Distribution of Gb3 Immunoreactivity in the Mouse Central Nervous System

We have shown previously that neurons in the mouse spinal cord express Gb3. We show in this article that distribution of anti-Gb3-Ab reactivity occurs in many different types of neurons of different areas of the central nervous system (CNS). The immunoreactive neurons are in olfactory bulbs, cerebral cortex, hippocampus, striatum, amygdala, thalamus, hypothalamus, cerebellum, and medulla oblongata. In several different circumventricular organs where vessels do not have the blood-brain-barrier (BBB) structure, anti-Gb3-Ab is not positive for vessel structures, while neurons at these regions are positive. Also, within the ventricular area, ependymal cells in the third ventricle express Gb3, as revealed by anti-Gb3-Ab staining and intensity analysis.


Introduction
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 (Gb 3 ) is a known receptor of Shiga toxin (Stx), which is central to the intoxication and disease process [14]. It has been shown that a Gb 3 knockout mouse is resistant to Stx [15]. To understand target components within the CNS, determining which cell types express Gb 3 is essential. Previously, we reported that in the mouse CNS, Shiga toxin-2 acts on spinal cord neurons OPEN ACCESS which express Gb 3 , and leads to hindlimb paralysis [16]. Other mouse CNS cell types expressing Gb 3 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 Gb 3 , it may increase the chance of being the primary target in the CNS. In this article, Gb 3 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 Gb 3 , this also could be an entry point of Stx into the CNS parenchyma.

Animals
Specific pathogen-free C57BL/6 mice, male, 20-22 g body weight (b.w.) were purchased from Charles River (Wilmington, MA, USA). Mice were given food and water ad libitum. All procedures were approved by the University of Maryland School of Medicine Animal Care and Use Committee. A total of 5 mice were used in this study.

Tissue Harvesting
Mice were euthanized by CO 2 inhalation. Two mice were perfused with 20 mL saline, followed by 20 mL 4% paraformaldehyde/phosphate buffered saline (4% PFA/PBS). Brains were marked for the Bregma position (the crossing point of the coronal suture and the sagittal suture on the skull) with a knife incision. Brains and spinal cords were harvested, and further fixed in 4% PFA/PBS overnight at room temperature. Brains and spinal cords from 3 mice were fixed in the same manner without perfusion. Brains were trimmed to 2 mm thickness from the Bregma to both rostral and caudal ends. Spinal cords were trimmed into cervical, thoracic and lumbar segments. After incubating in 30% sucrose/PBS at 4 °C overnight, trimmed segments were sectioned to 50 m thickness using a sliding microtome (SM2000R, Leica Microsystems, Bannockburn, IL, USA). The positions of brain sections from the Bregma was determined with reference to a C57BL/6 brain atlas [23]. Sections were collected and held in PBS at 4 °C until use.

Intensity Analysis of Anti-Gb 3 Immunofluorescence
For line profile intensity analysis, a LSM510 software line profile function was used. Within one image, a fixed length line was used to collect intensity data. Three intensity samples of neuronal and ependymal cytoplasm were collected per image. Appearance of high intensity of 2000 or higher was expressed as percentage of total data collected from the line profile. The VL, V3 and central canal regions were analyzed. Six images from each area were taken randomly from 3 mice, which were selected from a total of 5 mice (either perfuse-fixed or none perfuse-fixed). Averages of high intensity (%) between neurons and ependymal cells were compared. For area intensity analysis, Image-Pro Plus software (MediaCybernetics, Inc., Silver Spring, MD, USA) was used. The image acquired by LSM510 was saved as Tagged Image File Format (TIFF) in RGB, and only green channel (Gb 3 -AlexaFluor488) was retained in the image using Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA). The TIFF image was converted to Gray scale, and a fixed area (m 2 ) of region of interest (ROI) was made using Image-Pro Plus. The intensity range higher than background pixels was chosen, and 3 ROI areas within neuronal or ependymal cytoplasm per image were taken as intensity samples. The equivalent regions and number of samples comparable to those used in the Line profile intensity analysis were analyzed by the area intensity analysis method. Averages of ROI areas between neurons and ependymal cells were compared.

Statistics
Data from line profile or area intensity analysis were analyzed by student t-test (paired two-tail test), and p-values less than 0.01 were determined as significant.

Results and Discussion
Anti-Gb 3 -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 Gb 3 positive (Figure 1). In the cerebrum, neurons in the cortex, including motor cortex were also Gb 3 positive (Figure 2a). Astrocytes in the corpus callosum which is close to the cerebral cortex were Gb 3 negative (Figure 2b,c). Hippocampus neurons, CA1, 2, 3 and dentate gyrus, were Gb 3 positive (Figure 2e). Also, the neurons in the striatum (Figure 2b    CVO areas in the mouse CNS were also tested for anti-Gb 3 -Ab reactivity. Within the tested CVO areas, none appeared to have a Gb 3 expressing vessel structure. In contrast, neurons of these areas were positive for Gb 3 . In the OVLT, neurons which have characteristic large nuclei, were shown to be Gb 3 positive (Figure 4a). In Figure 4c, the lining of V3 consisting of ependymal cells appears positive for Gb 3 . In Figure 4e, neurons in the SFO, as well as MH are Gb 3 positive, while the choroid plexus is Gb 3 negative. In Figure 4g, neurons in the medulla oblongata are Gb 3 positive including the AP area. Within this image, a central canal positioned in the center, appears very dim.
Ependymal cells were tested as a Gb 3 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 (m 2

Conclusions
Mouse CNS neurons from various areas exhibited anti-Gb 3 -Ab reactivity. Ependymal cells at V3 were also found to be positive for anti-Gb 3 -Ab reactivity. However, vessels at the CVO or other areas of the mouse CNS did not exhibit anti-Gb 3 reactivity.