3D Visualization of Dynamic Cellular Reaction of Pulpal CD11c+ Dendritic Cells against Pulpitis in Whole Murine Tooth

In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray’s clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.

Solid optical clearing agents based through-Intact-Skull (TIS) window technique for long-term observation of cortical structure and function in mice

In vivo cortical optical imaging needs to overcome the scattering of skull. Compared to the traditional transcranial surgery-based open-skull glass window and thinned-skull preparation, chemical tissue optical clearing techniques can provide a skull-remained optical access to the brain while maintaining its original environment. However, previously demonstrated skull optical clearing windows could only maintain transparency for a couple of hours and hardly capable for high-resolution monitoring of awake animals. Here, we developed a convenient and easy-handling chronic skull optical clearing technique, named "Through-Intact-Skull (TIS) window", which was compatible with long-term observation at high resolution, and yielded large imaging depth of 900 μm for cortical neurovascular visualization. In addition, our TIS window could monitor neuron activity in awake mice for a long term. Therefore, our bio-compatible and non-invasive TIS window is a new promising approach for intravital brain microscopy with great potential for basic research in neuroscience.

An Optical Clearing Technique to Visualize Internal Root Structure

BackgroundRoots play an important role in the foraging and uptake of nutrients and water from soil to support sessile plant growth. Research on root growth and development in plants is very limited due to the thickness and opacity of roots. We developed a tissue clearing technique that enables visualization of crop internal root structure.ResultsThe application of methyl salicylate reduced the time taken for root clearing and accelerated the dye permeation into the tissue; the whole procedure for root clearing was performed within 3 days. We applied our technique on the roots of monocotyledonous plants, such as rice (Oryza sativa L.), wheat (Triticum aestivum L.), and maize (Zea mays L.), and dicotyledonous plants, such as rape (Brassica napus L. var oleifera), tomato (Solanum lycopersicum), and soybean (Glycine max (Linn.) Merr.), and obtained clear root structure. It not only shortens the time for root tissue clearing but also keeps the cell structure of intact roots. The transparent sample can be preserved for more than one year without any structural deformation.ConclusionsOur technique can create a 3-D reconstruction of the entire root structure. In summary, this method is a useful tool for visualizing the structures of thick root tissues and will be a valuable tool for research on root growth and development in plants.

Root. Canal. Morphology. of Mandibular. First. Permanent. Molars. in a Pakistani. Sub-population

Aim : To investigate the number and. patterns of. root canals. of mandibular. first. molars.. Methods.: Descriptive observational study to investigate the canal morphology using clearing technique. The duration of the study was one year from 1st February 2020 to 1st February 2021.Non purposive sampling technique was used and 200 mandibular first molar were collected. Data collected was analysed using the SPSS Vr 10. Study variables include Number and pattern of the roots in mandibular teeth. Descriptive statistics were used; frequencies of the root canals and their canal pattern were calculated. Results: In the mesial root, 14 teeth had a one root canal,186 possessed double root canals were observed. Distal root of 160 teeth possessed one canal, 40 teeth possessed two root canals with type 1 pattern mostly observed. Conclusion: Like other populations Pakistani population may have a diverse root canal system in the mandibular molars that ultimately affect endodontic therapy. Keywords: Roots, canals, anatomy, tooth clearing technique, demineralization

Investigation of PRDM10 and PRDM13 Expression in Developing Mouse Embryos by an Optimized PACT-Based Embryo Clearing Method

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.

Using the Fat-Clearing Technique to Improve Lymph Node Retrieval in Colorectal Cancer

College of American Pathologists recommended that at least 12 lymph nodes should be harvested for adequate staging of colorectal carcinoma. Lymph node harvesting is routinely performed by a manual technique of inspection and palpation, which is laborious and time-consuming. The study assessed the influence of the improved fat-clearing technique on the number of lymph nodes retrieved from colorectal cancer specimens and the clinical efficacy. Seventy colorectal cancer resection specimens were examined and assessed by 4 pathology residents. Thirty-five specimens were handled with the conventional manual technique by inspection and palpation, and the other 35 specimens with the improved fat-clearing technique to retrieve lymph nodes. As a result, compared with the conventional manual technique, the numbers of lymph nodes retrieved with the improved fat-clearing technique were significantly increased from 14.7 ± 6.2 lymph nodes to 20.8 ± 9.0 lymph nodes per specimen ( P < .05). Besides, the percentage of cases with at least 12 lymph nodes retrieved increased from 80% to 91%. The result of this study pointed out that using the improved fat-clearing technique to process colorectal specimens could increase the lymph node yield effectively, and was effective, practical, and suitable for routine gross examination.

Lipophilic dye-compatible brain clearing technique allowing correlative magnetic resonance/high-resolution fluorescence imaging in rat models of glioblastoma

In this work we optimized a novel approach for combining in vivo MRI and ex vivo high-resolution fluorescence microscopy that involves: (i) a method for slicing rat brain tissue into sections with the same thickness and spatial orientation as in in vivo MRI, to better correlate in vivo MRI analyses with ex-vivo imaging via scanning confocal microscope and (ii) an improved clearing protocol compatible with lipophilic dyes that highlight the neurovascular network, to obtain high tissue transparency while preserving tissue staining and morphology with no significant tissue shrinkage or expansion. We applied this methodology in two rat models of glioblastoma (GBM; U87 human glioma cells and patient-derived human glioblastoma cancer stem cells) to demonstrate how vital the information retrieved from the correlation between MRI and confocal images is and to highlight how the increased invasiveness of xenografts derived from cancer stem cells may not be clearly detected by standard in vivo MRI approaches. The protocol studied in this work could be implemented in pre-clinical GBM research to further the development and validation of more predictive and translatable MR imaging protocols that can be used as critical diagnostic and prognostic tools. The development of this protocol is part of the quest for more efficacious treatment approaches for this devastating and still uncurable disease. In particular, this approach could be instrumental in validating novel MRI-based techniques to assess cellular infiltration beyond the macroscopic tumor margins and to quantify neo-angiogenesis.