Transdiferenciación de células mesenquimales del tejido adiposo: hacia una terapia celular para enfermedades degenerativas de la retina / Transdifferentation of mesenchymal stem cells from adipose tissue: Through a cellular therapy for degenerative retinal diseases

Objetivos: Obtención de células troncales mesenquimales de tejido adiposo para trasplante y recuperación funcional de retinas degeneradas. Material y metodología: Extracción de la grasa inguinal de ratones transgénicos para proteína verde fluorescente y aislamiento de las células troncales mesenquimales. Estas células fueron implantadas utilizando inoculaciones intravítreas y subretinianas en un modelo murino (rd10) que asemeja la retinosis pigmentaria autosomal recesiva humana. Expresión de genes involucrados en el desarrollo de la retina mediante RT-PCR. Resultados: Nuestros resultados demuestran que en ambos abordajes quirúrgicos las células trasplantadas sobreviven, migran y se integran en diferentes capas de la retina de los ratones rd10. Además hemos identificado la expresión de genes en embriones y cuerpos embrionarios (Otx2, NF200,Pax6 periferina, IRBP) que pueden ser utilizados como activadores o potenciadores de la transdiferenciación. Conclusiones: las células mesenquimales del tejido adiposo reúnen las condiciones necesarias para su potencial uso en la terapia de enfermedades degenerativas de la retina. Los cuerpos embrionarios representan un modelo útil para estudiar los mecanismos reguladores de la diferenciación de las células retinianas (AU)

Objectives: To obtain adult mesenchymal stem cells from adipose tissue to be used in cellular therapies aimed to retinal regeneration. Material and Methods: isolation of adult mesenchymal stem cells from adipose tissue of green fluorescent protein transgenic mice. Recipient mouse eyes were transplanted with these cells by using intravitreal and subretinal procedures. We have used a mice model of human autosomal recessive retinitis pigmentosa (rd10). Gene expression by means of RT-PCR of mRNA isolated from embryo eyes (E9,5 to E18,5); post-natal eyes (P1 to P10) and adults eyes .That technique was also used in mouse embryoid bodies spontaneously differentiated from embryonic stem cells ES-D3. Results: In both surgical procedures we observed survival, migration and integration of mesenchymal stem cells into several retinal layers. We have also identified the expression of some retinal genes (Otx2, NF200,Pax6, peripherin,IRBP) wich may help to drive the mesenchymal stem cells transdifferentiation. Conclusions: Mesenchimal cells are very promising to be used in degenerative retinal diseases. We emphasize the usefulness of mouse embryoid bodies as a model to study the regulatory mechanisms of retinal neurons differentiation and its potential for obtaining retinal progenitors (AU)

Neuroprotective effect of intravitreal triamcinolone acetonide against photoreceptor apoptosis in a rabbit model of subretinal hemorrhage.

PURPOSE: To study photoreceptor apoptosis and iron migration as mechanisms of retinotoxicity in a rabbit model of subretinal hemorrhage (SRH) and to assess intravitreal triamcinolone acetonide (IVTA) for anti-apoptotic and neuroprotective effects. METHODS: In adult rabbits, eyes were studied histologically after subretinal injection of autologous blood. For comparisons of control eyes with eyes injected with 2 mg IVTA, morphometric analysis was performed with light microscopy, whereas apoptosis was quantified with terminal dUTP nick end labeling (TUNEL) and fluorescence microscopy. Localization of retinal iron was assessed with Perls' stain. RESULTS: Photoreceptor degeneration was initiated 48 hours after exposure to subretinal blood and progressed over 7 days. Increased TUNEL positivity demonstrating apoptotic cell death was associated with SRH and photoreceptor loss. VIP-Perls staining demonstrated iron in the photoreceptor layer and retinal pigment epithelium that correlated with photoreceptor degeneration. Treatment with IVTA enhanced photoreceptor cell survival by 11% at 48 hours and by 45% at 72 hours (P = 0.01) and reduced photoreceptor apoptosis ratios by 25% at 48 hours (P = 0.006). CONCLUSIONS: Photoreceptor toxicity caused by SRH occurs at least in part by apoptosis and is associated with iron migration to the photoreceptor layer. Treatment with IVTA reduced photoreceptor loss and apoptosis, indicating a neuroprotective action. Therapies to target SRH may augment anti-VEGF treatments in exudative age-related macular degeneration and other diseases of choroidal neovascularization.

Central corneal thickness and its relationship to intraocular pressure in mucopolysaccararidoses-1 following bone marrow transplantation.

PURPOSE: To examine the ocular findings in mucopolysaccaridoses-1 (MPS-1) and the relationship between intraocular pressure (IOP) and central corneal thickness following bone marrow transplantation. SETTING: clinical practice. SUBJECTS: 23 subjects with MPS-1 following bone marrow transplantation were examined. OBSERVATION PROCEDURES: age, sex, visual acuity, presence of strabismus, refractive error, fundus examination, intraocular pressure, and central corneal thickness were assessed for each individual. MAIN OUTCOME MEASURE: correlation of central corneal thickness with intraocular pressure. Only the right eye was used in correlations. RESULTS: Forty-six eyes of 23 subjects were examined. All subjects had been treated with successful bone marrow transplantation with mean follow-up of 8.5 years. Ages ranged from 17 months to 19 years (SD 5.03). Of the subjects, 60.8% were female; 85.5% were hyperopic; 8.6% had best-corrected visual acuity of >/=6/12; 34.5% had best-corrected visual acuity of <6/12 and >6/36 with 56.9% <6/36; 30% had strabismus. IOP ranged from 13 to 41 mm Hg with four children on antiglaucoma therapy. Central corneal thickness ranged from 484 microm to 705 microm and was moderately correlated with measured IOP in the right eye (r = 0.56). Corneal opacification moderately correlated with central corneal thickness (r = 0.57). Seventy percent had a normal disk evaluation; 30% had abnormal disk cupping. CONCLUSION: This is the first large case series of MPS-1 subjects demonstrating a correlation between measured IOP and central corneal thickness. All subjects had corneal opacification moderately correlating with corneal pachymetry.

Bioinformatic identification of novel putative photoreceptor specific cis-elements.

BACKGROUND: Cell specific gene expression is largely regulated by different combinations of transcription factors that bind cis-elements in the upstream promoter sequence. However, experimental detection of cis-elements is difficult, expensive, and time-consuming. This provides a motivation for developing bioinformatic methods to identify cis-elements that could prioritize future experimental studies. Here, we use motif discovery algorithms to predict transcription factor binding sites involved in regulating the differences between murine rod and cone photoreceptor populations. RESULTS: To identify highly conserved motifs enriched in promoters that drive expression in either rod or cone photoreceptors, we assembled a set of murine rod-specific, cone-specific, and non-photoreceptor background promoter sequences. These sets were used as input to a newly devised motif discovery algorithm called Iterative Alignment/Modular Motif Selection (IAMMS). Using IAMMS, we predicted 34 motifs that may contribute to rod-specific (19 motifs) or cone-specific (15 motifs) expression patterns. Of these, 16 rod- and 12 cone-specific motifs were found in clusters near the transcription start site. New findings include the observation that cone promoters tend to contain TATA boxes, while rod promoters tend to be TATA-less (exempting Rho and Cnga1). Additionally, we identify putative sites for IL-6 effectors (in rods) and RXR family members (in cones) that can explain experimental data showing changes to cell-fate by activating these signaling pathways during rod/cone development. Two of the predicted motifs (NRE and ROP2) have been confirmed experimentally to be involved in cell-specific expression patterns. We provide a full database of predictions as additional data that may contain further valuable information. IAMMS predictions are compared with existing motif discovery algorithms, DME and BioProspector. We find that over 60% of IAMMS predictions are confirmed by at least one other motif discovery algorithm. CONCLUSION: We predict novel, putative cis-elements enriched in the promoter of rod-specific or cone-specific genes. These are candidate binding sites for transcription factors involved in maintaining functional differences between rod and cone photoreceptor populations.

Crb1 is a determinant of retinal apical Müller glia cell features.

Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal blinding diseases, such as Leber congenital amaurosis and retinitis pigmentosa. In the previous studies we have shown that Crb1 resides in retinal Müller glia cells and that loss of Crb1 results in retinal degeneration (particularly in the inferior temporal quadrant of the mouse eye). Degeneration is increased by exposure to white light. Here, we studied the role of light and aging to gain a better understanding of the factors involved in the progress of retinal disease. Our data reveal that light is neither sufficient nor required to induce retinal disorganization and degeneration in young Crb1(-/-) mutant mice, suggesting that it rather modulates the retinal phenotype. Gene expression profiling showed that expression of five genes is altered in light-exposed Crb1(-/-) mutant retinas. Three of the five genes are involved in chromosome stabilization (Pituitary tumor transforming gene 1 or Pttg1, Establishment of cohesion 1 homolog 1 or Esco1, and a gene similar to histone H2B). In aged retinas, degeneration of photoreceptors, inner retinal neurons, and retinal pigment epithelium was practically limited to the inferior temporal quadrant. Loss of Crb1 in Müller glia cells resulted in an irregular number and size of their apical villi. We propose that Crb1 is required to regulate number and size of these Müller glia cell villi. The subsequent loss of retinal integrity resulted in neovascularization, in which blood vessels of the choroid protruded into the neural retina.

Alterations in photoreceptor-bipolar cell signaling following ischemia/reperfusion in the rat retina.

Studies of retinal ischemia/reperfusion indicate a disparity between the anatomical and functional results; while a large number of rod bipolar cells remain postischemia, there is a significant reduction in the amplitude of the scotopic b-wave of the electroretinogram (ERG). We investigated the alterations in photoreceptor-bipolar cell signaling following ischemia/reperfusion and suggest a mechanism for the decrease in b-wave amplitude. A cation channel probe (agmatine, 1-amino-4-guanidobutane, AGB) was used to assess cellular ion channel activity in neurochemically identified cells secondary to endogenous glutamate release or pharmacological manipulations. By applying the "neurochemical truth point" principle (Sun et al. [2007a] J Comp Neurol, this issue), we have been able to confirm the loss of specific subpopulations of neurons. ERG was used to assess gross retinal function, with parameters of the ERG model providing insight into changes in the phototransduction cascade and sensitivity of postreceptoral glutamate receptors. Following ischemia/reperfusion, rod bipolar cells maintained 2-amino-4-phosphonobutyric acid-responsive metabotropic glutamate receptors and displayed no change in sensitivity to flashes of light as assessed by ERG. Therefore, the loss in b-wave amplitude is likely due to alterations in photoreceptoral glutamate release detected as a change in postsynaptic AGB permeation into rod bipolar cells. Bipolar cell to amacrine cell signaling was also altered. The robust AGB entry into cholinergic amacrine cells was virtually absent in retinas that had undergone ischemia/reperfusion but remained in the AII amacrine cells. Such results suggest a loss of glutamate receptors and/or a change in receptor subunit expression in subpopulations of inner retinal neurons. Although many cells retain their characteristic neurochemical labeling following ischemia/reperfusion, caution should be used when assuming cells participate in functional retinal circuits based solely on the persistence of neurochemical labeling.

Intraperitoneal injection of Ginkgo biloba extract enhances antioxidation ability of retina and protects photoreceptors after light-induced retinal damage in rats.

PURPOSE: To investigate the effect of Ginkgo biloba extract EGb 761, a free-radical scavenger, on the antioxidation capability of retina after light-induced retinal damage in rats in an attempt to understand the mechanism by which EGb 761 protects the photoreceptors after light-induced retinal damage. METHODS: Seventy-two female Sprague-Dawley (SD) rats were evenly randomized into normal control group (NC group), light-induced retinal damage model group (M group), model + normal saline group (MN group), and model + EGb 761 group (ME group). Light-induced retinal damage model was induced via exposure to white light at 2740 +/- 120 lux for 6 hr. Rats in MN group and ME group were intraperitoneally injected daily with normal saline and 0.35% EGb 761 (100 mg/kg), respectively, 1 week before and 2 weeks after light exposure. The levels of malondialdehyde (MDA), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the retinal tissues were determined 24 hrs after light exposure; photoreceptor apoptosis was detected 4 days after light exposure. One and 2 weeks after light exposure, histopathologic examination was carried out, and the outer nuclear layer (ONL) thickness (number of nuclei) in the superior and inferior retina was counted. RESULTS: Twenty-four hours after exposure, the MDA levels in the other three groups were significantly higher than that in the NC group (p < 0.05); those in the M and MN groups were similar to each other (p > 0.05); and that of the ME group was significantly lower than those in the M and MN group (p < 0.05). The activities of T-SOD, GSH-Px, and CAT were similar in the M and MN groups (p > 0.05); the activities in the M and MN groups were significantly lower than those in the NC and ME groups (p < 0.05); and the activities in the ME group were significantly higher than those in the M and MN groups (p < 0.05). Four days after exposure, the apoptotic photoreceptors within the ONL in the ME group were obviously fewer than those in the M and MN groups. One week and 2 weeks after exposure, the ONL thickness (number of nuclei) in the ME group was more than that in the M and MN groups but less than that in the NC group. CONCLUSIONS: Intraperitoneal injection of EGb 761 can enhance the antioxidation ability of retina and partially inhibit the apoptosis of photoreceptors, thus exert a protective effect on photoreceptors.

Glutathione depletion induces differential apoptosis in cells of mouse retina, in vivo.

Oxidative stress affects numerous intracellular macromolecules, and may result in cell death unless precisely regulated. Unregulated oxidative stress can be controlled by various cellular defense mechanisms such as glutathione (GSH) which can critically counteract the damaging effects of oxidative stress in mammalian cells. We determined the effects of unregulated oxidative stress induced by GSH depletion on cells in mouse retina. Mice were intraperitoneally injected with buthionine sulphoximine (BSO) at 1.5 g/kg. After 0, 1, 4, and 7 days of BSO administration, retinas were excised and sections were subjected to GSH assay and terminal uridine deoxynucleotidyl nick end labeling (TUNEL) analysis. After 4 days of BSO administration, the number of TUNEL positive cells was significantly increased. However, after 7 days, TUNEL positive cells returned to the basal level. The retinal region most affected by the BSO treatment appeared to be the outer nuclear layer where the photoreceptor cells reside. Different from cells in other regions, retinal cells in the inner nuclear layer increased in their apoptosis even after the first day of BSO injection, and the increase was further potentiated after 4 days. Taken together, our studies suggested that GSH depletion may cause unregulated oxidative stress to the cells in the retina and indeed increased cell death in the retina. The cells in the inner nuclear layer seemed to be affected earlier than the cells in other layers of the retina. The GSH level in the retina may be a crucial therapeutic target in preventing blindness.

Identification of photoreceptor genes affected by PRPF31 mutations associated with autosomal dominant retinitis pigmentosa.

Several ubiquitously expressed genes encoding pre-mRNA splicing factors have been associated with autosomal dominant retinitis pigmentosa (adRP), including PRPF31, PRPF3 and PRPF8. Molecular mechanisms by which defects in pre-mRNA splicing factors cause photoreceptor degeneration are not clear. To investigate the role of pre-mRNA splicing in photoreceptor gene expression and function, we have begun to search for photoreceptor genes whose pre-mRNA splicing is affected by mutations in PRPF31. Using an immunoprecipitation-coupled-microarray method, we identified a number of transcripts associated with PRPF31-containing complexes, including peripherin/RDS, FSCN2 and other photoreceptor-expressed genes. We constructed minigenes to study the effects of PRPF31 mutations on the pre-mRNA splicing of these photoreceptor specific genes. Our experiments demonstrated that mutant PRPF31 significantly inhibited pre-mRNA splicing of RDS and FSCN2. These observations suggest a functional link between ubiquitously expressed and retina-specifically expressed adRP genes. Our results indicate that PRPF31 mutations lead to defective pre-mRNA splicing of photoreceptor-specific genes and that the ubiquitously expressed adRP gene, PRPF31, is critical for pre-mRNA splicing of a subset of photoreceptor genes. Our results provide an explanation for the photoreceptor-specific phenotype of PRPF31 mutations.

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 x 10(-3), 1 x 10(-1), and 1 x 10(1) microW 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.

Hyperoxia, hyperglycemia, and photoreceptor sensitivity in normal and diabetic subjects.

The aim of this study was to investigate the effects of an increase in the saturation of blood oxygen (SaO2) and/or serum glucose on photoreceptor sensitivity in normal subjects and in patients with diabetes mellitus. We monitored cone and rod sensitivity by recording dark-adaptation curves to both green and red test stimuli while inhaling either air (20% O2 + 80% N2) or 100% oxygen in 12 normal subjects and 12 diabetic patients with no (10) or mild (2) retinopathy. We also repeated the experiment in 10 of the normal subjects under hyperglycemia (mean serum glucose: 161 mg/dl). Results show that in normal subjects the dark-adapted cone sensitivity is improved by an increase in SaO2 or by hyperglycemia. Final rod sensitivity is unchanged during hyperoxia and during hyperglycemia when measured with a green test spot. However the kinetics of dark adaptation are altered during hyperglycemia, and an increase in final sensitivity is observed when measured with the red test spot. Inhalation of oxygen during hyperglycemia in normal subjects reduces cone sensitivity compared to that found during hyperglycemia alone (Pasteur effect). In diabetic subjects the dark-adapted cone threshold is comparable to that found in normal subjects, and sensitivity also increases with an increase in SaO2. The final rod threshold, however, is impaired compared to that of the control group, and rod sensitivity is improved by increasing the SaO2. The results suggest that the metabolism of rods and cones may differ in normal subjects: in cones, the rate of metabolism can be augmented by increasing the available oxygen or glucose, whereas rods appear more insensitive to increased blood oxygen saturation and hyperglycemia. In diabetic subjects, both cone and rod metabolism can be increased by supplemental oxygen, indicative of an early rod deficit. The study lends weight to the hypothesis that dark-adapted rods in diabetics are hypoxic before the onset of retinopathy.

Altered visual sensitivity in axial high myopia: a local postreceptoral phenomenon?

PURPOSE: The present study investigated retinal integrity in high myopia using spatial psychophysical tasks. METHODS: Ten axial high myopes (-8.5 to -11.5 D) and 10 age-matched control subjects (+/-1.0 D) were recruited. All participants underwent clinical examination and ocular biometry and demonstrated no visible macular disease with visual acuities better than 6/12. Foveal summation thresholds were determined for white and S-cone-isolating spots of various diameters up to 5.4 degrees and spatial contrast sensitivity to luminance sine wave gratings (0.5-9.7 cyc/deg). Data were analyzed after correction for the magnification induced by eye size and correcting lens power. RESULTS: Spatial summation for both white and S-cone-isolating spots showed a generalized loss of sensitivity at all spot sizes in myopes relative to control subjects (P = 0.01). Critical areas at maximum summation were significantly larger in myopes, for S-cone isolating spots only, after image size correction (P = 0.048). Sensitivity at maximum summation correlated negatively with vitreous chamber depth for both targets (P = 0.005). Sensitivities for S-cone and luminance spots also correlated (P < 0.001), indicating widespread dysfunction. Myopes displayed contrast sensitivity losses at high spatial frequencies (P

[Clinical and electroretinographic profile of commotio retinae]. / Perfil clínico e eletrorretinográfico da comoção retiniana.

PURPOSE: To investigate clinical, anatomic and electroretinographic changes in eyes that suffered blunt ocular trauma with commotio retinae. METHODS: Patients who presented commotio retinae after unilateral blunt ocular trauma less then 72 hours before were submitted to visual acuity testing, biomicroscopy, binocular indirect ophthalmoscopy, fluorescein angiography, optical coherence tomography and full-field electroretinography. Full-field ERG was repeated after 15 and 30 days. RESULTS: Sixteen patients were included in the study. On the first examination there was a statistically significant difference between affected and fellow eye in all response amplitudes, without b/a ratio alteration, and a delay in single-flash cone response and 30-Hz flicker implicit time. On the second examination, the difference between the eyes remained for oscillatory potentials, but disappeared on the last examination. In fluorescein angiography, all patients presented mottled hyperfluorescence and hypofluorescent areas, due to alterations in the pigment barrier. On optical coherence tomography, we found optically empty spaces at the site of the lesion. CONCLUSION: Found changes suggested photoreceptor and ganglion cells, but not Müller cell functional alterations, as well as pigment mobilization. These changes disappeared 30 days after the trauma.

Brain-derived neurotrophic factor gene delivery to muller glia preserves structure and function of light-damaged photoreceptors.

PURPOSE: To test the hypothesis that adenovirus (Ad)-mediated gene delivery of brain-derived neurotrophic factor (BDNF) to Müller cells can protect photoreceptors from light-induced retinal degeneration. METHODS: Adult Sprague-Dawley rats received an intraocular injection of Ad.BDNF, control Ad containing the green fluorescent protein (GFP) gene, or BDNF recombinant protein. Animals were then exposed to 5, 10, or 16 days of constant light. The effect of Ad.BDNF on photoreceptor survival was examined histologically, by measuring the outer nuclear layer (ONL) thickness, and functionally, by measuring the electroretinographic (ERG) response. RESULTS: Ad.BDNF mediated sustained expression of bioactive neurotrophin by Müller cells that lasted for at least 30 days after viral vector administration. BDNF gene delivery to Müller glia markedly increased the survival and structural integrity of light-damaged photoreceptors. For example, after 10 days of exposure to light, the average percentage of ONL preservation in the superior central retina of eyes that received Ad.BDNF was 71%, compared with 46% in eyes that received a control Ad.GFP or 15% in contralateral eyes. Of importance, retinas exposed to Ad.BDNF had more photoreceptor nuclei than retinas that received a single intraocular injection of BDNF recombinant protein. The neuroprotective effect of Ad.BDNF was accompanied by preservation of the ERG response of the treated eyes. CONCLUSIONS: These data provide proof of the concept that BDNF gene transfer into Müller cells is an effective strategy for preserving structure and function of photoreceptors in retinal degeneration.

Glial cell-mediated spread of retinal degeneration during detachment: a hypothesis based upon studies in rabbits.

In human subjects with peripheral retinal detachments, visual deficits are not restricted to the detached retina but are also present in the non-detached tissue. Based upon studies on a rabbit model of rhegmatogenous retinal detachment, we propose a glial cell-mediated mechanism of spread of retinal degeneration into non-detached retinal areas which may also have importance for the understanding of alterations in the human retina. Both detached and attached portions of the rabbit retina display photoreceptor cell degeneration and cystic degeneration of the innermost layers. An inverse mode of photoreceptor cell degeneration in the attached tissue suggests a disturbed support of the photoreceptor cells by Müller cells which show various indications of gliosis (increased expression of intermediate filaments, cell hypertrophy, decreased plasma membrane K(+) conductance, increased Ca(2+) responsiveness to purinergic stimulation) in both detached and attached tissues. We propose that gliotic alterations of Müller cells contribute to the degeneration of the attached retina, via disturbance of glial homeostasis mechanisms. A down-regulation of the K(+) conductance of Müller cells may prevent effective retinal K(+) and water clearance, and may favor photoreceptor cell degeneration and edema development.

The effects of indocyanine green and endoillumination on rabbit retina: an electroretinographic and histological study.

AIM: To evaluate the functional and morphological retinal toxicity associated with intravitreal injection of indocyanine green (ICG) dye in rabbit eyes during vitrectomy with endoillumination. METHODS: 20 eyes of 10 New Zealand pigmented rabbits were used in the study. All eyes underwent pars plana vitrectomy and removal of posterior vitreous cortex under endoillumination. In one eye of each rabbit, intravitreal injection of 0.1 ml of 2.5 mg/ml ICG was applied for 30 seconds followed by 10 minutes of endoillumination. The control eye had endoillumination only without ICG injection. Dark adapted and light adapted electroretinograms (ERGs) were performed before the surgery and 1 week after surgery for serial comparisons. Rabbits were killed 1 week after surgery and eyes were enucleated for histological examination. RESULTS: Serial ERG comparisons showed significant reduction in the light adapted a-wave amplitude (p = 0.037) and significant delays in the dark adapted and light adapted b-wave latencies (p = 0.020 and p = 0.038, respectively) in the ICG treated eyes. Histological examinations demonstrated loss of photoreceptor outer segments with focal absence of photoreceptors in some areas in the ICG injected eyes. CONCLUSIONS: Vitrectomy followed by intravitreal injection of 2.5 mg/ml ICG for 30 seconds with endoillumination may result in retinal toxicity causing functional and morphological retinal damages in rabbit eyes. The lowest concentration of ICG should be used if necessary for intraocular use to prevent potential retinal toxicity.

The genomic response to retinal disease and injury: evidence for endothelin signaling from photoreceptors to glia.

Regardless of proximal cause, photoreceptor injury or disease almost invariably leads to the activation of Muller cells, the principal glial cells in the retina. This observation implies the existence of signaling systems that inform Muller cells of the health status of photoreceptors. It further suggests that diverse types of photoreceptor damage elicit a limited range of biochemical responses. Using the mouse retina, we show by microarray, RNA blot, and in situ hybridization that the genomic responses to both light damage and inherited photoreceptor degeneration involve a relatively small number of genes and that the genes activated by these two insults overlap substantially with one another and with the genes activated by retinal detachment. Among the induced transcripts, those coding for endothelin2 (Edn2) are unusual in that they are localized to photoreceptors and are also highly induced in all of the tested models of photoreceptor disease or injury. Acute light damage also leads to a >10-fold increase in endothelin receptor B (Ednrb) in Muller cells 24 h after injury. These observations suggest that photoreceptor-derived EDN2 functions as a general stress signal, that EDN2 signals to Muller cells by binding to EDNRB, and that Muller cells can increase their sensitivity to EDN2 as part of the injury response.

Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats.

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft-host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft-host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.