Neuroinflamatorni procesi mozga u amiotrofičnoj lateralnoj sklerozi : studija na modelu hSOD1 G93A pacova

Abstract

Amiotrofična lateralna skleroza (ALS) je neurodegenerativno oboljenje koje pogađa motorne neurone u kičmenoj moždini i višim moždanim centrima. Bolest karakteriše vrlo brza progresija i kratak životni vek od pojave simptoma. Predloženo je više mehanizama koji opisuju patogenezu bolesti, ali je uzrok ALS i dalje nepoznat. Cilj ove studije je da okarakteriše promene u višim moždanim centrima u modelu hSOD1G93A pacova i pokuša da dodatno razjasni mehanizme bolesti. Primenom tehnike magnetno-rezonantnog oslikavanja lokalizovana su neurodegenerativna žarišta u mozgu hSOD1G93A pacova. Istom metodom je in vivo praćena infiltracija T-limfocita pomoću posebno dizajniranih antitela obeleženih ultra malim paramagnetnim partikulama gvožđe oksida. Pokazano je da se CD4+ T-limfociti infiltriraju u tkivo oko moždanih komora, a CD8+ T-limfociti u moždano stablo hSOD1G93A pacova. Praćenje stanja krvno-moždane barijere pomoću Gd-DTPA kontrastnog agensa je ukazalo na njenu narušenost u moždanim regionima gde je uočena infiltracija T-ćelija. Prisustvo aktivirane mikroglije i reaktivnih astrocita je uočeno u moždanom stablu hSOD1G93A pacova. Pored toga, reaktivna mikroglija je uočena u moždanom tkivu oko lateralnih komora i hipokampusu. Nastavci aktivirane mikroglije ostvaruju kontakt sa telom neurona što ukazuje na mogućnost njihove interakcije. Primenom Western blot metode je pokazano da je ekspresija akvaporina-4 (AQP4) povećana, a ulaznog kalijumovog ispravljača (Kir4.1) smanjena u moždanom stablu i moždanoj kori hSOD1G93A pacova. Imunohistohemija na zamrznutim moždanim presecima je pokazala povećanu AQP4 i smanjenu Kir4.1 imunoreaktivnost u nc. facialis, nc. trigeminus i motornoj kori hSOD1G93A pacova. Kada su ispitivani kortikalni astrociti u kulturi, uočena je povećana AQP4 i smnjena Kir4.1 imunoreaktivnost u ALS. Proučavanje funkcionalnih osobina Kir kanala je ukazalo na smanjenu gustinu struja i smanjenu specifičnu membransku provodljivost u ALS astrocitima u kulturi. Pored toga, blokiranje ovih struja dodavanjem 1 mM CsCl ili 100 μM BaCl2 u vanćelijski rastvor je pokazalo da su Cs+-senzitivne odnosno Ba2+-senzitivne struje značajno manje u ALS astrocitima. U ovoj studiji su lokalizovana žarišta neurodegenerativnih i neuroinflamatornih promena u mozgu hSOD1G93A pacova, uočeno je oštećenje krvno-moždane barijere, a posebno su istaknute promene u ekspresiji i funkciji astrocitnih proteina koji doprinose stabilnosti krvno-moždane barijere. Dobijeni podaci su pružili dodatna objašnjenja koja doprinose razumevanju mehanizama ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting lower and upper motor neurons. The disease shows fast progression and short lifetime following the disease onset. Several mechanisms contributing to ALS pathogenesis have been proposed, but the cause of disease remains unclear. The aim of this study was to characterize changes in the upper CNS regions of the hSOD1G93A rat model and to try to clarify disease mechanisms. Foci of neurodegeneration and neuroinflammation were localized in the brain of hSOD1G93A rat using magnetic resonance imaging. The same method was applied for in vivo monitoring of T-lymphocytes infiltration using specifically designed antibodies conjugated with ultrasmall superparamagnetic iron oxide particles. It was shown that CD4+ T-lymphocytes were infiltrated into the brain tissue around lateral ventricles while CD8+ T-lymphocytes were infiltrated into the brainstem of the hSOD1G93A rat. Using Gd-DTPA contrast, it has been shown that the blood-brain barrier was compromised in the brain regions invaded with T-cells. Activated microglia and reactive astrocytes were observed in the brainstem of the hSOD1G93A rat. In addition, reactive microglia was shown in the brain tissue around lateral ventricles and in the hippocampus. Processes of activated microglia were in contact with neuronal somata indicating possible interaction. Western blot results showed increased aquaporin-4 (AQP4) and reduced inwardly rectifying potassium channel (Kir4.1) expression in the brainstem and cortex of the hSOD1G93A rat. Immunohistochemistry on the frozen brain slices showed increased AQP4 and decreased Ki4.1 immunoreactivity in the nc. facialis, nc. trigeminus and motor cortex of the hSOD1G93A rat. Increased AQP4 and decreased Kir4.1 immunoreactivity were retained in cortical ALS astrocytes in culture. Examination of the functional properties of Kir channels revealed a decreased current density and diminished specific membrane conductance in ALS astrocytes in culture. Addition of 1 mM CsCl or 100 μM BaCl2 to the extracellular solution revealed significantly lower Cs+-sensitive and Ba2+-sensitive Kir currents in ALS. In this study we localized and characterized neurodegenerative nad neuroinflammatory changes in the brain of hSOD1G93A rat, confirmed the blood-brain barrier compromise and observed changes in the astrocytic proteins involved in the maintenance of the blood-brain barrier. Obtained data may contribute to further understanding of the mechanisms underlying ALS pathology.