Dystrophic Royal College Of Surgeons Research Articles

Intra- and intercellular Ca2+ signaling in retinal pigment epithelial cells during mechanical stimulation.

The intercellular communication (IC) was investigated between cultured rat retinal pigment epithelial (RPE) cells isolated from Long-Evans (LE) or dystrophic Royal College of Surgeons (RCS) rats and grown in solutions containing normal and high glucose concentrations, or after modulation of protein kinase C (PKC). This was performed by studying the conduction of the free Ca2+-concentration ([Ca2+]i) wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP). Mechanical stimulation of LE-RPE cells triggers Ca2+ influx, mediated by stretch-sensitive cation channels followed by intracellular Ca2+ release. A regenerative [Ca2+]i wave was found with a lower propagation rate in RCS-RPE cells. This rate could be increased by PKC down-regulation. Mechanical stimulation caused a [Ca2+]i increase in the mechanically stimulated (MS) cell followed after a delay by a [Ca2+]i rise in the adjacent cell layers. The intercellular [Ca2+]i wave propagation could be blocked by gap junction blockers such as halothane or PKC activation. An inhibition of the [Ca2+]i-wave propagation similar to that induced by halothane could be observed in cells grown in solutions containing 14 mM or higher concentrations of glucose. PKC down-regulated cells grown in glucose-rich medium did not develop this inhibitory effect on gap junction communication (GJC). FRAP experiments confirmed that the observed changes were consistent with a PKC-mediated inhibitory effect of high glucose concentrations on GJC.

Quantitative Analysis of Major Histocompatibility Complex Class II-Positive Cells in Posterior Segment of Royal College of Surgeons Rat Eyes

Potential antigen-presenting cells in the posterior segment of Royal College of Surgeons (RCS) rat eyes were analyzed quantitatively. Light microscopic immunohistochemistry was performed at postnatal days (P) 10, 20, 28, 42, 63, and 140 in the eyes of RCS rats and their congenic counterparts. Immunohistochemical studies were carried out using monoclonal antibodies against major histocompatibility complex (MHC) class II antigen (OX6), a cytoplasmic antigen in bone marrow-derived macrophages (ED1), a membrane antigen on resident tissue macrophages (ED2), and a microglia/macrophage marker (OX42). Some sections were stained by a double-labeling method using these antibodies. No MHC class II-positive cells were seen in dystrophic RCS rat retinas at P10. They were found, however, in the outer nuclear layer and debris of outer segments at P20. From P20 to P42 the number of cells increased, then decreased until P140. Congenic controls, however, showed no MHC class II-positive cells in the retina. Cells double-labeled with OX6 and ED1 were present in the outer nuclear layer at P42, but no OX6 or OX42 double-labeled cells were detected. Also, no ED2-positive cells were detected. Our results suggest that MHC class II-positive cells may play some role in retinal dystrophy.

Ganglion cell loss in RCS rat retina: A result of compression of axons by contracting intraretinal vessels linked to the pigment epithelium

In the dystrophic Royal College of Surgeons (RCS) rat retina, there is a progressive loss of photoreceptors. As a result, the retinal circulation becomes apposed to the retinal pigment epithelium (RPE) and neovascular formations develop. RPE and inner nuclear layer cells migrate along these vessels towards the retinal ganglion cell (RGC) layer. The retinal layers gradually become disrupted, and some of the RGC axon bundles involute into the retina. These bundles are always associated with blood vessels, and there is evidence of axon damage where they juxtapose. In wholemount preparations of dystrophic retinae (> or =6 months of age), abrupt changes are observed in the trajectory of RGC axon bundles, where they are crossed by circumferential vessels. Degenerative profiles can be seen at these locations. Visualisation of RGCs with Fluoro-gold shows wedge-shaped sectors in the dystrophic retina devoid of labelling, initially in the ventral retina but later spreading dorsally. It is hypothesised that the vessels supplying the neovascular formations contract and pull surface vessels into the retina, thus displacing any axon bundles that lie beneath them into the inner plexiform layer. The contractility may be an intrinsic property of the vessels or it may be conferred by the cells migrating along them. Axonal transport becomes blocked at the points of tension, thereby causing retrograde degeneration of the parent RGCs. Because RGC loss is also a feature of human retinitis pigmentosa, the RCS rat may provide a model to test interventions devised to prevent such loss following photoreceptor degeneration. This model also may be useful for testing methods designed to control blood vessel and matrix formation.

Ganglion cell loss in RCS rat retina: A result of compression of axons by contracting intraretinal vessels linked to the pigment epithelium

In the dystrophic Royal College of Surgeons (RCS) rat retina, there is a progressive loss of photoreceptors. As a result, the retinal circulation becomes apposed to the retinal pigment epithelium (RPE) and neovascular formations develop. RPE and inner nuclear layer cells migrate along these vessels towards the retinal ganglion cell (RGC) layer. The retinal layers gradually become disrupted, and some of the RGC axon bundles involute into the retina. These bundles are always associated with blood vessels, and there is evidence of axon damage where they juxtapose. In wholemount preparations of dystrophic retinae (≥6 months of age), abrupt changes are observed in the trajectory of RGC axon bundles, where they are crossed by circumferential vessels. Degenerative profiles can be seen at these locations. Visualisation of RGCs with Fluoro-gold shows wedge-shaped sectors in the dystrophic retina devoid of labelling, initially in the ventral retina but later spreading dorsally. It is hypothesised that the vessels supplying the neovascular formations contract and pull surface vessels into the retina, thus displacing any axon bundles that lie beneath them into the inner plexiform layer. The contractility may be an intrinsic property of the vessels or it may be conferred by the cells migrating along them. Axonal transport becomes blocked at the points of tension, thereby causing retrograde degeneration of the parent RGCs. Because RGC loss is also a feature of human retinitis pigmentosa, the RCS rat may provide a model to test interventions devised to prevent such loss following photoreceptor degeneration. This model also may be useful for testing methods designed to control blood vessel and matrix formation. J. Comp. Neurol. 392:58–77, 1998. © 1998 Wiley-Liss, Inc.

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.

Water escape performance of adult RCS dystrophic and congenic rats: a functional and histomorphometric study

The dystrophic Royal College of Surgeons (RCS) rat undergoes photoreceptor degeneration due to a hereditary defect in the retinal pigment epithelium. The congenic rat remains unaffected. Although the retinal degeneration is well characterized, few functional studies of this strain are available. We wished to compare the visual function of congenic and dystrophic RCS rats using a water escape paradigm that tested their ability to find a submersed, randomly placed platform using a light source as a clue. Three different behavioral experiments were sequentially performed on all animals: Experiment 1—The Light and Platform test measured the time to swim from the center of the pool to a platform located under a light clue. Each animal was given 10 trials. Experiment 2—The No Light with Platform test was performed as above except no light clue was used. Experiment 3—The Light and No Platform test was performed with a light clue but without a platform. The animal was allowed to swim for 2 min. All trials were videotaped. After the final experiment, the animals were sacrificed and a histomorphometric analysis of their retinas were performed. Expt. 1—The time to find the platform using light as a clue was greater for the dystrophic than for the congenic rats. Expt. 2—In the absence of light clue, there was no significant difference in performance between the congenic and dystrophic animals. Expt. 3—In the absence of a platform, a significantly greater amount of time was spent in the area indicated by the light clue by the congenic animals as compared to the dystrophic. Morphometric analysis revealed a mean number of 133 photoreceptor nuclear profiles/90 μm of retina for congenic rats as compared to 0.14 for dystrophic rats. This simplified version of the Morris water maze permits quantitative evaluation of visually-guided behavior in an important model of retinal degeneration.

Vasoactive intestinal polypeptide-containing nerve fibers are increased in abundance in the choroid of dystrophic RCS rats.

As photoreceptor degeneration progresses in Royal College of Surgeons (RCS) rats, a variety of morphological and physiological alterations occur in the outer retina. Since the choriocapillaris responds to changes in the outer retina in other retinopathies, we examined the possibility that changes in the choroidal vasculature also occur in RCS rats. The choroidal and choriocapillary vessels in RCS and control (RCS-rdy+) rats were examined during the period after which photoreceptor loss and retinal vascular changes had occurred (7-mos to 28-mos). Light microscopic (LM) morphometry and electron microscopic (EM) examination showed no significant differences between these groups in the number, size or morphology of these vessels. However, EM image analysis revealed that nerve fibers and bundles were twice as abundant in the RCS choroid than in the control. Using immunohistochemical techniques at the LM level combined with image analysis we found that vasoactive intestinal polypeptide positive (VIP+) fibers were significantly increased in the RCS choroid compared with control choroid. In contrast, the abundance of immunoreactive fibers labelled for substance P and dopamine beta hydroxylase appeared similar in both the control and RCS choroid. Since VIP is a potent vasodilator, the increased abundance of nerve fibers in the RCS choroid in conjunction with the unaltered number and size of these vessels suggests that choroidal blood flow may be increased. It is uncertain whether this increase is a response to the outer retinal pathology or contributes to it.

Retinovitreal neovascularization in the Royal College of Surgeons rat

Retinovitreal blood vessels, which we have previously reported in the dystrophic retinas of approximately 20% of older (greater than 15 months of age) spontaneously hypertensive (SHR) rats, also occur in about 20% of retinal dystrophic Royal College of Surgeons (RCS) rats greater than 1 year of age. We have demonstrated previously that these vessels, in the SHR rat, have the anatomic characteristics of retinovitreal neovascularization as it has been described in vasoproliferative retinopathies in humans (1). We now provide strong evidence that the endothelial cells and pericytes of the retinovitreal vessels that occur in RCS rats are proliferating, since they demonstrate by autoradiography significantly increased nuclear labeling with [3H]-thymidine over intra-retinal or choroidal vessels, or the abnormal vessels that grow within the retinal pigment epithelium in these dystrophic retinas. One important difference between the neovascularization that occurs in these rats and that which is observed in various human retinal vascular diseases is the frequent association of the new vessels in dystrophic rat retinas with surrounding cords of proliferating retinal pigment epithelium. The retinovitreal vessels in these strains of dystrophic rats represent a new animal model that may be useful for studying the fundamental processes underlying new blood vessel growth in the retina.