A translational murine model of sub-lethal intoxication with Shiga toxin 2 reveals novel ultrastructural findings in the brain striatum.

Infection by Shiga toxin-producing Escherichia coli causes hemorrhagic colitis, hemolytic uremic syndrome (HUS), acute renal failure, and also central nervous system complications in around 30% of the children affected. Besides, neurological deficits are one of the most unrepairable and untreatable outcomes of HUS. Study of the striatum is relevant because basal ganglia are one of the brain areas most commonly affected in patients that have suffered from HUS and since the deleterious effects of a sub-lethal dose of Shiga toxin have never been studied in the striatum, the purpose of this study was to attempt to simulate an infection by Shiga toxin-producing E. coli in a murine model. To this end, intravenous administration of a sub-lethal dose of Shiga toxin 2 (0.5 ηg per mouse) was used and the correlation between neurological manifestations and ultrastructural changes in striatal brain cells was studied in detail. Neurological manifestations included significant motor behavior abnormalities in spontaneous motor activity, gait, pelvic elevation and hind limb activity eight days after administration of the toxin. Transmission electron microscopy revealed that the toxin caused early perivascular edema two days after administration, as well as significant damage in astrocytes four days after administration and significant damage in neurons and oligodendrocytes eight days after administration. Interrupted synapses and mast cell extravasation were also found eight days after administration of the toxin. We thus conclude that the chronological order of events observed in the striatum could explain the neurological disorders found eight days after administration of the toxin

Intravenous administration of Stx2 causes synaptic modifications.

<p>Electron micrograph showing an apparently normal synapse (s) and pre-synaptic vesicles (v) in the striatum (A). Electron micrograph showing an increased number of pre-synaptic vesicles together with an incipient astrocytic process (*) in close proximity to a synaptic space (S) in the lower left corner (B). Electron micrograph showing full astrocytic interposition in the synaptic space (*) while the pre-synaptic terminal is full of vesicles (C). Chart illustrating the process in the synaptic space (right panel). Percentage of interrupted synapses in the striatum from Stx2-treated mice vs. vehicle-treated ones (D). An asterisk denotes statistical significance, p<0.05. Results are expressed as a percentage of the total number of synapses in an area of 3721 µm². The scale bar in A applies to all micrographs.</p

Behavioral motor test.

<p>Plotting of SHIRPA scores to standardize results of four motor behavioral tests (spontaneous motor activity, gait, pelvic elevation and hind limb activity). Arbitrary units (a.u.) are the mean summation of the four independent tests for each day and treatment (vehicle or Stx2). Normal behavior = 0; abnormal behavior = 1. The vehicle-treated group (vehicle) shows no abnormal behavior while the Stx2-treated group (Stx2) shows significant abnormal behavior (*) on day 8 of toxin treatment (p<0.05).</p

Intravenous administration of Stx2 causes neuronal damage.

<p>Conserved striatal neuron after i.v. vehicle (saline) administration (A); pale nucleus (N) and intact cytoplasm (c) and membranes (m); intact mitochondrion (arrow). After 2 days of treatment: vacuolated neuron with contrasted cytoplasm (c) and nucleus (N) (B); heterochromatin condensation (h). After 4 days: more contrasted nucleus (N) with increased heterochromatin (h) condensation and prominent indentation with loss of membranes (arrows) (C); vacuolated perinuclear mitochondrion (*) (C). Neuron with edema and loss of regular nuclear shape on day 8 (D). Disorganized endoplasmic reticulum (R) in the cytoplasm with edema (E) at higher magnification; loss of regular shape in the nucleus (F) of a neuron with edema (E); affected neuronal nucleus (N) with irregular shape and no apparent surrounded cytoplasm; (Ol, oligodendrocyte) (G). These features are absent in striatal neurons of the vehicle-treated group (A). Quantification of the percentage of damaged neurons with edema (H). Significant results are observed starting on day 4. Maximum number of neurons with edema in Stx2-treated mice observed on day 8 (*) (H). Results are expressed as a percentage of the total number of neurons in an area of 3721 µm². Data are mean ±SEM of 6–8 experiments (F and G). An asterisk denotes statistical significance, p<0.05. The scale bar in A applies to micrographs B–D and F–G.</p

Intravenous administration of Stx2 causes ultrastructural alterations at the blood brain barrier level.

<p>Electron micrograph showing a conserved endothelial cell with conserved endothelial nucleus that constitutes a microvessel from a striatal brain slice after i.v. administration of vehicle; microvessel surrounded by conserved synapses (arrows), dendrites (d) and myelinated axons (a) (A). Perivascular edema (e) 2 days after i.v. administration of Stx2 (B). After 4 days: more pronounced perivascular edema (e) (C). After 8 days: infarcted microvessel (arrow) with perivascular edema (*) near a neuron (N) (D). No cytoplasmic membrane is observed in the neuron at this stage. Quantification of the percentage of damaged microvessels by perivascular edema (E). Damage in microvessels begins to be significant on day 2 and is maximum on day 8 (*). Results are expressed as a percentage of the total number of microvessels in an area of 3721 µm². Data are mean ±SEM of 6–8 experiments (G). An asterisk denotes statistical significance, p<0.05. The scale bar in A applies to micrographs B–D.</p