Pathways to clinical CLARITY: volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease

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.

Islet–immune interactions in type 1 diabetes: the nexus of beta cell destruction

“…They have also provided insights into potential mechanisms behind the loss of tolerance [17,18], and allowed for in vivo assessment of phenotype and function of human cells [19,20]. Recent studies have illuminated interspecies differences in islet cell morphology and function that may contribute to the lack of success in translating therapeutics from mouse models to human patients [21][22][23][24]. Indeed, therapies targeting effector T cells for depletion [25] and those inhibiting T cell co-stimulation [26] successfully prevented immune cell infiltration of the pancreatic islets and symptomatic diabetes in NOD mice hundreds [27] of times and reversed in a handful of studies [28][29][30].…”

“…In addition to β-cell defects, α-cell dysfunction (e) contributes to impaired glucose homeostasis in T1D through hyperglucagonemia or failed counterregulatory responses. Figure created as in pediatric T1D tissues [23]. Cleared tissues offer compatibility with new methods of single mRNA transcript visualization [115], allowing measurement of localization of transcripts within the whole organ.…”

Rebuttal letter in response to Professor R.H. Anderson's letter ‘Evolution of the vertebrate heart’

“…; Hsueh et al. ) but we have not yet reached the critical point of applying robustly these techniques in a living organ. The proposed alternative models by the Anderson group (‘a continuous 3D meshwork’, MacIver et al.…”

“…The various techniques of imaging used in the field to understand ventricular cardiac shape in vivo still have a many shortages and caveats, before leading to definitive and convincing results; of course it is difficult (or not yet possible) to obtain 3D data from living embryonic heart and living adult hearts that integrate histology, contraction, morphology and cell tracking techniques. New innovative techniques are promising (Tomer et al 2014;Li et al 2016;He et al 2017;Hsueh et al 2017) but we have not yet reached the critical point of applying robustly these techniques in a living organ. The proposed alternative models by the Anderson group ('a continuous 3D meshwork', MacIver et al 2018a) or the 'nested layers' structure proposed by Hoffman (Hoffman, 2017) have good potential and 'raison d' etre', but we feel that the HVMB is still a viable and useful model and that the various proposed alternative models could be complementary rather than contradictory.…”

Unravelling the Mysteries of the Human AV Node

“…Over the past few years, we have tested and validated several new antibodies that might work well for this purpose, especially with the advent of the novel CLARITY method, which allows protein mapping in 3D anatomical structures without the need for tissue sectioning. 7 This topic is an on-going collaborative project in our laboratory and we hope to have data that directly answer this question in the near future.…”