Irregular retinal and RPE damage after pressure-induced ischemia in the rabbit

PURPOSE: Pressure-induced ocular ischemia is a frequent model for the investigation of the mechanisms and therapy of retinal ischemic damage. It is important to know whether the tissue damage in such experiments is uniform or irregular. METHODS: We reviewed histologic features of Dutch rabbit eyes after 60-80 min of pressure-induced ischemia. The eyes were enucleated 4 hr, 1 day, or 1 wk after circulation was restored, at which times the electroretinogram b-wave was moderately reduced. RESULTS: Light microscopy showed an irregular distribution of damage involving all retinal layers and retinal pigment epithelium. Some regions of damage (or preservation) were several millimeters wide; others were as small as a few cell widths. Correlation with electroretinogram reduction in individual eyes was difficult. CONCLUSIONS: These results show that pressure-induced ischemic damage in the rabbit, sufficient to reduce the electroretinogram, has a patchy and irregular effect on retina and retinal pigment epithelium. Erroneous judgments may be made about ischemic damage, or therapeutic intervention, if only small or selected regions of retina are examined histologically.     

Repair and adhesion mechanisms of the cryotherapy lesion in experimental retinal detachment

Cryotherapy to the pigment epithelium and retina induced a proliferation and metaplasia of pigment epithelial cells, Mueller cell hypertrophy, and proliferation of astrocytes. When cryotherapy was applied to the pigment epithelium and to the retina during retinal detachment surgery, a strong adhesion developed, characterized by the occurrence of true cell junctions between pigment epithelium and retinal cells. When only the pigment epithelium was treated, the adhesion appeared weak due to the absence of microvillous interdigitations normally present between pigment epithelium and retina.     

Light-induced apoptosis: differential timing in the retina and pigment epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

Immunocytochemical study of retinal diode laser photocoagulation in the rat

AIM: To determine the nature of the cellular infiltrate, alterations in cell adhesion molecules, and MHC II antigen expression in the rat retina following diode laser retinal photocoagulation. METHOD: 20 normal Lister rats underwent diode laser photocoagulation of the retina. Frozen sections from eyes enucleated at 0, 1, 5, 13, and 33 days post laser were examined for T cells (R7.3), CD4 T cells (W3/25), activated CD4 T cells (OX-40), CD8 T cells (OX-8), B cells (OX-33), and macrophages (OX-42), MHC II antigen (OX-6), and E-Selectin-1, VCAM-1, and ICAM-1. RESULTS: Retinal diode laser photocoagulation stimulated a wound healing response in the outer retina and choroid. The cellular infiltrate included macrophages and activated CD4 T cells at 13 and 33 days post laser. Glial cells in the inner plexiform and inner nuclear layers expressed MHC II antigen at 24 hours only. ICAM-1 antigen was induced in RPE cells and in Muller cells in the inner retina at all time intervals post laser and intense staining for ICAM-1 was present around intraretinal migrated cells at 13 and 33 days post laser. VCAM-1 antigen expression was induced in the choroidal vascular endothelium and RPE at 13 and 33 days after laser as was E-Selectin-1 antigen expression which was also evident focally at the external limiting membrane in association with migrated cells adjacent to the burn. CONCLUSIONS: These results suggest that alterations in cell adhesion molecules may regulate the migration and activation of retinal pigment epithelium, macrophages and CD4 T cells at the outer blood-retinal barrier and choroid following diode laser photocoagulation of the normal Lister rat retina.     

Tendon interposition in a juxta-epiphyseal fracture of the proximal phalanx

PURPOSE: To report the clinicopathologic features of intraocular osseous production in association with proliferative vitreoretinopathy. METHOD: The clinical and histopathologic features of two patients with proliferative vitreoretinopathy and intraocular bone formation are reviewed. RESULTS: Preretinal osseous tissue incorporated in the proliferative vitreoretinopathy was surgically removed in one patient, and osseous tissue was present in the proliferative vitreoretinopathy in the enucleated eye of the other patient. CONCLUSIONS: Bone formation, presumably from metaplastic retinal pigment epithelium, may be present in proliferative vitreoretinopathy tissue. The intraocular bone is present internal rather than external to the neurosensory retina.     

Elucidation of the mechanism of retinal degeneration and regeneration and the prospects for its clinical application

In order to obtain the basic knowledge necessary to develop therapeutical intervention for blindness due to the damaged retina and optic nerve, the mechanism of retinal degeneration and regeneration in an amphibian model, Cynops pyrrhogaster, was studied. In the retinal degenerative process following enucleation and reimplantation of the eye ball, evidence was found for active cell death of neural retinal cells. As the degeneration proceeded, Musashi, an ribonucleic acid (RNA)-binding protein, started its expression in the daughter cells of proliferating retinal pigment epithelium (RPE) cells, messenger RNA (mRNA) expression of proneural genes with basic helix-loop-helix motif was then detected in the newly developing retina. These results suggest that transdifferentiation of RPE cells to neural retina involves at least partial cascade, if not entirely, of neural induction from uncommitted ectodermal tissue. Search for genes that are required for transdifferentiation of RPE cells to neural retinal cells, in addition to those mentioned above, will provide the basic knowledge for successful retinal transplantation and retinal regeneration in higher vertebrates.     

The S1(1A1)-S0(1A1) Electronic Transition of Jet-Cooled o-Difluorobenzene

Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +15% fetal calf serum+basic fibroblast growth factor (1 ng ml-1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml-1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4-6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6-8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.     

Albumin movement out of the subretinal space after experimental retinal detachment

PURPOSE: The subretinal fluid of serous retinal detachments contains protein, but little is known about its origin and fate. The authors designed experiments to study the rate and route of albumin movement out of the subretinal space. METHODS: Experimental retinal detachments were made in Dutch rabbits by injecting Hanks' balanced salt solution containing serum levels (approximately 30 mg/ml) of fluorescein isothiocyanate (FITC) albumin into the subretinal space through a micropipette. Subretinal, vitreous, and serum fluid samples were withdrawn 0 to 4 hours later through a similar micropipette and were analyzed for osmolality, FITC albumin content (by fluorophotometry) and FITC+native albumin content (by gel electrophoresis). Sodium iodate was injected intravenously in some rabbits to damage the retinal pigment epithelium (RPE). RESULTS: Albumin injected into the subretinal fluid diffused steadily into the vitreous, and its concentration decreased by approximately 5% per hour. This rate was unaffected by RPE damage. Albumin did not move into the bloodstream unless the RPE was damaged with sodium iodate, and then it crossed the RPE at approximately 25% of the rate at which it moved into the vitreous. Subretinal fluid osmolality remained within the range of 293 to 294 mOsm/kg despite protein movement and the continual absorption of fluid from the detachments. CONCLUSIONS: These results show that albumin in the subretinal space diffuses readily into the vitreous, and subretinal osmolality changes are rapidly equilibrated with the vitreous. Albumin does not cross normal RPE, and it crosses iodate-damaged RPE more slowly than it crosses retina. Thus, there must be a constant supply of albumin if high subretinal concentrations are to be sustained in clinical serous detachments.     

Intraocular silicone oil tamponade. A clinico-pathologic study of 36 enucleated eyes

BACKGROUND: Previous histological studies have shown that intraocular silicone oil induces irreversible changes in ocular tissues, especially the retina. The purpose of this study was to analyze, in a larger group of enucleated eyes, changes in intraocular tissue after silicone oil injection, dependent on intraocular pressure, how long the oil was in the eye, and the viscosity of intraocular silicone oil. PATIENTS AND METHODS: We did histological examinations on 36 enucleated globes with intraocular silicone oil after vitreoretinal surgery and compared them with 68 enucleated globes treated with buckle and encircling band using immunohistochemistry (n = 5) and electron microscopy (n = 7). For statistical evaluation we used the chi(2) test and analysis of variance. RESULTS: After silicone oil injection we observed a more pronounced reduction in corneal endothelial cells (58%), more frequent closed chamber angle (86%), atrophy of the ciliary body (80%) (P < 0.05), proliferative vitreoretinopathy (89%), and glaucomatous atrophy of the optic nerve (56%) (P < 0.01). The retinae showed independent of the use of silicone oil a loss of inner and outer segments of photoreceptors and of ganglion cells and thinning and rareficaton of all other retinal layers. Globes with silicone oil revealed vacuoles both free and incorporated by macrophages in all layers of the retina. Similar vacuoles were seen in the optic nerve, choroid, retinal pigment epithelium, ciliary body, iris, chamber angle and the corneal endothelium. Silicone oil vacuoles were seen in the retina and optic nerve by 1 month after surgery in two eyes with high intraocular pressure (42 mmHg). Six of eight eyes with normal intraocular pressure levels showed retinal vacuoles, 3 of them after 2 months. Vacuoles in the optic nerve were found in eight of nine eyes with intraocular instillation of 1000 mPa silicone oil. There was no clinicohistopathological correlation between the presence of vacuoles in the retina or optic nerve and the duration and viscosity of intraocular silicone oil. CONCLUSIONS: This study suggests that vacuoles in eyes with silicone oil instillation can be found in the retina after 4 weeks. The period of intraocular silicone oil should be limited to 3-6 months.     

Sensitivities of ocular tissues to acute pressure-induced ischemia

Intraocular pressure was artificially elevated for eight hours in eight owl monkeys. The first permanent effect (produced at a perfusion pressure of plus 15 mm Hg) was partial necrosis of iris stroma and ciliary processes, associated with microscopic lesions in the photoreceptors and retina pigment epithelium around the disc and in the retinal periphery. At a slightly higher pressure, visual nerve fibers in the retina and optic nerve and their ganglion cells were affected. Simultaneously, the outer retinal layers showed damage to the pigment epithelium, photoreceptors, and other nuclear layers. At even higher pressures, nearly all the other intraocular tissues were affected except for Müller cells, astroglia in the optic nerve head, epithelium of the pars plana, and the pigment cells of the choroid. The possibility is raised of a nonischemic pressure-induced mechanism for destruction of disc astrocytes in human chronic glaucoma.     

In vivo and in vitro developmental toxicity in LPS-induced zinc-deficient rabbits

Adenosine, produced from the decomposition of adenosine triphosphate, is believed to provide protective effects during ischemia. On the other hand, adenosine metabolites may serve as precursors for oxygen free radical formation. The time course of formation of adenosine and its purine metabolites was studied during retinal ischemia in rats. Concentrations of adenosine and its purine nucleoside metabolites inosine, hypoxanthine, and xanthine in the retina-choroid of ketamine/xylazine-anesthetized rats were measured during retinal ischemia using high performance liquid chromatography. Quantitative measurements were made possible in the small tissue mass through the use of internal standards. Ischemia was induced by ligation of the central retinal artery. In each rat, one eye was ischemic while the other served as a non-ischemic control. Eyes were frozen in situ at 1, 5, 10, 20, 30, 60, and 120 min of ischemia. The retina-choroid was then removed from the frozen eyes and analysed. Significant increases in the concentrations of adenosine, inosine, and hypoxanthine in ischemic compared to control retina-choroid were detectable within 1 to 5 min of the onset of ischemia, and within 10 min for xanthine. Increase in adenosine concentration in ischemic relative to control retina-choroid plateaued at 30 min of ischemia, while inosine and hypoxanthine concentrations increased continuously. The increase in xanthine concentration was exponential throughout the measurement period. This study documented the time-related changes in purine nucleoside concentration during ischemia. Prolonged ischemia results in ongoing production of xanthine, which by serving as a precursor for oxygen free radical formation, could be a pathogenic factor in prolonged retinal ischemia.     

Effects of intravenous iodoacetate and iodate on pH outside rod photoreceptors in the cat retina

PURPOSE: Effects of intravenous iodoacetate (a glycolysis inhibitor) and iodate (a metabolism inhibitor selective to retinal pigment epithelium) on light-evoked alkalinizations and hypoxia-induced acidifications were studied in the dark-adapted cat retina, in vivo, to learn about pH regulation. METHODS: pH was recorded in the extracellular space surrounding rod photoreceptors with double-barrelled H(+)-selective microelectrodes. RESULTS: Intravenous infusion of 5 mg/kg iodoacetate-induced alkalinizations in the outer nuclear layer and suppressed both light-evoked and hypoxia-induced pH responses immediately. Iodate injection (30 mg/kg) produced acidifications in the subretinal space and affected light-evoked alkalinizations gradually but not hypoxia-induced acidifications. CONCLUSIONS: These results suggest that rods glycolysis plays an important role in both light-evoked and hypoxia-induced pH responses. And the retinal pigment epithelium may have little concern with light-evoked alkalinizations except that it plays an important role in regenerating the rhodopsin to be needed for the light responses of photoreceptors. Furthermore, the finding of the intravenous-iodoacetate-induced alkalinization in the outer nuclear layer supports that acid production by rods in the dark is originated from glycolysis to support the dark current. The iodate-induced acidification in the subretinal space indicators that the retinal pigment epithelium might actively transport acids from the subretinal space to the choroid.     

Localization of vascular endothelial growth factor in human retina and choroid

OBJECTIVE: To examine the distribution and relative levels of vascular endothelial growth factor (VEGF) in the nondiabetic and preproliferative diabetic human retina and choroid. METHODS: Immunohistochemical localization was performed on frozen sections from cryopreserved postmortem human tissue using a polyclonal antibody against VEGF and a streptavidin peroxidase system. Eyes from 5 subjects without diabetes and 8 subjects with diabetes were examined and analyzed using a 7-point immunohistochemical grading system. RESULTS: In subjects without diabetes, weak or no VEGF immunoreactivity was associated with retinal blood vessels. In subjects with diabetes, we found significantly increased immunoreactivity in the retinal vascular endothelium and blood vessel walls. Vascular endothelial growth factor immunoreactivity was also associated with intravascular leukocytes in subjects with and without diabetes. In the choroid of subjects without diabetes, immunoreactivity was almost exclusively associated with intravascular leukocytes, whereas in diabetic subjects, immunoreactivity was localized within choriocapillaris endothelium, choroidal neovascular endothelium, and migrating retinal pigment epithelium cells. CONCLUSIONS: The observed increase in VEGF immunoreactivity in the diabetic retina and choroid suggests that VEGF may contribute to 2 well-documented events during retinopathy: increased vascular permeability and angiogenesis.     

The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.     

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.     

Optic nerve damage following argon laser photocoagulation in a human eye

Argon laser photocoagulation of diabetic neovascular formations overlying the optic disc risks injury to the nerve fibers. Isolated reports of postoperative field defects have appeared but extensive pathologic studies of the effects on nerve fibers are lacking. In an eye with a malignant melanoma nasally, laser photocoagulation was directed at the superior temporal artery on the disc. Applications were made repeatedly until spasm was produced at two sites. The following day, the eye was enucleated. Histopathologic examination revealed loss of endothelial cells and absence of nuclei in the media of the treated artery, and coagulative necrosis of nerve fibers around the vessel. Extensive damage to the peripapillary outer segments and retinal pigment epithelium also was demonstrated.     

Post-mortem changes in the rabbit retina. A study by light microscopy

The course of post-mortem changes in rabbit retina has been followed. Short post-mortem periods are accompanied by degenerative changes limited mainly to the visual cells and retinal pigment epithelium. Long post-mortem periods are associated with degenerative changes throughout the retina. Retinal tissue maintained at room temperature was less affected than that kept at body temperature (37degreesC). Post-mortem changes are similar to those observed following periods of pressure induced ischaemia and it is thought that the mechanical effects of pressure on retinal tissue are minimal at the level of resolution afforded by light microscopy.     

Cellular and molecular biology of ischemic retina

We introduce our studies on the retinal ischemia in light of both pre- and post-Noell viewpoints. For several years now, we have employed in vivo intraretinal microelectrodes for this field of experiments. This series of studies on the cat eye revealed that the sensory retina as well as the retinal pigment epithelium is severely damaged after only a ten-minute stoppage of blood flow. This phenomenon in usually masked in the routine electroretinogram, a mass electrical response of the retina monitored from the ocular surface. Our studies, employing cultured amacrine cells from embryonic rat eyes, revealed that ischemic changes in neural cells are induced by an increase in extracellular glutamate. Among the glutamate analogs, N-methyl-D-aspartate (NMDA) is responsive to this change. An influx of calcium through NMDA receptor channels activates nitric oxide synthase (NOS), inducing intracellular nitric oxide (NO) in selected amacrine cells. Nitric oxide reacts with free radicals in the cell and induces peroxinitrite, which is toxic. This cascade triggered by ischemia is interrupted by extracellular zinc, magnesium, hemoglobin, nitroprusside, s-nitrosocysteine, and some NMDA antagonists. In terms of clinical application, there is a possibility that dihydroxyphenylalanine (DOPA), superoxide dismutase (SOD), and catalase (CAT), as well as vitamins B6 and B12, are important candidates for administration before an ischemic attack for prevention of damage to the retinal neurons. Gene expression of NOS, interleukin (IL)-1, IL-6, tumor necrosing factor (TNF), and transforming growth factor (TGF)-beta in the ischemic retina was investigated in order to discover reaction substances common to ischemic change and inflammation.     

Fourth module of Xenopus interphotoreceptor retinoid-binding protein: activity in retinoid transfer between the retinal pigment epithelium and rod photoreceptors

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.