In Shiga-toxin producing Escherichia coli (STEC) infections, a broad spectrum of central nervous system (CNS) symptoms occurs (abbreviations used in this article are listed in Table 1). Those symptoms include cortical blindness, poor fine-motor coordination, seizures and coma [1,2,3,4,5,6,7,8,9,10,11,12,13]. Globotriaosylceramide (Gb₃) is a known receptor of Shiga toxin (Stx), which is central to the intoxication and disease process [14]. It has been shown that a Gb₃ knockout mouse is resistant to Stx [15]. To understand target components within the CNS, determining which cell types express Gb₃ is essential. Previously, we reported that in the mouse CNS, Shiga toxin-2 acts on spinal cord neurons which express Gb₃, and leads to hindlimb paralysis [16]. Other mouse CNS cell types expressing Gb₃ have not been described in detail.

The trafficking route of Stx into the CNS is as important as determining its target. In human STEC patients’ brain magnetic resonance imaging (MRI), regions as the basal ganglia and also thalamus, cerebellum and brain stem, are found positive for increased permeability of fluid [17,18,19,20]. In a rabbit model, MRI showed enhanced permeability in the area surrounding V3 after Stx injections [21]. However, precise Stx trafficking routes and the mechanisms involved are still in question. Circumventricluar organs (CVO) are known to be devoid of the blood-brain-barrier (BBB), thus exchange of substances between the plasma and the CNS parenchyma is relatively easy [22]. The CVO is situated around the V3 (OVLT, SFO, ME, posterior pituitary, pineal gland and SCO) as well as the V4 (AP). Also, the choroid plexus located at both V3 and V4, is sometimes considered as the CVO. If the vessels at the CVO are expressing Gb₃, it may increase the chance of being the primary target in the CNS. In this article, Gb₃ expression in the CVO is addressed. Ependymal cells form a lining of the ventricle, which separates cerebro-spinal fluid (CSF) and parenchyma. As the choroid plexus makes CSF from serum and secretes it into the ventricles, there is a possibility of Stx2 in serum being transferred to the ventricle. If ependymal cells express Gb₃, this also could be an entry point of Stx into the CNS parenchyma.

Anti-Gb₃-Ab reactive neurons were seen throughout the mouse CNS. In the olfactory bulb, where Mitral cells accept input from nasal epithelium cells to sense smell, and interneurons such as periglomerular cells and granule cells reside, all neurons were Gb₃ positive (Figure 1). In the cerebrum, neurons in the cortex, including motor cortex were also Gb₃ positive (Figure 2a). Astrocytes in the corpus callosum which is close to the cerebral cortex were Gb₃ negative (Figure 2b,c). Hippocampus neurons, CA1, 2, 3 and dentate gyrus, were Gb₃ positive (Figure 2e). Also, the neurons in the striatum (Figure 2b), amygdala (Figure 4i), thalamus (Figure 4e) and hypothalamus were Gb₃ positive. The hypothalamic nuclei (ARH) neurons are shown to be Gb₃ positive in Figure 4c. In the cerebellum, Perkinje cells as well as granule cells were also Gb₃ positive (Figure 3). Neurons throughout the mouse CNS appeared to express Gb₃, hence, depending on the CNS entry site of Stx, all these neurons could become targets of the toxin.

CVO areas in the mouse CNS were also tested for anti-Gb₃-Ab reactivity. Within the tested CVO areas, none appeared to have a Gb₃ expressing vessel structure. In contrast, neurons of these areas were positive for Gb₃. In the OVLT, neurons which have characteristic large nuclei, were shown to be Gb₃ positive (Figure 4a). In Figure 4c, the lining of V3 consisting of ependymal cells appears positive for Gb₃. In Figure 4e, neurons in the SFO, as well as MH are Gb₃ positive, while the choroid plexus is Gb₃ negative. In Figure 4g, neurons in the medulla oblongata are Gb₃ positive including the AP area. Within this image, a central canal positioned in the center, appears very dim.

Ependymal cells were tested as a Gb₃ expressing cell type and intensity analysis was performed comparing high intensity pixels in neuron and ependyma in the ventricle area as V3, VL and central canal. A square shaped region of interest (ROI) was used to detect the pixel area which is higher than background intensity. The average area (μm²) of neurons vs. ependymal cells in V3, VL and central canal was; 1.857 ± 0.039 vs. 1.773 ± 0.197 (p = 0.275), 1.567 ± 0.085 vs. 0.226 ± 0.262 (p = 0.0097), 1.7739 ± 0.085 vs. 0.137 ± 0.275 (p = 0.0014), respectively. P values less than 0.01 were considered to be statistically significant, and shown as (*) in Figure 5a. The line profile intensity analysis revealed a similar result that averages of high intensity percent of neurons vs. ependyma in V3, VL and central canal were; 79.23 ± 23.29 vs. 71.41 ± 32.03 (p = 0.289), 66.31 ± 26.46 vs. 1.156 ± 2.997 (p = 4.029E-10), 77.73 ± 30.39 vs. 4.346 ± 4.954 (p = 1.042E-08). The results are graphed in Figure 5b. In both analyses, the intensity level of Gb₃ positive neurons and ependymal cells in the V3 region was similar, thus ependymal cells in this area were Gb₃ positive. In contrast, VL and central canal ependymal cells were Gb₃ negative.

As the CVO is naturally leaky, serum Stx2 might travel through this route where vessels do not express Gb₃. As ependymal cells at V3 express Gb₃, this could serve as an entry point of Stx, if Stx enters into CSF in the mouse CNS. Trafficking of Stx in the CNS needs to be further investigated.

Mouse CNS neurons from various areas exhibited anti-Gb₃-Ab reactivity. Ependymal cells at V3 were also found to be positive for anti-Gb₃-Ab reactivity. However, vessels at the CVO or other areas of the mouse CNS did not exhibit anti-Gb₃ reactivity.