Royal College Of Surgeons Rats Research Articles

Long-term Vision Rescue by Human Neural Progenitors in a Rat Model of Photoreceptor Degeneration

As a follow-up to previous studies showing that human cortical neural progenitor cells (hNPC(ctx)) can sustain vision for at least 70 days after injection into the subretinal space of Royal College of Surgeons (RCS) rats, the authors examined how functional rescue is preserved over long periods and how this relates to retinal integrity and donor cell survival. Pigmented dystrophic RCS rats (n = 15) received unilateral subretinal injections of hNPC(ctx) at postnatal day (P) 21; control rats (n = 10) received medium alone and were untreated. All animals were maintained on oral cyclosporine A. Function was monitored serially by measuring acuity (using an optomotor test) and luminance thresholds (recording from the superior colliculus) at approximately P90, P150, and P280. Eyes were processed for histologic study after functional tests. Acuity and luminance thresholds were significantly better in hNPC(ctx)-treated animals than in controls (P < 0.001) at all time points studied. Acuity was greater than 90%, 82%, and 37% of normal at P90, P150, and P270, whereas luminance thresholds in the area of best rescue remained similar the whole time. Histologic studies revealed substantial photoreceptor rescue, even up to P280, despite progressive deterioration in rod and cone morphology. Donor cells were still present at P280, and no sign of donor cell overgrowth was seen. Long-term rescue of function and associated morphologic substrates was seen, together with donor cell survival even in the xenograft paradigm. This is encouraging when exploring further the potential for the application of hNPC(ctx) in treating retinal disease.

Ionic Dysregulatory Phenotyping of Pathologic Retinal Thinning with Manganese-Enhanced MRI

To test the hypothesis that manganese-enhanced MRI (MEMRI) provides a sensitive and robust measure of an important retinal ionic dysregulatory phenotype in pathologic retinal thinning. Four hours after intraperitoneal MnCl(2) injection, high-resolution MEMRI data were collected from overnight dark-adapted male control Sprague-Dawley and albino Royal College of Surgeons rats before (at development stage postnatal day [P] 17) and during photoreceptor degeneration (P36 and P57). In separate experiments, control rats, with and without repetitive hypoxic preconditioning, were subjected to high IOP (100 mm Hg) for 60 minutes followed by 24 hours or 7 days of reperfusion (e.g., ischemia/reperfusion). Central retinal thickness and intraretinal ion activity were measured from the MEMRI data. Histology examination was also performed to confirm retinal damage. In two different neurodegenerative models, MEMRI revealed first-time evidence for changes (P < 0.05) in intraretinal ion regulation before and during pathologic, but not (P > 0.05) developmental, retinal thinning. This phenotype was significantly altered by a neuroprotective repetitive hypoxic preconditioning protocol. MEMRI and a nontoxic systemic dose of MnCl(2) provided an objective, noninvasive measure of an ionic deregulatory phenotype that appears useful for improved early diagnosis and treatment prognosis in a range of neurodegenerative diseases and their treatment.

Fate and protective effect of marrow stromal cells after subretinal transplantation

Engraftment of marrow stromal cells (MSCs) has been proposed as a therapeutic approach for degenerative diseases. In this study we investigated the fate and dynamic progress of grafted MSCs in living retina with the aim of evaluating the use of transplanted MSCs to treat retinal degeneration. Approximately 1x10(5) gfp-MSCs in 2 microl phosphate-buffered saline were injected into the subretinal space of adult Sprague-Dawley rats. Two weeks later, approximately 0.174%+/-0.082% of the transplanted cells had survived and diffused into the subretinal space. Nine weeks after transplantation the surviving gfp-MSCs accounted for 0.049%+/-0.023% of the number of cells injected and were mainly located at the injection site. The same number of MSCs were transplanted into the left eye subretinal space of 3-week-old hereditary retinal degenerative Royal College of Surgeons rats, and phosphate-buffered saline was injected into their right eyes as a control. Five weeks after transplantation, the amount of rudimentary photoreceptors was more significantly increased in grafted eyes than in control eyes. The results indicated that grafted MSCs could survive and rescue retinal degeneration.

Chondroitin Sulfate Proteoglycans and Microglia Prevent Migration and Integration of Grafted Müller Stem Cells into Degenerating Retina

At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68-positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5-week-old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N-terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5-week-old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell-based therapies to treat human retinal disease.

Morphological and Functional Rescue in RCS Rats after RPE Cell Line Transplantation at a Later Stage of Degeneration

It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19. Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression. Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors. RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Photopic ERG Negative Response from Amacrine Cell Signaling in RCS Rat Retinal Degeneration

The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity, which declines more slowly than the b-wave with degeneration. The absence of this response in the P23H rat indicates that the inner retinal cone pathway pathology is different in the two models. A relative increase in Kir4.1 channels on Müller cells of RCS retina may contribute to the enhanced negative ERG response in the RCS rat.

Transcorneal Electrical Stimulation Promotes the Survival of Photoreceptors and Preserves Retinal Function in Royal College of Surgeons Rats

To determine whether transcorneal electrical stimulation (TES) has neuroprotective effects on photoreceptors and preserves retinal function in Royal College of Surgeons (RCS) rats. Three-week-old RCS rats received TES through a contact lens electrode on the left eye weekly for 2 to 6 weeks. The right eyes received sham stimulation on the same days. Electroretinograms (ERGs) were recorded from the rats at 3 weeks (before TES), and at 5, 7, and 9 weeks of age. After the ERG recordings, the rats were killed for morphologic analyses of the retina. Morphologic analyses showed that the mean thickness of the outer nuclear layer (ONL) at each time point was significantly thicker in eyes treated with TES of 100 muA than in eyes with sham stimulation (TES 100 muA versus sham: 5, 7, and 9 weeks of age; P < 0.001). ERG studies showed that TES also significantly preserved retinal function up to 7 weeks of age, but did not preserve retinal function at 9 weeks of age. TES prolongs the survival of photoreceptors and delays the decrease of retinal function in RCS rats. Although further investigations are necessary before using TES on patients, these findings indicate that TES may be a therapeutic treatment for some patients with diseases of the photoreceptors such as retinitis pigmentosa.

Prolonged Protective Effect of Basic Fibroblast Growth Factor–Impregnated Nanoparticles in Royal College of Surgeons Rats

To investigate the protective effect of intravitreal injection of basic fibroblast growth factor-impregnated nanoparticles (bFGF-NPs) against photoreceptor degeneration in Royal College of Surgeons (RCS) rats. Three-week-old RCS rats received intravitreal injection of PBS, blank NPs, bFGF (2.5 microg), or bFGF-NPs (2.5 microg). Eyes were assessed by morphologic, immunohistochemical, and physiological analyses for the following 8 weeks. Cell death was examined using the TUNEL assay, and bFGF protein levels in the retina were measured by Western blot analysis. Rhodamine (Rh)-labeled bFGF-NPs were injected intravitreally and visualized by confocal microscopy to determine the localization of the nanoparticles in the retina. Intravitreally injected Rh-labeled bFGF-NPs were found in the outer nuclear layer 6 and 8 weeks after injection. ERG a- and b-wave amplitudes in bFGF-NP-treated retinas were greater than amplitudes in retinas receiving other treatment. Immunocytochemical analysis showed consistently greater opsin preservation in bFGF-NP-treated retinas, and a significantly higher number of photoreceptors and significantly fewer TUNEL-positive cells were present after bFGF-NP treatment than after bFGF treatment. Western blot analysis showed a significant increase in the bFGF level in bFGF-NP-treated retinas. The results suggest that intravitreally injected bFGF-NPs prevent photoreceptor degeneration by inhibiting apoptosis in the RCS rat retina because of targeting and sustained release of bFGF. This novel drug delivery system for bFGF may serve as a potential short-term treatment for photoreceptor degeneration in humans.

Preservation of outer retina and its synaptic connectivity following subretinal injections of human RPE cells in the Royal College of Surgeons rat

We have examined how transplantation of an RPE cell line to the subretinal space of RCS rats affects the distribution of synaptic connectivity markers in the outer plexiform layer of the retina. Using markers of pre- and post-synaptic profiles (bassoon and synaptophysin as presynaptic markers and mGluR6 for postsynaptic profiles) we found that the normal orderly patterns seen between photoreceptors and rod and ON-cone bipolar cells were severely disrupted in dystrophic rats. In areas in which injected cells preserved photoreceptors, more normally appearing pairing of pre- and post-synaptic markers was seen for both rods and cones. The degree of normality correlated with the amount of photoreceptor rescue. The secondary changes that are normally seen in bipolar and horizontal cells were prevented by the photoreceptor preservation. ERG recordings in the animals subsequently studied morphologically showed that both a- and b-waves could be rescued by grafting, albeit with lower amplitudes than normal. Together these anatomical and physiological studies indicate that besides the integrity of outer nuclear layer cells and phototransduction processes, relay circuitry through the outer retina was rescued by cell grafts.

The Effect of Intravitreal N-Methyl-dl-Aspartic Acid on the Electroretinogram in Royal College of Surgeons Rats

To investigate how the third-order neuronal response contributes to shaping the electroretinogram (ERG) in the Royal College of Surgeons (RCS) rat. Full-field ERGs were recorded from dystrophic RCS rats (n = 30) at 4, 6, 8, 10, 12, or 14 weeks of age in response to different stimulus intensities (maximum intensity, 0.84 log cd-s/m(2)). N-methyl-DL: -aspartic acid (NMDA, 5 mM) was injected into the vitreous cavity of the right eyes to eliminate the third-order neuronal response. The left eyes received the vehicle and served as controls. The third-order neuronal response was isolated by digitally subtracting waveforms of the NMDA-injected eyes from those of the control eyes. The ERG a- and b-waves deteriorated with the age of the rat. The third-order neuronal response was preserved to a greater degree than the b-wave despite progression of photoreceptor degeneration. Intravitreal injection of NMDA attenuated the a-wave and enhanced the b-wave across the stimulus range from low to middle intensities. This tendency became more pronounced with advancing rat age. In aged dystrophic RCS rats this phenomenon was seen even at maximum intensity. The difference between NMDA-injected and vehicle-injected eyes was larger for the threshold than for the maximum amplitude at each examined time point (P < 0.001). Intravitreal injection of NMDA decreased implicit times of the a- and b-waves after the rats reached 8 weeks of age (P < 0.005 for the a-wave). With advancing photoreceptor degeneration, the third-order neuronal response made a greater contribution to shaping the a- and b-waves in dystrophic RCS rats.

Clock gene expression in the rat retina: Effects of lighting conditions and photoreceptor degeneration

Previous studies have shown that, in the Royal College of Surgeon rat, circadian rhythms in the retinal dopaminergic and melatonergic systems are still present after the photoreceptors have degenerated, thus demonstrating that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors. The aim of the present study was to investigate the pattern of expression of the clock genes in the retina of the Royal College of Surgeons rat under different lighting conditions. Expression of clock genes was investigated in the retina of normal and dystrophic Royal College of Surgeons rats under 12 h of light/12 h of dark (LD), constant darkness (DD) and constant light (LL) using Real Time Quantitative RT-PCR. Our data indicate that, in control animals, Period1, Period2, Cryptochrome1, Cryptochrome2, Clock, Rora, Rev-Erbα and Npas2 mRNA levels showed a significant variation over the sampling period in LD cycles and in DD, whereas Bmal1 mRNA did not show any significant variation. In LL, the transcripts for Per1, Per2, Clock and Rev-Erbα showed significant temporal variations. In the dystrophic retina, only Per1 and Per2 mRNA levels showed a temporal variation over the 20-h period. Our work indicates that degeneration of the photoreceptor cells dramatically affected the expression levels and patterns of many clock genes. Finally, the present study suggests that investigating the expression pattern of clock genes using the whole retina or animals with photoreceptor degeneration may not provide any definitive answers about the working of the retinal circadian clock system.

MIO-M1 Cells and Similar Müller Glial Cell Lines Derived from Adult Human Retina Exhibit Neural Stem Cell Characteristics

Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor-like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor-2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including betaIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S-opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell-based therapies to restore retinal function. Disclosure of potential conflicts of interest is found at the end of this article.

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.

Preservation of visual cortical function following retinal pigment epithelium transplantation in the RCS rat using optical imaging techniques

The aim of this study was to determine the extent of cortical functional preservation following retinal pigment epithelium (RPE) transplantation in the Royal College of Surgeons (RCS) rat using single-wavelength optical imaging and spectroscopy. The cortical responses to visual stimulation in transplanted rats at 6 months post-transplantation were compared with those from age-matched untreated dystrophic and non-dystrophic rats. Our results show that cortical responses were evoked in non-dystrophic rats to both luminance changes and pattern stimulation, whereas no response was found in untreated dystrophic animals to any of the visual stimuli tested. In contrast, a cortical response was elicited in most of the transplanted rats to luminance changes and in many of those a response was also evoked to pattern stimulation. Although the transplanted rats did not respond to high spatial frequency information we found evidence of preservation in the cortical processing of luminance changes and low spatial frequency stimulation. Anatomical sections of transplanted rat retinas confirmed the capacity of RPE transplantation to rescue photoreceptors. Good correlation was found between photoreceptor survival and the extent of cortical function preservation determined with optical imaging techniques. This study determined the efficacy of RPE transplantation to preserve visual cortical processing and established optical imaging as a powerful technique for its assessment.

Protection of Visual Functions by Human Neural Progenitors in a Rat Model of Retinal Disease

BackgroundA promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat.Methodology/Principal FindingsAnimals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90–100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed.Conclusions/SignificanceWild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in vivo.

Effect of photoreceptor degeneration on circadian photoreception and free-running period in the Royal College of Surgeons rat

The study of how the retina processes the photic information required for the entrainment of the circadian system is an exciting new topic in retinal neurobiology. We have recently shown that in RCS/N- rdy rats melanopsin mRNA levels are dramatically reduced (about 90%) and melanopsin immunoreactivity cannot be detected in the retina of these rats at 60 days of age. Although RCS/N- rdy rats are a widely used model to investigate mechanisms of photoreceptor degeneration, no study has investigated circadian photoreception in these animals. The aim of this study was to examine circadian photoreception in RCS/N- rdy + ( rdy + ) rats homozygous for the normal rdy allele and age-matched RCS/N- rdy ( rdy) homozygotes with retinal dystrophy. No differences between RCS/N- rdy and rdy + were observed in light-induced phase shift of locomotor activity at the three light intensities used (1 × 10 − 3 , 1 × 10 − 1 , and 1 × 10 1 μW cm − 2 ). Surprisingly, we observed that in RCS/N- rdy the free-running period of the circadian rhythm of locomotor activity was shorter ( P < 0.01) than in rdy + , thus suggesting that photoreceptor degeneration may affect the free-running period of the locomotor activity rhythm.

Stimulation via a Subretinally Placed Prosthetic Elicits Central Activity and Induces a Trophic Effect on Visual Responses

Subretinal prosthetics are designed to electrically stimulate second-order cells, replacing dysfunctional photoreceptors in diseases such as retinitis pigmentosa (RP). For functional vision to occur, this signal must also reach central visual structures. In the current study, a subretinally implanted prosthetic was evaluated in the Royal College of Surgeons (RCS) rat model of RP, to determine its capacity to activate the retinotectal pathway. Prosthetic implants were placed in RCS and wild-type (WT) rats at 4 weeks of age and evaluated 3 months later. Control rats underwent sham surgery, implantation with inactive prosthetics, or no treatment. Implant- and visible-evoked responses were isolated and evaluated in the superior colliculus (SC). In WT and RCS rats with active prosthetics, implant-driven responses were found in 100% of WT and 64% of RCS rats and were confined to a small SC region that corresponded to the retinal sector containing the implant and differed from visible-evoked responses. In addition, visible-evoked responses were more robust at sites that received implant input compared to sites that did not. These effects were not seen in WT rats or RCS control animals; although a general trophic effect on the number of responsive sites was observed in all RCS rats with surgery compared to untreated RCS rats. Direct activation of the retina by a subretinal implant induces activity in the SC of RCS rats, suggesting that these implants have some capacity to replace dysfunctional photoreceptors. The data also provide evidence for implant-induced neurotrophic effects as a consequence of both its presence and its activity in the retina.

Prolonged Rhodopsin Phosphorylation in Light-Induced Retinal Degeneration in Rat Models

The effects of various light-induced stresses on the retina were examined in the retinal degenerative rat model. Retinal morphology and electroretinograms (ERGs) were analyzed after application of light-induced stress of several intensities (650, 1300, 2500, or 5000 lux). For evaluation of rhodopsin (Rho) function, the kinetics of Rho regeneration and dephosphorylation were studied by spectrophotometric analysis and immunofluorescence labeling with antibodies specifically directed toward the phosphorylated residues (334)Ser and (338)Ser in the C terminus of Rho. Retinal cGMP concentration was determined by ELISA. Expression levels of neurotrophic factors (FGF2, brain-derived neurotrophic factor [BDNF], platelet-derived growth factor [PDGF], and ciliary neurotrophic factor [CNTF]) were evaluated quantitatively by RT-PCR. Light intensity-dependent deterioration of ERG responses and thinning of the retinal outer nuclear layer were observed in wild-type and Royal College of Surgeons (RCS) rat retinas. Under dark adaptation after light-induced stress, the kinetics of Rho regeneration were not different between wild-type and RCS rat retinas. Rho dephosphorylation at (334)Ser and (338)Ser was extremely delayed in RCS rat retinas compared with wild-type without light-induced stress, but Rho dephosphorylation at those sites became slower in both RCS and wild-type rat retinas. In terms of expression of neurotrophic factors, almost no significant changes were observed between the animals after light-induced stress. The present study indicates that light-induced stress causes intensity-dependent deterioration in retinal function and morphology in wild-type and RCS rat retinas. Disruption of the phototransduction cascade resulting from slower kinetics of Rho dephosphorylation appears to be involved in retinal degeneration.

Structural and functional MRI reveals multiple retinal layers

MRI is a noninvasive diagnostic modality that reveals anatomy, physiology, and function in vivo without depth limitation or optical interference. MRI application to the retina, however, remains challenging. We improved spatial resolution to resolve layer-specific structure and functional responses in the retina and confirmed the laminar resolution in an established animal model of retinal degeneration. Structural MRI of normal rat retinas revealed three bands corresponding histologically to (i) the combined ganglion cell layer/inner nuclear layer plus the embedded retinal vessels, (ii) the avascular outer nuclear (photoreceptor) layer and its photoreceptor segments, and (iii) the choroidal vascular layer. Imaging with an intravascular contrast agent (gadolinium-diethylene-tri-amine-pentaacetic acid) enhanced the retinal and choroidal vascular layers bounding the retina, but not the avascular outer nuclear layer and the vitreous. Similarly, blood-oxygen-level-dependent (BOLD) functional MRI revealed layer-specific responses to hyperoxia and hypercapnia. Importantly, layer-specific BOLD responses in the two vascular layers were divergent, suggesting the two vasculatures are differentially regulated. To corroborate sensitivity and specificity, we applied layer-specific MRI to document photoreceptor degeneration in Royal College of Surgeons rats. Consistent with histology, layer-specific MRI detected degeneration of the outer nuclear layer. Surprisingly, MRI revealed increased thickness in the choroidal vascular layer and diminished BOLD responses to hyperoxia and hypercapnia in the Royal College of Surgeons rat retinas, suggesting perturbation of vascular reactivity secondary to photoreceptor loss. We conclude that MRI is a powerful investigative tool capable of resolving lamina-specific structures and functional responses in the retina as well as probing lamina-specific changes in retinal diseases.

Adenovirally Transduced Bone Marrow Stromal Cells Differentiate into Pigment Epithelial Cells and Induce Rescue Effects in RCS Rats

To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats. Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected. In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments. Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.