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Biomedical subjects

P Wiedemann

Publications and source records attributed to P Wiedemann.

At least 19 recordsLinked to original sources
PubMed2001

Hypoxia: modulation of endothelial cell proliferation by soluble factors released by retinal cells.

A devastating complication of ischemic retinopathies is retinal neovascularization. We studied the impact on retinal endothelial cell proliferation of soluble factors released from cultured retinal glial (Müller) cells and from retinal explant cultures. Hypoxia strongly stimulated VEGF release by all types of cultures but endothelial cell growth was not further increased by the corresponding conditioned media if compared to supernatants obtained under normoxia. When the final concentration of the hypoxia-conditioned media was adjusted to the VEGF level of normoxia-conditioned media, they even inhibited endothelial cell proliferation. Inhibition may be exerted by TGF-beta 2 but TGF-beta 2 mRNA and protein expression in Müller cells were found to be down-regulated under hypoxia. We conclude that retinal endothelial cell proliferation is controlled by the balance of the amount and/or efficacy of several stimulatory and inhibitory factors.

Animals↗
PubMed2001

Direct measurement of VEGF-induced nitric oxide production by choroidal endothelial cells.

Vascular endothelial growth factor (VEGF) and nitric oxide (NO) seem to be involved in the process of angiogenesis, but their interactions are not clearly understood. The aim of this study was to investigate the influence of VEGF on NO production of choroidal endothelial cells (CEC) and its importance in angiogenesis. Experiments were performed using cultured bovine CEC. Basal NO release of unstimulated CEC was measured and compared to NO release of VEGF-stimulated CEC (1, 10, and 100 ng/ml). Further, cells were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 and 2 mM) and incubated with and without VEGF (10 ng/ml) to investigate the effect of blocking NO synthase. NO release into the medium was assessed by an amperometric NO sensor. To show the importance of NO in angiogenesis, proliferation and migration of CEC were measured after VEGF stimulation and in the presence or absence of L-NAME (1 and 2 mM). Unstimulated CEC continuously produced low levels of NO. Stimulation of the cells with VEGF resulted in a dose-dependent increase in NO release. The time course after stimulation with 10 ng/ml VEGF was characterized by a prompt initial rise up to 140% of unstimulated levels and a subsequent sustained increase over 120 min. Pretreatment with L-NAME attenuated the VEGF-induced response. L-NAME incubation alone led to a reduction in basal NO release. L-NAME also significantly diminished the VEGF-enhanced CEC proliferation and migration. The results demonstrate that VEGF enhances the formation of NO in cultured CEC. The blockade of NO production reduces CEC proliferation and migration, an effect which may be important for controlling angiogenesis, especially in reducing neovascularization in age-related macular degeneration in the eye.

Aged↗
PubMed2001

[Retinal degeneration. Apoptosis as pathomechanism and therapy strategy].

Molecular techniques in ophthalmology and related subjects have led in recent years to the identification of many genes expressed in photoreceptor cells and have allowed the characterization of mutations leading to distinct phenotypes of retinal degeneration. Programmed cell death, or apoptosis, has been identified as the final common pathway in this disease group. A cascade of events has evolved, starting with specific stimuli and developing over different mediators and regulators (e.g., Fas ligand, proteins of the Bcl-2 family, p53) to effector enzymes (caspases). The ever increasing data of this pathway serve as a basis for new therapeutic strategies. We review the current knowledge on apoptosis in retinal degeneration.

Animals↗
PubMed2001

The influence of pro-inflammatory cytokines on human retinal pigment epithelium cell receptors.

PURPOSE: To investigate the mRNA expression of the receptors for tumour necrosis factor alpha (TNFRp55, TNFRp75), interferon gamma (IFN gamma R alpha, IFN gamma R beta), interleukin 10 (IL-10R, CRFB4) and transforming growth factor beta (TGF beta RII) on human retinal pigment epithelium (RPE) cells and to modulate this expression with the pro-inflammatory cytokines TNF-alpha and IFN-gamma as stimulators. METHODS: The cells were cultured in the presence of TNF-alpha (10 ng/ml), IFN-gamma (1000 U/ml) or a combination of both for 24 h, 48 h and 72 h. The total RNA was prepared, and the receptor mRNA expression was investigated by the reverse-transcription polymerase chain reaction method. The changes in mRNA expression during the modulation were quantified by the ribonuclease protection assay. RESULTS: The mRNA for TNFRp55, TNFRp75, IFN gamma R alpha, IFN gamma R beta, CRFB4 and TGF beta RII was constitutively expressed in vitro. IL-10R mRNA was detected in neither unstimulated nor stimulated RPE cells. Especially the mRNA of the TNF-Rp75 was up-regulated, mainly by IFN-gamma or the combination of both stimulators. CONCLUSION: Our results demonstrate that human RPE cells express the mRNA of different cytokine receptors and the expression may be partially modulated by pro-inflammatory cytokines. This may show that RPE cells act as corresponding cells not only in vitro, but also in inflammation and immunological processes in the eye. In this connection it could be hypothesised that activated RPE cells play a stimulating role in addition to the known suppressive one.

Adult↗
PubMed2001

Molecular and cellular evidence for T-cell stimulation by allogeneic retinal pigment epithelium cells in vitro.

PURPOSE: The rejection of retinal pigment epithelium (RPE) allografts is a major barrier to long-term success after retinal transplantation. The aim of this study was to investigate the action of RPE cells on allogeneic T cells in coculture with or without macrophages. For the detection of T-cell activation the interleukins IL-1beta and IL-2, typical for this process, were investigated. METHODS: Human RPE cells (6 x 10(5) cells/flask) were used as stimulator cells. To investigate the influence of MHC class II molecules the RPE cells were pre-incubated with different concentrations of interferon-gamma (IFN-gamma; 0, 50, 100, or 250 U/ml) for 4 days. This was followed by coculture with either 6 x 10(6) T cells or, in a second trial, the T cells plus 6 x 10(5) macrophages. The mRNAs of the cytokines under study were detected using a reverse-transcriptase polymerase chain reaction and were quantified by colorimetry after 6 h. The cytokine protein content in the supernatants was measured after 20 h using specific enzyme-linked immunoabsorbent assays. RESULTS: Cytokine-specific mRNAs and proteins were found in all samples. After coculture the level of IL-1beta mRNA was higher and that of cytokine-specific protein was significantly increased. Furthermore, the addition of macrophages led to increased cytokine secretion but a general influence of the pre-activation with interferon could not be found. Similar results were detected for IL-2; at the highest dose, IFN-gamma preactivation and, in combination with macrophages, a significant increase in the protein level could be found. CONCLUSION: These results show that RPE cells are able to activate allogeneic T cells in vitro. Professional antigen-presenting cells may promote this process, as may pre-treatment with IFN-gamma. The circumstances modelled here are involved in the rejection process after RPE transplantation in humans and help to explain this immune response.

Adult↗
PubMed2001

Pars plana lentectomy for treatment of congenital cataract.

BACKGROUND: Congenital cataract surgery can be performed using a pars plicata/plana or a limbal approach, if placement of an intraocular lens at the time of cataract removal is not a consideration. Because of the high incidence of secondary cataract formation in children the operation should be combined with capsulotomy and anterior vitrectomy. METHODS: The series consisted of 30 eyes from 20 consecutive children who suffered from congenital cataract and underwent cataract surgery between May 1995 and June 2000. The inclusion criterion was congenital cataract affecting the visual axis. We performed the operations as lens aspiration with anterior and posterior capsulotomy and anterior vitrectomy via the pars plana or plicata. We used contact lenses to rehabilitate vision. All patients received intensive orthoptic and pleoptic treatment. RESULTS: This surgical technique provided in all eyes a clear visual axis. During follow-up of 3 months to 4.5 years, secondary cataract developed in five eyes. Retinal detachment, glaucoma and endophthalmitis did not occur. One patient developed contact lens intolerance and a secondary intraocular lens was placed in the ciliary sulcus. DISCUSSION: Lentectomy via a pars plana or pars plicata approach is a suitable and safe method for treating cataract in children. Our chosen method of lentectomy is an alternative to early implantation of an intraocular lens. It is possible to perform uncomplicated secondary implantation of an intraocular lens in the ciliary sulcus.

Aphakia, Postcataract↗
PubMed2001

Retinal pigment epithelium melanin granules are phagocytozed by Müller glial cells in experimental retinal detachment.

The ability of retinal Müller glial cells to perform phagocytosis in vivo is studied in a rabbit model of experimental retinal detachment where pigment epithelial cells are occasionally detached together with the neural retina. While macrophages and/or microglial cells phagocytoze most of the cellular debris at the sclerad surface of the detached retinae, some Müller cells accumulate melanin granules. The granules are virtually intact at the ultrastructural level, and are surrounded by a membrane. They are often located close to the sclerad end of the cells, but some are distributed throughout the outer stem process up to the soma. It is concluded that rabbit Müller cells in vivo are capable of phagocytosis and of transporting the phagocytozed material within their cytoplasm.

Animals↗
PubMed2001

Immunosuppression by IL-10-transfected human retinal pigment epithelial cells in vitro.

PURPOSE: Retinal pigment epithelium (RPE) transplantation seems to be a possible therapy for restoring vision in the case of retinal degeneration. As there is a risk of allergic rejection, a gene-transfer of immunosuppressive cytokines into the graft may diminish this reaction. Therefore, we investigated the transfer of interleukin-10 (IL-10) into an immortalised human RPE cell line (hTERT-RPE1) and its effect on the proliferation of allogeneic immune competent cells. METHODS: The hTERT-RPE1 cells were transiently transfected with the cDNA of human IL-10 using a lipid-based transfection reagent. The expression of IL-10 mRNA was ana-lysed by reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay measured the secretion of the cytokine over 7 days. The effect of the secreted IL-10 on the proliferation of allogeneic T cells with and without homologous macrophages was investigated colorimetrically. To enhance this reaction, RPE cells were pre-activated with interferon-gamma (IFN-gamma). Anti-IL-10 antibodies were used in a neutralising assay. RESULTS: A transfection efficiency of 23.3 +/- 9.03% was achieved. IL-10 mRNA could only be shown in IL-10-transfected hTERT-RPE1 cells. The same was found for the level of cytokine, with a maximum on day 3 (10.34 +/- 0.09 ng/ml). A significant suppressive effect of the secreted IL-10 on T-cell proliferation was detectable on days 5 and 6. This effect could be significantly abolished with anti-IL-10 antibodies. CONCLUSIONS: The IL-10-producing hTERT-RPE1 cells had an immunosuppressive action on T-cell proliferation in vitro. A gene-transfer into RPE allografts before transplantation may be able to promote graft survival.

Cell Division↗
PubMed2001

Histological findings of surgically excised choroidal neovascular membranes after photodynamic therapy.

AIM: To investigate effects of photodynamic therapy (PDT) on human choroidal neovascularisation (CNV). METHODS: Two patients with recurrences after PDT with verteporfin underwent surgical extraction of the CNV. Immediately after surgical excision the subfoveal neovascular membranes were divided for light microscopic and for electron microscopic processing. For light microscopy tissues were embedded in paraffin. Sections were stained with haematoxylin and eosin, and the periodic acid Schiff (PAS) reaction was performed to determine histological diagnosis and to ensure tissue quality. For electron microscopy the specimens were fixed in glutaraldehyde and embedded in epoxy resin. Semithin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope. RESULTS: Light microscopy showed thick fibrovascular membranes in both cases. On the outer surface remnants of retinal pigment epithelial cells resting on thickened inner aspect of Bruch's membrane were found. On the retinal side some outer segments were found. The membrane showed areas with irregularly shaped vessels. Electron photomicrographs showed occluded vessels within the CNV containing thrombotic masses and/or ultrastructural damage of the neovascular endothelium. Most of the vessels presented regressive changes with vacuolisation and fragmentation of the neovascular endothelium accompanied by disintegration of the endothelial cell layer. Extravasation of red blood cells was observed. Occasionally, vessels with normal endothelium containing intact red blood cells were observed. Some vessels contained immature endothelial cells. At some locations the retinal pigment epithelium cells (RPE) were metaplastic showing highly vacuolised cytoplasm. CONCLUSIONS: These findings suggest that the evidence of fluorescein leakage from the CNV and enlargement of the neovascular complex following PDT could be related to new vessel growth and recanalisation of occluded vessels. Additionally, RPE disturbances were observed in the specimens. This finding may be related to the original pathology or could indicate that PDT treatment may result in RPE atrophy.

Aged↗
PubMed2001

Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR.

PURPOSE: To determine the electrophysiological properties of Müller (glial) cells from experimentally detached rabbit retinas. METHODS: A stable local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Müller cells were acutely dissociated and studied by the whole-cell voltage-clamp technique. RESULTS: The cell membranes of Müller cells from normal retinas were dominated by a large inwardly rectifying potassium ion (K+) conductance that caused a low-input resistance (<100 M(Omega)) and a high resting membrane potential (-82 +/- 6 mV). During the first week after detachment, the Müller cells became reactive as shown by glial fibrillary acidic protein (GFAP) immunoreactivity, and their inward currents were markedly reduced, accompanied by an increased input resistance (>200 M(Omega)). After 3 weeks of detachment, the input resistance increased further (>300 M(Omega)), and some cells displayed significantly depolarized membrane potentials (mean -69 +/- 18 mV). When PVR developed (in 20% of the cases) the inward K+ currents were virtually completely eliminated. The input resistance increased dramatically (>1000 MOmega), and almost all cells displayed strongly depolarized membrane potentials (-44 +/- 16 mV). CONCLUSIONS: Reactive Müller cells are characterized by a severe reduction of their K+ inward conductance, accompanied by depolarized membrane potentials. These changes must impair physiological glial functions, such as neurotransmitter recycling and K+ ion clearance. Furthermore, the open probability of certain types of voltage-dependent ion channels (e.g., Ca2+-dependent K+ maxi channels) increases that may be a precondition for Müller cell proliferation, particularly in PVR when a dramatic downregulation of both inward current density and resting membrane potential occurs.

Animals↗
PubMed2001

Facilitation of artificial retinal detachment for macular translocation surgery tested in rabbit.

PURPOSE: For macular translocation surgery, the native attached retina has to be detached either locally or completely. Although different surgical techniques are used, there is a general search for supporting procedures that facilitate and accelerate the retinal detachment. METHODS: Pars plana vitrectomies were performed in pigmented rabbits. In all experimental groups, a local retinal detachment was created by infusing the test solution with a thin glass micropipette attached to a glass syringe. In control animals a standard balanced salt solution was used at room temperature, in combination with a standard vitrectomy light source. In two test groups, a calcium- and magnesium-free solution was used for the vitrectomy, under illumination by a standard light source in group I (solution at room temperature) and group II (solution heated up to body temperature). In group III the rabbits were dark-adapted for half an hour, and then, during surgery, a red filter was used in front of the light source (standard balanced salt solution at room temperature). After the rabbits were killed at the end of surgery, the adherence of the retinal pigment epithelium (RPE) to the neural retina in the detachment area was quantified microscopically, and the morphologic integrity of the detached retinal tissue was examined by light and electron microscopy. No electrophysiology was performed. RESULTS: In all four groups, it was possible to detach the retina. The maximum adherence of the RPE cells to the neural retina was observed in the control group. Virtually no decrease in adherence was found in test group II (36 degrees C solution without calcium and magnesium), whereas a significant decrease was seen in both group I (calcium- and magnesium-free solution at room temperature) and group III (dark adaptation-red light technique; standard balanced salt solution at room temperature). In none of the experimental groups was any obvious damage of the retinal structure observed, even after exposure to the test solutions for 60 minutes. CONCLUSIONS: Both dark adaptation (red illumination) and the use of a calcium chloride- and magnesium chloride-free solution (at room temperature) can facilitate retinal detachment in macular translocation surgery. Both techniques are proposed as a gentle support for the operation, because they protect an intact RPE cell layer and do not cause retinal damage at the ultrastructural level.

Animals↗
PubMed2000

VEGF release by retinal glia depends on both oxygen and glucose supply.

Isolated retinae or isolated Müller cells were cultured in vitro, and vascular endothelial growth factor (VEGF) was assayed as protein (by ELISA) and as mRNA (by semi-quantitative RT-PCR). In both types of cultures, hypoxia (5% O2) resulted in an upregulated VEGF release. While the unstimulated VEGF secretion was virtually independent of glucose (0.125 - 25 mM), elevated glucose concentrations (10 - 25 mM) blocked most of the stimulatory effect of hypoxia on VEGF mRNA synthesis (determined in Müller cell cultures) as well as on VEGF release (in both retina and Müller cell cultures). It is concluded that in retinal glial (Müller) cells, being responsible for retinal VEGF synthesis (and, thus, for undesirable neovascularization), the metabolic effects of hypoxia can be compensated by a surplus of glucose.

Animals↗
PubMed2000

P2X7 receptors in Müller glial cells from the human retina.

ATP has been shown to be an important extracellular signaling molecule. There are two subgroups of receptors for ATP (and other purines and pyrimidines): the ionotropic P2X and the G-protein-coupled P2Y receptors. Different subtypes of these receptors have been identified by molecular biology, but little is known about their functional properties in the nervous system. Here we present data for the existence of P2 receptors in Müller (glial) cells of the human retina. The cells were studied by immunocytochemistry, electrophysiology, Ca(2+)-microfluorimetry, and molecular biology. They displayed both P2Y and P2X receptors. Freshly enzymatically isolated cells were used throughout the study. Although the [Ca(2+)](i) response to ATP was dominated by release from intracellular stores, there is multiple evidence that the ATP-induced membrane currents were caused by an activation of P2X(7) receptors. Immunocytochemistry and single-cell RT-PCR revealed the expression of P2X(7) receptors by Müller cells. In patch-clamp studies, we found that (1) benzoyl-benzoyl ATP (BzATP) was the most effective agonist to evoke large inward currents and (2) the currents were abolished by P2X antagonists; however, (3) long-lasting application of BzATP did not cause an opening of large pores in addition to the cationic channels. By microfluorimetry it was shown that the P2X receptors mediated a Ca(2+) influx that contributed a small component to the total [Ca(2+)](i) response. Activation of P2X receptors may modulate the uptake of neurotransmitters from the extracellular space by Müller cells in the retina.

ATP-Binding Cassette Transporters↗
PubMed2000

R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil inhibit human retinal pigment epithelial cell contraction.

PURPOSE: To evaluate the effects of R-(+)-verapamil, S-(-)-verapamil, and the racemate on in vitro contraction of human retinal pigment epithelial cells. METHODS: RPE cells from human donor eyes were enzymatically dissociated and cultured on collagen gels. Transdifferentiated RPE cells (seventh and eighth passage) were used for experiments. The contraction assays were treated with different concentrations (10-1000 microM) of R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil. RESULTS: Verapamil inhibited the gel contraction of human RPE cells. R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil acted equally in a dose-dependent manner from 10-1000 microM. CONCLUSION: The results indicate the effectiveness of verapamil to inhibit the contraction of RPE cells. R-(+)-verapamil is recommended because it is associated with fewer systemic and local side effects.

Adolescent↗
PubMed2000

[Blue-yellow perimetry in rhegmatogenous retinal detachment. Studies before and after therapy in comparison with white-white perimetry].

INTRODUCTION: Disturbance of the blue light perceptance in rhegmatogenous retinal detachment was demonstrated by Köllner 1907 with the help of colour-perimetries. We examined the blue- and white-function after retinal reattachment by blue-on-yellow-perimetry compared with white-on-white-perimetry. PATIENTS AND METHODS: 10 Patients with rhegmatogenous retinal detachment and a visual acuity > or = 0.1 were examined preoperatively, 4 days and 6 weeks after successful buckling procedure. We performed a white-on-white-perimetry as also perimetries with blue stimuli on a yellow luminance background (program 30-2 of the Humphrey Field Analyzer 750). RESULTS: Four days after surgery the increase in sensitivity was significantly less for the blue-function compared with the white-function. Preoperatively and 6 weeks postoperatively the sensitivities were not significantly different. Between these time-points function recovered in average for both methods from MD -15 dB to MD -5 dB. CONCLUSION: After successful retinal detachment surgery the blue- and white-function increased. Blue-on-yellow-perimetry showed more retinal sensitivity loss than white-on-white-perimetry immediately after surgery. Blue-on-yellow-perimetry measured the same retinal sensitivity loss as white-on-white-perimetry preoperatively and 6 weeks postoperatively.

Adult↗