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Internal Medicine (Tokyo, Japan) 2023A 32-year-old man was admitted for the evaluation of proteinuria (5.69 g/day). A light microscopic examination showed markedly dilated glomerular capillary loops with...
A 32-year-old man was admitted for the evaluation of proteinuria (5.69 g/day). A light microscopic examination showed markedly dilated glomerular capillary loops with vacuolated areas in many glomeruli, and vacuolated areas were seen on peritubular capillaries in the tubulointerstitium. When electron microscopy specimens prepared by pre-fixation with glutaraldehyde and post-fixation with osmium tetroxide were used for oil red staining, the deposition was confirmed on the affected areas. A genetic analysis of apoE showed that the lipoprotein glomerulopathy was due to apoE-Sendai (Arg145Pro, p.R163P) heterozygosity, which was found in not only the patient but also his mother and twin brother.
Topics: Male; Humans; Adult; Apolipoproteins E; Kidney Diseases; Kidney Glomerulus; Proteinuria; Heterozygote
PubMed: 37532513
DOI: 10.2169/internalmedicine.0834-22 -
Anatomical Record (Hoboken, N.J. : 2007) Aug 2023The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve...
The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve dissection. Little data have been reported on the normality of these nerves. The vestibular nerve is composed of the superior vestibular, inferior vestibular, and branch nerves. This study aimed to study the microscopic anatomy of the superior vestibular nerve (SVN) of guinea pigs using light microscopy and to search for normality patterns for use in experimental models in basic otologic research. We used eight male albino guinea pigs (Cavia porcellus, English strain), weighing between 400 and 500 g. After anesthetizing, the animals were perfused with a fixative solution of 2.5% glutaraldehyde. Dissection was performed by the access method to the temporal bone, coming to the rock and exposing the cochlea and vestibular nerve. The NVS fragments were removed, postfixed in osmium tetroxide, and embedded in the epoxy plastic resin Poly/Bed 812® (Polysciences Inc., Warrington, PA). Semi-thin transverse serial sections (0.5 μm) were made using a microtome MT6000-XL, RMC, Inc. and stained with toluidine blue. Morphology and morphometry were described and evaluated using the KS 400 application (Kontron 2.0, EchingBei, Munich, Germany) by macro, a computer program specially designed and developed for the study of the VIII nerve. The SVN was found to be devoid of epineurium, with only a thin conjunctive tissue layer. The myelin sheath of guinea pigs is relatively thin compared to the sensory and motor nerves found in mammals. The average fascicular area SVN was 0.19 ± 0.05 mm , with the largest area found to be 0.24 mm and the lowest was 0.12 mm . The average number of fibers was 5,753.00 ± 538 fibers. The density of myelinated fibers reached 32,316.08 ± 11,375.29 fibers/mm . Its diameter ranged from 1.0 to 9 μm and its peak was 3 μm. The measured results confirm the results of another study, indicating that the methodology is appropriate and reproducible. These findings are important for the evaluation of injured nerves in experimental models of peripheral neuropathy and basic ear disease.
Topics: Animals; Guinea Pigs; Vestibular Nerve; Male; Myelin Sheath; Cochlea
PubMed: 37461264
DOI: 10.1002/ar.25053 -
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 -
Heliyon Jun 2023In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same...
In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λ ∼550 nm) and far-red (λ ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.
PubMed: 37389060
DOI: 10.1016/j.heliyon.2023.e17394 -
Microscopy (Oxford, England) Oct 2023Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have...
Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis.
Topics: Humans; Epoxy Resins; Microscopy, Electron; Microscopy, Fluorescence; Green Fluorescent Proteins; HeLa Cells
PubMed: 37217182
DOI: 10.1093/jmicro/dfad028 -
Microscopy (Oxford, England) Nov 2023Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we...
Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we report a novel method to prepare OMVs for TEM imaging. To preserve vesicular shape and structure, we developed a dual fixation protocol involving osmium tetroxide incubation prior to negative staining with uranyl acetate. Combining osmium tetroxide with uranyl acetate resulted in preservation of sub-50 nm vesicles and improved morphological stability, enhancing characterization of lipid-based nanoparticles by TEM.
Topics: Microscopy, Electron; Coloring Agents; Osmium Tetroxide; Bacterial Outer Membrane; Microscopy, Electron, Transmission; Staining and Labeling; Osmium
PubMed: 37148329
DOI: 10.1093/jmicro/dfad027 -
Methods in Molecular Biology (Clifton,... 2023Dysfunction in adipocyte expansion during the onset of obesity is associated with metabolic abnormalities. Determination of adipocyte size and number is an important...
Dysfunction in adipocyte expansion during the onset of obesity is associated with metabolic abnormalities. Determination of adipocyte size and number is an important measure for a comprehensive evaluation of the metabolic status of adipose tissue. Here, we describe three methods for the determination of adipocyte size that can be applied to tissue samples obtained from humans and rodent models. While the first method presented is more robust, it does require the use of osmium, a toxic heavy metal, which requires special handling and disposal precautions in addition to specialized equipment. Two additional methods are described that can be of use to most researchers.
Topics: Humans; Adipocytes; Adipose Tissue; Obesity
PubMed: 37076669
DOI: 10.1007/978-1-0716-3167-6_4 -
Molecules (Basel, Switzerland) Mar 2023Sharpless asymmetric dihydroxylation is an important reaction in the enantioselective synthesis of chiral vicinal diols that involves the treatment of alkene with osmium... (Review)
Review
Sharpless asymmetric dihydroxylation is an important reaction in the enantioselective synthesis of chiral vicinal diols that involves the treatment of alkene with osmium tetroxide along with optically active quinine ligand. Sharpless introduced this methodology after considering the importance of enantioselectivity in the total synthesis of medicinally important compounds. Vicinal diols, produced as a result of this reaction, act as intermediates in the synthesis of different naturally occurring compounds. Hence, Sharpless asymmetric dihydroxylation plays an important role in synthetic organic chemistry due to its undeniable contribution to the synthesis of biologically active organic compounds. This review emphasizes the significance of Sharpless asymmetric dihydroxylation in the total synthesis of various natural products, published since 2020.
Topics: Hydroxylation; Biological Products; Alkenes; Stereoisomerism
PubMed: 36985698
DOI: 10.3390/molecules28062722 -
Archives Italiennes de Biologie Dec 2022Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by...
PURPOSE
Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by apoptosis has been demonstrated in experimental models. Rosmarinic acid (RA) is a phenolic compound used for therapeutic purposes in many diseases. In this study, we investigated the therapeutic effect of Rosmarinic acid application on inflammation and apoptotic development after spinal cord injury.
METHODS
Male Wistar albino rats (n: 24) were assigned to three group: control, SCI and SCI+ RA. All rats were fixed on the operating table after anesthesia, the skin of the thoracic region was opened with a midline incision and the paravertebral muscles were dissected and T10-T11 laminas were exposed. A cylindrical tube of 10 cm length was fixed to the area to be laminectomy. A metal weight of 15 grams was left down the tube. Spinal damage was created, skin incisions were sutured. 50 mg/kg rosmarinic acid was given orally for 7 days after the spinal injury. Spinal tissues were fixed in formaldehyde solution and processed for paraffin wax tissue protocol and 4-5 μm sections were taken with microtome for further immunohistochemical examination. Caspase-12 and CHOP antibodies were applied to sections. Remaining tissues were carried out in glutaraldehyde for the first fixation then in osmium tetroxide for the second. Tissues were kept in pure araldite and thin sections were taken for transmission electron microscope.
RESULTS
Values of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH), neuronal degeneration, vascular dilation, inflammation, CHOP and Caspase-12 expression were increased in SCI group compared to control group. Only glutathione peroxidase content was decreased in SCI group. In SCI group, disruption of basement membrane structure in canalis ependymalis, degeneration in structures of unipolar bipolar and multipolar neurons, and apoptotic changes were seen with increased inflammation in the piamater region and positive CHOP expression in vascular endothelial cells. In SCI+RA group, reorganization of basement membrane pill in canalis ependymalis were observed with mild Caspase-12 activity in some canalis ependymal and glial cells. Also, moderate CHOP expression in multipolar and bipolar neurons and glia cells were observed.
CONCLUSIONS
The application of RA has a significant effect on preventing damage in SCI. It was thought that CHOP and Caspase-12 mediated oxidative stress could be a guide in showing the potential and therapeutic target to stop the apoptotic course after SCI injury.
Topics: Male; Rats; Animals; Rats, Wistar; Caspase 12; Endothelial Cells; Spinal Cord Injuries; Rosmarinic Acid
PubMed: 36881913
DOI: 10.12871/000398292022341 -
The Journal of Sexual Medicine Feb 2023It is frequently quoted in mainstream media that the clitoris has "8000 nerve endings." However, no study has yet quantified the number of nerve fibers (axons)...
INTRODUCTION
It is frequently quoted in mainstream media that the clitoris has "8000 nerve endings." However, no study has yet quantified the number of nerve fibers (axons) innervating the human clitoris. The dorsal nerves of the clitoris (DNCs) are the primary source of sensation and somatic clitoral innervation. Therefore, reporting the number of axons in the DNCs is an important step in our understanding of clitoral innervation and sexual response with implications for many fields of medical practice. The purpose of this study is to quantify the mean number of axons in the human DNCs and to report the approximate mean number of nerve fibers that innervate the human glans clitoris.
METHODS
DNC samples were obtained from 7 transmasculine patients undergoing gender-affirming phalloplasty surgery. At the time of nerve coaptation, a small excess of the DNC (5 mm) was collected for analysis at the proximal level of the clitoral body, just distal of the emergence of the DNCs from underneath the pubic symphysis. Samples were placed into 3% glutaraldehyde fixative, postfixed in 1% osmium tetroxide, and serially dehydrated in ethanol and toluene. Samples were then embedded in araldite, sectioned on an ultramicrotome into 1-μm cross sections, and counterstained with 1% toluidine blue. Histomorphometric evaluation was performed at 1000x magnification with a Leitz Laborlux S microscope and image analysis software (Clemex Vision Professional) to obtain an axon counts. Descriptive statistics were performed to yield a mean and standard deviation of the number of axons in the DNCs. Assuming anatomic symmetry between bilateral DNCs, mean total number of somatic nerve fibers innervating the human glans clitoris was obtained by doubling the mean count of the DNCs.
RESULTS
Seven sample DNCs were collected. Of those, 5 were analyzed as 2 did not have sufficient nerve tissue present. The mean number of nerve fibers in the human DNCs was 5140 (SD = 218.4). The mean number of myelinated nerve fibers innervating the human clitoris was 10,281 (SD = 436.8).
CONCLUSION
This study is the first to report the number of axons in the human DNC, at a mean 5140. Given the bilateral nature of clitoral innervation and symmetry of anatomic structures, the approximate mean number of myelinated axons that innervate the human glans clitoris is 10,280. When the uncaptured unmyelinated fibers and contributions from the cavernosal innervation are accounted for, it is clear that far Moree than 8000 axons innervate the human clitoris.
Topics: Female; Humans; Clitoris; Nerve Fibers; Nerve Tissue; Sensation; Sexual Behavior
PubMed: 36763957
DOI: 10.1093/jsxmed/qdac027