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 C.F., LOPEZ-COSTA J.J., BOCCOLI J., IBARRA C

Melbourne, Australia

Simposio; 6TH INTERNATIONAL SYMPOSIUM ON SHIGA TOXIN (VEROCYTOTOXIN) PRODUCING E.COLI INFECTIONS; 2006

Australian Society for Microbiology

Stx from STEC 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. 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 Stx2 altered neuroglial ultrastructure. A recombinant Stx2 was obtained by affinity chromatography, detected and semi-quantified by Western Blot. Two of Stx2 elutions caused a 50% fall of Vero cell viability (0.01 and 100 pg/ml). This was reversed by preincubating the toxin with an anti-Stx2B antibody. We intracerebroventricularly (icv) microinfused for the first time a Stx2 elution in corpus striatum brains of 250-300g SD rats. After 8 days the animals were anaesthetized and perfused with fixatives for electron, light or immunofluorescence microscopies. Immunoelectron microscopy images showed clustered DAB deposits of Stx2 on axon membranes. Neurodegeneration associated with apoptosis was confirmed by nuclear lateralization, fragmentation, and electrodense chromatin. Cytoplasmic edema and gliosis in astrocytes and unmyelinated oligodendrocytes were also observed. Next, a significant decrease of NOS positive neurons and NOS activity (p<0.01) were observed in the corpus striatum and cerebral cortex, while 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 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.