Treatment Of Retinal Degeneration Research Articles

Neuroprotective effect of NecroX-5 against retinal degeneration in rodents.

NecroX-5 is a derivative of cyclopentylamino carboxymethylthiazolylindole (NecroX), an inhibitor of necrosis/necroptosis. NecroX-5 has been shown to scavenge mitochondrial reactive oxygen and nitrogen species, and thus preventing necrotic cell death against various kinds of oxidative stress in several tissues, including the brain. To examine the effect of NecroX-5 on retinal degeneration (RD), RD was induced in Sprague-Dawley rats by an intraperitoneal injection of N-methyl-N-nitrosourea and in BALB/c mice by blue light-emitting diode exposure. Scotopic electroretinography recording was used to evaluate retinal function. For histological evaluation, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry were performed. Electroretinography recordings showed that a-waves and b-waves were significantly reduced in both RD rats and mice, whereas the amplitudes of both waves were significantly increased in both NecroX-5-treated RD rats and mice compared with untreated RD animals. In hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, the outer nuclear layer where photoreceptors reside appeared to be more preserved, and there were fewer apoptotic cells in NecroX-5-treated RD retinas than in untreated RD retinas. In addition, immunohistochemistry with antiglial fibrillary acidic protein and anti-8-hydroxy-2'-deoxyguanosine showed lower levels of retinal injury and oxidative stress in NecroX-5-treated RD retinas than in untreated RD retinas. These results indicated that NecroX-5 protects retinal neurons from experimentally induced RD, suggesting that NecroX-5 may have a potential for the treatment of RD as a medication.

Subpopulations of Bone Marrow Mesenchymal Stem Cells Exhibit Differential Effects in Delaying Retinal Degeneration.

Bone marrow mesenchymal stem cells (BMSCs) have a therapeutic role in retinal degeneration (RD). However, heterogeneity of BMSCs may be associated with differential therapeutic effects in RD. In order to confirm this hypothesis, two subsets of rat BMSCs, termed rBMSC1 and rBMSC2, were obtained, characterized and functionally evaluated in the treatment of RD of Royal College of Surgeons (RCS) rats. Both subpopulations expressed mesenchymal stem cells (MSC) markers CD29 and CD90, but were negative for hemacyte antigen CD11b and CD45 expression. In comparison with rBMSC2, rBMSC1 showed higher rate of proliferation, stronger colony formation, and increased adipogenic potential, whereas rBMSC2 exhibited higher osteogenic potential. Microarray analysis showed differential gene expression patterns between rBMSC1 and rBMSC2, including functions related to proliferation, differentiation, immunoregulation, stem cell maintenance and division, survival and antiapoptosis. After subretinal transplantation in RCS rats, rBMSC1 showed stronger rescue effect than rBMSC2, including increased b-wave amplitude, restored retinal nuclear layer thickness, and decreased number of apoptotic photoreceptors, whereas the rescue function of rBMSC2 was essentially not better than the control. Histological analysis also demonstrated that rBMSC1 possessed a higher survival rate than rBMSC2 in subretinal space. In addition, treatment of basic fibroblast growth factor, an accompanying event in subretinal injection, triggered more robust increase in secretion of growth factors by rBMSC1 as compared to rBMSC2. Taken together, these results have suggested that the different therapeutic functions of BMSC subpopulations are attributed to their distinct survival capabilities and paracrine functions. The underlying mechanisms responsible for the different functions of BMSC subpopulation may lead to a new strategy for the treatment of RD.

Stem cell therapy for retinal diseases.

In this review, we discuss about current knowledge about stem cell (SC) therapy in the treatment of retinal degeneration. Both human embryonic stem cell and induced pluripotent stem cell has been growth in culture for a long time, and started to be explored in the treatment of blinding conditions. The Food and Drug Administration, recently, has granted clinical trials using SC retinal therapy to treat complex disorders, as Stargardt's dystrophy, and patients with geographic atrophy, providing good outcomes. This study's intent is to overview the critical regeneration of the subretinal anatomy through retinal pigment epithelium transplantation, with the goal of reestablish important pathways from the retina to the occipital cortex of the brain, as well as the differentiation from pluripotent quiescent SC to adult retina, and its relationship with a primary retinal injury, different techniques of transplantation, management of immune rejection and tumorigenicity, its potential application in improving patients' vision, and, finally, approaching future directions and challenges for the treatment of several conditions.

Mesenchymal Stem Cell Transplantation for Retinal Degenerations and Dystrophies: Present and Future

Retinal degenerations are the main causes of irreversible blindness in developed countries. Up to date the main pathological mechanisms of these diseases are not fully understood and consequently there is no complete treatment option for those diseases. In this aspect stem cells have drawn attention of many researchers and health care professionals. Considering ethical issues, safety and facile isolation Mesenchymal Stem Cells (MSCs) are more preferable for practical use. They have been used for several preclinical and clinical trials. In general the results were promising, however broader practical use should be preceded by resolving many problems and questions. In this review we will describe mesenchymal stem cells, especially those derived from Bone-Marrow (BMSC), their main features, privilege, mechanisms of action and their potential use for the treatment of retinal degenerations. We will also discuss the results of several pre-clinical and clinical trials.

Approaches to cell delivery: substrates and scaffolds for cell therapy.

Retinal degeneration, associated with loss of photoreceptors, is the primary cause of permanent vision impairment, impacting millions of people worldwide. Age-related macular degeneration and retinitis pigmentosa are two common retinal diseases resulting in photoreceptor loss and vision impairment or blindness. Presently, available treatments can only delay the progress of retinal degeneration, and there are no treatments that can restore permanent vision loss. Research is underway to develop methods of regenerating the impaired retina by delivering photoreceptor precursor cells and retinal pigment epithelium to the subretinal space. Challenges to cell transplantation include limited survival upon implantation and the formation of abnormal cell architectures in vivo. Retinal tissue engineering shows immense promise and potential in treatment of retinal degeneration by employing scaffold-based delivery systems of retinal progenitor cells to the subretinal space. Scaffold delivery strategy has been shown to enhance the cell survival and direct cell differentiation in a variety of retinal degenerative models. In this chapter, we summarize the research findings on different scaffold- or substrate-based transplantation techniques used to deliver retinal progenitor/photoreceptor precursors and retinal pigment epithelial cells to the subretinal space.

Assessment of Tropism and Effectiveness of New Primate-Derived Hybrid Recombinant AAV Serotypes in the Mouse and Primate Retina

Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4−/− mouse which is a model for Stargardt disease and in the Pde6brd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.

Induced pluripotent stem cells and retinal degeneration treatment

Lesions of the retinal pigment epithelium and photoreceptors lead to different kinds of retinal dystrophies, which are characterized by the grade of visual loss and the character of retinal atrophy...

The Oral Iron Chelator Deferiprone Protects Against Retinal Degeneration Induced through Diverse Mechanisms

To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

The Oral Iron Chelator Deferiprone Protects Against Retinal Degeneration Induced through Diverse Mechanisms.

To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

Sleeping BeautyTransposon-Mediated Transfection of Retinal and Iris Pigment Epithelial Cells

Subretinal transplantation of retinal (RPE) or iris (IPE) pigment epithelial cells has been advocated as a treatment for retinal degeneration. However, to our knowledge, in patients with age-related macular degeneration no significant beneficial effects on vision have been shown. Since the transplanted cells did not appear to maintain a healthy avascular and neuroprotective environment, we postulate that it will be necessary to transplant cells that express elevated levels of anti-angiogenic and neuroprotective activities. In our study, we provide a protocol for the efficient stable gene transfer and sustained gene expression of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic and neuroprotective factor, using the nonviral Sleeping Beauty transposon system (SB100X). Pigment epithelial cells were electroporated with a Venus reporter or a PEDF encoding plasmid, controlled by either CMV or CAGGS promoters. Transfection efficiencies and protein expression stability were evaluated by flow cytometry and immunoblotting. Gene expression profiles were analyzed by RT-PCR. SB100X-based delivery resulted in efficiencies of 100% with the Venus gene and 30% with the PEDF gene. Cell sorting enabled establishment of pure PEDF-transfected ARPE-19 populations. Transfected RPE and IPE cells have been shown to maintain stable PEDF secretion for more than 16 and 6 months, respectively. Transfection using the nonviral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cell's genome, ensuring continuous expression and secretion of PEDF. Stable expression of the therapeutic gene is critical for the development of cell-based gene addition therapies for retinal degenerative diseases.

Network Deficiency Exacerbates Impairment in a Mouse Model of Retinal Degeneration

Neural oscillations play an important role in normal brain activity, but also manifest during Parkinson’s disease, epilepsy, and other pathological conditions. The contribution of these aberrant oscillations to the function of the surviving brain remains unclear. In recording from retina in a mouse model of retinal degeneration (RD), we found that the incidence of oscillatory activity varied across different cell classes, evidence that some retinal networks are more affected by functional changes than others. This aberrant activity was driven by an independent inhibitory amacrine cell oscillator. By stimulating the surviving circuitry at different stages of the neurodegenerative process, we found that this dystrophic oscillator further compromises the function of the retina. These data reveal that retinal remodeling can exacerbate the visual deficit, and that aberrant synaptic activity could be targeted for RD treatment.

Increased Müller Cell De-Differentiation After Grafting of Retinal Stem Cell in the Sub-Retinal Space of Royal College of Surgeons Rats

In several vertebrate classes, the Müller glia are capable of de-differentiating, proliferating, and acquiring a progenitor-like state in response to acute retinal injury or in response to exogenous growth factors. Our previous study has shown that Müller cells can be activated and de-differentiated into retinal progenitors during Royal College of Surgeons (RCS) rats' degeneration, although the limited proliferation cannot maintain retinal function. We now report that rat retinal stem cells (rSCs) transplanted into RCS rats slowed the progression of retinal morphological degeneration and prevented the functional disruption. Further, we found that retinal progenitor cells labeled with Chx10 were increased significantly after rSCs transplantation, and most of them are mainly from activated Müller cells. rSCs transplantation also enhances neurogenic potential by producing more recoverin-positive photoreceptors, which was proved coming from Müller glia-derived cells. These results provide evidence that stem cell-based therapy may offer a novel therapeutic approach for the treatment of retinal degeneration, and that Müller glia in mammalian retina can be activated and de-differentiated by rSC transplantation and may have therapeutic effects.

Retinal remodeling in the Tg P347L rabbit, a large‐eye model of retinal degeneration

Retinitis pigmentosa (RP) is an inherited blinding disease characterized by progressive loss of retinal photoreceptors. There are numerous rodent models of retinal degeneration, but most are poor platforms for interventions that will translate into clinical practice. The rabbit possesses a number of desirable qualities for a model of retinal disease including a large eye and an existing and substantial knowledge base in retinal circuitry, anatomy, and ophthalmology. We have analyzed degeneration, remodeling, and reprogramming in a rabbit model of retinal degeneration, expressing a rhodopsin proline 347 to leucine transgene in a TgP347L rabbit as a powerful model to study the pathophysiology and treatment of retinal degeneration. We show that disease progression in the TgP347L rabbit closely tracks human cone-sparing RP, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming. The relatively fast disease progression makes the TgP347L rabbit an excellent model for gene therapy, cell biological intervention, progenitor cell transplantation, surgical interventions, and bionic prosthetic studies.

Retinal Degenerations: Genetics, Mechanisms, and Therapies

Retinal Degenerations: Genetics, Mechanisms, and Therapies

A new system for the treatment of retinal degeneration

Recent clinical studies indicate that stimulation of the retina with tiny amounts of current can have a therapeutic effect on retinal diseases such as retinitis pigmentosa. We have therefore developed a preventive treatment system which stimulates the eye with a thread electrode; among other objectives, the aim is that even blind patients can administer the therapy themselves under physician's supervision.

Loss of photoreceptor potential from retinal progenitor cell cultures, despite improvements in survival

Retinal degeneration (RD) results from photoreceptor apoptosis. Cell transplantation, one potential therapeutic approach, requires expandable stem cells that can form mature photoreceptors when differentiated. Freshly dissociated primary retinal cells from postnatal day 2–6 (PN2–6) mouse retina can give rise, post-transplantation, to photoreceptors in adult recipients. Unfortunately, incorporation rates are low; moreover, photoreceptor potential is lost if the same PN2–6 cells are cultured prior to transplantation. We investigated the identity of the cells forming photoreceptors post-transplantation, using FACS sorted primary postnatal day (PN) 3–5 Rho-eGFP retinal cells. Higher integration rates were achieved for cells that were expressing Rho-eGFP at PN3–5, indicating that post-mitotic photoreceptor precursors already expressing rhodopsin form the majority of integrating rods. We then investigated improvement of cell culture protocols for retinal progenitor cells (RPCs) derived from PN3–5 retinal cells in vitro. We succeeded in improving RPC survival and growth rates 25-fold, by modifying retinal dissociation, replacing N2 supplement with B27 supplement minus retinoic acid (B27 − RA) and coating flasks with fibronectin. However, levels of rhodopsin and similar photoreceptor-specific markers still diminished rapidly during growth in vitro, and did not re-appear after in vitro differentiation. Similarly, transplanted RPCs, whether proliferating or differentiated, did not form photoreceptors in vivo. Cultured RPCs upregulate genes such as Sox2 and nestin, markers of more primitive neural stem cells. Use of these cells for RD treatment will require identification of triggers that favour terminal photoreceptor differentiation and survival in vitro prior to transplantation.

Generation of a Transgenic Rabbit Model of Retinal Degeneration

To generate a transgenic (Tg) rabbit model of retinal degeneration and to characterize the pattern of degeneration by using histology and electrophysiology. Rhodopsin Pro347Leu Tg rabbits were generated by BAC transgenesis. Tg rabbits were identified by Southern blot analysis, and the expression levels were measured by quantitative RT-PCR. Retinal histology was examined by light and electron microscopy and immunohistochemistry. Retinal function was assessed by full-field electroretinograms (ERGs). Six lines of Tg rabbits were generated, and two lines with higher levels of expression showed rod-dominant progressive retinal degeneration. Retinal histology indicated a marked regional variation in the loss of photoreceptors with the central retina more severely affected than the peripheral retina. The characteristics of the ERGs of transgenic rabbits indicated that the rod components of the ERGs were reduced to only 5% by 48 weeks, whereas the cone components remained at 35% in the wild-type at the same time point. The retinal ultrastructure of Tg rabbits showed a large number of small vesicles that accumulated in the extracellular space of the photoreceptors. To the best of the authors' knowledge, this is the first rabbit model of progressive retinal degeneration. Because rabbits have large eyes and are easy to handle and breed, they will provide a useful animal model for the study of the pathophysiology of and new treatments for retinal degeneration.

Visual complications of Stickler syndrome in paediatric patients with Robin sequence

The most frequent diagnosis in patients with syndromic Pierre Robin sequence is Stickler syndrome, which may be complicated by congenital high myopia and substantial risk of retinal detachment. However, cases of Stickler syndrome with probable visual complications are rarely identified among this group of patients by members of the cleft team. This study was designed to determine the prevalence of Stickler syndrome among the author's group of patients with Robin sequence, and to investigate the visual outcome among paediatric patients with Robin sequence and Stickler syndrome. Eight children (six male and two female) with Stickler syndrome and Robin sequence were referred to be followed up every 6 months in the Ophthalmologic Department because of high myopia at less than 10 years of age. Three patients came from the author's study group and five were referred by other cleft surgeons. They were examined with repeat ophthalmic and indirect fundus examinations including cycloplegic refraction, and slit lamp biomicroscope examinations. Laser photocoagulation (2 cases) treatment for retinal degeneration or operation (3 cases) for retinal detachment was performed once evidence of significant ophthalmologic finding was noted. Of the 91 cases of newborns with isolated cleft palate treated by the first author, eight patients had Robin sequence, and among these, three had Stickler syndrome. The prevalence of Stickler syndrome among this subgroup of patients was 37.5% (3/8). Among these three patients and the additional five referred by other cleft surgeons, the average spherical equivalents of the first cycloplegic refraction for the 16 eyes was -12.39+/-2.72 diopter (D) (range -8.75 to -18.5D). Of the eight patients, five did not need any therapy, two children had retinal degeneration in the left eye and retinal detachment in the right eye while one child had retinal detachment in the right eye only. Laser photocoagulation was performed in the two left eyes with retinal degeneration and was successful. Surgery was performed on the three eyes with retinal detachment, one was successful while two failed and the patients developed total blindness at ages four and six respectively. Out of these three children with retinal complications, only one child remained free of visual deterioration in both eyes during the follow-up period. Early identification of Stickler syndrome in children with Robin sequence by cleft surgeons is necessary to insure early referral to an ophthalmologist for detection of myopia, monitoring for retinal detachment, and prevention of visual complications.

Efficient generation of retinal progenitor cells from human embryonic stem cells

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cell-derived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.

Retinal degeneration is delayed by tissue factor pathway inhibitor-2 in RCS rats and a sodium-iodate-induced model in rabbits.

To investigate the in vivo effects of tissue factor pathway inhibitor 2 (TFPI-2), which stimulates proliferation of retinal pigment epithelial cells, but not the proliferation of fibroblast and vascular endothelial cells in vitro, on retinal degeneration using a sodium-iodate (SI)-induced model in rabbits and Royal Collage of Surgeons (RCS) rats. 79 microg of recombinant TFPI-2 (rTFPI-2) or vehicle alone was injected intravitreously to 18 eyes of 12 pigmented rabbits a day after 20 mg/kg of SI was intravenously administered. Retinal function was assessed 4, 7, 14, and 21 days after the injection by analysing amplitudes of the c-wave of a bright flash electroretinogram. Additionally, 10 microg of rTFPI-2 or vehicle alone was injected intravitreously to 11 eyes of RCS rats at both 3 and 4 weeks old, then the retina was examined histologically at 5 weeks old. The rTFPI-2-treated eyes in rabbits showed a significantly less decrease in the relative amplitude of the c-wave than control eyes on days 4 and 7. The thickness of the outer nuclear layer was significantly thicker and the vacuole in the photoreceptor layer was less frequently observed in the rTFPI-2-treated RCS rats than the controls. Intravitreal injection of TFPI-2 rescues SI-induced retinal degeneration in rabbits and naturally occurring retinal degeneration in RCS rats at least partly. These results may suggest that this compound can be utilized in the treatment of retinal degeneration.