Karakteristike promena, distribucija antigena i ekspresija proinflamatornih citokina u mozgu lisica prirodno inficiranih virusom besnila

Abstract

U ovoj disertaciji ispitani su uzorci mozga 10 odraslih ridih lisica (Vulpes vulpes), oba pola, prirodno inficiranih virusom besnila. Za dokazivanje prisustva virusa besnila u ispitivanom materijalu korišcene su metode direktne imunofluorescencije i RT-PCR. Patohistološka i imunohistohemijska analiza izvršena je na uzorcima kore velikog mozga, hipokampusa, malog mozga i produžene moždine, koji su fiksirani u puferizovanom 10% formalinu, obradeni u automatskom tkivnom procesoru i uklapani u parafin. Parafinski isecci debljine 4-5μm bojeni su hematoksilin-eozinom. Ispitivanje prisustva virusnog antigena i proinflamatornih citokina izvršeno je streptavidin-biotin (LSAB) metodom, uz upotrebu antitela za virusni antigen, interleukin 1 (IL1-) i tumor nekroticni faktor (TNF-). U proceni distribucije virusnog antigena po regijama mozga korišcena je semikvantitativna analiza. Morfometrijskom analizom utvrden je broj inficiranih nervnih celija, a ovi podaci su obradeni korišcenjem deskriptivnih statistickih parametara, ANOVA i Tukey testa. Prisustvo virusa besnila dokazano je kod svih 10 ispitivanih mozgova metodom direktne imunofluorescencije i RT-PCR. Patohistološkim pregledom ustanovljene su promene u tipu akutnog negnojnog encefalitisa, pre svega umnožavanje glija celija i perivaskularni infiltrati limfocita i makrofaga. Pored toga, uocene su i distroficne promene na neuronima, satelitoza, neuronofagija, endotelioza i leptomeningitis. Negrijeva tela su bila prisutna u piramidalnim neuronima kore velikog mozga i hipokampusa u 30% slucajeva. Ekspresija virusnog antigena je dokazana u perikarionu neurona, kao i u njihovim aksonskim i dendritskim produžecima. Njegova distribucija je bila veoma široka, a najintenzivnija ekspresija zabeležena je u hipokampusu i produženoj moždini. Ekspresija IL1- i TNF- utvrdena je u zonama sa zapaljenskim promenama, i to u mikroglija celijama, makrofagima i limfocitima perivaskularnih nakupina i meke moždanice. Uocena je direktna pozitivna zavisnost izmedu intenziteta ekspresije citokina i stepena zapaljenskih promena. Zavisnost izmedu distribucije virusnog antigena i proinflamatornih citokina nije uocena.

In this dissertation, brain samples of 10 adult red foxes (Vulpes vulpes), of both sexes, naturally infected with rabies, were examined. Direct immunofluorescence and RT-PCR method were used to prove the presence of rabies virus in the examined material. Histopathologic and immunohistochemical analysis were performed on samples of the cerebral cortex, the hippocampus, the cerebellum and the medulla, which were fixed in buffered 10% formalin, processed in an automatic tissue processor, and embedded into the paraffin. Four to five μm thick paraffin sections were stained with hematoxylin-eosin. Investigation of the presence of viral antigen and pro-inflammatory cytokines was performed with streptavidin-biotin (LSAB) method, with the use of an antibody to a viral antigen, interleukin 1 (IL1-), and tumor necrosis factor (TNF-). In assessing the distribution of viral antigen by regions of the brain semiquantitative analysis was used. The number of infected neurons was determined by morphometric analysis, and these data were analyzed using descriptive statistical parameters, ANOVA and Tukey test. The presence of rabies virus was confirmed in all of 10 examined brains by direct immunofluorescence and RT-PCR. Histopathological examination revealed lesions characteristic for acute nonsuppurative encephalitis, primarily multiplication of glial cells and perivascular infiltrates of lymphocytes and macrophages. In addition, dystrophic changes in neurons, satellitosis, neuronophagia, endotheliosis and leptomeningitis were observed. Negri bodies were present in pyramidal neurons of the cerebral cortex and hippocampus in 30% of cases. The expression of viral antigen has been demonstrated in neuronal perikaryon, axons and dendrites. Its distribution was very broad, and the most intense expression was observed in the hippocampus and medulla oblongata. Expression of IL1- and TNF- was found in areas with inflammatory lesions, in microglial cells, macrophages and in the lymphocytes of perivascular infiltrates and leptomeninges...