Intracerebroventricular administration of Shiga toxin type 2 altered neuroglial ultrastructures and changed the expression levels of nitric oxide synthase and glial fibrillary acidic protein

GOLDSTEIN J; PISTONE CREYDT V; LOIDL F; LOPEZ-COSTA J; BOCCOLI J; IBARRA C

Melbourne, Australia

Congreso; 6th International Symposium on Shiga Toxin (Verocytotoxin) – producing Escherichia coli infections; 2006

Shiga toxin (Stx) is the main cause of hemorrhagic colitis which may derive to Hemolytic Uremic Syndrome (HUS). Argentina is the first country with 400 new cases a year and 6 % of these cases derive to neurological injuries. The study of cellular metabolic disbalances produced by the toxin on affected brains is scarce. Thus we asked how Stx altered neuroglial ultrastructure. A recombinant Stx type 2 obtained by affinity chromatography was analyzed by Western Blot. Two eluted sample containing Stx2 caused a 50% fall of Vero cell viability (0.01 and 100 pg/ml) that was reversed by preincubating the toxin with an anti-Stx2B antibody. For the first time we intracerebroventricularly (icv) microinfused a Stx2 elution in corpus striatum brains of 250-300g SD rats. After 8 days the animals were anaesthetized and perfused with fixative solutions for electron, light and immunofluorescence microscopies. Immunoelectron microscopy images showed clustered DAB deposits of Stx2 on axon membranes. Neurodegeneration associated with apoptosis was observed by nuclear lateralization, fragmentation, and electrodense chromatin. Cytoplasmic edema and gliosis in astrocytes and unmyelinated oligodendrocytes were also observed. Next, significant decreases of nitric oxide synthase (NOS) positive neurons and NOS activity (p<0.01) in the corpus striatum and cerebral cortex were observed. An opposite effect was found in the hypothalamic paraventricular nucleus (p<0.01). Finally confocal miscrocopy images showed an increase in the expression of astrocytic glial fibrillary acidic protein (GFAP) that contacted positive Stx2 containing neurons. Colocalization of GFAP expression and Stx2 binding in astrocytes was also observed. We concluded that the icv microinfusion technique resulted original to study the direct action of Stx2 to selected cells of different brain areas, discarding possible systemic secondary effects. Neuronal and glial ultrastructural damage was associated with the observed pathological changes for NOS expression and activity and GFAP expression caused by direct Stx2 administration.