High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Abstract

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

“…Using this approach, human tracheal tissue compartments can be easily identified and intrinsic airway ganglia can be quantified ( Scott et al., 2014 ). The human pancreas has also been cleared to visualize the neuroinsular network ( Butterworth et al, 2018 ).…”

Optogenetic stimulation of cholinergic fibers for the modulation of insulin and glycemia

“…It has been reported in humans that cholinergic activation of β-cell insulin secretion occurs as a paracrine signal from α-cells which release acetylcholine 29,30 . However, cholinergic neural inputs do exist in human islets, although to a lesser extent than in rodent islets [31][32][33] . In humans, a complex picture is emerging of intra-islet cellular communication and signaling of hormone secretion 29,30,34 .…”

Islet sympathetic innervation and islet neuropathology in patients with type 1 diabetes

“…Optical clearing. Optical clearing was performed using two methods: (1) manually cut sections were embedded in acrylamide hydrogel and passively cleared as previously described (PACT) 74 or (2) vibratome sections were optically cleared by a modified iDISCO procedure (HSK-1, Visikol Inc.) (https:// dx. doi.…”