Human liver afferent and efferent nerves revealed by 3-D/Airyscan super-resolution imaging.

Neural regulation of hepatic metabolism has long been recognized. However, the detailed afferent and efferent innervation of the human liver has not been systematically characterized. This is largely due to the liver's high lipid and pigment contents, causing false-negative (light scattering and absorption) and false-positive (autofluorescence) results in in-depth fluorescence imaging. Here, to avoid the artifacts in three-dimensional (3-D) liver neurohistology, we embed the bleached human liver in the high-refractive-index polymer for tissue clearing and antifade 3-D/Airyscan super-resolution imaging. Importantly, using the paired substance P (SP, sensory marker) and PGP9.5 (pan-neuronal marker) labeling, we detect the sensory nerves in the portal space, featuring the SP+ varicosities in the PGP9.5+ nerve bundles/fibers, confirming the afferent liver innervation. Also, using the tyrosine hydroxylase (TH, sympathetic marker) labeling, we identify 1) condensed TH+ sympathetic nerves in the portal space, 2) extension of sympathetic nerves from the portal to the intralobular space, in which the TH+ nerve density is 2.6 ± 0.7-fold higher than that of the intralobular space in the human pancreas, and 3) the TH+ nerve fibers and varicosities contacting the ballooning cells, implicating potential sympathetic influence on hepatocytes with macrovesicular fatty change. Finally, using the vesicular acetylcholine transporter (VAChT, parasympathetic marker), PGP9.5, and CK19 (epithelial marker) labeling with panoramic-to-Airyscan super-resolution imaging, we detect and confirm the parasympathetic innervation of the septal bile duct. Overall, our labeling and 3-D/Airyscan imaging approach reveal the hepatic sensory (afferent) and sympathetic and parasympathetic (efferent) innervation, establishing a clinically related setting for high-resolution 3-D liver neurohistology.NEW & NOTEWORTHY We embed the human liver (vs. pancreas, positive control) in the high-refractive-index polymer for tissue clearing and antifade 3-D/Airyscan super-resolution neurohistology. The pancreas-liver comparison reveals: 1) sensory nerves in the hepatoportal space; 2) intralobular sympathetic innervation, including the nerve fibers and varicosities contacting the ballooning hepatocytes; and 3) parasympathetic innervation of the septal bile duct. Our results highlight the sensitivity and resolving power of 3-D/Airyscan super-resolution imaging in human liver neurohistology.

Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) with low microvessel density and fibrosis often exhibit clinical aggressiveness. Given the contribution of cancer-associated fibroblasts (CAFs) to the hypovascular fibrotic stroma in pancreatic ductal adenocarcinoma, investigating whether CAFs play a similar role in PNETs becomes imperative. In this study, we investigated the involvement of CAFs in PNETs and their effects on clinical outcomes. METHODS: We examined 79 clinical PNET specimens to evaluate the number and spatial distribution of α-smooth muscle actin (SMA)-positive cells, which are indicative of CAFs. Then, the findings were correlated with clinical outcomes. In vitro and in vivo experiments were conducted to assess the effects of CAFs (isolated from clinical specimens) on PNET metastasis and growth. Additionally, the role of the stromal-cell-derived factor 1 (SDF1)-AGR2 axis in mediating communication between CAFs and PNET cells was investigated. RESULTS: αSMA-positive and platelet-derived growth factor-α-positive CAFs were detected in the hypovascular stroma of PNET specimens. A higher abundance of α-SMA-positive CAFs within the PNET stroma was significantly associated with a higher level of clinical aggressiveness. Notably, conditioned medium from PNET cells induced an inflammatory phenotype in isolated CAFs. These CAFs promoted PNET growth and metastasis. Mechanistically, PNET cells secreted interleukin-1, which induced the secretion of SDF1 from CAFs. This cascade subsequently elevated AGR2 expression in PNETs, thereby promoting tumor growth and metastasis. The downregulation of AGR2 in PNET cells effectively suppressed the CAF-mediated promotion of PNET growth and metastasis. CONCLUSION: CAFs drive the growth and metastasis of aggressive PNETs. The CXCR4-SDF1 axis may be a target for antistromal therapy in the treatment of PNET. This study clarifies mechanisms underlying PNET aggressiveness and may guide future therapeutic interventions targeting the tumor microenvironment.

Multimodal 3-D/2-D human islet and duct imaging in exocrine and endocrine lesion environment: associated pancreas tissue remodeling.

Pancreatic intraepithelial neoplasia (PanIN) and islet cell microadenoma are exocrine and endocrine neoplasms of human pancreas that have been linked to pancreatic ductal adenocarcinoma (PDAC) and neuroendocrine tumor, respectively. However, in health and at the surgical margin of pancreatic cancer, it remains unresolved how to simultaneously characterize duct and islet remodeling to investigate the exocrine-endocrine association in the lesion microenvironment. Here, we develop a new vibratome-based approach to detect, confirm, and analyze the two types of pancreas remodeling via stereo/three-dimensional (3-D) and classic/two-dimensional (2-D) histology. Surgical margins of PDAC (n = 10, distal) and cadaveric donor pancreases (n = 10, consecutive cases) were fixed, sectioned by vibratome (350 µm), and surveyed for PanIN and microadenoma via stereomicroscopy. After lesion detection, PanIN and microadenoma were analyzed with 3-D fluorescence imaging and clinical microtome-based histology for confirmation and assessment of microenvironment. Multimodal imaging of PDAC surgical margins and cadaveric donor pancreases detected the peri-PanIN islet aggregation with duct-islet cell clusters. Organ-wide survey of cadaveric donor pancreases shows a marked 2.3-fold increase in the lesion size with the PanIN-islet association vs. without the association. In the survey, we unexpectedly detected the islet cell microadenoma adjacent to (<2 mm) PanIN. Overall, among the 53 early lesions in the cadaveric donor pancreases (PanINs and microadenomas), 81% are featured with the associated exocrine-endocrine tissue remodeling. Multimodal 3-D/2-D tissue imaging reveals local and simultaneous duct and islet remodeling in the cancer surgical margin and cadaveric donor pancreas. In the cadaveric donor pancreas, the peri-PanIN islet aggregation and PanIN-microadenoma association are two major features of pancreas remodeling in the early lesion microenvironment.NEW & NOTEWORTHY We develop a new multimodal 3-D/2-D imaging approach (matched stereomicroscopic, fluorescence, and H&E signals) to examine human duct-islet association in the PDAC surgical margin and cadaveric donor pancreas. In both conditions, peri-PanIN islet aggregation with duct-islet cell clusters was identified. The PanIN-islet cell microadenoma association was unexpectedly detected in the donor pancreas. Our work provides the technical and morphological foundations to simultaneously characterize human islets and ducts to study their association in health and disease.

Local islet remodelling associated with duct lesion-islet complex in adult human pancreas.

AIMS/HYPOTHESIS: Islets are thought to be stably present in the adult human pancreas to maintain glucose homeostasis. However, identification of the pancreatic intraepithelial neoplasia (PanIN)-islet complex in mice and the presence of PanIN lesions in adult humans suggest that similar remodelling of islet structure and environment may occur in the human pancreas. To identify islet remodelling in a clinically related setting, we examine human donor pancreases with 3D histology to detect and characterise the human PanIN-islet complex. METHODS: Cadaveric donor pancreases (26-65 years old, n = 10) were fixed and sectioned (350 µm) for tissue labelling, clearing and microscopy to detect local islet remodelling for 3D analysis of the microenvironment. The remodelled microenvironment was subsequently examined via microtome-based histology for clinical assessment. RESULTS: In nine pancreases, we identified the unique peri-lobular islet aggregation associated with the PanIN lesion (16 lesion-islet complexes detected; size: 3.18 ± 1.34 mm). Important features of the lesion-islet microenvironment include: (1) formation of intra-islet ducts, (2) acinar atrophy, (3) adipocyte association, (4) inflammation (CD45+), (5) stromal accumulation (α-SMA+), (6) increase in Ki-67 proliferation index but absence of Ki-67+ alpha/beta cells and (7) in-depth and continuous duct-islet cell contacts, forming a cluster. The duct-islet cell cluster and intra-islet ducts suggest likely islet cell neogenesis but not replication. CONCLUSIONS/INTERPRETATION: We identify local islet remodelling associated with PanIN-islet complex in the adult human pancreas. The tissue remodelling and the evidence of inflammation and stromal accumulation suggest that the PanIN-islet complex is derived from tissue repair after a local injury.

Heterogeneity and neurovascular integration of intraportally transplanted islets revealed by 3-D mouse liver histology.

Intraportal islet transplantation has been clinically applied for treatment of unstable type 1 diabetes. However, in the liver, systematic assessment of the dispersed islet grafts and the graft-hepatic integration remains difficult, even in animal models. This is due to the lack of global and in-depth analyses of the transplanted islets and their microenvironment. Here, we apply three-dimensional (3-D) mouse liver histology to investigate the islet graft microstructure, vasculature, and innervation. Streptozotocin-induced diabetic mice were used in syngeneic intraportal islet transplantation to achieve euglycemia. Optically cleared livers were prepared to enable 3-D morphological and quantitative analyses of the engrafted islets. 3-D image data reveal the clot- and plaque-like islet grafts in the liver: the former are derived from islet emboli and associated with ischemia, whereas the latter (minority) resemble the plaques on the walls of portal vessels (e.g., at the bifurcation) with mild, if any, perigraft tissue damage. Three weeks after transplantation, both types of grafts are revascularized, yet significantly more lymphatics are associated with the plaque- than clot-like grafts. Regarding the islet reinnervation, both types of grafts connect to the periportal nerve plexus and develop peri- and intragraft innervation. Specifically, the sympathetic axons and varicosities contact the α-cells, highlighting the graft-host neural integration. We present the heterogeneity of the intraportally transplanted islets and the graft-host neurovascular integration in mice. Our work provides the technical and morphological foundation for future high-definitional 3-D tissue and cellular analyses of human islet grafts in the liver.NEW & NOTEWORTHY Modern 3-D histology identifies the clot- and plaque-like islet grafts in the mouse liver, which otherwise cannot be distinguished with the standard microtome-based histology. The two types of grafts are similar in blood microvessel density and sympathetic reinnervation. Their differences, however, are their locations, severity of associated liver injury, and access to lymphatic vessels. Our work provides the technical and morphological foundation for future high-definitional 3-D tissue/cellular analyses of human islet grafts in the liver.

Adult enteric nervous system in health is maintained by a dynamic balance between neuronal apoptosis and neurogenesis.

According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Human pancreatic afferent and efferent nerves: mapping and 3-D illustration of exocrine, endocrine, and adipose innervation.

The pancreas consists of both the exocrine (acini and ducts) and endocrine (islets) compartments to participate in and regulate the body's digestive and metabolic activities. These activities are subjected to neural modulation, but characterization of the human pancreatic afferent and efferent nerves remains difficult because of the lack of three-dimensional (3-D) image data. Here we prepare transparent human donor pancreases for 3-D histology to reveal the pancreatic microstructure, vasculature, and innervation in a global and integrated fashion. The pancreatic neural network consists of the substance P (SP)-positive sensory (afferent) nerves, the vesicular acetylcholine transporter (VAChT)-positive parasympathetic (efferent) nerves, and the tyrosine hydroxylase (TH)-positive sympathetic (efferent) nerves. The SP+ afferent nerves were found residing along the basal domain of the interlobular ducts. The VAChT+ and TH+ efferent nerves were identified at the peri-acinar and perivascular spaces, which follow the blood vessels to the islets. In the intrapancreatic ganglia, the SP+ (scattered minority, ~7%) and VAChT+ neurons co-localize, suggesting a local afferent-efferent interaction. Compared with the mouse pancreas, the human pancreas differs in 1) the lack of SP+ afferent nerves in the islet, 2) the lower ganglionic density, and 3) the obvious presence of VAChT+ and TH+ nerves around the intralobular adipocytes. The latter implicates the neural influence on the pancreatic steatosis. Overall, our 3-D image data reveal the human pancreatic afferent and efferent innervation patterns and provide the anatomical foundation for future high-definition analyses of neural remodeling in human pancreatic diseases.NEW & NOTEWORTHY Modern three-dimensional (3-D) histology with multiplex optical signals identifies the afferent and efferent innervation patterns of human pancreas, which otherwise cannot be defined with standard histology. Our 3-D image data reveal the unexpected association of sensory and parasympathetic nerves/neurons in the intrapancreatic ganglia and identify the sympathetic and parasympathetic nerve contacts with the infiltrated adipocytes. The multiplex approach offers a new way to characterize the human pancreas in remodeling (e.g., fatty infiltration and duct lesion progression).

Pancreatic neuro-insular network in young mice revealed by 3D panoramic histology.

AIMS/HYPOTHESIS: It has been proposed that the neuro-insular network enables rapid, synchronised insulin secretion. However, to date, acquiring the pancreatic tissue map to study the neural network remains a challenging task as there is a lack of feasible approaches for large-scale tissue analysis at the organ level. Here, we have developed 3-dimensional (3D) panoramic histology to characterise the pancreatic neuro-insular network in young mice. METHODS: Pancreases harvested from young wild-type B6 mice (3 and 8 weeks old) and db/db mice (3 weeks old; db/db vs db/+) were used to develop 3D panoramic histology. Transparent pancreases were prepared by optical clearing to enable deep-tissue, tile-scanning microscopy for qualitative and quantitative analyses of islets and the pancreatic tissue network in space. RESULTS: 3D panoramic histology reveals the pancreatic neurovascular network and the coupling of ganglionic and islet populations via the network. This integration is identified in both 3- and 8-week-old mice, featuring the peri-arteriolar neuro-insular network and islet-ganglionic aggregation. In weaning hyperphagic db/db mice, the 3D image data identifies the associated increases in weight, adipose tissue attached to the pancreas, density of large islets (major axis > 150 µm) and pancreatic sympathetic innervation compared with db/+ mice. CONCLUSIONS/INTERPRETATION: 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.

Human pancreatic neuro-insular network in health and fatty infiltration.

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 deep-tissue 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 peri-lobular 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.

The Role of Accessory Cells in Islet Homeostasis.

PURPOSES OF REVIEW: Scattered throughout the pancreas, the endocrine islets rely on neurovascular support for signal relay to regulate hormone secretion and for maintaining tissue homeostasis. The islet accessory cells (or components) of neurovascular tissues include the endothelial cells, pericytes, smooth muscle cells, neurons (nerve fibers), and glia. Research results derived from experimental diabetes and islet transplantation indicate that the accessory cells are reactive in islet injury and can affect islet function and homeostasis in situ or in an ectopic environment. RECENT FINDINGS: Recent advances in cell labeling and tissue imaging have enabled investigation of islet accessory cells to gain insights into their network structures, functions, and remodeling in disease. It has become clear that in diabetes, the islet neurovascular tissues are not just bystanders damaged in neuropathy and vascular complications; rather, they participate in islet remodeling in response to changes in the microenvironment. Because of the fundamental differences between humans and animal models in neuroinsular cytoarchitecture and cell proliferation, examination of islet accessory cells in clinical specimens and donor pancreases warrants further attention.

mRNA destabilization improves glycemic responsiveness of transcriptionally regulated hepatic insulin gene therapy in vitro and in vivo.

BACKGROUND: Hepatic insulin gene therapy (HIGT) employing a glucose and insulin sensitive promoter to direct insulin transcription can lower blood sugars within 2 h of an intraperitoneal glucose challenge. However, post-challenge blood sugars frequently decline to below baseline. We hypothesize that this 'over-shoot' hypoglycemia results from sustained translation of long-lived transgene message, and that reducing pro-insulin message half-life will ameliorate post-challenge hypoglycemia. METHODS: We compared pro-insulin message content and insulin secretion from primary rat hepatocytes expressing insulin from either a standard construct (2xfur), or a construct producing a destabilized pro-insulin message (InsTail), following exposure to stimulating or inhibitory conditions. RESULTS: Hepatocytes transduced with a 2xfur construct accumulated pro-insulin message, and exhibited increased insulin secretion, under conditions that both inhibit or stimulate transcription. By contrast, pro-insulin message content remained stable in InsTail expressing cells, and insulin secretion increased less than 2xfur during prolonged stimulation. During transitions from stimulatory to inhibitory conditions, or vice versa, amounts of pro-insulin message changed more rapidly in InsTail expressing cells than 2xfur expressing cells. Importantly, insulin secretion increased during the transition from stimulation to inhibition in 2xfur expressing cells, although it remained unchanged in InsTail expressing cells. Use of the InsTail destabilized insulin message tended to more rapidly reduce glucose induced glycemic excursions, and limit post-load hypoglycemia in STZ-diabetic mice in vivo. CONCLUSIONS: The data obtained in the present study suggest that combining transcriptional and post-transcriptional regulatory strategies may reduce undesirable glycemic excursion in models of HIGT.

PanIN-associated pericyte, glial, and islet remodeling in mice revealed by 3D pancreatic duct lesion histology.

Pericytes and glial cells are accessory cells of neurovascular networks, which have been reported to participate in scar formation after tissue injury. However, it remains unclear whether similar reactive cellular responses occur in pancreatic intraepithelial neoplasia (PanIN). In this study we developed three-dimensional (3D) duct lesion histology to investigate PanIN and the associated pericyte, glial, and islet remodeling. Transparent mouse pancreata with a Kras(G12D) mutation were used to develop 3D duct lesion histology. Deep-tissue, tile-scanning microscopy was performed to generate panoramic views of the diseased pancreas for global examination of early stage and advanced duct lesion formation. Fluorescence signals of ductal and neurovascular networks were simultaneously detected to reveal associated remodeling. Significantly, in Kras(G12D)-mutant mice, when the low-grade PanINs emerge, duct lesions appear as epithelial buds with perilesional pericyte and glial activation. When PanINs occur in large scale (induced by cerulein injections to the mutant mice), the 3D image data identifies 1) aggregation of PanINs in clusters in space; 2) overexpression of the pericyte marker NG2 in the PanIN microenvironment; and 3) epithelial in-growth to islets, forming the PanIN-islet complexes. Particularly, the PanIN-islet complexes associate with proliferating epithelial and stromal cells and receive substantial neurovascular supplies, making them landmarks in the atrophic lobe. Overall, perilesional pericyte and glial activation and formation of the PanIN-islet complex underline cellular heterogeneity in the duct lesion microenvironment. The results also illustrate the advantage of using 3D histology to reveal previously unknown details of neurovascular and endocrine links to the disease.

Three-dimensional islet graft histology: panoramic imaging of neural plasticity in sympathetic reinnervation of transplanted islets under the kidney capsule.

Microscopic examination of transplanted islets in an ectopic environment provides information to evaluate islet engraftment, including revascularization and reinnervation. However, because of the dispersed nature of blood vessels and nerves, global visualization of the graft neurovascular network has been difficult. In this research we revealed the neurovascular network by preparing transparent mouse islet grafts under the kidney capsule with optical clearing to investigate the sympathetic reinnervation via three-dimensional confocal microscopy. Normoglycemic and streptozotocin-induced diabetic mice were used in syngeneic islet transplantation, with both groups maintaining euglycemia after transplantation. Triple staining of insulin/glucagon, blood vessels, and tyrosine hydroxylase (sympathetic marker) was used to reveal the graft microstructure, vasculature, and sympathetic innervation. Three weeks after transplantation, we observed perigraft sympathetic innervation similar to the peri-islet sympathetic innervation in the pancreas. Six weeks after transplantation, prominent intragraft, perivascular sympathetic innervation was achieved, resembling the pancreatic intraislet, perivascular sympathetic innervation in situ. Meanwhile, in diabetic recipients, a higher graft sympathetic nerve density was found compared with grafts in normoglycemic recipients, indicating the graft neural plasticity in response to the physiological difference of the recipients and the resolving power of this imaging approach. Overall, this new graft imaging method provides a useful tool to identify the islet neurovascular complex in an ectopic environment to study islet engraftment.

Plasticity of Schwann cells and pericytes in response to islet injury in mice.

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 intra-islet 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/INTERPRETATION: The 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.

3-D imaging and illustration of mouse intestinal neurovascular complex.

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.

Application of three-dimensional imaging to the intestinal crypt organoids and biopsied intestinal tissues.

Two-dimensional (2D) histopathology is the standard analytical method for intestinal biopsied tissues; however, the role of 3-dimensional (3D) imaging system in the analysis of the intestinal tissues is unclear. The 3D structure of the crypt organoids from the intestinal stem cell culture and intestinal tissues from the donors and recipients after intestinal transplantation was observed using a 3D imaging system and compared with 2D histopathology and immunohistochemistry. The crypt organoids and intestinal tissues showed well-defined 3D structures. The 3D images of the intestinal tissues with acute rejection revealed absence of villi and few crypts, which were consistent with the histopathological features. In the intestinal transplant for megacystis microcolon intestinal hypoperistalsis syndrome, the donor's intestinal tissues had well-developed nerve networks and interstitial cells of Cajal (ICCs) in the muscle layer, while the recipient's intestinal tissues had distorted nerve network and the ICCs were few and sparsely distributed, relative to those of the donor. The 3D images showed a clear spatial relationship between the microstructures of the small bowel and the features of graft rejection. In conclusion, integration of the 3D imaging and 2D histopathology provided a global view of the intestinal tissues from the transplant patients.

Potential targeting of the tumor microenvironment to improve cancer virotherapy.

In 2015, oncolytic virotherapy was approved for clinical use, and in 2017, recombinant adeno-associated virus (AAV) delivery was also approved. However, systemic administration remains challenging due to the limited number of viruses that successfully reach the target site. Although the US Food and Drug Administration (FDA) permits the use of higher doses of AAV to achieve greater rates of transduction, most AAV still accumulates in the liver, potentially leading to toxicity there and elsewhere. Targeting the tumor microenvironment is a promising strategy for cancer treatment due to the critical role of the tumor microenvironment in controlling tumor progression and influencing the response to therapies. Newly discovered evidence indicates that administration routes focusing on the tumor microenvironment can promote delivery specificity and transduction efficacy within the tumor. Here, we review approaches that involve modifying viral surface features, modulating the immune system, and targeting the physicochemical characteristics in tumor microenvironment to regulate therapeutic delivery. Targeting tumor acidosis presents advantages that can be leveraged to enhance virotherapy outcomes and to develop new therapeutic approaches that can be integrated with standard treatments.

Transparent tissue in solid state for solvent-free and antifade 3D imaging.

Optical clearing with high-refractive-index (high-n) reagents is essential for 3D tissue imaging. However, the current liquid-based clearing condition and dye environment suffer from solvent evaporation and photobleaching, causing difficulties in maintaining the tissue optical and fluorescent features. Here, using the Gladstone-Dale equation [(n-1)/density=constant] as a design concept, we develop a solid (solvent-free) high-n acrylamide-based copolymer to embed mouse and human tissues for clearing and imaging. In the solid state, the fluorescent dye-labeled tissue matrices are filled and packed with the high-n copolymer, minimizing scattering in in-depth imaging and dye fading. This transparent, liquid-free condition provides a friendly tissue and cellular environment to facilitate high/super-resolution 3D imaging, preservation, transfer, and sharing among laboratories to investigate the morphologies of interest in experimental and clinical conditions.

Oncogenic KRAS, Mucin 4, and Activin A-Mediated Fibroblast Activation Cooperate for PanIN Initiation.

Over 90% of patients with pancreatic ductal adenocarcinoma (PDAC) have oncogenic KRAS mutations. Nevertheless, mutated KRAS alone is insufficient to initiate pancreatic intraepithelial neoplasia (PanIN), the precursor of PDAC. The identities of the other factors/events required to drive PanIN formation remain elusive. Here, optic-clear 3D histology is used to analyze entire pancreases of 2-week-old Pdx1-Cre; LSL-KrasG12D/+ (KC) mice to detect the earliest emergence of PanIN and observed that the occurrence is independent of physical location. Instead, it is found that the earliest PanINs overexpress Muc4 and associate with αSMA+ fibroblasts in both transgenic mice and human specimens. Mechanistically, KrasG12D/+ pancreatic cells upregulate Muc4 through genetic alterations to increase proliferation and fibroblast recruitments via Activin A secretion and consequently enhance cell transformation for PanIN formation. Inhibition of Activin A signaling using Follistatin (FST) diminishes early PanIN-associated fibroblast recruitment, effectively curtailing PanIN initiation and growth in KC mice. These findings emphasize the vital role of interactions between oncogenic KrasG12D/+ -driven genetic alterations and induced microenvironmental changes in PanIN initiation, suggesting potential avenues for early PDAC diagnostic and management approaches.

3-D illustration of network orientations of interstitial cells of Cajal subgroups in human colon as revealed by deep-tissue imaging with optical clearing.

Morphological changes of interstitial cells of Cajal (ICC) have been proposed to characterize motility disorders. However, a global view of the network orientations of ICC subgroups has not been established to illustrate their three-dimensional (3-D) architectures in the human colon. In this research, we integrate c-kit immunostaining, 3-D microscopy with optical clearing, and image rendering to present the location-dependent network orientations with high definition. Full-depth colonic tissues were obtained from colectomies performed for nonobstructing carcinoma. Specimens of colon wall were prepared away from the tumor site. C-kit and nuclear fluorescent staining were used to identify the ICC processes and cell body. Optical clearing was used to generate transparent colon specimens, which led to panoramic visualization of the fluorescence-labeled ICC networks at the myenteric plexus (ICC-MY), longitudinal (ICC-LM) and circular (ICC-CM) muscles, and submucosal boundary (ICC-SM) up to 300 µm in depth via confocal microscopy with subcellular level resolution. We observed four distinct network patterns: 1) periganglionic ICC-MY that connect with ICC-LM and ICC-CM, 2) plexuses of ICC-LM within the longitudinal muscle and extending toward the serosa, 3) repetitive and organized ICC-CM layers running parallel to the circular muscle axis and extending toward the submucosa, and 4) a condensed ICC-SM layer lining the submucosal border. Among the four patterns, the orderly aligned ICC-CM layers provide an appropriate target for quantitation. Our results demonstrate the location-dependent network orientations of ICC subgroups and suggest a practical approach for in-depth imaging and quantitative analysis of ICC in the human colon specimen.