Riboflavin-UV--a crosslinking for fixation of biosynthetic corneal collagen implants.

PURPOSE: To evaluate riboflavin-UV-A crosslinking as an alternative suture-free fixation method for biosynthetic corneal collagen implants. METHODS: A range of cell-free corneal implants consisting of recombinant human collagen type III were examined. In vitro, the implants were crosslinked with different riboflavin solutions and irradiations. Ex vivo, the biosynthetic corneal implants were placed on the anterior cornea of porcine and rabbit eyes after performing deep anterior lamellar keratoplasty with a trephine, femtosecond laser, or excimer laser. UV-A crosslinking was performed with isotonic or hypotonic riboflavin at either standard or rapid procedure. The corneas were excised, fixed in PFA 4%, and embedded in paraffin. Crosslinking effects on the implants and the adhesion between implant and corneal bed were evaluated by slit-lamp biomicroscopy, optical coherence tomography (OCT) images, and histologically. RESULTS: After the crosslinking procedure, the implants showed different degrees of thinning. The accuracy of cutting the corneal bed was highest with the excimer laser. Good adhesion of the implant in the corneal bed could be demonstrated in OCT images. This was more accurate in porcine eyes than in rabbit eyes. Histologically, crosslinks between implant and corneal stroma were demonstrated. There was no difference between standard and rapid crosslinking procedures. CONCLUSIONS: Riboflavin-UV-A crosslinking as a fixation method for biosynthetic corneal collagen implants was demonstrated to be promising. It can reduce suture-related complications such as haze formation and surface irregularity. Stability of the implants, especially shrinkage after riboflavin-UV-A crosslinking, needs to be further evaluated. Biostability, integration, and long-term outcome are further evaluated in in vivo animal experiments.

Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.

The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes for the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500µm thick RHCIII-MPC constructs comprising over 85% water are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, unlike the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30µm wide fibronectin stripes enhanced cell attachment and showed the highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas.

Precision, morphology, and histology of corneal flap cuts using a 200-kHz femtosecond laser.

PURPOSE: Laser-assisted in situ keratomileusis (LASIK) requires precise corneal flap cutting. Especially the creation of thin flaps has recently gained importance for thin-flap LASIK. Currently, there is a trend towards faster femtosecond lasers that can produce flaps in a short period of time. We analyzed flaps created with a 200-kHz femtosecond laser concerning their cut precision, morphology, and histology. METHODS: Femtosecond laser flap cutting was performed on 36 porcine cadaver eyes using the prototype 200-kHz femtosecond laser UltraFlap (WaveLight GmbH, Erlangen, Germany). The eyes were assigned to 3 thickness groups, with a cut depth of 100 µm, 130 µm, or 180 µm, respectively. Additionally, flap diameters were varied, ranging from 8.0 mm to 9.5 mm. Flap thicknesses were determined with a micrometer gauge. Flap diameters were measured with a sliding caliper. Furthermore, flaps were created for histologic examination. RESULTS: There were no complications during flap creation. The mean flap thickness and standard deviation was (in micrometers) 96.33 ± 7.45 (intended thickness: 100), 134.67 ± 4.96 (intended thickness: 130), and 174.59 ± 9.35 (intended thickness: 180), respectively. The flap diameter revealed a mean (in mm) of 8.03 ± 0.15 (intended diameter: 8.0), 8.56 ± 0.10 (intended diameter: 8.5), 9.09 ± 0.10 (intended diameter: 9.0), and 9.54 ± 0.15 (intended diameter: 9.5), respectively. Histologic examination showed very little to almost no changes in the structure of the corneal stroma. CONCLUSIONS: Flap creation could be performed easily without any complications. The morphology and accuracy of the cuts were very reliable and precise. Histology showed a smooth cut.

Repeated transchoroidal implantation and explantation of compound subretinal prostheses: an exploratory study in rabbits.

PURPOSE: For human trials with retinal prostheses it is mandatory to develop procedures to safely explant and possibly reimplant the devices. This prompted us to investigate in a small exploratory study the safety of repeated transchoroidal implantation and explantation procedures of complex subretinal devices in laboratory animals. METHODS: Repeated transchoroidal surgery was performed in four rabbits. The rabbits were examined by clinical examination and funduscopy. Function was assessed by electroretinography and cortical recordings following light and subretinal electrical stimulation. Sections of the retina and of the implantation channel were examined by light microscopy. RESULTS: Using the same access route, repeated transchoroidal subretinal implantation surgery was successfully performed in all cases. Fixation of implants was stable for up to 13 months; retinas remained attached at all examination dates. Electroretinograms and visual evoked cortical potential proved retinal and visual pathway integrity. Subretinal electrical stimulation elicited retinal and cortical responses. While retinal morphology at earlier stages was found to be essentially unaltered, atrophic disorganization in the region of the subretinal channel was observed after 10 months and after subretinal electrical stimulation. CONCLUSIONS: Repeated transchoroidal surgery can be safely performed for implantation, explantation, and reimplantation of subretinal devices in rabbits. With modifications, we believe the technique can be applied in human surgery.

Effect of 3 excimer laser ablation frequencies (200 Hz, 500 Hz, 1000 Hz) on the cornea using a 1000 Hz scanning-spot excimer laser.

PURPOSE: To evaluate the possible side effects on human and porcine cadaver eyes induced by excimer laser ablation with 3 ablation frequencies. SETTING: Augenklinik, Klinikum rechts der Isar, Technische Universitaet Muenchen, Munich, Germany. METHODS: Central epithelial abrasion was performed on porcine and human donor eyes. Next, the porcine eyes were photoablated (-9.00 diopters) at 1 of 3 frequencies (200 Hz, 500 Hz, or 1000 Hz) using a prototype 1000 Hz excimer laser. The human eyes were ablated at 1000 Hz. The surface of the stroma as well as the structure and ultrastructure of the corneal cells and stroma were examined using light microscopy, transmission electron microscopy, and scanning electron microscopy (SEM). Special attention was given to the detection of potential thermal damage and the evaluation of corneal smoothness. RESULTS: Histopathologic examination showed very low to almost no differences between the 3 repetition rates. In all cases, SEM showed an equally smooth surface. CONCLUSIONS: The structural and ultrastructural evaluation of corneas treated with ablation frequencies of 200 Hz, 500 Hz, and 1000 Hz showed no specific side effects associated with the high repetition rates. The ablation quality was comparable in the 3 frequency groups. However, the treatment time was significantly less with a high repetition rate, indicating the clinical potential in refractive surgery of excimer lasers with a repetition rate of 1000 Hz. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes.

Epi-laser in situ keratomileusis: comparative evaluation of epithelial separation with 3 microkeratomes.

PURPOSE: To evaluate the cleavage plane, corneal cytoarchitecture, and cell vitality of separated corneal epithelial sheets created with 3 commonly used microkeratomes. SETTING: Laboratories of the Regensburg University Medical Center, Regensburg, Germany. METHODS: Mechanical separation of the epithelial layer in 10 porcine eyes and 2 human eyes was performed with 3 different microkeratomes (Amadeus II, Zyoptix XP, Epivision). Five of 10 porcine corneas and the 2 human corneas treated with each microkeratome were processed for histology, electron microscopy, and immunohistochemistry. In 5 of 10 porcine corneas, the corneal epithelial sheets were separated from the globe and cell vitality was assessed with the trypan blue dye vitality test. RESULTS: A reproducible epithelial separation with a smooth surface was achieved in all eyes. The cleavage plane was located between the lamina lucida and the lamina densa. Damage to epithelial cells was mainly limited to the cut margins. CONCLUSIONS: Mechanical separation of the epithelial sheet in epithelial laser in situ keratomileusis (epi-LASIK) was safe and reproducible with all evaluated microkeratomes. Immunohistochemistry and electron microscopy showed the cleavage plane in epi-LASIK was between the basal epithelium and the basement membrane at the level of the lamina lucida.

Clinical evaluation of experimentally induced choroidal neovascularizations in pigmented rabbits by subretinal injection of lipid hydroperoxide and consecutive preliminary photodynamic treatment with Tookad.

PURPOSE: Up to date several approaches have been undertaken to achieve an 'easy-to-handle' animal model of choroidal neovascularizations (CNVs) in rabbits; however, so far in none of the studies could healthy retinal tissue be maintained, which is mandatory to further investigate the effects of photodynamic therapy (PDT) or anti-vascular-endothelial-growth-factor treatments. It was our aim to reevaluate and verify the method of inducing experimental CNVs in rabbits using subretinally injected linoleic acid hydroperoxide (LHP) as proposed by Tamai et al. and to use it for experimental PDT. MATERIAL AND METHODS: In 33 eyes of Chinchilla breed rabbits LHP of two different concentrations (25 and 100 microg/50 microl) was injected into the subretinal space via a transvitreal approach under guidance of an operation microscope. Ophthalmoscopic and angiographic examinations were performed on days 3, 7, 14 and 28 after surgery. Preliminary PDT with different experimental parameter sets was performed in 3 eyes using the new photosensitizer Tookad. RESULTS: Using LHP in the higher concentration, an angiographically determined CNV induction was observed in 27% of all injection sites (n = 34) on days 14 and 28 revealing early well-demarcated and progressive leakage. No CNV was detected at the lower LHP concentration (60 injection sites). Subretinal CNV was verified histologically revealing vessel formation above the retinal pigment epithelium level. Herein, a significant damage to the outer retinal layers was always observed; however, the general structure of the choriocapillary layer was maintained. Tookad PDT was clinically able to completely stop leakage in 1 case and reduce leakage in 2 cases. Histologically the choriocapillary layer was occluded. CONCLUSION: Subretinal injection of LHP induces angiographically well-demarcated classic CNVs in rabbits; however, the CNV rate was low, and histology revealed severe damage of the outer retinal layers but not of the choriocapillary layer, which is important for studying PDT interactions. Preliminary experimental PDT could clinically stop or reduce leakage from angiographic CNV. Due to the small CNV rate and the significant collateral retinal tissue damage, this model seems to be only of partial suitability for investigating new treatment modalities in CNV.

Light-induced decomposition of indocyanine green.

PURPOSE: To investigate the light-induced decomposition of indocyanine green (ICG) and to test the cytotoxicity of light-induced ICG decomposition products. METHODS: ICG in solution was irradiated with laser light, solar light, or surgical endolight. The light-induced decomposition of ICG was analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. Porcine retinal pigment epithelial (RPE) cells were incubated with the light-induced decomposition products of ICG, and cell viability was measured by trypan blue exclusion assay. RESULTS: Independent of the light source used, singlet oxygen (photodynamic type 2 reaction) is generated by ICG leading to dioxetanes by [2+2]-cycloaddition of singlet oxygen. These dioxetanes thermally decompose into several carbonyl compounds. The decomposition products were identified by mass spectrometry. The decomposition of ICG was inhibited by adding sodium azide, a quencher of singlet oxygen. Incubation with ICG decomposition products significantly reduced the viability of RPE cells in contrast to control cells. CONCLUSIONS: ICG is decomposed by light within a self-sensitized photo oxidation. The decomposition products reduce the viability of RPE cells in vitro. The toxic effects of decomposed ICG should be further investigated under in vivo conditions.

Intrinsic tissue fluorescence in an organotypic perfusion culture of the porcine ocular fundus exposed to blue light and free radicals.

BACKGROUND: A wide variety of pathological pathways may result in age-related macular degeneration. Because of its complexity, there is no comprehensive model of the disease yet. One key feature is the accumulation of the autofluorescent pigment lipofuscin in the retinal pigment epithelium (RPE). Thus, we developed an organotypic perfusion culture model of the porcine ocular fundus, generating lipofuscin under exposure to blue light and hydrogen peroxide. METHODS: Porcine fundi (choroid, Bruch's membrane, RPE, and retina) were explanted in toto, transferred into a perfusion culture chamber, perfused with cell culture medium and kept at 37 degrees C. Free radical stress was induced by supplementation of H(2)O(2), and/or the specimens were exposed to blue light, or kept untreated as controls. After a culture period of 7 days, the specimens were subject to microscopic inspection, histology, fluorescence microscopy, and measurement of fluorescence spectra as well as fluorescence decay times. RESULTS: Histology showed atrophic ganglion cells and rod outer segments. All other tissue structures were morphologically intact. Compared to the controls, RPE and retina exposed to light showed increased fluorescence, which was shifted towards shorter wavelengths. The fluorescence spectra and decays resembled that of lipofuscin granules isolated from human donor eyes. HPLC analysis revealed the abundance of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2E), its precursor products, as well as two new, green-emitting fluorophores. CONCLUSIONS: Porcine ocular fundi were successfully preserved in an organotypic perfusion culture for 7 days, and exhibited remarkable autofluorescence after light and free radical exposure, making the model suitable for investigations of lipofuscinogenesis.

Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model.

The purpose of this study was to characterise an ex-vivo adult porcine retina-retinal pigment epithelium (RPE) perfusion organ culture model. Fresh porcine full-thickness retina-RPE-choroid tissue samples were clamped into tissue carriers and mounted in two-compartment containers. The retinal and choroidal sides were continuously perfused with culture medium. pO(2), [Na(+)], [K(+)], [Cl(-)], [glucose], [lactate], and pH were measured in the medium. Tissue samples were examined after 24h, 4, 7, and 10 days in culture. The morphology of the retina and the RPE was examined by light and electron microscopy (LM, EM). The retinal cellular integrity was further examined by immunohistochemistry (Ki 67, GFAP, rhodopsin, synaptophysin, syntaxin, NF 200, TUNEL-test). Fresh porcine full-thickness retina-RPE-choroid tissue samples and tissue samples in static organ culture served as controls. LM, EM, and immunohistochemistry showed intact retinal and RPE cytoarchitecture kept in perfusion culture. Photoreceptor outer segments showed first signs of degeneration after 24h, significant signs of apoptosis and necrosis appeared in the retina after 4 days in perfusion culture. Control tissue samples kept in static culture showed disintegration of the retinal cytoarchitecture after 4 days in culture. The data show that adult porcine retina-RPE tissue can be maintained morphologically intact in perfusion organ culture for at least 10 days. Although first signs of degeneration set in after 24h the structural preservation of the tissue in perfusion organ culture is superior to that in static culture. The perfusion culture model of the retina refines organotypic in vitro test systems and may help to reduce the number of necessary animal experiments in retina and RPE research. It offers new perspectives for the safety testing of substances designed for intraocular application.

Investigation of selective retina treatment (SRT) by means of 8 ns laser pulses in a rabbit model.

BACKGROUND: It has been shown that selective retina treatment (SRT) using a train of 1.7 microseconds laser pulses allows selective damage of the retinal pigment epithelium (RPE) while sparing the adjacent photoreceptors and thus avoiding laser scotoma. It was the purpose of this work to investigate SRT laser effects with Q-switched pulses of only 8 nanoseconds in duration by evaluating the angiographic and ophthalmoscopic damage thresholds and the damage range by histology in a rabbit model. MATERIALS AND METHODS: A flash lamp pumped frequency doubled (532 nm) Nd:YAG laser with 8 nanoseconds pulse duration was used. In total 210 laser lesions, each calculated to be 102 microm in diameter on retina, were applied through a slit lamp onto the fundus of six eyes of Chinchilla Bastard rabbits. The rabbits were irradiated with increasing energies with single pulses and a train of 10 laser pulses at 10 Hz. After treatment fundus photography and angiography were performed to determine the damage thresholds (ED(50)-probability of RPE cell damage and neurosensory retinal damage) as well as the safety range between both thresholds (ratio of angiographic ED(86) vs. ophthalmoscopic ED(14)). Selected histology was taken for single and repetitive pulse lesions after treatment. RESULTS: Angiographic and ophthalmoscopic ED(50)-thresholds decreased with increasing number of pulses. For single pulse application ophthalmoscopic and angiographic ED(50) were determined to 365 and 144 mJ/cm(2), respectively. Regarding 10 pulses 266 and 72 mJ/cm(2) were found. No retinal hemorrhages or disruptions were observed for both sets of parameters. The therapeutic window between angiographic and ophthalmoscopic threshold revealed a factor of 3.1 for single pulses and 2.3 for repetitive pulse irradiation. The safety range respectively had a factor of 0.8 (single pulses) and 1.7 (10 pulses). Histologic examination of laser lesions with single and repetitive pulses at radiant exposures within the therapeutic window-292 and 213 mJ/cm(2) respectively-revealed damaged RPE, intact Bruch's membrane and choriocapillaries. Photoreceptors were partly spared but also damaged to various extents. CONCLUSIONS: Short laser pulses of 8 nanoseconds pulse duration can damage the RPE without retinal hemorrhage or disruption. Selective damage of the RPE without affecting the photoreceptors can only rarely be achieved due to the small safety range. Thus, so far microsecond laser pulses for SRT seems favorable compared to nanosecond pulses in order to prevent unintentional photoreceptor damage.

Ascorbic acid modulates proliferation and extracellular matrix accumulation of hyalocytes.

Ascorbic acid is known to influence proliferation and functional properties of several cell types and is therefore widely used in tissue engineering. In this study, the effect of ascorbic acid on the proliferation and functional properties of hyalocytes was evaluated. Hyalocytes were cultured with different amounts of ascorbic acid in classical two-dimensional (2-D) cultures and a three-dimensional (3-D) pellet culture system. Ascorbic acid enhanced hyalocyte proliferation dose-dependently at concentrations between 0.1 and 3 microg/mL; proliferation was constant over a wide concentration range up to 150 microg/mL, concentrations of 500 microg/mL showed toxic effects. In 2-D hyalocyte culture, the accumulation of glycosaminoglycans (GAG) and collagens increased in response to ascorbic acid supplementation of 10 or 200 microg/mL. Normalized to the cell number, GAG production was not influenced, whereas collagen production increased. These results could be verified in a pellet-like 3-D culture system. Ascorbic acid also influenced hyalocytes on the mRNA level; the expression of COL11A1 was clearly enhanced by ascorbic acid. To conclude, ascorbic acid modulates proliferation and collagen accumulation of hyalocytes; it also influences mRNA expression of the cells. Taken together with the fact that ascorbic acid is present in high concentrations in the vitreous body, this vitamin seems to be an important factor for in vitro hyalocyte culture.

Histological evidence for revascularisation of an autologous retinal pigment epithelium--choroid graft in the pig.

BACKGROUND: Translocation of a free autologous graft consisting of retinal pigment epithelium (RPE), Bruch's membrane, choriocapillaris and choroid in patients with exudative age-related macular degeneration is currently being evaluated in clinical practice. Angiographic studies in these patients suggest that their grafts become revascularised. AIM: To investigate the histological evidence of revascularisation of the graft in a porcine model. METHODS: In 11 pigs (11 eyes), an RPE-choroid graft was translocated from the mid-periphery to an intact or an intentionally damaged RPE and Bruch's membrane at the recipient site. The eyes were enucleated 1 week or 3 months after surgery. Tissue sections were evaluated using immunohistochemistry. RESULTS: Bridging vessels between recipient layer and graft were identified from 1 week to 3 months after surgery. This reconnection occurred regardless of whether the Bruch's membrane of the recipient site was left intact or intentionally damaged at the time of transplantation. The vasculature of the graft appeared open and perfused. Vessels with transcapillary pillars and conglomerates of small new vessels were present in the graft. CONCLUSIONS: This study showed histological evidence for revascularisation by angiogenesis of a free autologous RPE-choroid graft.

Safety testing of indocyanine green in an ex vivo porcine retina model.

PURPOSE: To assess retinal toxicity of indocyanine green (ICG) in a porcine ex vivo perfusion organ culture model, and to measure intraretinal penetration of ICG applied to the retinal surface. METHODS: The retinal surface of fresh porcine retinal tissue was exposed to ICG 0.1% and 1% dissolved in glucose 5% for 1 and 30 minutes with and without concomitant illumination. Specimens were then kept in perfusion organ culture for 24 hours before examination by light microscopy and the TUNEL technique. Tissue samples treated with DNAse served as positive controls, and samples exposed to saline served as negative controls. Fluorescence microscopy was used to localize ICG at 1 minute, 60 minutes, 2 hours, and 3 hours after a 1-minute exposure of the retinal surface to ICG 1%. RESULTS: No increase in TUNEL-positive cells was observed after exposure to ICG 0.1% for 1 minute. Moderate apoptosis was found after 1-minute exposure to ICG 1% and 30-minute exposure to ICG 0.1%, and severe apoptosis was found after 30-minute exposure to ICG 1%. Concomitant application of light did not influence the degree of apoptosis. No signs of cell necrosis were found. After 1-minute exposure of the retinal surface, ICG 1% gradually penetrated the entire retina. CONCLUSIONS: ICG induced apoptosis but not necrosis in all nuclear retinal layers in a dose-dependent manner. Brief exposure to ICG 0.1% for 1 minute and illumination for 3 minutes simulated the intraoperative use of ICG. No retinal apoptosis or necrosis was observed. ICG briefly applied to the retinal surface gradually penetrated the entire retina.

Perfluorohexylethan (O62) as ocular endotamponade in complex vitreoretinal surgery.

PURPOSE: To investigate the safety and performance of perfluorohexylethan (O62), a partially fluorinated alkane, as an intraoperative tool and heavy ocular endotamponade in complex vitreoretinal surgery. PATIENTS AND METHODS: In a prospective clinical study, O62 was used as a postoperative ocular endotamponade in 11 eyes of 11 patients after pars plana vitrectomy for the following inferior pathologic conditions, proliferative vitreoretinopathy (n = 8), rhegmatogenous retinal redetachment with inferior tears (n = 1), and inferior giant tear (n = 2). The median duration of the O62 tamponade was 43 days (range, 17-55 days), and the median follow-up period after removal of the tamponade was 16 months. RESULTS: The initial postoperative retinal attachment rate was 100%. In 7 of 11 eyes, the retina remained attached during the O62 tamponade and after its removal. During the tamponade period, no epiretinal membrane formation or macular pucker was observed in these seven eyes. Recurrent retinal detachments with proliferative vitreoretinopathy developed in 4 of 11 eyes under the tamponade. The median follow-up after removal of O62 was 16 months. A secondary cataract developed in all five phakic eyes. Severe emulsification was noted in all patients starting in the second week after surgery causing a decrease of visual acuity and a significantly reduced funduscopic view. In the early postoperative period, a marked inflammatory reaction in the anterior segment was seen in all patients. Slightly whitish precipitates were noted in 2 of 11 eyes. A transient increase in intraocular pressure up to 35 mmHg was observed in 2 of 11 eyes. CONCLUSION: O62 showed good tamponade properties for the inferior retina over 6 weeks. However, its use as a postoperative retinal tamponade is limited by its severe emulsification propensity and unclear inflammatory potential.

Subretinal electrical stimulation of the rabbit retina with acutely implanted electrode arrays.

BACKGROUND: Subretinal implants intend to replace photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli from the subretinal space. This study intended to prove the feasibility of a newly developed transchoroidal surgery and, furthermore, of a subretinal electrode array, which closely resembles envisioned human implants to electrically stimulate the visual system in rabbits. METHODS: Five rabbits (ten eyes) were implanted with a 4x2-electrode array via a transchoroidal access to the subretinal space. The electrodes were connected to an arbitrary stimulus generator to apply voltage pulses. Retinae were accessed by light microscopy after stimulation with various intensities. RESULTS: The stimulating foil could be introduced into the subretinal space in all eyes. In seven of ten eyes electrically evoked cortical potentials following subretinal electrical stimulation could be elicited. Threshold voltages ranged from less than 0.1 to 2.38 V with a corresponding threshold charge of approximately 1.0 nC per electrode or 10 micro C/cm(2). Histology revealed localized retinal damage over some of the electrodes succeeding stimulation strengths of 2 V and consistent damage over all electrodes succeeding voltages of 3 V. CONCLUSIONS: The study demonstrates the feasibility of the transchoroidal surgical access to place subretinal implants in rabbit eyes and provides proof of successful cortical activation following subretinal electrical stimulation by an electrode array envisioned for human implantations.

Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation.

Our objective was to determine whether key properties of extracellular matrix (ECM) macromolecules can be replicated within tissue-engineered biosynthetic matrices to influence cellular properties and behavior. To achieve this, hydrated collagen and N-isopropylacrylamide copolymer-based ECMs were fabricated and tested on a corneal model. The structural and immunological simplicity of the cornea and importance of its extensive innervation for optimal functioning makes it an ideal test model. In addition, corneal failure is a clinically significant problem. Matrices were therefore designed to have the optical clarity and the proper dimensions, curvature, and biomechanical properties for use as corneal tissue replacements in transplantation. In vitro studies demonstrated that grafting of the laminin adhesion pentapeptide motif, YIGSR, to the hydrogels promoted epithelial stratification and neurite in-growth. Implants into pigs' corneas demonstrated successful in vivo regeneration of host corneal epithelium, stroma, and nerves. In particular, functional nerves were observed to rapidly regenerate in implants. By comparison, nerve regeneration in allograft controls was too slow to be observed during the experimental period, consistent with the behavior of human cornea transplants. Other corneal substitutes have been produced and tested, but here we report an implantable matrix that performs as a physiologically functional tissue substitute and not simply as a prosthetic device. These biosynthetic ECM replacements should have applicability to many areas of tissue engineering and regenerative medicine, especially where nerve function is required.

Clinical findings on the use of long-term heavy tamponades (semifluorinated alkanes and their oligomers) in complicated retinal detachment surgery.

BACKGROUND: Heavy tamponades for pathologies in the lower part of the retina are a new development, and different tamponades have recently come into clinical use: semifluorinated alkanes (F(6)H(6), F(6)H(8)) and their oligomers (OL62HV). METHOD: Nine patients had been operated on using F(6)H(8) (n=5) and by OL62HV (n=4). In all cases the reasons for using the tamponades were complicated retinal detachments in the lower part. In three cases the use was primary and in six cases tamponades were used after reoperations. In all cases the endotamponade was removed within 6 weeks. Fluorescein angiography (FLA) was performed in the F(6)H(8) group. RESULTS: In the F(6)H(8)group dispersion developed in two of the three aphacic patients. In two out of five cases soft epiretinal membranes and cellular material could be found between the substance and the lower periphery. In two membranes examined by light microscopy, cystic cells and amorphous material could be found. In one case (PDRP, aphacic) cyclophotocoagulation had to be performed because of persistent elevated IOP. FLA was unremarkable. In the OL62HV group, severe recurrent PVR reaction occurred in the lower periphery (2/4) and unusual precipitates were observed (4/4). In one case, after a normal postoperative period (VA 0.05 after 5 days) an extensive cellular reaction on the complete surface of the tamponade occurred. After 5 weeks VA was no light perception. During removal of the oligomer unusual adherent cellular components were found on the surface of the retina. The retina appeared necrotic, showed constricted retinal vessels and there was optic atrophy. Histologically, fluffy epiretinal material and a lens capsule obtained from one eye filled with OL62HV resembled the appearance with F(6)H(8). CONCLUSION: Heavy endotamponades on the basis of semifluorinated alkanes can lead to an unusual biological reaction and need further investigation before clinical use.

RPE in perfusion tissue culture and its response to laser application. Preliminary report.

PURPOSE: To study the effects of conventional laser application on the retinal pigment epithelium (RPE) in a perfusion tissue culture model of porcine retinal pigment epithelium without overlying neurosensory retina. METHODS: RPE with underlying choroid was prepared from enucleated porcine eyes and fixed in a holding ring (Minusheet). Specimens were then placed in two-compartment tissue culture containers (MinuCell & Minutissue, Bad Abbach, Germany) and were cultured during continuous perfusion with culture medium at both sides of the entire specimen, the upper RPE and the lower choroid (12 specimens out of 6 eyes). Cultures were kept for 1, 3, 7 and 14 days and were examined histologically. Laser treatment was performed on each tissue ring by application of 3 x 3 laser burns one day after culture began (argon ion laser, wavelength: 514 nm, pulse duration: 100 ms; spot size: 200 microm) using different energy levels (400-1,000 mW); (16 specimens out of 8 eyes). RESULTS: During laser treatment a marked lightening of the RPE with centrifugal spreading was observed. Using higher levels of energy, a contraction of the RPE towards the center of the laser spot was noticed. One day after laser photocoagulation histology revealed destruction of RPE; within 3-7 days of culture, migration and proliferation of neighboring cells was observed in several lesions. After 7 days the initial defect of the irradiated area was covered with dome shaped RPE cells and after 14 days multilayered RPE cells were showing ongoing proliferation. However, there were also cases without proliferation after laser treatment. The non-treated, continuously perfused RPE showed regular appearance in histological sections: during the first 7 days of culture, light microscopy revealed a normal matrix with a well-differentiated RPE monolayer. Subsequently proliferation even without treatment was observed and after 14 days the RPE became multilayered. CONCLUSION: It was possible to study the early healing response to the effect of laser treatment using the permanently perfused tissue culture system. A marked proliferation and repair of the laser defect could be observed in several but not all lesions. After 14 days even without laser treatment a proliferative multilayered RPE was present. Although this limits the use of the system for longer than 7 days, it seems to be useful for investigation of RPE-related disorders.

Outflow facilities through Descemet's membrane in rabbits.

BACKGROUND: The outflow pathway in viscocanalostomy, a new procedure in glaucoma surgery, is unclear; however, outflow through Descemet's membrane has been postulated. This study evaluates outflow rates through Descemet's membrane at different IOP levels in rabbits. METHODS: 51 Descemet's membranes without endothelium from enucleated rabbit eyes were installed in a double-ring system, the Minuth sheet. Different intraocular pressure levels (20, 25, 30, 40, 50 mmHg) were applied to one side of the system. The system was filled with balanced salt solution. The total amount of fluid percolating through Descemet's membrane was measured after 12 h. Based on this, flow rates were calculated. The area of Descemet's membrane was 6.9 mm2. RESULTS: At the pressure of 20 mmHg the flow rate was less than 0.003 microl/min. At pressures above 30 mmHg flow rates ranged from 0.04 microl/min to 0.15 microl/min with a mean of 0.09 microl/min. To achieve pressure control at high pressures, an area of at least 150 mm2 of Descemet's membrane would be needed. CONCLUSION: Descemet's membrane provides good outflow resistance in rabbit eyes. Based on our results for pressure control by outflow through Descemet's membrane only, at least the whole corneal area is needed. If the same is true in humans, additional outflow sources are necessary in cases of viscocanalostomy.