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Frontiers in Neurology 2024Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the...
BACKGROUND
Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac.
MATERIALS AND METHODS
Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM.
RESULTS
Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base.
DISCUSSION
The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.
PubMed: 38817543
DOI: 10.3389/fneur.2024.1355785 -
Basic & Clinical Pharmacology &... Nov 2020Osmium tetroxide is a strong oxidizing agent used in electron microscopy. Eye exposure may cause severe burns, and after inhalation or ingestion damage to the...
Osmium tetroxide is a strong oxidizing agent used in electron microscopy. Eye exposure may cause severe burns, and after inhalation or ingestion damage to the respiratory or gastrointestinal tract occurs. Exposure to osmium and its compounds is extremely rare. We present a case of a 32-year-old female stained by 9 mL of 2% osmium tetroxide in acetone during an accident in the laboratory, with rare dermal and ocular findings. Due to lack of data in toxicological databases and the absence of antidote, the therapy was symptomatic. Osmium was detected in serum 19 hours later (0.22 μg/L) and in urine during the 15-hour collection (three samples-7.05, 1.65 and 8.45 μg/L). In blood serum on admission, after 1 and 2 days after exposure, the levels of iron (28.2, 39.8 and 50.5 μmol/L; reference range 5.8-34.5 μmol/L) and transferrin receptor/ferritine were elevated. To our knowledge, this is the first paper documenting a significant absorption from the skin and potentially from the eye conjunctiva, based on serum and urine analysis. The relationship between increased iron in blood and exposure has not been described yet, and the mechanism remains unknown. The patient is being followed up for the unknown long-term effects.
Topics: Adult; Eye; Female; Humans; Osmium Tetroxide; Skin
PubMed: 32524772
DOI: 10.1111/bcpt.13450 -
Membranes Dec 2021The recovery of osmium from residual osmium tetroxide (OsO) is a necessity imposed by its high toxicity, but also by the technical-economic value of metallic osmium. An...
The recovery of osmium from residual osmium tetroxide (OsO) is a necessity imposed by its high toxicity, but also by the technical-economic value of metallic osmium. An elegant and extremely useful method is the recovery of osmium as a membrane catalytic material, in the form of nanoparticles obtained on a polymeric support. The subject of the present study is the realization of a composite membrane in which the polymeric matrix is the polypropylene hollow fiber, and the active component consists of the osmium nanoparticles obtained by reducing an alcoholic solution of osmium tetroxides directly on the polymeric support. The method of reducing osmium tetroxide on the polymeric support is based on the use of 10-undecenoic acid (10-undecylenic acid) (UDA) as a reducing agent. The osmium tetroxide was solubilized in -butanol and the reducing agent, 10-undecenoic acid (UDA), in -propanol, -butanol or -decanol solution. The membranes containing osmium nanoparticles (Os-NP) were characterized morphologically by the following: scanning electron microscopy (SEM), high-resolution SEM (HR-SEM), structurally: energy-dispersive spectroscopy analysis (EDAX), Fourier transform infrared (FTIR) spectroscopy. In terms of process performance, thermal gravimetric analysis was performed by differential scanning calorimetry (TGA, DSC) and in a redox reaction of an organic marker, -nitrophenol (PNP) to -aminophenol (PAP). The catalytic reduction reaction with sodium tetraborate solution of PNP to PAP yielded a constant catalytic rate between 2.04 × 10 mmol s and 8.05 × 10 mmol s.
PubMed: 35054577
DOI: 10.3390/membranes12010051 -
Microscopy (Oxford, England) Aug 2022Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone...
Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone pigments in Boraginaceae. The new fixation method combined glutaraldehyde containing malachite green, imidazole-osmium and p-phenylenediamine treatments, and cells were then observed with a transmission electron microscope. The method prevented the extraction of lipids, including shikonin derivatives, and improved the visualization of subcellular structures, especially the membrane system, when compared with that of conventional fixation. The improved quality of the transmission electron micrographs is because malachite green ionically binds to the plasma membrane, organelles and lipids and acts as a mordant for electron staining with osmium tetroxide. Imidazole promotes the reaction of osmium tetroxide, leading to enhanced electron staining. p-Phenylenediamine reduces osmium tetroxide bound to cellular materials and increases the electron density. This protocol requires only three additional reagents over conventional chemical fixation using glutaraldehyde and osmium tetroxide.
Topics: Glutaral; Imidazoles; Lipids; Microscopy, Electron; Microscopy, Electron, Transmission; Osmium Tetroxide; Plant Cells
PubMed: 35388424
DOI: 10.1093/jmicro/dfac018 -
Histochemistry and Cell Biology Apr 2022High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid...
High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.
Topics: Freeze Substitution; Freezing; Lipids; Osmium Tetroxide; Potassium Permanganate
PubMed: 34984524
DOI: 10.1007/s00418-021-02070-0 -
Academic Forensic Pathology Sep 2019Fat embolism is common following trauma and is a common autopsy finding in these cases. It may also be seen in non-traumatic cases and is seen in children as well as... (Review)
Review
Fat embolism is common following trauma and is a common autopsy finding in these cases. It may also be seen in non-traumatic cases and is seen in children as well as adults. In comparison fat embolism syndrome (FES) only occurs in a small number of trauma and non-trauma cases. Clinical diagnosis is based on characteristic clinical and laboratory findings. Fat embolism exerts its effect by mechanical blockage of vessels and/or by biochemical means including breakdown of fat to free fatty acids causing an inflammatory response. Fat embolism can be identified at autopsy on microscopy of the lungs using fat stains conducted on frozen tissue, including on formalin fixed but not processed tissue. With FES fat emboli can be seen in other organs including the brain, kidney and myocardium. Fat can also be identified with post-fixation staining, typically with osmium tetroxide. Scoring systems have been developed to try and determine the severity of fat embolism in lung tissue. Fat embolism is also common following resuscitation. When no resuscitation has taken place, the presence of fat on lung histology has been used as proof of vitality. Diagnosis of fat embolism syndrome at autopsy requires analysis of the history, clinical and laboratory findings along with autopsy investigations to determine its relevance, but is an important diagnosis to make which is not always identified clinically. This paper reviews the history, clinical and laboratory findings and diagnosis of fat embolism and fat embolism syndrome at autopsy.
PubMed: 32110249
DOI: 10.1177/1925362119896351 -
BioRxiv : the Preprint Server For... Sep 2023Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial...
Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial section electron microscopy, via membrane staining using osmium tetroxide, is ideal for visualizing cells and synaptic connections but, in whole brain samples, faces significant challenges related to chemical treatment and volume changes. These issues can adversely affect both the ultrastructural quality and macroscopic tissue integrity. By leveraging time-lapse X-ray imaging and brain proxies, we have developed a 12-step protocol, ODeCO, that effectively infiltrates osmium throughout an entire mouse brain while preserving ultrastructure without any cracks or fragmentation, a necessary prerequisite for constructing the first comprehensive mouse brain connectome.
PubMed: 37808722
DOI: 10.1101/2023.09.26.558265 -
ELife Oct 2022Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks...
Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high-quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here, we present an in situ time-lapsed X-ray-assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method, we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray-assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray-assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys, or humans.
Topics: Humans; Mice; Animals; Osmium Tetroxide; Osmium; X-Rays; Staining and Labeling; Microscopy, Electron
PubMed: 36263931
DOI: 10.7554/eLife.72147 -
Current Eye Research Aug 2018To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology...
PURPOSE
To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology as a function of retinal eccentricity.
METHODS
Electron microscopy was used to describe and quantify two major organelles in elderly monkey retinal epithelium, lipofuscin bodies and melanosomes, at different retinal loci extending from the macula to the peri-macula, equator, periphery and ora serrata. Osmium tetroxide was used to distinguish lipofuscin bodies from melanosomes.
RESULTS
Lipofuscin bodies and melanosomes diminished in number with advanced age but there was an inverse relationship between these two organelles. Lipofuscin bodies were more numerous in the macula and melanosomes more numerous in the peripheral retina. Three types of lipofuscin bodies were identified: 1) smaller and tending to locate in the middle third of the epithelial cell, 2) larger, less common, and located more basally, and 3) extremely rare, melano-lipofuscin, containing a melanosome. When osmicated, all lipofuscin bodies contained electron dense materials. When osmium tetroxide was not used for fixation, the first two types of lipofuscin bodies lost their electron densities while the third type retained its electron density due to the melanosome it contained.
CONCLUSION
As previously reported for human retina, lipofuscin is most abundant in the macular and peri-macular epithelium and least abundant in the periphery, whereas melanosomes show the opposite relationship. This distribution pattern could contribute to the macula's greater vulnerability to photo-toxicity. Three types of lipofuscin bodies are found in aging monkey retinal epithelium. All types contain electron dense material, but the most prominent two types lose their densities in the absence of osmium tetroxide during fixation. Most of the electron densities in lipofuscin bodies must contain a material that binds strongly to osmium tetroxide such as polyunsaturated fatty acids.
Topics: Aging; Animals; Electron Probe Microanalysis; Female; Lipofuscin; Macaca mulatta; Male; Melanosomes; Microscopy, Electron; Models, Animal; Osmium Tetroxide; Retinal Pigment Epithelium
PubMed: 29641909
DOI: 10.1080/02713683.2018.1464194 -
Methods in Cell Biology 2023Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies,...
Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy. The chemistry of this protocol was modified from an approach developed for improved and uniform staining of large brain volumes. Briefly, primary fixation was in paraformaldehyde and glutaraldehyde with malachite green followed by secondary fixation with osmium tetroxide, potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide and finally uranyl acetate and lead aspartate staining. Samples were then dehydrated in acetone with a propylene oxide transition and embedded in a hard formulation Quetol 651 resin. The samples were trimmed and mounted with silver epoxy, metal coated and imaged via serial block-face scanning electron microscopy and focal charge compensation for charge suppression. High-contrast plant tobacco and duckweed leaf cellular structures were readily visible including mitochondria, Golgi, endoplasmic reticulum and nuclear envelope membranes, as well as prominent chloroplast thylakoid membranes and individual lamella in grana stacks. This sample preparation protocol serves as a reliable starting point for routine plant volume electron microscopy.
Topics: Volume Electron Microscopy; Osmium Tetroxide; Staining and Labeling; Glutaral; Microscopy, Electron, Scanning
PubMed: 37451777
DOI: 10.1016/bs.mcb.2023.04.008