Karl Engelsberg

Development of the Embryonic Porcine Neuroretina in vitro.

Purpose: The objective of this study was to investigate the survival and morphology of embryonic porcine full-thickness neuroretina in culture. Methods: Porcine fetuses were taken out by cesarian section, and the eyes were enucleated. Neuroretinas were explanted on culture plate inserts and were kept for 0–42 days in vitro under standard culture conditions. Green nucleic acid (Sytox) was used for measuring the extent of cell death, and 4,6-diaminidine-2-phenylindoldihydrochloride was used as a marker for the cellular layers. The explants were examined as whole-mount preparations and vertical sections. Sectioned tissue was stained with hematoxylin-eosin and labeled for immunohistochemistry with photoreceptor-specific antibodies raised against transducin and recoverin. Results: In explants kept for 0–5 days in vitro, the developing retina consisted of multiple rows of neuroblastic cells and a more defined, but multilayered ganglion cell layer (GCL). Older explants revealed a more differentiated appearance with ultimately all normal retinal layers present, even after 42 days in vitro. Transducin- and recoverin-labeled photoreceptors were seen in these specimens, but no outer segments were found. The whole-mount preparation revealed extensively Sytox-labeled cells in the GCL at 2 days in vitro, but very few cells were labeled in older explants. Conclusion: This study shows that cultured fetal porcine full-thickness neuroretina can survive and develop according to its intrinsic timetable for at least 6 weeks in vitro. The in vitro system for culturing of the full-thickness retina may be useful in experiments involving retinal transplantation.

Effects of Glial Cell Line-derived Neurotrophic Factor on the Cultured Adult Full-thickness Porcine Retina.

Abstract Background: The tissue culture system offers a possibility to study factors involved in neuronal survival which may be important in a transplantation paradigm. The use of adult tissue in this setting poses specific challenges since traditionally mature neurons survive poorly in vitro. In the current paper, we have explored effects of glial cell line-derived neurotrophic factor (GDNF) on cultures of adult porcine retina. Methods: Full-thickness retinal sheets were isolated from adult porcine eyes and were cultured for 5 or 10 days under standard culture conditions with or without GDNF added to the culture medium. The grafts were analyzed morphologically using hematoxylin and eosin staining, immunohistochemistry and transferase dUTP nick end labeling (TUNEL) labeling. Retinas derived from normal adult porcine eyes were used as controls. Results: After 5 d in vitro (DIV), cultures without GDNF showed dissolving retinal lamination while specimens cultured with GDNF displayed the normal laminated morphology. At 10 DIV, the untreated cultures had been reduced to a degenerated cell mass, while the GDNF-cultured specimens retained thin but distinguishable retinal layers. TUNEL labeling confirmed these results. Immunohistochemical labelings and outer nuclear layer thickness measurements showed an increased preservation of photoreceptors and horizontal cells in the GDNF-treated group. Conclusions: The procedure of culturing retina involves several steps causing severe traumatic effects on the tissue, such as ganglion cell axotomy, interruption of the blood flow as well as separation from the retinal pigment epithelium (RPE). In this paper, we have shown that addition of GDNF in the culture medium attenuates the effect of these steps, resulting in enhanced preservation of several retinal neuronal subtypes. The results may be of importance for research in retinal transplantation where storage time of the donor tissue prior to transplantation is a critical issue.

Transplantation of cultured adult porcine full-thickness retina.

In this study we wanted to examine how an adult neuroretina from an animal with an eye similar to the human one survives in vitro. We also wanted to investigate how the culture process affects the adult retina when used in a transplantation paradigm. Full-thickness neuroretinal sheets from adult porcine eyes were dissected into pieces measuring 3 mm in diameter. These were kept in culture for 1–3 days. After this time, the explants were fixed or transplanted subretinally to adult pigs, which were killed after 72–74 days. Transplanted eyes, as well as tissue kept in culture only, were processed for hematoxylin and eosin staining and immunohistochemistry. Explants kept 1 day in vitro (DIV) displayed the normal morphology. In these specimens, single pyknotic cells were evident in the outer nuclear layer (ONL) and ganglion cell layer, but were more frequent in the inner nuclear layer (INL). After longer times in vitro, severe degenerative changes appeared. Transplanted explants kept 1 DIV prior to transplantation exhibited normal retinal lamination in two out of four specimens. Transducin and recoverin labeling revealed photoreceptors with inner segments in these grafts. Rod bipolar cells displayed a normal morphology. Vertically arranged Mùller cells were also seen in the laminated grafts. Two of the three transplants kept 2 DIV displayed minimal lamination. Eyes with transplants kept 3 DIV prior to transplantation displayed degenerated grafts in all eyes. This study shows that adult porcine neuroretinal explants kept in culture for 1 day display a normal morphology in their major part. Additionally, 1-day explants can survive transplantation with retained morphology even after several months. This indicates the possibility of storing adult donor tissue between harvest and transplantation. The culture system may also be used in the future as a tool for manipulating retinal donor tissue prior to transplantation.

Isolation of photoreceptors in the cultured full-thickness fetal rat retina.

PURPOSE To create a retina consisting mainly of photoreceptors for future use as donor tissue in retinal transplantation. METHODS Fetal full-thickness neuroretinas from Sprague-Dawley embryonic day (E) 17 or E20 rats were placed in culture for 7 or 14 days. Explants and age-matched control retinas were examined by light microscopy and with a panel of immunohistochemical markers labeling all seven of the major retinal cell types. RESULTS E17 and E20 control retinas displayed vimentin-labeled Müller cells, NF160-labeled ganglion cells, and synaptic vesicles labeled with synaptophysin. The remaining cell types were found in control specimens of postnatal age 2 days and older. After 7 or 14 days in culture, all explants were significantly thinner than their aged-matched controls and displayed multiple rows of cells organized in a single layer. Within this layer, they contained rhodopsin-labeled rod photoreceptors, presynaptic vesicles, and vertically arranged Müller cells. Transducin-labeled cone photoreceptors were found in all but the youngest explants. Scattered PKC-labeled rod bipolar cells and calbindin-labeled horizontal cells were found in the inner part of most explants, whereas beta-III-tubulin-labeled ganglion cells and parvalbumin-labeled amacrine cells were seen only sporadically. No NF160-labeled ganglion cells were found. CONCLUSIONS Fetal full-thickness rat retina in vitro develops into a retina consisting of predominantly synapse-containing cone and rod photoreceptors embedded in a scaffold of well-organized Müller cells. These explant retina characteristics are well adapted for use as donor tissue in future retinal transplantation experiments.

Ocular phenotype analysis of a family with biallelic mutations in the BEST1 gene.

PURPOSE To investigate the genetic cause and perform a comprehensive clinical analysis of a Danish family with autosomal recessive bestrophinopathy; to investigate whether Bestrophin may be expressed in normal human retina. DESIGN Retrospective clinical and molecular genetic analysis and immunohistochemical observational study. METHODS setting: National referral center. participants: A family with 5 individuals and biallelic BEST1 mutations, and enucleated eyes from 2 individuals with nonaffected retinas. observation procedures: Molecular genetic analysis included sequencing of BEST1 and co-segregation analysis. Clinical investigations included electro-oculography, full-field electroretinography, multifocal electroretinography, spectral-domain optical coherence tomography, and fundus autofluorescence imaging. Immunohistochemical analysis was performed. main outcome measures: BEST1 mutations, imaging findings, electroretinography amplitudes, and implicit times. RESULTS The index case was compound heterozygous for p.A195V and a novel 15 base pair deletion leading to p.Q238L. The index case at age 10 demonstrated multifocal vitelliform changes that were hyperautofluorescent, cystoid macular edema in the inner nuclear layer, no light rise in the electro-oculography, and a reduced central but preserved peripheral retinal function by multifocal electroretinography. Full-field electroretinography demonstrated a reduced rod response and inner retina dysfunction. Retinal structure was normal in all 3 family members who carried a sequence change in BEST1. Electro-oculography light peak was reduced in both the mother and sister (heterozygous for p.Q238L). Immunohistochemistry could not confirm the presence of Bestrophin in normal human retina. CONCLUSIONS Because of a relatively well preserved retinal function, autosomal recessive bestrophinopathy may be a suitable first candidate, among the BEST1-related ocular conditions, for gene replacement therapy.

Scaffolding the retina: The interstitial extracellular matrix during rat retinal development.

To examine the expression of interstitial extracellular matrix components and their role during retinal development.

Early development of retinal subtypes in long-term cultures of human embryonic retina.

Purpose: To study early signs of neuronal and glial differentiation in the human embryonic retina. Materials and Methods: 6.5-to 8-week-old human embryos were obtained from elective abortions. The neuroretinas were kept in culture as full-thickness sheets for 7–42 days. Results: The control retinas consisted of a neuroblast cell layer and a thin marginal zone. Most explants displayed presence of retinal lamination, but also contained regions of disorganization. Vimentin labeling showed vertically arranged Müller cells in all explants. Recoverin-labeled photoreceptors appeared in explants kept 14 days and longer. By labeling with antibodies against PKC and parvalbumin, rod bipolar cells and amacrine cells could be seen in explants kept for 42 days in culture.Conclusions: We have shown that the embryonic full-thickness neuroretina can survive in a culture environment for at least 6 weeks, and can develop several types of the retina-specific neuronal and glial cells.

Human Retinal Development in an in situ Whole Eye Culture System.

Phenotypic characterization of human retinogenesis may be facilitated by use of the tissue culture system paradigm. Traditionally, most culture protocols involve isolation of retinal tissue and/or cells, imposing various degrees of trauma, which in many cases leads to abnormal development. In this paper, we present a novel culture technique using whole embryonic eyes to investigate whether the retina in situ can develop normally in vitro. All procedures were carried out in accordance with the Declaration of Helsinki. Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion. A total of 19 eyes were enucleated. The ages of the embryonic retinas were 6–7.5 weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 17 eyes were placed on culture plates and divided into 3 groups kept for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the specimens were processed for hematoxylin and eosin staining, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; rod bipolar cells) and vimentin (Müller cells) were used. TUNEL was used to detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. Specimens kept 7–14 DIV had a similar appearance, while 28-day specimens consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured retinas displayed completely normal lamination without rosettes or double folds. Pyknotic cells were found at the inner margin of the retinas, and the proportion of these cells increased with time in vitro. TUNEL staining revealed a few scattered cells in 7-DIV specimens, and the amount of stained cells in the inner part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin labeling showed cells arranged in a vertical pattern in all retinas. Labeling with recoverin revealed photoreceptors in 4 of the retinas kept for 14 DIV, and in all retinas kept for 28 DIV. After 28 DIV, 2 of the eyes labeled with PKC contained rod bipolar cells with minimal axons. Here we showed that human embryonic retinas can be kept in culture in situ within the eye for at least 4 weeks. Abnormal lamination is not as frequent as in isolated full-thickness retinas, indicating that physical and biochemical contact with surrounding tissues is vital for proper development. Several types of the retina-specific neuronal and glial cells were seen to differentiate according to the in vivo schedule. The results are important for future studies of retinal development, and the technique can also be used for testing the effects of various drugs on the immature retina.

Large Eyelid Defect Repair Using a Free Full-Thickness Eyelid Graft

Summary: Large eyelid full-thickness defects are traditionally repaired using flaps with a blood-supplying pedicle, for the reconstruction of the anterior or posterior lamella or both. This is a 2-stage procedure involving occlusion of vision in the affected eye for 4–8 weeks, as the flap pedicle is not divided until vascularization is deemed adequate. However, the importance of using a flap with a pedicle to ensure adequate perfusion of the graft has recently been questioned.

Clinical, optical coherence tomography, and fundus autofluorescence findings in patients with intraocular tumors

Purpose To describe clinical, optical coherence tomography (OCT) and fundus autofluorescence (FAF) findings in patients with intraocular tumors and determine if OCT and FAF could be helpful in the differential diagnosis and management of different choroidal tumors. Methods Forty-nine patients with untreated, macular, midperipheral, and extrapapillary intraocular tumors were included. All patients underwent ophthalmic examination: best-corrected visual acuity, slit-lamp biomicroscopy, funduscopy, and standardized B mode, and if possible A mode, ultrasonography, and OCT and FAF imaging of the surface of the intraocular tumors. Results Of the 49 patients studied, 19 had choroidal nevi, ten had indeterminate choroidal melanocytic lesions (IMLs), ten had malignant melanomas, and ten had other choroidal tumors. The choroidal nevi revealed subretinal fluid (SRF) on OCT in only 11%. FAF detected isoauto-fluorescence in 42%, hypoautofluorescence in 37%, patchy FAF pattern in 16%, and a diffuse FAF pattern in 5%. Seventy percent of patients with IML showed SRF on OCT and 20% showed tumor growth on follow-up, detected only by OCT and FAF imaging. FAF revealed a patchy pattern in 50% and a diffuse pattern in 40% of cases with IML. Ninety percent of the patients with choroidal melanoma had SRF on OCT and FAF revealed a patchy pattern in 60% and a diffuse pattern in 40%. Patients with other choroidal tumors had SRF on OCT in 30% of cases and no characteristic pattern on FAF. Conclusion Both OCT and FAF were helpful in the differential diagnosis of choroidal nevi versus IMLs, choroidal melanomas, and other choroidal tumors. Also, detailed and periodical clinical evaluation of patients with intraocular tumors using OCT and FAF imaging for the detection of both SRF and FAF patterns overlying the tumor can be useful for detection of tumor growth.