Pharmakologisch-induzierte Degeneration der Retina in vitro und in vivo

Zusammenfassung:
In diesem Projekt werden selektiv Zellen der Retina mit Hilfe von chemischen Verbindungen geschädigt und die Auswirkungen auf Funktion und Integrität der Netzhaut untersucht. Dies erfolgt sowohl in der Zellkultur als auch im Tier-modell. Das geschädigte Gewebe wird dabei mit zell- und molekularbiolo-gischen Methoden analysiert um über den Verlauf der Degeneration und die dabei ablaufenden Vorgänge innerhalb der Zelle Aussagen treffen zu können. Mit Methyl-Nitroso-Urea (MNU; schädigt spezifisch Photorezeptoren) und Natriumjodat (NaIO3; wirkt selektiv auf RPE-Zellen) kann ausserdem im Tiermodell eine dosisabhängige Verschlechterung des Sehvermögens induziert, welche mittels verhaltensphysiolgischer Methoden quantifiziert wird. Die gewonnenen Ergebnisse sollen zu einem besseren Verständnis degenerativer Erkrankungen im Menschen führen und dazu dienen, Therapien für Krankheiten wie Retinitis pigmentosa oder altersabhängiger Makuladegeneration zu entwickeln.

Project description:
In vitro models:
In an organotopic retinal culture system the effect of sodium iodate (NaIO3) and methylnitrosourea (MNU) on the mammalian retina will be investigated in vitro. The main focus is on the reaction of Müller cells in response to the toxins.  Müller cells are retinal glial cells which exert neuroprotecive function. However, excessive Müller cell activation results in gliosis, a feature that is detrimental to the retina by inhibition of regenerative processes. To investigate Müller cell activation we will stain control and NaIO3 treated explants for GFAP and glutamine synthetase. Furthermore, we want to validate the hypothesis that the retinal pigment epithelium (RPE) is secreting protective factors (e.g., PEDF) after NaIO3 treatment by comparing retinal explants with and without RPE in co-culture.

In vivo models:
Part 1) In this study, we compare decrease in visual function in pigmented C57BL/6 mice after systemic treatment with low doses of NaIO3 using different functional measurements and correlate these changes with structural alterations in the neural retina. Especially, optokinetic response has been contrasted with water maze behavior over a time period of three months. Hereby, we could show that measurement of the optokinetic reflex (OKR) is not only more sensitive in detecting changes in visual function but provides addi-tionally the possibility to accurately detect visual acuity thresholds. To investigate the in-volved cell types of the retina morphometric and electrophysiological measurements are performed. Significant changes in the retinal layering as well as in the electroretinogram involving retinal pigment epithelium (RPE) and photoreceptors (PR) are detected. RPE cells undergo necrosis and induce apoptosis in the PR. There, immunohistochemical and PCR results show caspase-dependent cell death, especially the main apoptotic pathway via caspase 3.
Part 2) Herein, we perform the same approach as above but induce photoreceptor degenera-tion by systemic injection of MNU. Application of MNU leads to an instant decrease in visual function and a more delayed decrease in thickness of the retinal outer nuclear layer without affecting the RPE. Assessment of involved apoptotic effectors does not show caspase 3 activity, but the upregulation of other cell death pathways. Molecular measurements revealed e.g. a possible involvement of inflammation by caspase 1 and ER-mediated apoptosis by caspase 12.

Both models of retinal degeneration will also be used to test efficacy of stem cell-based cell replacement therapy for AMD (see here).