Bimodal Whole-Mount Imaging Of Tendon Using Confocal Microscopy And X-Ray Micro-Computed Tomography

Abstract

9BACKGROUND: 3-dimensional imaging modalities for optically dense connective tissues 10 such as tendons are limited and typically have a single imaging methodological endpoint. Here, 11we have developed a bimodal procedure that utilises fluorescence-based confocal microscopy 12 and x-ray micro-computed tomography for the imaging of adult tendons to visualise and 13 analyse extracellular sub-structure and cellular composition in small and large animal species. 14 RESULTS: Using fluorescent immunolabelling and optical clearing, we visualised the 15 expression of the basement membrane protein laminin-α4 in 3D throughout whole rat Achilles 16 tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex 17 network of laminin-α4 within the tendon core that predominantly localises to the interfascicular 18 matrix compartment. Furthermore, we implemented a chemical drying process capable of 19 creating contrast densities enabling visualisation and quantification of both fascicular and 20 interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also 21 demonstrated that both modalities can be combined using reverse clarification of fluorescently 22 labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. 23 CONCLUSIONS: Whole-mount imaging of tendon allowed us to identify the presence of an 24 extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated 25 using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed 26 tomography imaging of tendon using chemical drying is not only simple and rapid, but also 27 markedly improves on previously published methods. Combining these methods provides the 28 ability to gain spatio-temporal information and quantify tendon substructures to elucidate the 29 relationship between morphology and function. 30

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations

“…However it is yet to be established whether these are two distinct cell populations, or whether vascular-associated CD146 cells are able to migrate away from the vasculature. The interconnected network of CD146 forms structures which were similar those seen in 3D imaging of LAMA4 in SDFT (Marr et al, 2020). The colocalisation of CD146 and LAMA4 in the current study further reinforces the putative ligand-receptor interaction that CD146 and LAMA4 share, which has been demonstrated in previous studies (Ishikawa et al, 2014;Wragg et al, 2016).…”

“…3D immunolabelling of SDFT segments was performed as previously described (Marr et al, 2020). All steps were performed with orbital agitation.…”

The tendon interfascicular basement membrane provides a vascular niche for CD146+pericyte cell subpopulations

“…However, we did observe some spe-cies-specific differences; for example, there was no detection of Nestin in equine TDCs, which is abundant in mice and human Achilles tendons, particularly in the IFM [21]. Another explanation for the discrepancy in population proportions in this study is the removal of the epitenon in the current study, which is known to house CD146 + cells [36]. MACS was successfully employed to enrich CD146 populations, with approximately 65% of cells positive for CD146 post-sorting as determined by flow cytometry.…”

“…In addition, our recent studies have established that CD146 + cells migrate to sites of injury in the rat Achilles tendon, which is accompanied by increased LAMA4 [30]. 3D imaging of tendon revealed an interconnected network of CD146 labelling within the IFM; structures which were similar those seen in 3D imaging of LAMA4 in SDFT [36]. The colocalisation of CD146 and LAMA4 in the current study further reinforces the putative ligand-receptor interaction that CD146 and LAMA4 share, which has been demonstrated in previous studies [49,50].…”

“…3D immunolabelling of SDFT segments was performed as previously described [36]. All steps were performed with orbital agitation.…”