Ndp view2

The thickness of mucus layer was measured in medium and distal colonic tissue sections using the ruler tool of the NDP view + 2.50.19 software (Hamamatsu) in both PAS and PAS-AB stained sections. 20 different measures were performed in two different tissue sections per stain in both medium and distal colon. Mucus layer thickness has been evaluated in all the samples that presented faecal pellets.

The HE stained sections were digitalized with a slide scanner (Hamamatsu Photonics Europe GmbH Herrsching, Germany) and viewed with the NDP.view 2.7.52 software (Hamamatsu Photonics). The analysis was performed with ImageJ 1.52p software (Wayne Rasband, National Institute of Health, Bethesda, MD, USA). Two representative fields of 40× magnification from the first third (initiating with the enthesis) of each supraspinatus tendon were chosen. To contrast the surrounding tissue, the tenocyte nuclei were selected manually with the freehand tool. The following parameters were analyzed: number, area, perimeter, circularity, minor and major dimension of the nuclei.

Once mounted, slides were scanned with a digital scanner NanoZoomer (HAMAMATSU, Réf: 2.0-RS: C10730-13) to obtain high resolution virtual slides. Digitalized slides were analyzed with NDP View 2.0 software (HAMAMATSU). Morphometric investigation was carried out by two observers (M. B. Delisle & J. Ceccom) to determine the numerical density of amyloid deposits and of neurons expressing SphK1 or SPL at two levels: negative or mild (+) and strong (++) among the different cortical layers. Columns constituted of contiguous microscopic fields, from the pial surface to the white matter were drawn on each slide. As the fields were examined at a magnification of x400, each field was 300 μM × 150 μM in size. As the thickness of the cortex appeared to be variable between the different sections, after the counting step, the columns were standardized to 10 fields [43 (link)]. Field 1 corresponded to the cortex immediately under the pial surface and field 10 reached the white matter (Additional file 1). In each field, the number of profiles of Aβ deposits, of neurons and of neurons expressing low level (+) and high level (++) of SphK1 and of SPL was counted and reported on a database. For Aβ deposits, focal and diffuse plaques were recorded separately according to published discriminating features [1 (link)].

Within 30 min after the last session of electrical stimulation, the rats were sacrificed, and bladders were immediately dissected. The specimen of the anterior bladder wall near the bladder base at the level of the trigone was obtained (8- to 10-mm width, 3- to 5-mm height), and the vesical fascia was preserved to minimize the damage on the integrity of the wall. Bladder tissues of 1 mm³ were immersed in 3% cacodylate buffered glutaraldehyde (0.1 M, pH 7.2) for transmission electron microscopy (TEM). Other specimens were fixed in 10% formaldehyde solution, dehydrated in alcohol, diaphanized in xylene, and embedded in paraffin. In order to get a comparable histological morphology and minimize the error and bias, the tissues' direction was adjusted to the same. Subsequently, 3-μm tissue sections were cut from these paraffin blocks. All of the bladder specimens were stained using H&E staining and Masson's trichrome. Images were captured using a NanoZoomer-S360 digital slide scanner (Hamamatsu, Hamamatsu City, Japan) with a 40× lens, and the quality of the images was manually checked before application of the digital algorithm. Morphometric analyses were performed using NDP.view 2.0 software (Hamamatsu, Hamamatsu City, Japan) and ImageJ software (National Institutes of Health, Bethesda, Maryland) by 2 authors (TC and BX).