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Review
. 2021 Apr 26:12:644826.
doi: 10.3389/fendo.2021.644826. eCollection 2021.

Pancreas Optical Clearing and 3-D Microscopy in Health and Diabetes

Martha Campbell-Thompson  1 , Shiue-Cheng Tang  2
Affiliations
Review

Pancreas Optical Clearing and 3-D Microscopy in Health and Diabetes

Martha Campbell-Thompson et al. Front Endocrinol (Lausanne). .

Abstract

Although first described over a hundred years ago, tissue optical clearing is undergoing renewed interest due to numerous advances in optical clearing methods, microscopy systems, and three-dimensional (3-D) image analysis programs. These advances are advantageous for intact mouse tissues or pieces of human tissues because samples sized several millimeters can be studied. Optical clearing methods are particularly useful for studies of the neuroanatomy of the central and peripheral nervous systems and tissue vasculature or lymphatic system. Using examples from solvent- and aqueous-based optical clearing methods, the mouse and human pancreatic structures and networks will be reviewed in 3-D for neuro-insular complexes, parasympathetic ganglia, and adipocyte infiltration as well as lymphatics in diabetes. Optical clearing with multiplex immunofluorescence microscopy provides new opportunities to examine the role of the nervous and circulatory systems in pancreatic and islet functions by defining their neurovascular anatomy in health and diabetes.

Keywords: CLARITY; Schwann cell; adipocyte; autonomic (vegetative) nervous system; confocal 3-D microscopy; islet; lightsheet microscopy; organoid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Optical clearing of human pancreas by PACT and iDISCO. (A) Fixed pancreas sample from a control donor before and after clearing using passive CLARITY (PACT) showing the degree of sample transparency achieved with this method. (B) Representative example of 2-photon imaging for a 0.8 mm x 0.8 mm x 0.25 mm region (X, Y, Z axes) containing an islet immunostained for glucagon (red) and glial fibrillary acidic protein (GFAP, green). (C) GFAP-stained Schwann cells overlay an islet and cell projects were analyzed using the ImageJ neurite tracer program. (D) The traced Schwann cells are shown by Neurite tracer skeleton diagram.
Figure 2
Figure 2
Mouse and human islet Schwann cells. (A) A fixed frozen section (40µm) from a C57BL/6 mouse pancreas was stained for GFAP (green) using whole mount staining. A single 2-D slice (7th of 13 slices) and the 3-D maximum intensity projection (MIP, 12 µm stack) are shown to demonstrate the increase in cellular information obtained with a z-stack. (B) A human control pancreas sample (~1 mm3) was cleared by iDISCO and immunolabeling with glucagon (red) and GFAP (green) before confocal 3-D imaging (maximum intensity projection 50 µm). (C) The region identified by white box in (A) shows Schwann cells at the periphery of the islet that extended along nerves to islet interiors traveling along afferent vessels. (D) A single Schwann cell shows a clear nuclear region (white arrow) and numerous extensions with a termination at an alpha-cell (asterisk). See also Supplementary Video 1 for (A).
Figure 3
Figure 3
Intrapancreatic ganglia and neuroinsular complexes in adult human pancreas. Pancreas samples were studied in control donors using passive CLARITY (PACT) and immunostaining with primary antibodies for PGP9.5 (white) and GFAP (green) to delineate nerve fibers and supporting Schwann cells, respectively ( Table 2 ). PGP9.5 also stained islet endocrine cells although with much less intensity (asterisks). Intrapancreatic ganglia represent post-ganglionic neurons and fibers of the parasympathetic efferent system and 3-D imaging shows how ganglia are interconnected (A) and also extend to islets (B). (C) Intrapancreatic ganglia contained varying numbers of neurons and small collections of neurons were also found widely scattered with efferent and afferent axons in both interlobular and intralobular regions. (D) Imaging for PGP9.5 and islet alpha-cells (GCG, green) demonstrate close association of clustered alpha-cells with neurons at a small ganglion, also known as a neuro-insular complex II. Scale bars: 500µm (A), 50µm (B), 25 µm (C, D). See also Supplementary Video 2 for (D).
Figure 4
Figure 4
Human pancreas vasculature. 3-D extended projection of human pancreas exocrine and endocrine vasculature are shown with X, Y and Z axes in mm (Scale bar 200 µm). (A) The extensive nature of the human pancreas vascular system is demonstrated by immunolabeling with monoclonal anti-CD31 (red) and islets are shown stained with monoclonal anti-glucagon (green) antibodies. (B) An islet identified by yellow-dotted line in (A) is shown at higher resolution in (B) (Scale bar 50 µm). (C) The islet microvasculature is shown without the glucagon overlay (Scale bar 50 µm). See also Supplementary Video 3 for (A–C).
Figure 5
Figure 5
3-D projection human primary pancreas exocrine cells in culture. Isolated human exocrine cells were obtained from a pancreas donor non-islet fractions and following filtration to remove islets and clumps, exocrine cells were plated in 1:1 DMEM:F12 and Matrigel and grown for 6 days. A maximum projection image shows ductal cells (cytokeratin 19, green), acinar cells (amylase, red) and nuclei (blue). Cells were kindly provided by Dr. Thomas Schmittgen, College of Pharmacy, University of Florida. See also Supplementary Video 4 for entire 3-D z-stack (12 µm).
Figure 6
Figure 6
Human pancreatic fatty infiltration. Images were derived from tile scanning of optically cleared pancreatic specimens. (A, B) Normal lobule of human pancreas. Adipocytes are clearly seen around the blood vessel and inside the lobule (magnified, arrows). Green, nuclear staining; red, CD31. (C, D) Acinar atrophy of diseased lobule. This view was acquired 2-cm distal to the pancreatic ductal adenocarcinoma. Overlay of transmitted light and fluorescence signals identifies the fatty infiltration.
Figure 7
Figure 7
Panoramic and high-resolution images of optically cleared NOD mouse pancreas with insulitis. (A) Map of 8-week NOD mouse pancreas. Overlay of transmitted light and fluorescence signals identifies the Lyve1+ lymph node (filled with CD3+ T lymphocytes and surrounded by fats) and locations of insulitic islets (yellow arrows). Two islets (cyan box) are magnified in (B). (B) Islets with insulitis are shown with blood vessels (red), lymphatic vessels (magenta), and nuclei (white). CD3+ T lymphocytes are identified around the islets and congregated in the lymphatic vessels (asterisk; vascular compartment vs. extravascular domain). This feature is further magnified in (C). (C) Peri-islet aggregation of T lymphocytes and their vascular association shown as well as the peri-islet vasodilation and lymphocytic infiltration. See also Supplementary Video 5 for (B).
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