Noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse.

PURPOSE: Optical coherence tomography (OCT) is a high-resolution imaging technique that measures the intensity of backscattered light from biological microstructures in living tissue. The objective was to evaluate OCT as a routine, noninvasive technique for quantitative measurements of retinal thickness and detachment in small animal models of retinal degenerative diseases. METHODS: An OCT scanning unit was designed and built to visualize retinal tissue from rodents at high resolution in vivo. Several normal and retinal degeneration (rd) mouse strains with different pigmentation, as well as a transgenic mouse strain that carries a wild-type beta-PDE gene in an rd/rd background, were analyzed at different ages. Retinal detachment was induced by subretinal injection of saline. Retinal function was evaluated by full-field ERG, and then each retina was cross-sectionally scanned by OCT. OCT image analysis and measurements of retinal thickness were performed. Animals were then killed and retinal histology was documented. RESULTS: OCT images of the mouse retina revealed structural landmarks allowing assignment of retinal structures. There was no difference in the OCT pattern between pigmented and nonpigmented mice. Changes in the retinal thickness measured by OCT correlated very well with the loss in function measured by ERG and histology in rd/rd and rd/rd/tg(+) transgenic mice at a variety of ages. In addition, retinal detachment caused by surgery was easily visualized and observed by OCT imaging. CONCLUSIONS: OCT imaging is applicable to the mouse retina. There is excellent agreement between the retinal thickness measured by OCT, ERG amplitude, and retinal histology, thus validating OCT imaging as a sensitive and noninvasive tool for monitoring the structural progression of retinal diseases in rodent models. OCT also appears useful for visualizing retinal detachments in the mouse.

Subretinal injections in rodent eyes: effects on electrophysiology and histology of rat retina.

PURPOSE: To describe a reliable and fast method for subretinal injection in rodents and to assess the effect of the procedure on retinal function and histology. METHODS: Corneas of rodents were punctured with a 28 gauge hypodermic insulin needle avoiding the lens. The injection procedure can be observed with the aid of a dissecting microscope and methylcellulose solution on the eye. A 33 gauge blunt needle was inserted into the eye through the corneal puncture and guided toward the subretinal matrix. Addition of fluorescein to the injection mixture facilitated immediate evaluation of the injection. Rat eyes were either non-injected (controls), received only a corneal puncture or were injected with fluorescent microspheres or PBS-fluorescein mixture. Retinal function and integrity were assessed through electroretinographic (ERG) analysis and postmortem histology. RESULTS: The anterior injection procedure provided a fast and simple method for subretinal injections. In rats a successful subretinal delivery was achieved in more than 90%, with less than 5% of the injected eyes developing cataracts. No significant differences in b-wave ERG amplitudes in rodent eyes over a five-week period were observed between non-injected control eyes and subretinally injected eyes (1 to 10 microl of PBS-fluorescein or 2 microl fluorescent microspheres). Histological analysis revealed that re-attachment of the rat retina occurred in approximately 1 day post-injection and the phagocytotic ability of RPE cells remained intact. CONCLUSIONS: This method was easily learned and required a minimum of equipment and animal preparation. With experience, 10 to 30 eyes could be injected per h. Furthermore, the injection procedure did not compromise the lens, retina or retinal pigment epithelium (RPE).

Rod photoreceptor maturation does not vary with retinal eccentricity in mammalian retina.

PURPOSE: Test the hypothesis that the development of mammalian rod outer segments (ROS) varies with retinal eccentricity. METHODS: During the period of photoreceptor cell development, ROS lengths, opsin mRNA and (rhod)opsin were measured in central and peripheral retina of cows and pigmented rats. Published ROS length and/or rhodopsin data from albino rats, cows and monkeys were re-analyzed. Logistic growth curves were fitted to the newly obtained and published data. Within a species, growth in central and peripheral regions was compared. RESULTS: The logistic growth curves fit all the data well and provide an excellent view of the developmental increases in ROS length, opsin mRNA and (rhod)opsin in each retinal region. Within a species, the growth curves for ROS length, opsin mRNA and (rhod)opsin concentration are superimposable. The age at which ROS length reaches 50% of its adult value is invariant with eccentricity. An exception to this pattern is the simian parafoveal ROS, which appears to have a delayed course of development. CONCLUSIONS: The hypothesis is disproved. Unlike rod photoreceptor cell genesis, ROS development is invariant with retinal eccentricity. Primate parafoveal ROS appear to have a different pattern of development.

Ribozyme-targeted destruction of RNA associated with autosomal-dominant retinitis pigmentosa.

PURPOSE: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa. METHODS: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction. RESULTS: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA. CONCLUSIONS: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.

Topographical regulation of cone and rod opsin genes: parallel, position dependent levels of transcription.

RNase protection assays were used to follow rhodopsin and red cone opsin mRNA levels during bovine fetal development as a function of retinal position. Following induction, an equivalent radial gradient of rod and cone opsin mRNA is present in the fetal retina. This gradient is maintained in the adult retina even though no corresponding gradient in rod or cone cell density is present. Since the mRNA expression gradient does not progress radially, position dependent levels of photoreceptor-specific transcription is suggested.

Fetal topography of bovine rhodopsin mRNA suggests retinotopographically determined gene expression.

PURPOSE: To understand the developmental processes in the differentiating bovine retina, topographic accumulation of rhodopsin mRNA in staged fetal and adult retinas was analyzed. METHODS: Isolated retinas were spread on a nylon membrane with the photoreceptor cells facing the membrane and dissected into 25-mm square tissue segments, sometimes with as many as 150 segments/eye. Subsequent to disruption of the tissue in each segment, rhodopsin and beta-actin mRNA levels were quantitated with a solution hybridization assay. Slight variations in RNA extraction efficiency and retinal segment size were corrected using beta-actin mRNA as an internal standard. RESULTS: Analysis of multiple fetal and adult bovine retinas revealed a relatively static central-to-peripheral gradient of rhodopsin mRNA level that appears at the time of transcriptional induction (6 to 6.5 months of gestation) and persists into adulthood. After induction of rhodopsin mRNA expression, increase of rhodopsin mRNA levels was detected simultaneously in all retinal segments. Furthermore, the rate of increase in rhodopsin mRNA levels in peripheral and central regions was identical. CONCLUSIONS: Fetal induction of rhodopsin mRNA expression occurs simultaneously in all photoreceptor cells across the retina, but the levels are set according to a topographically predetermined pattern. This suggests that regulation of accumulation of rhodopsin mRNA during development is determined according to spatial coordinates before gene induction, most likely in a nonphotoreceptor retinal cell type.

Transcription of photoreceptor genes during fetal retinal development. Evidence for positive and negative regulation.

Rod photoreceptor outer segments are elaborated at approximately 6 months gestation in the cow coinciding with a dramatic increase in mRNAs encoding many visual transduction and associated proteins. Nuclear run-on determination of relative transcription rates demonstrates that gene expression follows three distinct patterns. Opsin, S-antigen, and transducin are all minimally detectable at 5.2 months gestation and increase throughout development. Only opsin demonstrates an additional sharp increase in transcriptional activity which resembles a positive gene-specific enhancer that is first effective between 6.3 and 7.4 months gestation. In contrast, interphotoreceptor retinoid binding protein (IRBP) transcription is already at 43% of its adult level at 5.2 months gestation. To further understand these differences, the relative contributions of initiation and elongation to nuclear run-on signals were examined using either Sarkosyl or ammonium sulfate. Transcriptional rates for S-antigen and transducin were not affected, however, opsin was reduced approximately 4-fold and IRBP was increased approximately 2-fold. Opsin is therefore likely to be initiated de novo during the run-on reaction and responds to a gene-specific positive regulator. The increase in IRBP transcription rate suggests the removal of an elongation inhibitory factor and supports the idea that a negative regulatory element may be involved in controlling IRBP expression.

Synthesis and stability of retinal photoreceptor mRNAs are coordinately regulated during bovine fetal development.

Steady state photoreceptor specific mRNA levels in bovine retina were studied during fetal maturation for five gene transcripts, including rhodopsin, arrestin (S-antigen), rod alpha-subunit of transducin, interphotoreceptor retinoid-binding protein (IRBP) and rod alpha-subunit of cGMP phosphodiesterase in order to understand mechanisms of gene regulation during photoreceptor development. A 10-15-fold increase in each transcript level begins between 5.5 and 6 months of gestation for each gene, suggesting a single coordinate induction event at this time. Quantitative analysis of transcriptional rates for each gene by nuclear run-on also reveals a coordinate increase at approximately the same time, demonstrating that induction is achieved by transcriptional activation. Interestingly, however, gene specific transcription rates in the pre-induction retina do not appear to parallel mRNA steady state levels. During fetal development neither the transcript level of each gene relative to the others nor relative mRNA turnover rates change substantially after the induction event at 5.5-6.0 months. However, at earlier times all genes exhibit higher mRNA turnover, implying that differential mRNA stability may also play an important role in determining steady-state levels.

Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina.

Differentiation and maturation of the photoreceptor outer segments are key steps in the development of the visual system. Morphological studies presented here show that the cow and human are nearly identical in the timing of outer segment appearance during fetal development, implying that the bovine retina is a good model system for the final stages of human photoreceptor development. To study photoreceptor maturation, rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) were quantified by ELISA in a developmentally staged series of fetal bovine retinas. In addition, their localization within these retinas was determined by immunogold electron microscopy. Rhodopsin, as detected by antibodies directed against either the N- or C-terminal portions of the molecule, is first found at about 5.5 months gestation. It is first detected on the plasma membrane of the immature cilia and on the earliest emergent outer segment membrane, even before organized disk membranes are apparent. In contrast, whereas rhodopsin levels and outer segments are nearly undetectable before 5 months gestation, IRBP accumulates to a significant level (4-5% of the adult) as early as 3 months gestation. Immunogold electron microscopy confirmed this finding, with localization of IRBP predominantly in the subretinal space.

Uptake and isomerization of all-trans retinol by isolated bovine retinal pigment epithelial cells: further clues to the visual cycle.

The site and substrate for all-trans to 11-cis isomerization in the visual cycle have remained obscure for several decades. Only recently studies on a subcellular level have begun to shed some light on these phenomena. We have addressed this system on a cellular level by utilizing intact isolated bovine retinal pigment epithelial cells, maintained during short-term incubation in vitro. Supplementation with labeled all-trans retinol incorporated in a lipid vesicle carrier, in a range of 1-6 nmol per 10(6) cells, resulted in a rapid uptake of retinol. The majority of the internalized retinol was processed prior to mixing with endogenous retinoid pools and most of it was converted into all-trans retinylester. Up to 10% of the incorporated label was isomerized yielding 11-cis retinol, 11-cis retinaldehyde and 11-cis retinylester. The kinetics of the 11-cis retinoid formation indicated that 11-cis retinol is the first isomerization product. The level of 11-cis retinol apparently 'triggered' further processing into other 11-cis retinoids. An updated model with discussion topics is presented for the retinoid pathway relevant to the visual cycle.

A comparison of some photoreceptor characteristics in the pineal and retina. II. The Djungarian hamster (Phodopus sungorus).

A rod-specific antiserum was used to immunolabel elements within the retina and pineal of the adult Djungarian hamster and Welsh Mountain sheep. In the retina immunostaining was localized to the outer segments and perikarya of photoreceptor cells, while in the pineal limited numbers of labelled pinealocytes were scattered throughout the gland. An enzyme-linked immunosorbent assay (ELISA) was then used to obtain a quantitative measure of rod opsin in total eye and pineal extracts from the Djungarian hamster. Total rod opsin (+/- SEM) in the eye was measured by absorbance spectroscopy (1.88 +/- 0.10 nmoles opsin/eye) and by using the ELISA (1.75 +/- 0.02 nmoles opsin/eye). The opsin content from a total of 56 pineals gave a mean value of 0.34 +/- 0.01 pmoles opsin/pineal. Since a functional photopigment should be coupled in a 1:1 ratio to a chromophore, we investigated whether we could identify 11-cis and/or all-trans retinaldehydes in the pineal extracts by quantitative extraction and HPLC analysis as the oximes. No evidence of 11-cis or all-trans retinaloxime could be found, the chromatograms were indistinguishable from those produced by extracts of cortical brain tissue. We conclude that the opsin present within the adult hamster pineal is not coupled to the common vertebrate retinaldehyde chromophore, and as a result, is unlikely to be part of a functional photopigment.

A rapid versatile microassay for cellular retinol-binding protein using Lipidex-1000 microcolumns.

A new, rapid and versatile microassay for cellular retinol-binding protein has been developed based on separation of bound and free ligand by means of Lipidex-1000, a hydrophobic Sephadex derivative. This requires quantitative manipulation of retinol in aqueous solution. The tendency of retinol to adhere to glass and plastic surfaces was overcome by addition of the detergent Ammonyx LO, which yields a micellar dispersion. Detergent concentrations up to 10 mM did not interfere with binding of retinol to Lipidex-1000 or binding protein. The binding capacity of Lipidex-1000 was found to exceed 400 nmol of retinol per ml of gel. Retinal pigment epithelium (RPE) cells were used as a source for cRBP (cellular retinol-binding protein). The binding protein is saturated with ligand by incubation for 60 min at room temperature at concentrations of free retinol over 180 nM. Separation of protein-bound retinol from free retinol is achieved via Lipidex-1000: protein-bound (specific and nonspecific) retinol is not retained and is eluted by buffer with the protein fraction. Free retinol is retained by Lipidex and is subsequently recovered by elution with methanol. Total recovery of ligand approaches 100%. Analysis time is about 4 hr for a maximum of ca. 50 samples. Nonspecific protein binding can be determined equally effectively either by incubation with 3 mM PCMBS or by addition of a 100-fold molar excess of nonlabeled retinol.(ABSTRACT TRUNCATED AT 250 WORDS)

A new isolation procedure for retinal pigment epithelium.

A new isolation procedure for bovine retinal pigment epithelial cells has been developed. It is based on perfusion of the whole bovine eye via the central ophthalmic artery with a cold, buffered isotonic salt solution free of divalent cations for 15 min. The perfusion both weakens the association of the pigment epithelial cells with Bruch's membrane and the adhesion between retina and pigment epithelium. The retina then is removed carefully, after which the pigment epithelial cells are detached from the Bruch's membrane by gentle jets of buffer solution. The perfusion technique provides a high yield of intact retinal pigment epithelial cells, which show good viability in subsequent cell culture. Hence, cells isolated in this way are not only very well suited for long-term cell culture but also for direct biochemical analysis and short-term incubation studies.