Aims/hypothesis Identification of a pancreatic neuro-insular network in mice suggests that a similar integration of islets and nerves may be present in the human pancreas. To characterise the neuro-insular network and the intra-pancreatic ganglia in a clinically related setting, we examined human pancreases in health and with fatty infiltration via 3-dimensional (3D) histology and compared the human pancreatic microenvironment with its counterpart in mice. Methods Human pancreatic specimens from individuals with normal BMI, high BMI (≥ 25) and type 2 diabetes were used to investigate the neuro-insular network. Transparent specimens were prepared by tissue clearing for transmitted light and deeptissue fluorescence imaging to simultaneously visualise infiltrated adipocytes, islets and neurovascular networks. Results High-definition images of human islets reveal that both the sympathetic and parasympathetic nerves enter the islet core and reside in the immediate microenvironment of islet cells. Around the islets, the neuro-insular network is visualised with 3D histology to identify the intra-pancreatic ganglia (peri-lobular and intra-parenchymal ganglia) and the islet-ganglionic association. In humans, but not in mice, pancreatic fatty infiltration (BMI dependent) features adipocytes infiltrating into the parenchyma and accumulating in the perilobular space, in which the peri-lobular ganglia also reside. We identified the formation of adipose-ganglionic complexes in the peri-lobular space and enlargement of ganglia around adipocytes. In the specimen from the individual with type 2 diabetes, an increase in the number of nerve projections from the intra-parenchymal ganglia is associated with severe fatty infiltration. Conclusions/interpretation We present new perspectives of human pancreas and islet innervation via 3D histology. Our results strongly suggest that fatty infiltration in the human pancreas creates a neurotrophic microenvironment and promotes remodelling of pancreatic innervation.

Aims/hypothesis Islet Schwann (glial) cells and pericytes are the microorgan's accessory cells positioned at the external and internal boundaries facing the exocrine pancreas and endothelium, respectively, adjacent to the endocrine cells. Plasticity of glial cells and pericytes is shown in the glial scar formation after injury to the central nervous system. It remains unclear whether similar reactive cellular responses occur in insulitis. We applied three-dimensional (3D) histology to perform qualitative and quantitative analyses of the islet Schwann cell network and pericytes in normal, streptozotocin-injected (positive control of gliosis) and NOD mouse models. Methods Vessel painting paired with immunostaining of mouse pancreatic tissue was used to reveal the islet Schwann cells and pericytes and their association with vasculature. Transparent islet specimens were prepared by optical clearing to facilitate 3D confocal microscopy for panoramic visualisation of the tissue networks.Results In-depth microscopy showed that the islet Schwann cell network extends from the peri-islet domain into the core. One week after streptozotocin injection, we observed intraislet perivascular gliosis and an increase in pericyte density. In early/moderate insulitis in the NOD mice, perilesional gliosis occurred at the front of the lymphocytic infiltration with atypical islet Schwann cell morphologies, including excessive branching and perivascular gliosis. Meanwhile, pericytes aggregated on the walls of the feeding arteriole at the peri-and intralesional domains with a marked increase in surface marker density. Conclusions/interpretationThe reactive cellular responses demonstrate plasticity and suggest a stop-gap mechanism consisting of the Schwann cells and pericytes in association with the islet lesion and vasculature when injury occurs.

Our work provides insight into the neuro-insular integration at the organ level and demonstrates a new approach for investigating previously unknown details of the pancreatic tissue network in health and disease.

Because of the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and three-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with micrometer-level resolution. Four examples are given: 1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intraganglionic at myenteric plexus; 2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface; 3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts; and 4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.

We have established a useful approach for 3-D imaging, panoramic illustration, and quantitation of the enteric glia in the human colon mucosa to help characterize their roles with mucosal components in health and disease.