Revisiting nestin expression in retinal progenitor cells in vitro and after transplantation in vivo.

The purpose of this study is to characterize the co-expression of nestin--a neuroectodermal stem cell and a reactive glial marker-with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all-trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1-4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C alpha (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCalpha, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.

Visual functional effects of constant blue light in a retinal degenerate rat model.

Retinal degenerative conditions increase susceptibility to light damage, but rapid retinal degeneration (RD) models show less susceptibility to cyclic dim light. We investigated whether constant blue light (BL) exposure can eliminate the residual visual responses in a comparatively rapid RD rat model. Pigmented rhodopsin mutant S334ter line-3 rat pups (21 days old) were exposed for 5-6 consecutive days to constant BL. Visual behavior was evaluated with an optokinetic head tracking apparatus. Electrophysiological recordings were made from the superior colliculus (SC). S-antigen, red-green opsin and rhodopsin immunoreactive residual photoreceptors were counted. Following BL exposure, head tracking was significantly reduced at 0.25 cycles degree(-1) in 38-day-old line 3 rats. With a 0.125 cycles degree(-1) stimulus, the head tracking performance of 80-day-old BL rats were similar to that of 220-day-old no-BL-treated line-3 rats. SC recordings also revealed a significant decrease in the residual photoreceptor activity. Histological evaluation showed reduction of the rod population in the central area of the light-damaged retina. Exposure to constant BL considerably reduces the residual visual responses in a rapid degenerating RD rat model.

A new model of experimental subretinal neovascularization in the rabbit.

Existing animal models of choroidal neovascularization (CNV) present several problems: they are hard to reproduce, they are inefficient, and the CNV created is not sustainable. The purpose of this study is to develop a highly efficient, reliable, sustainable rabbit model of CNV to facilitate the study of anti-angiogenic and anti-proliferative therapies for ocular diseases. Twenty-two pigmented rabbits were used in this study. Eleven rabbits received subretinal injections of either 10 microl of Matrigel with 500 ng of vascular endothelial growth factor (VEGF) or 20 microl of Matrigel with 750 ng of VEGF; eight rabbits received subretinal injections of either 10 or 20 microl of Matrigel only; three rabbits used as controls received subretinal injections of 20 microl phosphate-buffered saline (PBS) alone. Fundus photography, fluorescein angiography, optical coherence tomography, and histologic examinations were performed 1, 2, 4, and 9 weeks after injection. All experimental eyes showed angiographic leakage within this localized area 1 week after injection. The amount of leakage usually increased at weeks 2 and 4 and, in most cases, persisted at week 9. Control eyes demonstrated no leakage at any time point. Optical coherence tomography of treated eyes showed subretinal fluid and the presence of a lesion, possibly vascular or fibrotic, at the site of the leakage. Histologic analysis confirmed the presence of new subretinal blood vessels in the areas of Matrigel deposit. In conclusion, this novel method provides a highly reproducible, reliable, and sustainable rabbit model of experimental choroidal neovascularization. Such a model may prove useful for screening new anti-angiogenic therapies in a larger animal eye.

Implantation of an inactive epiretinal poly(dimethyl siloxane) electrode array in dogs.

The aim of this study is to investigate the long-term, mechanical biocompatibility of a polymer microtechnology that can be used to position electrodes in close proximity to the retina. Poly(dimethylsiloxane) (PDMS) arrays were manufactured by soft-lithography at Lawrence Livermore National Laboratory. The PDMS implant measured 4 mm x 40 mm x 55-60 microm and included 4-8 electrodes. Micromolded ribs were placed at the perimetry for strength and ease of manipulation. The PDMS arrays were implanted epiretinally in four normal dogs, with a single retinal tack used in each case to hold the device on the retina. The mechanical effects of the implant were followed up after surgical implantation by photography, fluorescein angiography, optical coherence tomography (OCT), and electrophysiologic tests. An intraoperative retinal tear occurred in the first implanted dog, causing retinal detachment and necessitating termination. The remaining dogs experienced no gross complications secondary to the array implantation procedure. During the follow-up period of 2 months in one eye and 6 months in three eyes, OCT demonstrated that the arrays were in close contact with the retina. Fluorescein angiography showed good perfusion of the retina under the array. At the end of 6 months, there was no statistical difference from baseline in mean retinal thickness under the array (P=0.43) or peripapillary retinal nerve fibre layer thickness corresponding to the implanted area (P=0.34). The mean distance between the array and the retinal surface varied from 32 to 68 microm throughout the follow-up. Histopathologic evaluation of the retinal implantation site in eyes followed for 6 months showed a general preservation of the normal, layered retinal structure, except for some localized retinal thinning in two eyes, where the array frame had been in direct retinal contact. The PDMS substrate micro array is a new and promising technology that can be scaled to support a high-density retinal stimulating array. Its implantation and handling is surgically manageable, and it forms a mechanically stable, acceptable interface with the inner retinal surface.

Retinal transplants evaluated by optical coherence tomography in photoreceptor degenerate rats.

Optical coherence tomography (OCT), a non-invasive method, was used for qualitative assessment of fetal retinal sheet transplants by non-invasive imaging. Rhodopsin-mutant S334ter-line-3 rats with fast retinal degeneration (28-37-day old) were transplanted with fetal retinal sheets from embryonic day (E) 18-19 pigmented normal rats. Retinal thickness measurements from transplanted (n = 51), no surgery control (n = 8), and normal pigmented rat eyes (n = 6) were obtained using a Zeiss stratus OCT-3 scanning instrument. Frozen retinal sections were stained with hematoxylin/eosin. S334ter-line-3 rats showed significant reduction in OCT retinal thickness (p<0.001) compared to normal pigmented rats at the age of 21 days. In 62% of the transplanted rats, OCT scanning revealed the presence of a subretinal graft, which was confirmed by subsequent histology. Retinal thickness in the transplant area was significantly increased compared to the area outside the transplant and to non-transplanted eyes (p<0.001). While most of the transplants with single-band OCT images (87%) had rosetted transplants, a considerable proportion of transplants having a multi-band OCT image were found to have well-laminated areas in the graft after histological evaluation. Following retinal transplantation in rodents, OCT imaging data correlated mostly with transplant morphology. OCT is a useful technique for in vivo screening and evaluation of retinal transplants. This technique determines surgical outcomes at a much earlier stage.

Development, implantation, in vivo elution, and retrieval of a biocompatible, sustained release subretinal drug delivery system.

A biocompatible, sustained-release subretinal drug-delivery platform was developed to overcome the therapeutic accessibility limitations of current retinal disease treatments. The prototype implants were fabricated by coating nitinol, poly(methyl methacrylate) or chromic gut core filaments, with a drug-eluting polymer matrix. The polymer coatings are manufactured and coated by SurModics. The coating is a mixture of poly(butyl methacrylate) and poly(ethylene-co-vinyl acetate). The drug is either triamcinolone acetonide or sirolimus. The rods were successfully implanted into the subretinal space of 20/24 rabbits. Four rabbits were lost to early surgery from a dysfunctional infusion line and hemorrhage. No serious complications were observed during the 4-week follow-up period. Slight conjunctival redness was reported in all rabbits by 1-day follow-up, but the redness had subsided by the following week. Intraocular lens touch occurred in six rabbits during the implantations; of these, four had a lensectomy at the time of surgery, and the remaining two developed cataract. Corneal edema developed in three rabbits by 1-week follow-up, but subsided within 2 weeks. Initial observations of the implantation and elution characteristics revealed that the implants are well tolerated by the retinal tissue and that the implant can elute triamcinolone acetonide for a period of at least 4 weeks without eliciting an inflammatory response or complications. There were adverse clinical indications with the sirolimus-loaded implants at the delivered dose. Device retrieval required an uncomplicated surgical procedure, and revealed no associated or adherent tissue. Implant drug content analysis and opacity changes to the polymer matrix coating following retrieval demonstrated the sustained elution of the drug.

Nanobiolistic delivery of indicators to the living mouse retina.

The development of a technique to load functional indicators into living neurons is an ongoing challenge in retinal neurophysiology. In a number of live-cell preparations, fluorescence-based indicators have been of particular importance for investigating ionic concentrations, protein localization, and other physiological parameters. In the present study, we demonstrate a novel technique that uses a modified gene gun to propel silver nanoparticles coated with indicators into live retinal neurons, and we highlight the advantages of using this technique to deliver these functional indicators.

Long-term stimulation by active epiretinal implants in normal and RCD1 dogs.

An epiretinal prosthesis, consisting of an extraocular microelectronic stimulator and an intraocular electrode array, was implanted in one eye of three blind and three sighted dogs. Three dogs (2 blind, 1 normal) were stimulated for 120 days, and two dogs (both normal) for 60 and 103 days respectively for 8-10 h/day at levels of 0.1 mC cm(-2) and 0.05 mC cm(-2), with each stimulus level presented to half of the array. One blind dog was kept as an inactive implant control. During the study period, electroretinograms (ERG) and fundus photographs were recorded. At the end of the study period, the dogs were sacrificed and histological and morphometric evaluation was made of the retina. No inflammatory reaction, neovascularization or hemorrhage was observed during the follow-up examinations. ERGs were unchanged. Stimulus levels used were of sufficient amplitude to elicit cortical evoked potentials. Histological evaluation showed no inflammatory infiltrates or changes in retina morphometry related to electrical stimulation when compared to the unstimulated control eye. Morphometric analysis revealed no consistent differences relating to electrical stimulation. In summary, chronic electrical stimulation of the dog retina at up to 0.1 mC cm(-2) with an epiretinal prosthesis does not appear to adversely affect the retina.

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.

The mechanism of increasing outflow facility during washout in the bovine eye.

PURPOSE: To investigate the relationship between outflow facility and separation between the inner wall of the aqueous plexus and the juxtacanalicular connective tissue (JCT) during washout in the bovine eye. METHODS: Facility was recorded during 3 hours of anterior chamber perfusion at 15 mm Hg in eight pairs of bovine eyes. One eye of each pair was then lowered to 0 mm Hg for 1 hour, whereas the fellow eye was kept at 15 mm Hg. After a brief perfusion at 15 mm Hg, both eyes were perfusion fixed and processed for electron microscopy. Micrographs of the inner wall were analyzed for separation from the JCT. To study the role of cellular adhesion between the inner wall and JCT, 12 additional pairs were perfused with integrin-binding peptide (RGD: Arg-Gly-Asp) or sham control peptide (RGE: Arg-Gly-Glu) at 2 micro M to 2 mM, before IOP was reduced. RESULTS: During the first 3 hours, facility increased in both eyes because of "washout." However, after 1 hour of 0 mm Hg, facility decreased by 13% (P < 0.006), whereas facility increased by 20% (P < 0.001) in the fellow eyes maintained at 15 mm Hg. Two types of separation were observed between the inner wall and JCT: cell-matrix separation between the endothelial cell and basal lamina and matrix-matrix separation between the basal lamina and JCT. A significant positive correlation (P = 0.042) was found between the degree of matrix-matrix separation and the change in outflow facility after 1 hour of 0 mm Hg. Compared with RGE control, RGD had no apparent effect on outflow facility (P > 0.35) or on the change in outflow facility after 1 hour at 0 mm Hg (P > 0.15). CONCLUSIONS: The increase in outflow facility that occurs during washout in the bovine eye is reversible and correlates with the degree of separation between the basal lamina of the inner wall endothelium and the JCT. Therefore, adhesions tethering the inner wall to the JCT may be important ultrastructural features involved in the regulation of aqueous humor outflow resistance.

[Experimental studies of effects of retinoic acid on the proliferation of retinal cells].

OBJECTIVE: To determine whether the application of exogenous retinoic acid (RA) may induce the proliferation of retinal cells in adult rat. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into 4 groups. In Group 1 and Group 2, all-trans RA (5 microliter, 0.001 mol/L) was injected into the subretinal space. In Group 3 and Group 4, all-trans RA (10 microliter, 0.001 mol/L) was injected into epiretinal vitreous space. In the Group 1 and Group 3, transient ischemic-reperfusion injuries of the experimental eyes were induced by ligating ophthalmic artery prior to RA treatment. In the control group, 5 SD rats were treated by ischemia-reperfusion injuries but no exogenous RA application. The treated eyes were enucleated for light microscopic analysis and immunohistochemical assays after 2 - 4 weeks of RA application. RESULTS: In group 1, the number of the retinal cells expressing rod-specific opsin marker in the subretinal space was significantly increased and the thickness of inner nuclear layer was also increased after the RA treatment for 16 days. However, no cell proliferation was detected in group 2. There were also no changes within population of retinal cells in Group 3 and Group 4 in which RA was injected into epiretinal vitreous space no matter under the ischemia-reperfusion or non-ischemia-reperfusion. In the control group, there were no significant morphological changes within the neural retinal layers as well as photoreceptor proliferation. CONCLUSIONS: The application of RA in the subretinal space can induce photoreceptor proliferation in adult rat under an ischemic-reperfusion injury condition. It will provide a new idea for the regeneration of neural retinal cells.

[A pilot study of bone marrow stromal cells intraocular transplantation in the S334 transgenic rats and Sprague-Dawley rats].

PURPOSE: To investigate the survival and differentiation of human bone marrow stromal cells (BMSC) intraocular transplantation in newborn S334 retinal degeneration transgenic rats and (Sprague-Dawley)SD rats. METHODS: Human bone marrow stromal cells line was grown on the adhesive substrate in the condition media including a-Modified Eagle medium (a-MEM)/10% fetal bovine serum. The experiments were divided into four groups: Group 1: BMSC + (Retinoid Acid) RA transplanted in S334 transgenic rats (n = 5); Group 2: BMSC transplanted in S334 transgenic rats (n = 5); Group 3: BMSC + RA transplanted in SD rats (n = 5); Group 4: BMSC transplanted in SD rats (n = 5). 2 microl cell suspension (about 4 x 10(4) cells) was injected into the vitreous space in the transgenic rats and normal SD rats at Postnatal 1 (P1) respectively. The right eyes were treated eyes and the left eyes were used as control. At P14 and P23, the rats were killed and enucleated for histological assays using plastic section. RESULTS: In Group 1, the transplanted cells were well survived. They could continue to differentiate and participate in late-stage retinal development. The number of inner nuclear layer increased. Moreover, the host retina increased their thickness, but photoreceptor cells were not rescued from transplant. In Group 2, at P14, the BMSC continue to differentiate toward their linage cell fate and formed into hemorrhage island structures with few neurons if RA was not applied. Group 3, BMSC could survive, migrate. The number of inner nuclear layer increased also. In Group 4, it revealed that host retina structures were disorganized and transplant cells formed atypical proliferating mass. CONCLUSIONS: This pilot experiment indicated that bone marrow stromal cells could survival, differentiate and participate in the retinal development after transplanted into vitreous space in the new born transgenic rats and SD rats. Histological assays showed that transplanted cells integrated with inner nuclear layer of host retina. Thus, bone marrow stromal cells may be a useful vehicle for auto- transplantation for the therapy of variety of retinal degenerative disorders.