Feasibility of combined upper and lower gastrointestinal endoscopic biopsy in the common marmoset (Callithrix jacchus) to evaluate gastrointestinal diseases

Background: Chronic gastroenteropathies, including gluten sensitivity and marmoset wasting syndrome, frequently occur in captive colonies of common marmosets (Callithrix jacchus). Early identification and diagnosis of affected animals are desirable. Endoscopic examination of the colon in marmosets is described, but the small intestine can harbor significant mucosal lesions not representing those in the colon. Evaluating the small intestine currently requires invasive surgical biopsies due to the small patient size, carrying a risk of severe complications. Methods: Endoscopic intubation and multisite biopsy of the duodenum/proximal jejunum are demonstrated in 10 marmosets under general anesthesia. Results: Esophagogastroduodenoscopy with colonoscopy efficiently aid in examining the gastrointestinal tract and obtaining an antemortem histologic diagnosis in marmosets with chronic gastrointestinal signs. Conclusions: This minimally invasive technique is feasible in marmosets. Future investigations into the pathogenesis of chronic gastroenteropathies will benefit from these data, leading to improved animal welfare and better individual and colony health management

Intrinsic innervation patterns of the smooth muscle in the rumen and reticulum of lambs

The rumen and reticulum of sheep serve as a fermentation chamber. Both compartments exhibit specific motility patterns. With developmental changes, the size of the reticulorumen dramatically increases when newborn lambs mature to adult sheep. This makes it possible to investigate the intrinsic innervation of the reticuloruminal muscles in lambs by taking the entire reticulum and rumen into account. The aim of the study was to analyse the projections and neurochemistry of myenteric neurons in the rumen and reticulum, which project to the inner or outer muscle layer, respectively. Therefore, we applied retrograde tracing with the fluorescent dye 1,1′-didodecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (Dil) and subsequent immunohistochemical detection of choline acetyltransferase (ChAT), substance P (SP) and vasoactive intestinal peptide (VIP). In both compartments innervation of both the inner and the outer muscle layer consisted mainly of cholinergic neurons (65–70%). The majority of them co-localized SP. The non-cholinergic neurons projecting to the muscle expressed immunoreactivity for VIP. Polarized innervation of the muscle layers was found neither in the rumen nor in the reticulum. Consequently, intrinsic innervation patterns for the smooth muscle layers in the rumen and reticulum differ from all gastrointestinal regions examined thus far

Architecture and Chemical Coding of the Inner and Outer Submucous Plexus in the Colon of Piglets

<div><p>In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm² and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm²) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.</p></div

Culture of vibrating microtome tissue slices as a 3D model in biomedical research

Abstract The basic idea behind the use of 3-dimensional (3D) tools in biomedical research is the assumption that the structures under study will perform at the best in vitro if cultivated in an environment that is as similar as possible to their natural in vivo embedding. Tissue slicing fulfills this premise optimally: it is an accessible, unexpensive, imaging-friendly, and technically rather simple procedure which largely preserves the extracellular matrix and includes all or at least most supportive cell types in the correct tissue architecture with little cellular damage. Vibrating microtomes (vibratomes) can further improve the quality of the generated slices because of the lateral, saw-like movement of the blade, which significantly reduces tissue pulling or tearing compared to a straight cut. In spite of its obvious advantages, vibrating microtome slices are rather underrepresented in the current discussion on 3D tools, which is dominated by methods as organoids, organ-on-chip and bioprinting. Here, we review the development of vibrating microtome tissue slices, the major technical features underlying its application, as well as its current use and potential advances, such as a combination with novel microfluidic culture chambers. Once fully integrated into the 3D toolbox, tissue slices may significantly contribute to decrease the use of laboratory animals and is likely to have a strong impact on basic and translational research as well as drug screening

General architecture of the ISP (A) and OSP (B) in the porcine colon.

<p>Specimens were immunohistochemically stained for the pan-neuronal marker Hu C/D. The ISP consists of a dense network composed of ganglia that are highly variable in size and shape, whereas the OSP comprises a wider meshwork consisting of significantly fewer ganglia per cm² and significantly smaller ganglia than the ISP. Scale bar = 0.5 mm.</p

Proportions of neuronal populations in tissues incubated with colchicine and untreated tissues.

<p>means ± SEM, n = number of tissues, N = number of animals. * = significant difference (p < 0.05) between treated and untreated tissues</p><p>Proportions of neuronal populations in tissues incubated with colchicine and untreated tissues.</p

Absolute numbers of immunoreactive neurons per ganglion in the ISP and OSP.

<p>means ± SEM, n = number of tissues, N = number of animals. n.s. = non-significant</p><p>Absolute numbers of immunoreactive neurons per ganglion in the ISP and OSP.</p

Relative distribution of neurochemically-defined subpopulations in the submucous plexuses of the porcine colon.

<p>Specimens of the ISP and OSP were immunohistochemically stained for ChAT, nNOS, SP, VIP and the pan-neuronal marker Hu C/D. The numbers of neurons per ganglion for the respective subpopulations are presented as mean percentage ± SEM of Hu C/D-immunoreactive neurons per ganglion. ISP versus OSP ** <i>P</i> ≤ 0.001; N = 4, n = 12. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133350#pone.0133350.s003" target="_blank">S1 Table</a> for significant differences between subpopulations within the ISP or OSP.</p