Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Three-dimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.
Microglial activation is a component of neurodegenerative pathology. Here, we examine whether activated microglia participate in age-related dopaminergic (DA) cell death in the substantia nigra pars compacta (SNc) of the zitter (zi/zi) rat, a mutant characterized by deletion of the attractin gene. Confocal microscopy with double-immunohistochemical staining revealed activated microglia-formed cell-clusters surrounding DA neurons in the SNc from 2 weeks after birth. An immunoelectron microscopic study showed that the cytoplasm of activated microglia usually contains phagosome-like vacuoles and lamellar inclusions. Expression levels of the pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were increased in the midbrain of 2-month-old zi/zi rats. Chronic treatment with the anti-inflammatory agent minocycline altered the morphology of the microglia, reduced cluster formation by the microglia, and attenuated DA cell death in the SNc, and reduced the expression of IL-1β in the midbrain. These results indicate that activated microglia, at least in part and especially at the initial phase, contribute to DA cell death in the SNc of the zi/zi rat.
Minamata disease is a methylmercury poisoning caused by consumption of marine food contaminated by man-made methylmercury environmental pollution, and its most prominent feature is marked pathological changes in the central nervous system. Morphological alterations are less pronounced in the liver and the kidney, although their mercury levels are higher than those of the brain. In marine mammals, methylmercury is known to be easily converted to inorganic mercury and it combines with selenium forming mercury selenide, which may counteract the toxicity of mercury. However, little is known about the formation of mercury and selenium complex in human organs. In the present study, we examined the cerebrum, cerebellum, liver, and kidney of a Minamata disease case to study the mercury and selenium localization using electron probe microanalysis. Our results indicated the mercury and selenium localization in the specified tissue of the brain, liver, and kidney such as glial cells, Kupffer cells, and renal tubules.