Natalia Helena Bryniarska-Kubiak, MSc - 2022

Stroke is the second most common cause of death in the world after cardiovascular disease. About 85% of all strokes are ischemic, the molecular mechanisms of which are still not fully understood. Recently, the involvement of neuroimmune mechanisms in the course of this disease has been indicated, with a particular interest in chemokine and its receptors. It has been shown that these proteins can modulate the activity of nervous system cells, mediate neuronal-glial communication and be important in the course of inflammatory processes and the regulation of homeostasis. Therefore, the overall aim of this dissertation was to evaluate the contribution of chemokines and/or chemokine receptors to the pathophysiology of cerebral ischemia. The studies were carried out in an ex vivo model using organotypic hippocampal culture (OHC) established from 6-7 days old offspring of Sprague-Dawley rats. It should be emphasized that the OHC model preserves the structure of the hippocampus, the relationships between the nervous and immunoendocrine systems, and functional interactions between individual cells. The oxygenglucose deprivation (OGD) procedure was used as a model of ischemia. Three chemokines belonging to different subfamilies were selected for the study: CX3CL1, CXCL12, and CCL2, and their receptors CX3CR1, CXCR4, and CCR2, respectively. Modulation of these receptors was performed with the use of appropriate antagonists: AZD8797, AMD3100, and Irbesartan. Moreover, an attempt was made to elucidate the molecular mechanisms behind the observed neuroimmune and biomechanical changes. All the results of colorimetric, biochemical, immunofluorescence, and biomechanical tests presented in the study were obtained by conducting comparative experiments in control and OGD cultures. In the first part of the work, the OGD model was verified. The OGD procedure has been shown to exert a cytotoxic effect and also increases the level of HIF-1α - a marker of hypoxia. Using confocal fluorescence microscopy, it was shown that the most robust damage in the OGD model was localized in the CA1 region of the hippocampus. Moreover, the OGD procedure led to changes in the organization of the actin cytoskeleton and the depolymerization of microtubules followed by the formation of tubulin aggregates in OHC. At the same time, atomic force microscopy operating in the force spectroscopy mode was applied to investigate the mechanical properties of OHCs. OGD was shown to induce significant stiffening of OHCs. The latest scientific reports indicate the presence of relationships between biomechanics (tissue stiffness) and inflammatory processes. Measurements of the level of the important inflammation mediators showed that OGD significantly enhances the synthesis of pro-inflammatory factors, including cytokines (IL-6, IL-18, TNF-α) and chemokines (CCL , CCL5, and C CL ) with a simultaneous decrease in the production of the anti-inflammatory cytokine IL-10. The next stage of the experiments was focused on the assessment of the effect of OGD on the role of chemokines and their receptors in the neuroinflammation process induced by OGD in OHC. In the case of the CX3CL1-CX3CR1 system, no effect of OGD on the level of endogenous CX3CL1 was observed. Also, exogenous administration of CX3CL1 did not affect the amount of LDH released after OGD. Interestingly, AZD8797, an antagonist of the CX3CR1, attenuated the cytotoxic effect induced by OGD. This observation was the basis for the research in which cultures with "silenced" microglia were used. The obtained results indicate that the elimination of microglia cells from OHC enhances the cytotoxicity induced in the OGD model and modulates the pro-and anti-inflammatory cytokine secretion profile. The effect of the OGD procedure on the CXCL12-CXCR4 system was also assessed. The increase in expression of the CXCL12 protein was shown in OHC after OGD. At the same time, it was observed that the compound AMD3100, which is an antagonist of the CXCR4 receptor, significantly increases the cytotoxicity and enhances the synthesis of the proinflammatory cytokine (IL-18) in the OHC after the OGD procedure. Interesting results were also provided by studies on the assessment of the role of the CCL2- CCR2 system in the OGD model. It was found that the expression of the CCL2 was increased after OGD. At the same time, it was shown that the use of Irbesartan - a CCR2 antagonist - significantly reduced the release of LDH after OGD and had a strong anti-inflammatory effect by decreasing the synthesis of pro-inflammatory cytokines (IL-6, IL- β) and chemokines (CCL3, CCL5, CXCL10). In the next stage of the study, it was shown that the protective and anti-inflammatory effect of rbesartan was mediated by the activation of the PP γ transcription factor. The antagonist of PP γ – GW9662 reduced the protective effect of Irbesartan in the OGD model. Importantly, experiments carried out using atomic force microscopy provided evidence that the anti-inflammatory effect of Irbesartan was accompanied by a change in OHC biomechanics since Irbesartan limited changes in culture stiffness in the OGD model. The second part of this dissertation presents the results of analyzes and biochemical tests carried out in patients with acute ischemic stroke. The analysis of computed tomography images and data from the medical interview were presented. This information allowed to assessment demographic data (age, gender) and risk factors, including comorbidities. Moreover, the determination of the concentration of CXCL12 and CCL2 chemokines in the plasma of patients was performed. Although the obtained results did not show differences in the concentration of CXCL12 and CCL2 in the plasma of patients with ischemic stroke compared to the values of the concentrations in the control group, the tests performed showed a tendency to increase the concentrations of these chemokines in patients with moderate and/or severe disease, which were assessed based on the NIHSS scale. In conclusion, this study shows that the use of the oxygen and glucose deprivation model in hippocampal organotypic cultures allows for the study of neuroimmune mechanisms of ischemic stroke, with particular emphasis on chemokines and their receptors. At the same time, it should be emphasized that in the OGD model, not only significant deficits in the immune component were observed, but also changes in the architecture of the cytoskeleton and biomechanical disorders. Based on the results obtained, it can be postulated that modulation of some chemokine-chemokine receptor systems, mainly CCL2-CCR2, has a protective effect in the model used, which gives grounds to consider this strategy as a useful tool in the potential therapy of ischemic stroke.