Tissue-Clearing Technique and Cutaneous Nerve Biopsies: Quantification of the Intraepidermal Nerve-Fiber Density Using Active Clarity Technique-Pressure Related Efficient and Stable Transfer of Macromolecules Into Organs

BACKGROUND AND PURPOSE: Cutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies. METHODS: Five healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD). RESULTS: The mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm², respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm², respectively, and 3.06, 2.87, and 47.21 fibers/mm², respectively, in the patients with PHN. CONCLUSIONS: This study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

An Immuno-Electron Microscopic Study for Apoptosis and Expression of p53 on the Synovium of the Osteoarthritic Knee in Human / 대한해부학회지

This study was designed to observe the apoptosis and expression of p53 in the osteoarthritic synovial membrane compared with normal synovial membrane of human. The collected normal and osteoarthritic synovia were dissected and fixed for two hours (in 4% paraformaldehyde and 0.1% glutaraldehyde solution). In this study, TUNEL staining and immunocytochemical gold labeling techniques were used. In the immunocytochemical gold labeling techniques, primary antibodies which was to be monoclonal mouse anti-p53 were used. Donkey anti-mouse IgG tagged with 6 nm colloidal gold particles was used as the secondary antibody. The tissues were observed under JEOL 1200 EX-II transmission electron microscope. The results were as follows. 1. On TUNEL staining, normal synovium were not seen TUNEL positive signal cells. But, in the osteoarthritic synovium, few TUNEL positive cells were seen in synovial membrane and subsynovial layers. 2. On the transmission electron microscopic observation, normal synovium had 1~3 synovial cell layers, which had phagocytic synovial cells and secretory synovial cells. The osteoarthritic synovium had 2~5 synovial cell layers, which consisted with abnormally proliferated secretory synovial cells. These cells had heterochromatin in nucleus and well developed endoplasmic reticulum in the cytoplasm. 3. On the normal synovium of the human knee joint, p53 positive cells were not identified. But, in the osteoarthritic synovium of the human knee joint, p53 positive cells were identified. These cells were recognized secretory synovial cells and apoptotic cells. In the secretory synovial cells, the distributions of p53 were mitochondria and rough endoplasmic reticulum. In the apoptotic cells, p53 were marked on rough endoplasmic reticulum, which showed secretory synovial cells. On the basis of above findings, it is obvious that osteoarthritic synovial membrane has identified the apoptotic cells compared with normal synovium. These apoptotic cells might be identified as mainly secretory synovial cells and a few phagocytic synovial cells. The immunogold of p53 was marked at rough endoplasmic reticulum and in nucleus of apoptotic cells. Apoptosis in the osteoarthritic synovium seemed to be developed through p53 negative dependent pathway.

An Immunoelectronmicroscopic Study on the Cellular Expression of Extracellular Matrix Components during Skin Regeneration after Damage in the Mouse / 대한해부학회지

To observe the cellular expression of extracellular matrix components during mouse skin regeneration, the wounded skin samples were processed by immunoelectronmicroscopic methods, using primary antibodies for fibronectin, collagen type IV and laminin. The tissues were observed under transmission electron-microscope. The results were summarized as follows. 1. The granulation tissues and x-cells were observed in the wound margin at 18 hr post injury. The number of fibroblasts was increased in the granulation tissues at 1 day post injury. 2. The expression of fibronectin was observed in x-cells at 18 hr post injury, and in fibroblasts at 1 day post injury. In x-cells, after 1 day post injury, the expression of fibronectin was decreased. 3. At 1 day post injury, the expression of collagen type IV was increased in fibroblasts whereas not in x-cells. 4. The expression of laminin was increased by 18 hr post injury, but decreased after 1 day post injury. On the basis of above findings, in mouse, the regenerations of wounded skin were faster than other animals. The first step, infiltration response, was processed till 18 hr or 1 day post injury. The second step, fibroblast proliferation phase, began at 1 day post injury. In the regenerations of wounded skin, x-cells migrated to the wound region and activated, earlier than fibroblast. Thereafter, x-cells which appeared to be transformed into fibroblasts, played an important role in the synthesis of fibronectin and collagen type IV, and the formation of granulation tissue, with migrated fibroblasts to the wound region.

An Immuno -Electron Microscopic Study for Perlecan in the Synovium of Degenerative Arthritis of Knee / 대한해부학회지

This study was designed to observe the expression of perlecan in the normal and degenerative arthritic synovial membrane. By using the immunohistochemical staining and immuno -electron microscopical gold labeling techniques, we observed five materials of normal and degenerative arthritic synovia each. The results were as follows. 1. By the immunohistochemical methods, perlecan -positive staining was seen on the 1 ~2 cell layers of the normal synovial membrane. But, a weaker staining compared to that seen in the normal synovial membrane was found in the degenerative arthritic synovial membrane. 2. Under the electron microscopic observation, perlecan was largely distributed in the rough endoplasmic reticulum of the secretory synovial cell, and in the vacuoles of the phagocytic synovial cell on the normal synovium of the human knee joint. It was also found in the extracellular matrix of the synovial membrane. 3. Perlecan -positive cells were also identified on the degenerative arthritic synovium of the human knee joint. However, fewer perlecan was observed here than that found in the normal synovium. In conclusion, perlecan is synthesized by the secretory synovial cells and degraded by the phagocytic synovial cells. And it, known as a major component of the basement membrane, also proven to exist in the extracellular matrix of the synovial membrane having no basement membrane. From the fact that less perlecan was observed in the degenerative arthritis, perlecan is might to play a major role in the degenerative process.