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Wnt-signaling is a key regulator of stem cell homeostasis, extensively studied in the intestinal crypt and other metazoan tissues. Yet, there is hardly any data available on the presence of Wnt-signaling components in the adult enteric nervous system (ENS) . Therefore, we employed RNAscope HiPlex-assay, a novel and more sensitive hybridization technology. By amplifying target specific signals, this technique enables the detection of low abundance, tightly regulated RNA content as is the case for Wnt-signaling components. Additionally, we compared our data to previously published physiological single cell RNA and RiboTag-based RNA sequencing analyses of enteric gliosis using data-mining approaches. Our descriptive analysis shows that several components of the multidi-mensional regulatory network of the Wnt-signaling pathway are present in the murine ENS. The transport and secretion protein for Wnt-ligands Wntless as well as canonical (Wnt3a and Wnt2b) and non-canonical Wnt-ligands (Wnt5a, Wnt7a, Wnt8b and Wnt11) are detectable within submucosal and myenteric plexus. Further, corresponding Frizzled receptors (Fzd1, Fzd3, Fzd6, and Fzd7) and regulatory signaling mediators like R-Spondin/DKK ligands are present in the ENS of the small and large intestine. Further, data mining approaches revealed, that several Wnt-related molecules are expressed by enteric glial cell clusters and are dynamically regulated during the inflammatory manifestation of enteric gliosis. Our results suggest, that canonical and non-canonical Wnt-signaling has a much broader impact on the mature ENS and its cellular homeostasis in health and inflammation, than previously anticipated.

Background Braak’s hypothesis states that sporadic Parkinson’s disease (PD) follows a specific progression of pathology from the peripheral to the central nervous system, and this progression can be monitored by detecting the accumulation of alpha-Synuclein (α-Syn) protein. Consequently, there is growing interest in understanding how the gut (commensal) microbiome can regulate α-Syn accumulation, as this could potentially lead to PD. Methods We used 16S rRNA and shotgun sequencing to characterise microbial diversity. 1 H-NMR was employed to understand the metabolite production and intestinal inflammation estimated using ELISA and RNA-sequencing from feces and the intestinal epithelial layer respectively. The Na + channel current and gut permeability were measured using an Ussing chamber. Immunohistochemistry and immunofluorescence imaging were applied to detect the α-Syn protein. LC-MS/MS was used for characterization of proteins from metabolite treated neuronal cells. Finally, Metascape and Ingenuity Pathway Analysis (IPA) bioinformatics tools were used for identification of dysregulated pathways. Results We studied a transgenic (TG) rat model overexpressing the human SNCA gene and found that a progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young TG rats. Interestingly, this ratio then increased with ageing. The dynamics of Lactobacillus and Alistipes were monitored and reduced Lactobacillus and increased Alistipes abundance was discerned in ageing TG rats. Additionally, the SNCA gene overexpression resulted in gut α-Syn protein expression and increased with advanced age. Further, older TG animals had increased intestinal inflammation, decreased Na + current and a robust alteration in metabolite production characterized by the increase of succinate levels in feces and serum. Manipulation of the gut bacteria by short-term antibiotic cocktail treatment revealed a complete loss of short-chain fatty acids and a reduction in succinate levels. Although antibiotic cocktail treatment did not change α-Syn expression in the enteric nervous system of the colon, however, reduced α-Syn expression was detected in the olfactory bulbs (forebrain) of the TG rats. Conclusion Our data emphasize that the gut microbiome dysbiosis synchronous with ageing leads to a specific alteration of gut metabolites and can be modulated by antibiotics which may affect PD pathology.

Evidence is mounting that the novel corona virus SARS-CoV2 inflicts neurological symptoms in a subgroup of COVID-19 patients. While plenty of theories on the route of neuroinvasion have been proposed, little histological evidence has been presented supporting any of these hypotheses. Therefore, we carried out immunostainings for ACE2 and TMPRSS2, two proteinases crucial for the entry of SARS-CoV2 into host cells, in the human enteric nervous system (ENS), as well as in the choroid plexus of the lateral ventricles. Both of these sites are important, yet often neglected entry gates to the nervous system. We found that ACE2 and TMPRSS2 are expressed by enteric neurons and glial cells of the small and large intestine, as well as choroid plexus epithelial cells, indicating that these cells meet the molecular requirements for viral entry. Together, our results are fundamental histological evidence substantiating current theories of neuroinvasion by SARS-CoV2.

Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet- and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain’s Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis and contribute to the technical discussion of ultrastructural tissue-retention.

The choroid plexus (CP) is a structure in the brain ventricles that produces the main part of the cerebrospinal fluid (CSF). It is covered with specialized cells which show epithelial characteristics and are the site of the blood–CSF barrier. These cells form a contiguous cell sheet with ventricle-lining ependymal cells which are known to express aquaporin-4 (AQP4). In contrast, CP epithelial cells express aquaporin-1 (AQP1) apically. We investigated the expression patterns of aquaporins in the CP-ependyma transition from human body donors using immunofluorescence and electron microscopy. Ependymal cells and subependymal astrocytes at the base of the CP showed a particularly high AQP4 immunoreactivity. Astrocytic processes formed a dense meshwork or glial plate around the blood vessels entering the CP. Interestingly, some of these astrocytic processes were in direct contact with the CP stroma, which contains fenestrated blood vessels, separated only by a basal lamina. Electron microscopy confirmed the continuity of the subastrocytic basal lamina with the CP epithelium. We also probed for components of the AQP4 anchoring dystrophin–dystroglycan complex. Immunolabeling for dystrophin and AQP4 showed an overlapping staining pattern in the glial plate but not in previously reported AQP4-positive CP epithelial cells. In contrast, dystroglycan expression was associated with laminin staining in the glial plate and the CP epithelium. This suggests different mechanisms for AQP4 anchoring in the cell membrane. The high AQP4 density in the connecting glial plate might facilitate the transport of water in and out of the CP stroma and could possibly serve as a drainage and clearing pathway for metabolites.

Neurogenesis in the postnatal enteric nervous system (ENS) is controversially discussed. Yet, deciphering the regenerative potential of the ENS is essential for our understanding and therapy of human enteric neuropathies. Dickkopf1 (DKK1) is a Wnt-antagonist and involved in the homeostasis of various tissues. We hypothesize that DKK1 could function as a negative regulator on the proliferation of ENS-progenitors in the postnatal gut of mice and human infants. Here, we provide evidence that DKK1 is expressed in the murine and human ENS. If applied to ENS-progenitors in vitro, DKK1 leads to an increased proliferation, however, followed by extensive apoptosis. Yet, once we block apoptosis, DKK1-stimulation markedly increases enteric neurogenesis in murine and human ENS-progenitors. Thus, DKK1 is a strong, ambivalent regulator of the ENS-progenitor cell pool in mice and humans. These results are fundamental steps to reshaping our understanding of the homeostasis of the ENS in health and disease. The Wnt-antagonist Dickkopf1 (DKK1) is an ambivalent regulator of progenitors of the murine and human enteric nervous system. It induces enteric neurogenesis and gliogenesis if its apoptotic function is blocked.

Traditional anatomical preparations as they are often found in university collections are both historical treasure and moral responsibility of the hosting institutions. These remains not only represent the tradition and history of anatomical science and education, but above all are the remains of human beings and thus should be treated with dignity. While the discussion on public display of historical human remains is ongoing, the responsibility to keep them in an optimal state of preservation is not in question. However, most time-honored anatomical collections do or did not have the financial and/or technical capacity to keep these high standards, underlining the need for exchange of technical know-how between anatomical institutes and the support of restoration/conservation professionals. Here, we present a state-of-the-art conservation and restoration of a fetal natural skeleton from the early twentieth century along with the professional documentation of restoration-measures. Therefore, we used modern photo- and video documentation, including photogrammetry, as well as UV- and x-ray-examinations to record historical and new restoration procedures. We carried out measures to reconstitute the structural stability of the skeleton. Moreover, we replaced lost parts of the left foot skeleton, and critically discuss the legitimacy of body part replacement in historical specimens. We are convinced, that similar cases of insufficiently preserved specimens can be found in most anatomical collections. Therefore, this article is not only intended to document the state-of-the-art conservation and restoration of a natural skeleton, but also serve as an inspiration for similar campaigns in other institutions in the future.

Anatomical dissection courses are central to medical education. Despite advances in fixation techniques, cadavers remain vulnerable to microbial contamination. Thus, we aimed to assess microbial entry pathways and discuss mitigation strategies in a dissection course setting. Microbial load assessments were performed using sedimentation and contact plate sampling at various points, including doorknobs, gown sleeves, and air inside the dissection hall. Airborne microbial loads were significantly lower under laminar airflow systems compared to areas without controlled air supply, particularly near sinks and entryways (median 17.0 vs. 51.0 CFUs/4 h). The airborne microbial load was significantly lower during unoccupied times compared to dissection hours (median 2.5 vs. 44.0 CFUs/4 h). The dissection hall doorknob showed increased microbial counts after use on 4 of 6 days, though levels remained below critical thresholds. Gown sleeves had a variable but overall low microbial load. Our study suggests that controlled air supply is a key factor for reduced microbial loads. In contrast, the risk of microbial transmission via the doorknobs is negligible. The microbial load on gown sleeves was comparable to that of clean hospital laundry. Future studies should monitor microbial loads on cadaver surfaces to clarify contamination dynamics and refine hygiene protocols.

The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74–91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

PurposeCompromised swallowing, speaking, and local complications are the major disadvantages of established approaches to the posterior tongue and oropharynx. The mandibular split involves an esthetically unpleasant bipartition of the lower lip and is prone to bony non-union or sequestration. The conventional pull-through technique on the other hand lacks the secure reattachment of the lingually released soft tissues.MethodsThe feasibility of a new modified pull-through approach was tested on three anatomical specimens. CAD/CAM cutting guides were used to design a retentive bone flap to properly refixate the genioglossus and geniohyoid muscles after the procedure. The radiographic assessment and treatment planning was performed on 12 cadavers. The entire procedure was tested surgically via dissection in three of those cases. This procedure was then applied in a clinical case.ResultsPrecise repositioning and dynamic compression of bony segments was possible reproducibly and without injury to adjacent structures. In all dissected cases, a median lingual foramen was found and in two cases vessels entering it could be dissected Radiologic anatomical landmarks were sufficient in all 12 cases to perform the clinical planning procedure. Clinically, the osteotomized segment demonstrated good blood supply and plateless repositioning was verified postoperatively via cone beam scan.ConclusionThe method presented is safe and easy to perform. Individual cutting guides improve the safety and accuracy of the procedure, potentially eliminating the need for osteosynthesis. We provide the anatomical and radiologic basis for clinical evaluation of this pedicled bone flap procedure and present the clinical application of this modified pull-through approach.