Abstract
Three-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology. Moreover, while recent progress has been made with intact methods for clearing, labeling, and imaging whole organs such as the mature brain, these approaches are generally unsuitable for soft, irregular, and heterogeneous tissues that account for the vast majority of clinical samples and biopsies. Here we develop a biphasic hydrogel methodology, which along with automated analysis, provides for high-throughput quantitative volumetric interrogation of spatially-irregular and friable tissue structures. We validate and apply this approach in the examination of a variety of developing and diseased tissues, with specific focus on the dynamics of normal and pathological pancreatic innervation and development, including in clinical samples. Quantitative advantages of the intact-tissue approach were demonstrated compared to conventional thin-section histology, pointing to broad applications in both research and clinical settings.
Highlights
Three-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology
We found that when published CLARITY methods – which had been developed for solid organs with fixed architecture, such as the brain – were applied to irregular or fragile tissues such as clinical biopsies, significant difficulties were encountered
The firmness of the hydrogel composite created difficulties with friable tissues, resulting in clumping, shearing, and poorly-controlled gelation following hydrogel embedding. These effects led to adverse consequences including optical aberrations, reduced antibody penetration, high levels of tissue-gel composite expansion, and significant tissue damage, in organs with irregular surface features or cavities where expansion could lead to rupture of enclosing structures
Summary
Three-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology. Volumetric imaging techniques such as optical coherence tomography enable organ-wide analysis of three-dimensional structures, but sacrifice spatial resolution and molecular information, losing detailed features at the cellular level[2]. This tradeoff is severely limiting in the peripheral and enteric nervous systems, which play critical roles in development, mature physiology, and diseases including diabetic neuropathy and gastroparesis[3]. While development of numerous clearing methodologies, including CLARITY, iDISCO, CUBIC, uDISCO, and SWITCH8–12, has shown that solid mouse tissues such as brain, lung, heart, and kidney, can be cleared and labelled, several key challenges remain: these methods are not widely suitable for soft, fragile, and irregular tissue targets such as those commonly found in clinical settings; material changes to the tissue rendered them incompatible with existing clinical analysis; and the use of specialized (and in some cases corrosive) chemical tools or customized devices represent practical barriers to adoption in clinical settings
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