Material and Methods NIH mice (20-25g) from both sexes were subjected to i.v. s-l administration of Stx2 (1/3500; DL 50 =1/1500), Stx2+LPS (s-l 1/5000; DL 50 =1/3000), LPS from purified wild type Stx2 (s-l 1/5000) or vehicle for morphologic and behavioral experiments. They were anesthetized, perfused and their brains processed for confocal (binding of biotinylated lectins to study the changes of the microvasculature profile (number and size of microvessels) and Fluorojade-B to determine neurodegeneration) or transmission electron microscopy (TEM). Another group of mice were subjected to the behavioral Shirpa (standard neurologic test), Object recognition (memory) and inclined plane (motor and balance) tests. NEUROVASCULAR AND BEHAVIORAL CHANGES AFTER SUB-LETHAL ADMINISTRATION OF INTRAVENOUS STX2 AND/OR LPS IN MOUSE BRAINS Carla Tironi-Farinati 1, Maria Laura Cejudo 1, Alipio Pinto 1, Guilherme Monteiro-Gomes 2, Patricia Geoghegan 3, Adriana Cangelosi 3, Mariana Jacobsen 1, Luciana D’ Alessio 4, C. Fabián Loidl 5, Maribel Rubin 2, Jorge Goldstein 1. 1 Laboratorio de Neurofisiopatología, Dep. Fisiología, Fac. de Medicina, Universidad de Buenos Aires, Argentina. 2 Universidad Federal De Santa Maria, Dept. de Neurofarmacologia, Santa Maria, Brazil 3 CNCBB, ANLIS, Malbrán. 4 Hospital Ramos Mejía, Buenos Aires, Argentina. 5 Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis”, Fac.de Medicina, Universidad Buenos Aires, Argentina. Gb3 Stx2 Hoescht Merge Vehicle Results from SHIRPA screen that show differences between treated animals with Stx 2 or Stx 2 +LPS (n=6) vs. vehicle ones (n=6) at 24 or 48 hs post- inyection. Spontaneous activity level gives information about activity and motor function; air puff startle response about autonomic function; finger approach, suspended hindlimb splay and horizontal rotation response about sensorimotor function; catalepsy and negative geotaxis about neuromuscular function. NN 24 hs 48 hs Introduction and Objectives The CNS is usually affected in patients that suffer from hemorrhagic colitis and HUS by STEC infection. We previously observed that intracerebroventricular Stx2 causes neuronal and astrocytic damage, and an increased neuronal Gb 3 receptor in rat brains. The study of pathological changes that occur throughout the neurovascular unit by Stx2 including LPS from EHEC should be considered. The objective of the present work is to determine whether the sub-lethal (s-l) administration of intravenous (i.v.) Stx2 or Stx2 with LPS changes i) the brain microvasculature profile, ii) the ultrastructure of neurovascular cells, and/or iii) a set of behavioral tests. Results C B Mice lost weight since 24 hs post-injection of a lethal dose of Stx 2 or Stx 2 +LPS (A). Increased levels of blood urea (B) and creatinine (C) were observed 48 hs post-injection of a lethal dose of Stx 2+ LPS, but only creatinine levels were augmented when animals were treated with a lethal dose of Stx 2. * vs. vehicle p<0,05 ** vs. 24 hs p<0,05 A Changes in body weight, blood urea and creatinine The area observed in this study is located in the mouse hippocampus (A and B). Confocal immunofluorescence micrographs show that the ev injection of a sublethal dose of Stx 2 increased the area and the intensity of the microvessels inmunolabeled with LEA at 24 and 48 hs post injection (D and E respectively) compared to the mice injected with the vehicle (C). Cuantification was made using the Image J. software. (F, G, and H). * p< 0.05 H G F C D E A B An increase in the microvasculature profile is determined after 2 days of iv s-l Stx2 in the hippocampus An increase in the microvasculature profile is determined after 2 days of iv s-l Stx2 in the striatum **** * * * * **** * * * **** * * * * Comparation of different treatments on day 4. Number of microvessels, Area of ​​ microvessels, and fluorescence intensity. Results showed the significant increase in the number, area and fluorescent intensity of the microvasculature of LPS, stx2 and Stx2+LPS compared with vehicle *(p<0,05). And a significant decline is shown in those treated with LPS and Stx2+LPS compared with Stx2 **(p< 0.05). Veh.LPSStx2 Stx2+LPS Analysis by confocal miscroscope of brain striatal cryostat sections treated with iv vehicle, LPS, Stx2 and Stx2+LPS, after 4 days post-injection. The microphotographs are reproduced by the ImageJ softtware (inlet) to assay for differences in the number, area and intensity of staining in the cerebral microvasculature between the different treatments with LEA. 4 days Analysis by confocal miscroscope of brain striatal cryostat sections treated with iv vehicle, LPS, Stx2 and Stx2+LPS, after 7 days post-injection. The microphotographs are reproduced by the ImageJ softtware (inlet) to assay for differences in the number, area and intensity of staining in the cerebral microvasculature between the different treatments with LEA. Stx2+LPSVeh.LPSStx2 7 days * * * * * * * * * ** * ** * Comparation of different treatments on day 7. Number of microvessels (A) Area of microvessels (B) and fluorescence intensity (C). Results showed a significant increase in the number of microvessels between Stx2 and Stx2+LPS compared with the control and treated only with LPS *(P<0,05). It can also be observed a significant increase in the area represented by the microvessels of Stx2+LPS and the other treatments *(p<0,05), as well as an increase in fluorescence intensity between Stx2, Stx2+LPS compared with other treatments *(p<0,05), and Stx2+LPS also compared with other treatments **(p<0,05). Conclusions The i.v. s-l Stx2 is enough to change the microvasculature starting at 2 days. This correlates well with the cell damage revealed by Fluorojade-B and TEM, and with the observed behavioral dysfunctions (memory, motor and balance). LPS exacerbates the microvasculature profile done by Stx2. The microvasculature profile could be a suitable marker to study the neuropathogenic condition by STEC in animal models and in patients. Neurodegeneration is confirmed by the histofluorescent Fluorojade-B method in neurons of striatum (a, d). The histofluorescent Fluorojade-B method is negative in striatal brain areas treated with i.v. vehicle (c). The number of neurons stain with the Fluorojade-B is higher than the neurons stain with the vehicle and this is statistically significant (p<0.05). Scale bar in c applies to micrograph d. Administration of i.v. Stx2 causes neurodegeneration Administration of i.v. Stx2 causes ultrastructural alterations in neurons and in the blood brain barrier I.v. administration of Stx2 causes neuronal alterations in dorsal striatum of mice brains. An electron micrograph shows a neuron with edema that loses its regular nuclear shape (b). This is not observed in the same striatal neurons when injected i.v. with a vehicle (saline) (a). In another micrograph it can be observed in higher magnification disorganized endoplasmic reticulum in the cytoplasm with edema (c). Another electron micrograph shows a neuron that loses its regular nuclear shape; this nuclear form suggests fragmentation and it may lead to an apoptosis state (d). Affected neuronal nuclei by i.v. Stx2 are also observed with contrasted and irregular shape and no apparent surrounded cytoplasm (e). These features are not observed in striatal neurons of the vehicle (a). Scale bar in a applies to micrographs b, d and e. A 3 um I.v. administration of Stx2 causes ultrastructural alterations at the BBB level in dorsal striatum of mice brains. An electron micrograph shows a conserve endothelial cell that forms a microvessel after the i.v. administration of the vehicle. A conserve endothelial nucleus is observed. The microvessel is surrounded by conserved dendrites and myelinated axons (a). Another electron micrograph shows, in contrast, an infarcted microvessel with perivascular (asterisk) and intracytoplasmic (arrow) edema near a neuron (N) in the endothelium following the i.v. administration of Stx2 (b). Finally another electron micrograph shows a complete collapsed microvessel in which its lumen is difficult to note (c). Scale bar in a applies to micrograph b. * The inclined plane test shows reduced motor ability and balance after 5 days (p<0.05). Cognitive object recognition test shows significant differences found between 4 and 7 days