Kate Singletary
Faculty Sponsor: Dr. Multhaup
Background: Optical tissue clearing (OTC) is a novel technique that removes light-scattering molecules in inherently translucent or opaque tissues, such as solid organs, rendering them transparent to light, while retaining 3-dimensional integrity in the cleared tissue (1). We have optimized OTC techniques for solid organs (eg. lung, heart, brain) to enable high quality imaging of structural, morphological, and compositional features in treated tissues compared to conventional histology (2). Here in, we evaluated for first time, the efficacy of our OTC techniques [uDISCO and index matched clear imaging for tissue evaluation (inCITE)] in clearing mouse facial flap (with eyeball) and mouse hind limb tissues that included skin (with hair/adnexa), skeletal muscle, vessels, nerve, fat, cartilage, bone, and bone marrow components. Methods: Murine limb and face flap explants were obtained (n=5) following perfusion fixation with paraformaldehyde (PFA). Untreated controls (n=2) were compared to OTC treatment (n=3). Of treated samples, half were limbs and half were face flaps. Bone tissue in limbs was decalcified with formic acid and EDTA for 2-4 days. Following hydrogel fixation, samples were cleared with either uDISCO or inCITE techniques. All cleared samples were analyzed using histological and immunohistochemical staining/analysis for specific markers (skin, muscle, bone, nerve and vessel), and 3-dimensional confocal imaging was performed to compare cleared versus uncleared (stained/unstained) tissues treated with either technique. Results: Analysis of the composite tissues revealed that in comparison to controls, cleared tissues allowed for higher quality staining and imaging because of enhanced tissue transparency. Both uDISCO and inCITE techniques successfully cleared limb and face flaps. uDISCO technique was fast and efficient in clearance of composite facial flaps and limbs. However, the harsher chemical process compromised structure and planar composition resulting in distorted imaging and immunohistochemical analysis versus control treatment. In comparison, the milder chemical process of the inCITE successfully cleared composite tissues with preserved microanatomic as well as 3-dimensional integrity due to the hydrogel matrix fixation. This allowed high quality imaging (compared to controls or uDISCO) and preserved sensitive cellular elements (including bone marrow) and molecular as well as morphological features (including tissue interfaces) of composite tissues. Conclusion: We successfully confirmed the effectiveness of OTC techniques in composite tissues including osteo-myo-cutaneous and multiple germ-layer derived components. We established a bone decalcification protocol for limb tissues that retained integrity of surrounding tissues while rendering transparency for high quality imaging. Our protocol has potential applications in 3-dimensional imaging analysis of vascularized composite allografts such as face or limb transplants. Further optimization of OTC techniques could allow high quality imaging delineation of bone marrow engraftment, bone healing, nerve regeneration, immune cell infiltration, or graft vasculopathy (as in chronic rejection) in these transplants. This technique could hold promise in regenerative medicine applications to evaluate tissue interfaces of engineered composite tissue constructs to determine the in-vivo dynamics of healing, engraftment and vascularization.
