Your browser does not support JavaScript!

Home    Collections    Type of Work    Doctoral theses  

Doctoral theses

Search command : Author="Παπαδάκης"  And Author="Νικόλαος"

Current Record: 6 of 2410

Back to Results Previous page
Next page
Add to Basket
[Add to Basket]
Identifier 000459147
Title Investigation of the role of NADPH oxidase inhibitors in experimental animal models of retinopathies
Alternative Title Μελέτη του ρόλου των αναστολέων των NADPH οξειδασών σε πειραματικά ζωικά μοντέλα αμφιβληστροειδοπαθειών
Author Διονυσοπούλου, Σταυρούλα
Thesis advisor Θερμού Κυριακή
Reviewer Τσιλιμπάρης, Μιλτιάδης
Χαραλαμπόπουλος, Ιωάννης
Αντωνίου, Κατερίνα
Γραβάνης, Αχιλλέας
Σιδηροπούλου, Κυριακή
Βεργίνης, Παναγιώτης
Abstract The retina constitutes a particularly complex tissue in which, the direct interaction and functional coupling of neurons, glial cells (macro/microglia) and vessels forms the Neurovascular Unit (NVU), important for the maintenance of retinal physiological function. Various pathological factors affect some or all of the components of the NVU, disrupting its function and causing the manifestation of retinal diseases (retinopathies). Diabetic Retinopathy (DR), the most common complication of diabetes, affects a significant percentage of diabetes patients worldwide. Neurodegeneration, neuroinflammation and vasculopathy have been identified as the main components involved in the onset and development of DR, disrupting the function of the NVU. On the other hand, oxidative stress and glutamate excitotoxicity are interrelated mechanisms, directly involved in the manifestation of retinal disorders, including DR. NAPDH oxidases are enzymatic complexes (NOX family, seven known isoforms: NOX1-5, DUOX1/DUOX2) that represent one of the major sources of oxidative stress under pathological conditions in the retina, due to their sole role being the production of Reactive Oxygen Species. In different retinal cell types, the main isoforms NOX1,2 and 4 are expressed, which, in pathological conditions, have been associated with neurotoxic effects in the tissue. The aim of this PhD thesis was to evaluate the role of NADPH oxidases in the development of retinal disorders associated with excitotoxicity and diabetes, as well as the efficacy of specific NADPH oxidase inhibitors against neurodegeneration, neuroinflammation and vasculopathy, that characterize DR, essentially limiting the disruption of NVU and, therefore constituting a complete therapeutic approach for DR. For this purpose, two in vivo models were utilized in rats (Sprague-Dawley): 1) the model of AMPA induced excitotoxicity and 2) the model of streptozotocin (STZ) induced DR, in two paradigms, two and five weeks. In vivo model of AMPA excitotoxicity: the specific NOX2 inhibitor, GLX7013170, or the specific NOX4 inhibitor, GLX7013114, were co-administered with AMPA (8.4mM) intravitreally at the dose of 10-4M. Twentyfour hours (24 h) after the injections, eyes were isolated and tissues were collected for immunohistochemical studies. In vivo model of STZ induced DR: diabetes was induced by intraperitoneal injection of STZ (70mg/ml) and treatment included the topical administration as eye drops (20μl/drop) of each substance tested (pan-NOX inhibitor: VAS2870, NOX4 inhibitor : GLX7013114, NOX2 inhibitor: GLX7013170), at the concentration of 10mg/ml, either 1) once daily, for a total duration of 14 days, starting 48 hours after STZ administration (two-week paradigm) or 2) once daily, for 7 days in total, starting 4 weeks after STZ administration (five-week paradigm). Twentyfour hours after the last day of Ph.D. Thesis | Stavroula Dionysopoulou 13 treatment, tissues were collected for further use in various techniques, which included: immunohistochemistry, western blot, Real Time PCR, ELISA and Evans-Blue analysis, as well as Pattern Electroretinogram (PERG). Moreover, a pharmacokinetic study of the NOX4 inhibitor, GLX7013114, was performed, where the levels of GLX7013114 in the retina of healthy rats were determined through LC-MS/MS analysis. In the AMPA excitotoxicity model, AMPA administration was associated with the induction of oxidative damage in retinal tissue, which was significantly reduced by the NOX4 inhibitor. In addition, both NOX2/NOX4 inhibitors examined, GLX7013170 and GLX7013114, respectively, significantly reduced macro/microglia activation, while only the NOX2 inhibitor provided protection to the NOS positive amacrine cells against AMPA induced excitotoxicity. In the model of STZ induced DR, diabetes caused an increase in NOX2 and NOX4 isoform mRNA expression in the retina in the two-week paradigm, while in the five-week paradigm the expression of the NOX2 isoform was increased, in contrast with the significant reduction of NOX1 isoform mRNA. In the same paradigm, general NOX inhibition (VA2870) was associated with protection of ganglion cell axons, as well as reduced activation of micro/macroglia, an effect also observed in the case of NOX2 inhibitor, GLX7013170. However, the NOX2 inhibitor did not provide any protection at the level of ganglion cell axons, although it was found to regulate Bcl-2 protein expression in the retina of diabetic rats. Administration of the NOX4 inhibitor, GLX7013114, resulted in: 1) reduction of oxidative damage, caspase-3 expression, the loss of NOS amacrine cells and ganglion cell axons and micro/macroglial activation in both paradigms examined. GLX7013114 also significantly reduced the expression of TNF-α in the two-week paradigm, while in the five-week paradigm it reduced the expression of VEGF and IL-1β/IL-6 (mRNA), vascular leakage and protected ganglion cell function as revealed by PERG analysis of amplitudes recorded. In addition, the pharmacokinetic study of the NOX4 inhibitor, GLX7013114, confirmed that this inhibitor, administered topically as eye drops, reaches the retina and remains at detectable levels for up to 8 hours, justifying its therapeutic effects. In conclusion, NADPH oxidases are involved in the onset of excitotoxic phenomena in the retina, while at the same time their expression in retinal tissue is differentially affected by diabetes (NOX1,2 and 4). AMPA excitotoxicity is correlated with the induction of oxidative damage in the retina. NOX inhibitors can act as neuroprotective (NOX2 inhibitor) and anti-inflammatory agents (NOX2 and NOX4 inhibitor) against AMPA excitotoxicity. NOX2 and NOX4 isoforms are both involved in the development of retinal complications in the early stages of DR, which makes their inhibitors promising therapeutic targets. In particular, the extensive study of NOX4 inhibitor, GLX7013114, in both paradigms of DR, in combination with the pharmacokinetic analysis, support its efficacy to act against neurodegeneration, neuroinflammation and vasculopathy, constituting a complete therapeutic approach for the treatment of the early stage of DR.
Language English
Subject Diabetic retinopathy
Excitotoxicity
Neurodegeneration
Neuroinflammation
Therapeutic agents
Vasculopathy
Αγγειοπάθεια
Διαβητική αμφιβληστροειδοπάθεια
Διεγερτοτοξικότητα
Θεραπευτικοί παράγοντες
Νευροεκφύλιση
Φλεγμονή
Issue date 2023-12-08
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/4/1/8/metadata-dlib-1696501094-886804-28655.tkl Bookmark and Share
Views 848

Digital Documents
No preview available

No permission to view document.
It won't be available until: 2024-06-08