Protektivno dejstvo modulatora oksidativnog i nitrozativnog stresa u neuroinflamaciji - eksperimentalna i klinička studija

Abstract

Neuroinflammation is recognised as the leading mechanism in development of many of neurological diseases, including multiple sclerosis (MS). Recently published papers suggest biphasic MS pathogenesis which is based on neuroinflammation, which prevails in the early MS phase, and also neurodegeneration, which predominates in the later stage of the disease, at the time when accumulated neurological disability occurs. The invasion of central nerve system (CNS) by activated immune cells is established as the main part of disease initiation, while oxidative and nitrosative stress participation, through their mediators, as well as glutamatergic excitotoxicity, although recognised as important factors in disease pathophysiology, are still insufficiently tested. Starting with weak and conflicting results, found in recently performed studies, which have evaluated oxidative and nitrosative stress participation in neuroinflammation, the general aim of the actual investigation was to more accurately define the role of named factors in acute attacks of experimental and clinical neuroinflammation, at the same time investigating mutual correlations of those biomarkers with morphological and neurological characteristic of neuroinflammatory acute attacks. In the experimental part, the stated model of neuroinflammation – the acute model of experimental autoimmune encephalomyelitis (EAE), was used. The EAE animals were treated with selective inhibitor of inducible nitric oxide synthase (iNOS), aminoguanidine (AG), and, also, with antioxidant, N acetyl L cysteine (NAC). The potential protective effects of those agents were tested according to biochemical (concentration NO2 i NO3, MDA, GSH, SOD), immunohistochemical (GFAP, EAAT1, OX42, ED1 and iNOS expression) and neurological characteristics of neuroinflammation. On the other side, in the clinical part, the values of nitrosative and oxidative stress parameters (concentration of NO2 i NO3, MDA, AOPP, SH groups, SOD) were evaluated in hemolysates, plasma and cerebrospinal fluids of patients in acute attacks of different clinical phenotypes of neuroinflmamation, defined as clinically isolated syndrome of CNS (CIS) and early defined relapse remitting multiple sclerosis (RRMS). The obtained values of named parameters were correlated with radiological, clinical and paraclinical findings of neuroinflammation. The obtained data show increased oxidative stress intensity in all parts of CNS during accute EAE as well as in all tested media obtained from CIS and RRMS patients during their relapses. The closed correlations between parameters of oxidative stress and morphological and neurological findings of neuroinflammation suggest the relevancy of oxidative stress in pathogenesis of accute attacks in both experimental and clinical neuroinflammation. In compliance with expected, the higher values of antioxidative capacity were demonstrated in CIS, compared to RRMS patients. However, the higher level of oxidative disorders, based on obtained values of MDA and AOPP, were also demonstrated in these patients. These results are understood as the consequences of higher reactive adequacy of the body and CNS, in CIS patients (due to shorter disease duration). Thus, the prooxidative processes are more pronounced in these patients as an adaptive phemonenon in suppression of neuroinflammation (through some of the known physiological functions), but due to intensive, uncontrolled generation of reactive oxygen species (ROS), detrimental effects are accumulated. Inversely, the general and CNS antioxidative capacity were decreased in RRMS patients due to their continuous exposition to ROS, which explains oxidative stress intensity and worse radiological and clinical findings, which DOKTORSKA DISERTACIJA was confirmed by the obtained correlations. The obtained results show increased level of nitric oxide (NO.) during neuroinflammation attacks, when NO. exerts direct or indirect pathological effects on CNS tissues. The CNS disorders in neuroinflammation are also mediated by glutamatergic excitotoxicity, caused by increased level of glutamate as the consequence of astrocytes activation. The increased expression of iNOS, and other parameters of glial cell activity (GFAP, OX42, ED1) as well as glutamatergic excitotoxicity (EAAT1) were found in imunohistochemical analysis of forebrain section obtained from EAE animals. The protective biochemical, imunohistochemical and neurological effects were demonstrated after AG administration and thiol supplementation by NAC. Also, in the clinical part, the oxidative and nitrosative stress importance in the pathogenesis of acute neuroinflammatory attacks were demonstrated, while both general and CNS oxidative and nitrosative profile were different in patients with different clinical phenotypes of neuroinflammation. The significant correlations between tested parameters and clinical and radiological features, as well as disease duration, were obtained in CIS and RRMS patients. These data suggest the closed correlations between disease duration and the worse radiological and neurological score, and decreased antioxidative and antinitrosative profile, as well as increased, general and CNS, oxidative and nitrosative stress. The similar correlations were obtained between all tested parameters and other general parameters of biological and biochemical syndrome of inflammation, in both, CIS and RRMS group. The obtained results give an advanced insight into the roles and imoprtances of oxidative and nitrosative stress during neuroinflammation and offer the posibility for antioxidative and antinitrosative treatments in accute attacks of neuroinflammation. In this way, the diseases caused by neuroinflammation might be controlled in early phases whose characteristic is reversibility, at the same time delaying later phases which are accompanied with irreversible neurological disabilities. Although there are not ideal biomarkers of neuroinflammation, some of here tested oxidative and nitrosative stress parameters might serve as surogat biomarkers for the earliest diagnosis, tracking and assessing neuroinflammation intensity, and its radiological and clinical correlates.