Transplantation of full-thickness retina in the rhodopsin transgenic pig

PURPOSE: To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease. METHODS: Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium. After a survival time of 4 months, eye specimens were studied by light and electron microscopy as well as with immunohistochemical markers. RESULTS: One eye developed endophthalmitis in the immediate postoperative period and was terminated. Laminated grafts with correct polarity were found in 13 of the remaining 19 eyes. In most cases, these grafts had well-developed organized photoreceptors with outer segments apposed to the host retinal pigment epithelium. The inner layers of the graft contained mostly Müller cells. Both eyes of the hosts had a reduction of photoreceptor cells in most of the retina, while inner layers remained relatively intact. CONCLUSIONS: Full-thickness neuroretinal grafts can be transplanted to a large animal host with photoreceptor degeneration. The transplantation procedure is relatively atraumatic to both graft and host tissue, and the grafts survive well for at least 4 months. The graft and host retina does not seem to form extensive neuronal contacts, and future work must be directed at stimulating such activity without disrupting the retinal neuronal organization.     

Full-thickness retinal transplants: a review

Embryonic full-thickness rabbit neuroretinal sheets were transplanted to the subretinal space of adult hosts. This was accomplished by using a new transplantation technique involving vitrectomy and retinotomy. The grafts were followed from 10 to 306 days after surgery and were then examined by different histological techniques. In the light microscope, the transplants were seen to develop the normal retinal lamination and fusion with the host retina, especially after long survival times. Ultrastructurally, normal photoreceptor outer segments, well integrated with the host retinal pigment epithelium, were found. Growth cones were present in the zone of fusion between graft and host retina. Immunohistochemical labeling revealed many of the normal retinal components not previously found in retinal transplants, and graft-host connections between neurons in the rod pathway were seen. The morphology of vibratome-sectioned neuroretinal sheets as well as adult full-thickness grafts was also examined. These transplantation types showed less of the normal morphology compared with embryonic full-thickness grafts. The immunogenicity of embryonic full-thickness and fragmented grafts was compared using major histocompatibility complex immunolabeling. Fragmented grafts elicited a response from the host immune system similar to a chronic transplant rejection. This reaction was absent in the full-thickness grafts which is in accordance with their good long-term survival.     

Transplantation of full-thickness retina in the normal porcine eye: surgical and morphologic aspects

PURPOSE: To report a surgical technique for transplantation of full-thickness neuroretinal sheets into the subretinal space of a large animal with a vascularized retina and to establish the light microscopic morphology of such specimens. METHODS: Twelve normal pigs underwent transplantation of a neuroretinal sheet from a neonatal donor into the subretinal space by means of a vitrectomy-based technique. After a survival of 33 to 72 days, eye specimens were studied with a light microscope. RESULTS: In most eyes, the transplants displayed a laminated morphology, with photoreceptor outer segments facing the host retinal pigment epithelium. These grafts had normal outer retinal layers, while the inner layers were less developed. The host retina straddling the graft showed evidence of photoreceptor degeneration, but the inner layers were well preserved. CONCLUSION: Full-thickness neuroretinal sheets can be transplanted to the subretinal space of a large animal eye with a vascularized retina. The grafts survive well and display mostly photoreceptors, which in combination with the well-preserved host inner retina may be of importance in attempts at reconstructing the retina in photoreceptor degenerative disease.     

Retinal detachment in children and adolescents

During a 4-year period 22 eyes with rhegmatogenous retinal detachment in children and young adults up to 20 years of age were operated on. This group comprised 6.3 percent of all rhegmatogenous retinal detachments operated on during the same period. In 9 eyes (40.9%) the detachment was due to a direct ocular trauma. In the remaining cases the main etiological factors were: myopia with or without lattice degeneration (5 eyes), retinopathy of prematurity (2 eyes), hereditary vitreoretinal degeneration (1 eye), uveal coloboma (2 eyes), aphakia after congenital cataract (1 eye), sex-linked juvenile retinoschisis (1 eye), and central retinal vein occlusion (1 eye), Conventional surgical procedures using episcleral or intrascleral implants or an encircling band combined with cryotherapy were used. After a follow-up of 7 months to 3.3 years (mean 1.5 years) the retina was flat in 18 cases (82%) and still detached in those with uveal coloboma (2 eyes), hereditary vitreoretinal degeneration (1 eye), and central retinal vein occlusion (1 eye). All traumatic detachments were flat.     

Survival of full-thickness retinal xenotransplants without immunosuppression

BACKGROUND: A study was carried out to explore the survival of xenogeneic full-thickness retinal transplants in the subretinal space of hosts without immunosuppression. METHODS: Nine adult rabbits received a complete immature rat neuroretina in the subretinal space. No immunosuppression was given, and the animals were followed up for 15 or 34 days. The eyes were then examined histologically with hematoxylin and eosin staining as well as with antibodies against major histocompatibility complex (MHC) classes I and II, and the retinal pigment antigen RPE-65. RESULTS: Surviving grafts were found in five out of nine eyes. Three grafts displayed the laminated appearance of a normal retina, and two had developed into rosettes. In four of the five specimens with surviving grafts, the host retinal pigment epithelium (RPE) was continuous, and MHC labeling showed no or minimal upregulation. In four specimens, no graft was found. Three of these displayed RPE defects and an increase in MHC class I- and II-labeled cells in the host choroid, subretinal space and host neuroretina. CONCLUSIONS: Full-thickness xenogeneic neuroretinal grafts can survive for at least 34 days in an adult host without immunosuppression. Immature grafts can develop the laminated appearance of a normal retina. The integrity of the host RPE seems to correlate with graft survival. We conclude that xenogeneic retinal grafts can survive and develop if the integrity of the donor tissue is intact and if damage to the RPE is minimal.     

Encapsulated cell-based intraocular delivery of ciliary neurotrophic factor in normal rabbit: dose-dependent effects on ERG and retinal histology

PURPOSE: ERG and histologic changes were investigated in normal rabbits after intravitreal implantation of encapsulated cell technology (ECT) devices releasing ciliary neurotrophic factor (CNTF). METHODS: Fifteen adult New Zealand White albino rabbits had ECT devices secreting CNTF at 22, 5, or 0 ng/d implanted in the superior temporal quadrant of the left eye. The low dose has been shown to produce substantial rescue of photoreceptors in the rcd1 canine model of retinal degeneration. Right eyes were untreated. Ganzfeld dark- and light-adapted ERGs and clinical observations were performed at 5, 15, and 25 days after implantation. Rod a-waves and rod and cone b-waves and outer nuclear layer (ONL) morphology were evaluated at 25 days. RESULTS: Clinical examination showed minimal changes in a few CNTF-treated eyes, including vitreous membranes and engorgement of iris vessels at day 25. Retinas appeared normal. CNTF did not significantly affect the rod a- or b-waves, although the b-wave amplitude tended to be larger in CNTF-treated retinas at low flash intensities. The cone b-wave amplitude was significantly reduced in high-dose eyes at some flash intensities. The ONL area in high-dose eyes was significantly greater because of increased thickness than in fellow retinas. ONL cell size was significantly increased, and staining density decreased in CNTF-treated retinas. CONCLUSIONS: CNTF, given by intravitreal ECT device at doses that protect photoreceptors in a canine model of retinal degeneration (5 ng/d), did not adversely affect either rod or cone ERG function of normal rabbit retina. The cone ERG was more sensitive to suppression being reduced, at low flash intensities, by 22 ng/d. Dose-related changes in the ONL and photoreceptor cell nuclei did not represent a toxic effect, because they were not associated with deficits in the rod ERG over a broad range of intensities.     

Impact of ozone therapy on the electrophysiological parameters of the retina in patients operated on for its rhegmatogenous detachment

The impact of ozone therapy on changes in the data of maximum combined electroretinography (ERG), macular ERG (MERG), and rhythmic ERG (RERG) at 12, 32, and 40 Hz was studied in the rehabilitative period in patients operated on for rhegmatogenous retinal detachment. The integral and local glial indices K(g) and K(c) were calculated. Studies were conducted before, within 1 week and 1 and 2 months after a course of therapy. With ozone therapy, ERG and RERG depended on the specific features of changes available in the fundus of the eye before surgery. In the early rehabilitative period after surgery (circlage with subretinal fluid removal), ozone therapy normalized the glial indices, by decreasing the supernormal activity of Muller's glial cells and drastically increasing their reduced activity. The maximum effect was revealed for the function of phororeceptors (except for the eyes showing pronounced myopic dystrophic changes in the fundus). There were increases in MERG, ERG b-wave, and low-frequency RERG by 12 Hz, i.e. improved functions of the macular region and distal neurons of the retinal rod cell system. A latent period was established for the manifestation of a positive effect of ozonwe therapy on the functional of neurons of the internal retinal nuclear layer, as evidenced by RERG changes. Ozone therapy positively affect retinal electrogenesis in cases of total retinal detachment accompanied by a pronounced reduction in the glial index Kg before surgery. The therapy was found to have a positive impact on the functional activity of the retina of pair eyes, by developing within a one-moth latent period.     

Changes of cone electroretinograms to colour flash stimuli after successful retinal detachment surgery

AIM: To examine the changes in the short wavelength (S) and mixed long (L) and middle (M) wavelength sensitive cone (L,M-cone) electroretinograms (ERGs) after successful retinal detachment surgery. METHODS: Cone ERGs elicited by different colour flashes were recorded from 19 eyes with unilateral rhegmatogenous retinal detachment treated successfully by conventional buckling surgery. Ganzfeld colour flashes on a bright white background were used to elicit S-cone and L,M-cone ERGs. The ratio (operated eye/fellow eye) of the S-cone b-wave elicited by a 450 nm stimulus and the ratio (operated eye/fellow eye) of the L,M-cone b-wave elicited by a 633 nm stimulus were evaluated preoperatively and 1, 3, and 6 months after surgery. RESULTS: Preoperatively, no significant difference was observed between the ratio of the S-cone ERG amplitudes and the ratio of the L,M-cone ERG amplitudes. Postoperatively, the ratio of the L,M-cone ERGs increased significantly over the preoperative value (p=0.001) but the ratio of the S-cone ERG did not improve. There were significant differences between the ratios of the S-cone and the L,M-cone ERGs at 1, 3, and 6 months after surgery. The postoperative recovery of the S-cone ERG was significantly greater in eyes treated within 4 weeks after the onset of the detachment than in eyes treated later than 4 weeks. CONCLUSIONS: These results indicate that the impairment of the L,M-cone system caused by retinal detachment may be reversible. However, the S-cone system may have more profound permanent damage.     

Retinal pigment epitheliopathy and neuroretinal degeneration in ascarid-infected eyes

The generalized choroidal and retinal response to a focal nonreplicating infection of the eye with ascarid larvae was examined in an animal model. Intravitreal injection of Ascaris suum larvae in guinea pigs induced a diffuse eosinophilic choroiditis, retinal pigment epitheliopathy and neuroretinal degeneration, distant from focal reactions about larvae. As the choroiditis progressed, inflammatory cells separated the choriocapillaris from Bruch's membrane, and the endothelial cells lost their fenestrations. Focal disruption of the elastic and outer collagenous layers of Bruch's membrane occurred, but inflammatory cells rarely invaded the retina. Progressive generalized degenerative and proliferative RPE changes produced a multilayered RPE with loss of cell polarity, RPE basal infoldings and apical microvilli, formation of multiple giant cystic spaces, and proliferation of subretinal fibroblasts. Early loss of photoreceptor outer segments progressed to a generalized disruption of the outer neural retina and cystoid retinal degeneration. Eosinophil mediators and alterations of the choriocapillaris may contribute to the generalized progressive retinal degeneration distant from a parasite larva in ascarid-infected eyes.     

The neural retina in retinopathy of prematurity

Retinopathy of prematurity (ROP) is a neurovascular disease that affects prematurely born infants and is known to have significant long term effects on vision. We conducted the studies described herein not only to learn more about vision but also about the pathogenesis of ROP. The coincidence of ROP onset and rapid developmental elongation of the rod photoreceptor outer segments motivated us to consider the role of the rods in this disease. We used noninvasive electroretinographic (ERG), psychophysical, and retinal imaging procedures to study the function and structure of the neurosensory retina. Rod photoreceptor and post-receptor responses are significantly altered years after the preterm days during which ROP is an active disease. The alterations include persistent rod dysfunction, and evidence of compensatory remodeling of the post-receptor retina is found in ERG responses to full-field stimuli and in psychophysical thresholds that probe small retinal regions. In the central retina, both Mild and Severe ROP delay maturation of parafoveal scotopic thresholds and are associated with attenuation of cone mediated multifocal ERG responses, significant thickening of post-receptor retinal laminae, and dysmorphic cone photoreceptors. These results have implications for vision and control of eye growth and refractive development and suggest future research directions. These results also lead to a proposal for noninvasive management using light that may add to the currently invasive therapeutic armamentarium against ROP.     

Evaluation of the rhodopsin knockout mouse as a model of pure cone function

PURPOSE: To determine a time window in the rhodopsin knockout (Rho(-/-)) mouse during which retinal function is already sufficiently developed but cone degeneration is not yet substantial, thus representing an all-cone retina. METHODS: Electroretinograms (ERGs) were obtained from 14 homozygous Rho(-/-) mice and eight C57Bl/6 control mice. The same individuals were tested every 7 days, beginning as early as postnatal day (P)14. The ERG protocols included flash and flicker stimuli, both under photopic and scotopic conditions. Retinal and choroidal morphology was observed in animals of comparable age. RESULTS: Functionally, the developmental phase lasted until postnatal week (PW)3 in both the Rho(-/-) mice and the control animals. During PW4 to 6, the Rho(-/-) mice showed a plateau in ERG parameters with normal or even supernormal cone responses and complete absence of rod contributions. At PW7, there was a marked onset of degeneration, which progressed so that no ERG signals were left at PW13, when the control eyes still had normal ERG responses. Microscopically, cone degeneration paralleled the functional changes, beginning at approximately PW6 and almost complete at PW13, whereas retinal pigment epithelium (RPE) and choroid did not show any abnormalities. CONCLUSIONS: From PW4 to 6, Rho(-/-) mice appear to have normal cone and no rod function. Despite the missing rod outer segment (OS), the structure of retina, RPE, and choroid remained unchanged. Therefore, the Rho(-/-) mice can serve during this age period as a model for pure cone function. Such a model is particularly useful to evaluate rod-cone interaction and to dissect rod- from cone-mediated signaling pathways in vivo.     

Progress in retinal sheet transplantation

The aim of retinal transplantation is to prevent blindness and to restore eyesight, i.e. to rescue photoreceptors or to replace damaged photoreceptors with the hope of re-establishing neural circuitry. A promising experimental paradigm is the sub-retinal transplantation of sheets of fetal retina, with or without its attached retinal pigment epithelium (RPE), into recipient rats with retinal degeneration. Sheets of fetal retina have already developed their primordial circuitry. Such transplants can develop lamination resembling a normal retina dependent on the presence of healthy RPE either from the host or from the graft. In several retinal degeneration models, transplants have been shown to restore visually evoked responses in an area of the superior colliculus corresponding to the placement of the transplant in the retina. The functional effect of transplants may be due to transplant/host connectivity and/or rescue of host photoreceptors. In summary, sheets of fetal retina can morphologically repair an area of a degenerated retina, and there is evidence to suggest that transplants form synaptic connections with the host and restore visual responses in rats with retinal degeneration.     

Dissecting the dark-adapted electroretinogram

Although gross recordings of the ganzfeld flash-evoked electroretinogram (ERG) can potentially provide information about the activity of many, if not all, retinal cell types, it is necessary to dissect the ERG into its components to realize this potential fully. Here we describe various procedures that have been used in intact mammalian eyes to identify and characterize the contributions to the dark-adapted ERG of different cells in the retinal rod pathway. These include (1) examination of the very early part of the response to a flash (believed to reflect directly the photocurrent of rods), (2) application of high-energy probe flashes to provide information about the underlying rod photoreceptor response even when this component is obscured by the responses of other cells, (3) pharmacological suppression of responses of amacrine and ganglion cells to identify the contribution of these cells and to reveal the weaker responses of bipolar cells, (4) use of pharmacological agents that block transmission of signals from rods to more proximal neurons to separate responses of rods from those of later neurons, (5) examination of the ERG changes produced by ganglion-cell degeneration or pharmacological block of nerve-spike generation to identify the contribution of spiking neurons, (6) modeling measured amplitude-energy functions and timecourse of flash responses and (7) using steady backgrounds to obtain differential reductions in sensitivity of different cell types. While some of these procedures can be applied to humans, the results described here have all been obtained in studies of the ERG of anaesthetized cats, or macaque monkeys whose retinas are very similar to those of humans. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dark adaptation in locally detached retina

Nonrhegmatogenous retinal detachments were formed in the eyes of Dutch rabbits by subretinal injection of Hanks' balanced salt solution. The electroretinogram (ERG) was recorded locally from the acutely detached retina, and simultaneously from the surrounding attached retina (vitreal ERG [VERG]), before and after exposure to diffuse intense irradiation. Light adaptation elevated b-wave threshold for both the local ERG (LERG) and VERG by about 3 log units; thresholds for both responses recovered fully within 60-90 min after the irradiation. The normal time course of dark adaptation of the LERG suggests the occurrence of substantial rhodopsin regeneration in the rod photoreceptors of nonrhegmatogenously detached retina. These results differ from reports that visual pigment regeneration is slow in central serous chorioretinopathy, possibly because our detachments were studied within hours of formation, whereas some photoreceptor degeneration may be present in older clinical detachments.     

The quantity of rhodopsin in human eyes

The content of rhodopsin in the eyes of 15 donors (30 eyes) was determined. Both retinal and pigment epithelial fractions were collected from each globe, extracted using 1% CTAB, and the rhodopsin difference spectrum of each fraction was obtained separately. The total amount of rhodopsin, obtained by summing the amounts recovered from the retinal and PE fractions, ranged from 2.00 to 11.94 (median: 6.40) nmoles/eye. Previously reported mean values of about 3.5 to 4.0 nmoles per retina have been obtained using a variety of methods. The present higher values, perhaps largely dependent on procedural details described herein, appear plausible given the known concentrations of rhodopsin in rod outer segments, rod outer segment volumes, and number of rods in the human retina.     

Retinal degenerations in the dog: IV. Early retinal degeneration (erd) in Norwegian elkhounds

A new early onset hereditary retinal degeneration is characterized in Norwegian elkhound dogs. This disease, termed early retinal degeneration (erd), was studied in 10 affected dogs, from 30 days- to 7 years old, clinically, by electroretinography, and by light- and electron-microscopic morphology. Control studies were performed on 49 non-affected dogs. Affected dogs are initially nightblind, and become totally blind between 12- and 18 months of age. The postnatal development of their rod and cone photoreceptors is abnormal both structurally and functionally. Morphologically, rod and cone inner- and outer-segment growth occurs but appears uncoordinated. Adjacent rods become very disparate in the size and proportions of their inner- and outer segments. Prominent villiform processes extend from the inner segments of rods and, to a lesser extent, cones. Synaptic terminals of rods and cones fail to develop properly. The b-wave of the electroretinogram fails to develop and the electroretinogram (ERG) remains a-wave-dominated. Subsequent to these abnormalities of development, the rods and cones degenerate, rapidly at first and later more gradually. In normal dogs, development of the ERG a- and b-waves is shown to follow, respectively, morphologic development of the photoreceptor outer segments and synaptic terminals. Similarly the abnormal development and subsequent degeneration of photoreceptor outer segments and synaptic terminals in affected dogs, correspond in time course to development and degeneration of the ERG a- and b-waves.     

MHC expression in fragment and full-thickness allogeneic embryonic retinal transplants

Background: The study was carried out to evaluate the expression of major histocompatibility complex (MHC) molecules in retinal transplants with different tissue integrity. Methods: Twelve adult rabbits received an allogeneic subretinal neuroretinal transplant, in the form of either fragmented embryonic cells or a complete full-thickness embryonic retina. A controlled transvitreal approach was used for both transplantation types. The grafts were examined histologically after 31 or 49 days with hematoxylin and eosin staining and immunohistochemical analysis of MHC class I and class II expression. Results: All five fragment transplants developed into rosettes. Two of them displayed MHC class I-labeled cells, and four MHC class II-labeled cells. The cells were concentrated on the scleral side of the graft, and there was also a marked increase of labeled cells in the choroid. MHC labeling was often associated with defects in the retinal pigment epithelium. Six of the seven full-thickness grafts displayed a laminated morphology with well-developed retinal layers. The seventh consisted of rosettes. None of these grafts displayed MHC class I- or class II-labeled cells. Conclusions: The findings suggest that host immune response against fragmented and intact neuroretinal grafts is different, indicating tissue integrity as one factor affecting graft-host immune interactions. The absence of immune response in full-thickness grafts is encouraging and important in the struggle to find therapies for retinal degenerative disease. Received: 17 November 1999 Revised: 6 January 2000 Accepted: 18 January 2000     

The effect of a retinal lesion on the distribution of B wave potentials on the sclera

In the experiments presented here we have used the isolated perfused eye technique to investigate the distribution of ERG potentials on the sclera over local retinal lesions induced by xenon arc photocoagulation. Three lesioned dog eyes were examined. In two cases the lesion was allowed 28 days to stabilize, whilst in the third the experiment was performed two hours after treatment. Immediately after enucleation, the eyes were placed in a perfusion system capable of maintaining retinal function for many hours. The scleral ERG profile was then measured on the scleral surface over both treated and untreated areas of retina in the same eye. It was found that the ERG amplitudes were consistently lower over the treated areas of retina when compared to the untreated side of the same eye. More surprising was the discovery that the ERG distribution on the untreated side was significantly different to that observed in control eyes. In particular it was found that the limbal areas of sclera on the untreated side now had significant ERG amplitudes present, whereas the control eyes had close to zero amplitude in this region. It is concluded that the induced retinal lesions caused a widespread redistribution of ERG potentials around the entire surface of the globe. The potential applications of these results in the assessment of local retinal function is discussed. A schematic model is presented for the distribution of ERG potentials on the surface of control and lesioned eyes.     

Insulin-like growth factor-I and its receptor in neovascular age-related macular degeneration

PURPOSE: The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). METHODS: Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. RESULTS: In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. CONCLUSIONS: The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.     

氩激光治疗视网膜周边裂孔和变性

目的 探讨氩激光光凝视网膜周边裂孔和变性的疗效。方法 对１５３例（１６５眼）视网膜周边裂孔和变性进行氩激光治疗。结果 随访２月至１年半者５０例（５３眼）中治愈４７眼占８８．７％,好转５眼占９．４％,无效１眼占１．９％视网膜脱离再次手术。５２眼无视网膜脱离,效果满意。结论 氩激光光凝是治疗视网膜周边裂孔和变性的安全有效方法。