is fenestrated, it is the RPE that forms the outer blood-retinal barrier and regulates the environment of the outer retina. Like all epithelia and endothelia, the ability of RPE to regulate transepithelial transport depends upon two properties: apical tight junctions to retard diffusion through the paracellular spaces of the monolayer, and an asymmetric distribution of proteins to regulate vectorial transport across the monolayer. During development, these properties form gradually. Initially, the tight junctions are leaky, and the RPE exhibits only partial polarity. As the neural retina and choriocapillaris develop, there are progressive changes in the composition of the apical junctional complexes, the expression of cell adhesion proteins, and the distribution of membrane and cytoskeletal proteins. Development can be used to dissect the multiple mechanisms that establish and maintain polarity and barrier function. These mechanisms are regulated by the interactions that develop between the RPE and its neighboring tissues. This review discusses the remodeling of the apical, lateral and basal plasma membranes of RPE that occurs during normal development, and establishes a framework to integrate the data obtained from multiple species. It examines the progress in understanding how environmental interactions regulate this development.     

Transplanted and repopulated retinal pigment epithelial cells on damaged Bruch's membrane in rabbits

AIMS: The authors studied how artificially damaged Bruch's membrane influenced growth and differentiation of transplanted embryonic retinal pigment epithelial (RPE) cells and of host RPE cells in rabbits. METHODS: Embryonic RPE cells obtained from pigmented rabbits were transplanted into the subretinal space of adult albino rabbits. The host RPE was removed with a silicone cannula, and Bruch's membrane was damaged by scratching with a microhooked 27 gauge needle under the detached retina in closed vitrectomy. The transplantation sites were examined 3, 7, and 14 days after surgery by light and electron microscopy. RESULTS: Varying degrees of damage in Bruch's membrane were observed. Pigmented and hypopigmented RPE cells showed a normal polarity and tight junctions were seen at the sites of mild to moderate damage 3-7 days after the surgery. In contrast, fibroblast-like cells with no such features of RPE cells formed multiple layers at the sites of severe damage involving the full thickness of Bruch's membrane and the choriocapillaris even 14 days after the surgery. Without transplantation, host RPE cells repopulated the damaged areas in the same way as transplanted RPE cells. CONCLUSIONS: Transplanted embryonic RPE cells as well as host RPE cells grew and differentiated on the moderately damaged Bruch's membrane, while the severely damaged Bruch's membrane did not allow differentiation of RPE cells although these cells could grow and cover the damaged areas.     

Choroidal vascular lesions in serous retinal detachment viewed with indocyanine green angiography

In serous retinal detachment due to damaged retinal pigment epithelium (RPE), fluorescein angiography shows dye leakage into the subretinal space from the choroid. We performed indocyanine green (ICG) angiography in 110 eyes with serous retinal detachment comprising 71 eyes with central serous chorioretinopathy (CSC), 19 with bullous retinal detachment, 18 with Harada's disease, and 2 with toxemia of pregnancy. Choroidal tissue staining was present around the site of subretinal leakage in late-phase ICG angiograms from 63 eyes with CSC and 18 with bullous retinal detachment. ICG angiography also showed leakage from choroidal vessels in 16 eyes with Harada's disease and 2 with toxemia of pregnancy. As a common feature, ICG angiography showed choroidal vascular hyperpermeability in various types of serous retinal detachment. Choroidal circulation was delayed in Harada's disease and toxemia of pregnancy. Choroidal hypoperfusion and hyperpermeability of choroidal vessels probably contribute to the damage of RPE, and choroidal vascular hyperpermeability probably provides fluid pressure to move fluid into the subretinal space from the choroid.     

Fluorescent dots in fluorescein angiography and fluorescein leukocyte angiography using a scanning laser ophthalmoscope in humans

PURPOSE: The purpose of the study is to disclose the nature of fluorescent dots and segments traditionally observed with fluorescein angiography (FA) using a scanning laser ophthalmoscope (SLO 101; Rodenstock, München, Germany). The authors developed a new method, called fluorescein leukocyte angiography (FLA), to display directly the movement of leukocytes in human retinal vessels. METHODS: Fluorescein angiography was performed on two normal volunteers using a scanning laser ophthalmoscope and fluorescent dots and segments were observed. Fluorescein leukocyte angiography, using an injection of fluorescent buffy coat layer from which the fluorescent plasma and nonfluorescent erythrocytes have been removed externally, was performed on seven normal volunteers. Injection fluid smears were examined through a fluorescent microscope. Peripheral blood smears taken during midphase of FA and FLA also were examined. In addition, 15 early-phase FAs of central serous chorioretinitis (CSC) were studied retrospectively. RESULTS: In the FAs of normal volunteers, fluorescent dots were detected only in perimacular capillaries at early phase. Eight of the 15 CSC FAs examined showed both fluorescent dots and segments. In the FLAs, fluorescent dots were detected in whole retinal vessels for more than 30 minutes. Fluorescent segments were observed in FA but not in FLA. Injected fluid smears from one FLA showed fluorescent leukocytes and small platelets. However, in peripheral blood smears of the FLA, leukocytes and platelets were more visible and exhibited higher contrast than those of an FA due to background plasma fluorescence. The mean velocity of 21 flowing leukocytes in perifoveal capillaries was 1.37 +/- 0.35 mm/second in 2 FAs and that of 89 flowing leukocytes was 1.41 +/- 0.29 mm/second in 7 FLAs. CONCLUSIONS: The authors' observations suggest that fluorescent dots in scanning laser ophthalmoscope imaging are fluorescein-stained leukocytes, whereas fluorescent segments are the hyperfluorescent plasma that is located between rouleaux formations of erythrocytes. The velocity of the fluorescent dots could be measured in the perimacular capillaries by either FA or FLA; however, only FLA can display the flow of fluorescent leukocytes in large vessels.     

Physical and linkage mapping of human chromosome 17 loci to dog chromosomes 9 and 5

We performed fluorescein and indocyanine green (ICG) angiographies in 56 patients with central serous chorioretinopathy, and studied the choroidal lesions. In the early phase, choroidal filling with ICG was delayed in 77% in the area including focal leakage. Hypofluorescent findings around the site of focal leakage persisted through the phase in 23%, and we think this finding was caused by filling defect of the choriocapillaris. In the late phase, choroidal tissue staining by ICG was present in 82% in the area including focal leakage. Multiple areas of choroidal staining were also present in unaffected areas in 43% and in 62% of fellow eyes. Choroidal tissue staining by ICG was revealed in 48% in the area of choroidal filling delay, and this finding persisted after focal leakage had disappeared following photocoagulation. We think this finding was caused by choroidal vascular hyperpermeability. These findings suggest that choroidal circulatory disturbance and choroidal vascular hyperpermeability play a causative role in damage to the retinal pigment epithelium in central serous chorioretinopathy.     

Fine structure of the retinal pigment epithelium in the Port Jackson shark (Heterodontus phillipi)

The structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the Port Jackson shark (Heterodontus phillipi). In this elasmobranch the RPE consists of a single layer of low cuboidal cells which show basal (scleral) infolding and apical (vitreal) processes that enclose photoreceptor outer segments. Laterally these epithelial cells are joined by a series of apically located tight junctions. The RPE cells display a large vesicular nucleus, abundant smooth endoplasmic reticulum as well as numerous polysomes and mitochondria. Phagosomes are present, rough endoplasmic reticulum is scarce and myeloid bodies were not observed. Melanosomes are absent over the choroidally located tapetum lucidum, but are not abundant even in extratapetal areas. This paucity of melanosomes probably makes retinomotor movements unimportant. Bruch's membrane or complexus basalis is a pentalaminate structure. The endothelium of the choriocapillaris is thin but minimally fenestrated.     

Immunoreactivity for calcium-binding proteins in the claustrum of the monkey

The sequence of degenerative changes in the retinal pigment epithelium (RPE) and the choroid of retinal degeneration (rd)-mice was studied in correlation with photoreceptor changes. Three weeks to 26-month-old animals were investigated using light and transmission electron microscopy, enzyme histochemistry and quantitative morphology. Changes in the choriocapillaris (CC) were additionally studied by scanning electron microscopy of corrosion cast preparations. In 3-week-old mice, in which most of the outer segments of photoreceptors in the central portion of the retina had disappeared but remnants of the cells were still present, the RPE was enlarged and showed elongated microvilli. In 8-week-old animals, the photoreceptors were completely absent in large areas of the posterior pole region. In these areas the RPE was also completely lost. Quantitative evaluation performed in histological serial sections showed that loss of RPE measured as length of RPE-free Bruch's membrane, continuously increased up to the age of 20 months. In 8-week-old animals, CC adjacent to degenerating RPE showed loss of fenestration. In 10-week-old animals, the CC disappeared in those areas where the RPE was already lacking. The loss of CC increased with increasing age and in 20-month-old animals 5-10% of the entire CC was lacking. Loss of the related arterioles and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive nerve fibers occurred only in approximately 2-year-old rd-mice. Compared to other animal models, RPE and CC defects in rd-mice are relatively large. The rd-mice might therefore provide a good tool to study factors involved in CC degeneration.     

Correlation between scanning laser ophthalmoscope microperimetry and anatomic abnormalities in patients with subfoveal neovascularization

PURPOSE: The purpose of the study is to identify the anatomic abnormalities associated with an absolute scotoma and the location and stability of fixation in patients with subfoveal neovascularization in age-related macular degeneration, presumed ocular histoplasmosis syndrome, and other disorders. METHODS: Scanning laser ophthalmoscope microperimetry was superimposed on color fundus photographs and fluorescein angiograms of 21 eyes with subfoveal neovascular membranes secondary to age-related macular degeneration (14 eyes) and presumed ocular histoplasmosis syndrome (7 eyes). The authors determined the location and the area occupied by the absolute scotoma and each of the following subretinal lesions: subretinal hemorrhage, neurosensory retinal detachment, retinal pigment epithelial (RPE) atrophy, RPE hyperplasia, atrophy of the choriocapillaris, hard exudates, and the subfoveal neovascular membrane. The area of absolute scotoma determined by scanning laser ophthalmoscope microperimetry was superimposed on the anatomic lesions. The authors calculated the relative risk ratio (RR) of an absolute scotoma occurring in regions corresponding to each anatomic abnormality, and determined the preferred location and stability of fixation in each eye. RESULTS: An absolute scotoma was present in areas of chorioretinal scar (RR = 107.61), RPE atrophy (RR = 9.97), subretinal hemorrhage (RR = 2.88), and the neovascular membrane (RR = 1.86). Fixation was stable in all patients with presumed ocular histoplasmosis syndrome but only 29% of patients with age-related macular degeneration. Fifty-five percent of patients with stable fixation fixated over an area of RPE hyperplasia. CONCLUSION: The relative risk of an absolute scotoma is highest over areas of chorioretinal scars, RPE atrophy, subretinal hemorrhage, and the neovascular membrane. Fixation is more stable in patients with subfoveal neovascularization from presumed ocular histoplasmosis syndrome than with age-related macular degeneration and frequently is present over an area of RPE hyperplasia.     

Harvest and storage of adult human retinal pigment epithelial sheets

PURPOSE: To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RPE) cells. METHODS: Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 50% gelatin containing 300 mM sucrose and stored at 4 degrees C. The viability of the cells, as well as their ability to proliferate in vitro, was studied for 96 hours after harvesting. Light microscopy (LM), transmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclude contamination. RESULTS: LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connections. The initial viability of intact RPE sheets was 86%, with a progressive decline in viability with increased storage time. Cells harvested within 24 hours after death maintained greater viability than those harvested after 24 hours (p < 0.05). Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vitro. CONCLUSION: Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplantation if harvested within 24 hours of death and maintained 82% viability for as long as 48 hours if stored at 4 degrees C.     

Adenoid cystic carcinoma of the breast: value of histologic grading and proliferative activity

Using vitreous fluorophotometry and quantitative fluorescence microscopy the authors studied the permeability of the blood-retinal barrier (BRB) to fluorescein in control and in 8 days streptozotocin-induced diabetic rats. Vitreous fluorophotometry showed that fluorescein permeates BRB in control and in diabetic rats. However, in diabetic rats the permeability to fluorescein was significantly increased as compared to control rats. The vitreous penetration ratio (VPR) values for total and free fluorescein at 60 min, were higher for diabetic rats (231.2+/-12.9 min-1 for total fluorescein and 1299.24+/-58.0 min-1 for free fluorescein) than for control rats (95.5+/-3.5 min-1 for total fluorescein and 646.6+/-55. 0 min-1 for free fluorescein) (P<0.05). Quantitative confocal fluorescence microscopy confirmed these findings and identified the site of leakage across the BRB by comparing the relative importance of the fluorescein leakage across the outer and inner BRB. In control rats the fluorescence levels remained relatively low in the photoreceptor layer, next to the outer BRB but in the inner nuclear layer, next to the inner BRB reached values that were almost ten times higher. These results suggest that in retinas of control rats fluorescein penetrates predominantly through the inner BRB. In diabetic rats the fluorescence levels in the photoreceptor and in the inner nuclear layer were significantly increased as compared to the fluorescence levels in controls rats. Nevertheless, in the inner nuclear layer the fluorescence levels were also generally higher than the fluorescence levels at the photoreceptor layer. The rates of fluorescence levels between the inner nuclear layer and the photoreceptor layer were apparently 3:1, 60 min after the single intravenous injection of fluorescein. Also, the fluorescein penetration in the inner nuclear layer of the diabetic rats is higher than that observed in the inner nuclear layer of the control rats (P<0.001). These findings suggest that the permeability to fluorescein of both components of the BRB is increased 8 days after the induction of diabetes by streptozotocin and that the permeability of the retinal vasculature is preferentially affected.     

Recombinant GM2-activator protein stimulates in vivo degradation of GA2 in GM2 gangliosidosis AB variant fibroblasts but exhibits no detectable binding of GA2 in an in vitro assay

OBJECTIVE: To examine choroidopathy in patients with Beh?et disease. DESIGN: Prospective clinical study. PARTICIPANTS: Thirty-three patients (63 eyes) with Beh?et disease. INTERVENTION: Patients underwent simultaneous indocyanine green (ICG) and fluorescein angiography with a double detector of scanning laser ophthalmoscopy. MAIN OUTCOME MEASURES: Angiographic findings recorded on videotapes were evaluated. The relation of angiographic findings with systemic activity and aqueous inflammation was also analyzed. RESULTS: Fluorescein angiography showed leakage in varying degrees from retinal vessels in 30 patients (53 eyes, 84%). The ICG angiographic findings were choroidal vascular wall staining in 16 eyes (25%), hyperfluorescent spots in 42 eyes (66%) and hypofluorescent plaques in 22 eyes (35%), both of which were not evident with fluorescein, leakage from choroidal vessels in 3 eyes (5%), and irregular filling of choriocapillaris in 11 eyes (17%). These findings did not have a statistically significant correlation with the presence or absence of aqueous inflammation or oral aphthous ulcerations. CONCLUSIONS: The patients with Beh?et disease showed choroidal abnormalities, which could be revealed only by ICG angiography, but not with funduscopy or fluorescein angiography. Simultaneous ICG and fluorescein angiography would be useful for examining choroidal lesions in Beh?et disease.     

Host response to mycobacterial infection in the alcoholic rat: male and female dimorphism

BACKGROUND: The intravitreal injection of ornithine produces selective damage to the retinal pigment epithelium (RPE) and results in a loss of RPE, choriocapillaris and photoreceptor cells. To elucidate the mechanism of secondary retinal atrophy, we investigated the presence of apoptotic cells in a rat model of ornithine-induced retinopathy. METHODS: At 6 and 12 h and 1, 2, 4, 7, 14 and 28 days after an intravitreal injection of L-ornithine hydrochloride in rat eyes, we removed the eyes and subjected them to histopathological examination. We detected apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL) assay, which stains the 3'-OH ends of fragmented DNA. We used electron microscopy to detect the apoptotic cells morphologically. RESULTS: RPE cells were selectively damaged immediately after ornithine administration. TUNEL-positive photoreceptor cells appeared exclusively in the photoreceptor cell layer 12 h after ornithine administration. The number of TUNEL-positive cells increased throughout the 2 days following the injection, then decreased markedly. TUNEL-positive cells remained until 28 days, when the photoreceptor cells had disappeared. The ganglion cell layer, inner nuclear layer and damaged RPE cells were negative for TUNEL staining during all stages. The electron microscopic study also revealed the pyknotic nuclei of apoptotic photoreceptor cells. CONCLUSION: An intravitreal injection of ornithine caused primary damage to the RPE, and subsequently some of the photoreceptor cells revealed apoptosis by TUNEL assay. These findings suggest the dysfunction of the RPE causes photoreceptor cell death according to the intrinsic program of an apoptotic mechanism.     

Indocyanine green angiography in choroidal osteoma

BACKGROUND: Choroidal osteoma is a rare choroidal tumor; knowledge of its indocyanine green characteristics is limited. METHODS: The fundus photographs and the fluorescein and indocyanine green angiograms of three patients were reviewed. Each patient was examined at least twice with a follow-up varying from 10 to 60 months. RESULTS: Late-phase fluorescein angiograms allow assessment of the extension of the osteoma as it is variably hyperfluorescent due to tumor staining combined with a variable degree of overlying retinal pigment epithelial changes. The hypofluorescent area observed in the early phase of the indocyanine green angiogram corresponds with the extent of the osteoma but the borders may be difficult to demarcate. In the late phase of the indocyanine green angiogram, hypofluorescence due to choriocapillaris loss and hyperfluorescence due to leakage from abnormal choroidal vessels are combined. Infrared angiography high-lights abnormal choroidal vessels and vascular spiders present on the tumor surface. It is difficult to differentiate these choroidal vascular anomalies from subretinal neovascularization. CONCLUSIONS: We find no homogeneous pattern either on fluorescein or on infrared angiography. The findings may change with follow-up, indicating changes within the tumor or the surrounding tissue that are still poorly understood.     

The effects of prostaglandins on the blood-retinal barrier

The effects of prostaglandin (PG), a novel PG-related compound, and epinephrine on the blood-retinal barrier (BRB) in the rabbit eye were examined by ophthalmoscopy, fundus photography, fluorescein angiography (FAG), vitreous fluorophotometry (VFPM), light and electron microscopy, and the horseradish peroxidase tracer. Intravitreal injection of PGE2 produced retinal vasodilation and large increase in a vitreous fluorescein leakage in VFPM. Intravitreal injection of PGF2 alpha produced a small increase in vitreous fluorescein leakage in VFPM, but no retinal vasodilation. Intravitreal injection of epinephrine produced retinal vasodilation and a small increase in vitreous fluorescein leakage in VFPM. But intravitreal injection of latanoprost (PhXA41) produced no retinal vasodilation and no increase in vitreous fluorescein leakage in VFPM. After intravitreal injection of PGE2, morphological changes in the retina were found, but intravitreal injection of PhXA41 did not induce morphological changes in the BRB or the retina. PhXA41 was less destructive to the BRB and the retina than PGE2, PGF2 alpha, and epinephrine.     

Photoreceptor allografts in a feline model of retinal degeneration

BACKGROUND: Photoreceptor transplants provide a potential means to restore function in a degenerate retina and/or rescue degenerating host photoreceptors by trophic influences. We have examined photoreceptor allografts in the Abyssinian cat model of hereditary photoreceptor degeneration to determine the viability and influence of such transplants on the host retina. METHODS: Small pieces of 3- to 5-day-old normal kitten retina containing undifferentiated photoreceptors were injected into the subretinal space of adult Abyssinian cats at an early stage of retinal degeneration using standard vitreo-retinal surgical techniques. The retinas were examined by ophthalmoscopy and fundus photography, then by light and electron microscopy at different times after surgery. RESULTS: Such allografts survive for at least 6 months after surgery. The photoreceptors develop outer segments, invariably in rosettes. The transplants gradually integrate with the host retina but detach the host photoreceptor layer from the retinal pigment epithelium (RPE), which tends to reduce the number of host photoreceptors over the transplant. There is no slowing of the photoreceptor degeneration in neighboring non-detached retina. Inflammation or rejection was not detected. CONCLUSION: Undifferentiated, neonatal photoreceptor allografts survive and develop outer segments in the subretinal space of the Abyssinian mutant feline retina. The allografts gradually integrate with the host neural retina without inducing rejection. In the vicinity of the transplant there is increased loss of host photoreceptors, considered to be due to their detachment from the RPE layer. There is no evidence of any rescue of host photoreceptors elsewhere in this mutant retina.     

Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite

Decreased retinal blood flow has been measured in streptozotocin (STZ)-induced diabetes of 1 week's duration, and primary insulin intervention was effective in maintaining normal retinal blood flow in diabetic rats. Retinal blood-flow abnormalities precede clinical diabetic retinopathy in both diabetic animals and patients. An important characteristic of diabetic retinopathy is the difficulty of reversibility once it has been established. Because altered retinal hemodynamics is a possible marker of early diabetic retinopathy, we investigated in this study whether retinal blood-flow changes in rats can be normalized by secondary insulin intervention following short and chronic periods of untreated STZ-induced diabetes. Subcutaneous insulin pumps were placed into diabetic rats for 1 week after 1 week of diabetes (2-week group) and after 3 weeks of diabetes (4-week group). Retinal circulatory parameters were determined using image analysis of video fluorescein angiogram recordings. For the 2-week group, retinal blood flow was significantly (P < 0.05) reduced in the untreated diabetic rats compared with nondiabetic and insulin-treated diabetic rats (80.6+/-29.2, 131.9+/-50.1, and 151.3+/-54.0 pixels2/s respectively). Retinal blood flow was also significantly (P < 0.05) reduced in the 4-week untreated diabetic rats compared with nondiabetic rats (95.7+/-22.2 vs. 125.7+/-29.5 pixels2/s). In contrast to the shorter-duration group, insulin treatment for 1 week after 3 weeks of diabetes did not totally normalize retinal blood flow (117.5+/-32.4 pixels2/s). These results suggest that vascular abnormalities could become more resistant to normalization following short-term (1 week) insulin treatment after longer periods of untreated diabetes.     

Tissue effects of subclinical diode laser treatment of the retina

OBJECTIVE: To determine whether consistent tissue effects are obtained when laser lesions are produced with a commercially available diode laser that are near the limit of clinical detection at the time of treatment. METHODS: Continuous-wave or micropulse diode laser was used to produce clinically undetectable (subthreshold) or barely detectable (threshold) retinal lesions in pigmented rabbits. Tissue effects at intervals after treatment were determined in retinal pigment epithelial (RPE) whole mounts by fluorescence microscopy, and in sections of retina and RPE by light and electron microscopy. RESULTS: Continuous-wave and micropulse laser lesions that were originally clinically undetectable were detectable as zones of pigment mottling after 5 days. By microscopy, compaction and/or swelling was seen in the outer retina, and cells in the RPE monolayer became heterogeneous in size, shape, and pigmentation, but the tissue responses in the outer retina and RPE were variable even within and among lesions in the same eye. CONCLUSIONS: Subthreshold energies used to create both continuous-wave and micropulse laser lesions produced variable effects on the RPE and the overlying neurosensory retina. It appears that, near the minimum effective dose of laser irradiation, individual RPE cell heterogeneity becomes detectable as variability in sensitivity to laser injury. CLINICAL RELEVANCE: As laser energy is reduced to limit collateral tissue damage in clinical applications, it may be difficult to generate reproducible lesions because of heterogeneity among individual cells.     

Changes in choroidal innervation in Royal College of Surgeons rats with hereditary retinal degeneration

In Royal College of Surgeons (RCS) rats with hereditary retinal degeneration loss of retinal pigmented epithelium (RPE) and choriocapillaris is most pronounced in the upper-temporal quadrant. To investigate whether changes in choroidal vasodilative innervation might be involved in the RPE degeneration, we analyzed whole mount preparations of the retina and choroid stained for nitric oxide synthase and for NADPH-diaphorase (d) of 19 dystrophic RCS rats and 24 age-matched congenic controls of different age groups. Density of NADPH-d-positive nerve fibers was quantitatively evaluated in the upper-temporal and lower-nasal quadrant. Our results revealed that even in control animals there were much less positively stained nerve fibers in the upper-temporal than in the lower-nasal quadrant. Nerve fiber density in both quadrants increased for up to 3 months and remained nearly constant throughout life. In the dystrophic animals up to 3 months of age nerve fiber density was similar to that seen in the controls. In dystrophic animals older than 3 months nerve fiber density in the upper-temporal quadrant decreased significantly, whereas density in the lower-nasal quadrant revealed nearly the same values as in the age-matched controls. Decrease of NADPH-d stained nerve fibers in this quadrant occurred prior to the vascular changes in the choriocapillaris. In the retina of RCS dystrophic rats an increase of NADPH-d-positive amacrine cells was found only in 3-month-old animals. Most of these cells were located in the vicinity of irregularly arranged branches of the central retinal artery. In animals 5 months of age and older the number of cells decreased to the same values found in controls, so that we assume that increase of NADPH-d-positive amacrine cells is involved in capillary degeneration or sprouting.     

Photoreceptor recovery in retinoid-deprived rats after vitamin A replenishment

Dietary deficiency in the retinoid precursors of the visual pigment chromophore 11-cis retinal results in the synthesis of photoreceptor outer segments containing opsin in excess of the vitamin A available for rhodopsin regeneration. This suggests that vitamin A-free opsin may be incorporated into newly synthesized outer segment disc membranes. If this opsin is functionally intact, it should be possible convert it to rhodopsin in vivo by providing the appropriate retinoids, and the resulting rhodopsin should should be able to mediate visual transduction. Experiments were conducted to evaluate this possibility and to identify the rate-limiting steps in photoreceptor recovery from retinoid depletion. Rates were maintained on diets either containing or lacking retinoid precursors of 11-cis retinal for 23 weeks, at which time outer segment opsin content greatly exceeded the availability of visual cycle retinoids in the retina. The retinoid-deprived animals were then each given a single intramuscular injection of all-trans retinol. At various time intervals after retinol administration, electroretinograms (ERGs) were recorded on some rats, and retinal rhodopsin contents were determined in others. At similar time intervals, blood and retinal pigment epithelial (RPE) retinoid levels and photoreceptor outer segment size were also determined. No significant increase in retinal rhodopsin content was observed up to 8 hr after injection, despite the fact that by 3 hr, blood retinol levels had recovered to more than 30% of normal. By 1 day after injection, however, rhodopsin levels had recovered to 30% of normal and ERG responses showed increases in visual sensitivity commensurate with the recovery of rhodopsin. The lag in rhodopsin recovery was apparently due to delayed uptake of retinol from the blood by the RPE. Photoreceptor outer segment size was reduced by over 50% in the retinoid- deprived rats and did not begin to recover by 1 day. By 1 week, however, outer segment size had returned to an average of 65% of normal. Commensurate with this regrowth of the outer segments, both rhodopsin levels and visual sensitivity increased between 1 and 7 days after vitamin A administration. Because the rates of recovery in rhodopsin levels and visual sensitivity greatly exceeded the normal rate of new opsin synthesis at short time intervals after vitamin A repletion, it appears that the opsin incorporated into the disc membranes of retinoid-deprived rats is able to form functional rhodopsin in vivo when the chromophore is supplied. Regrowth of the outer segments back to their normal size is required for full recovery of visual sensitivity.