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Ultramicroscopy Nov 2022Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde...
Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde and osmium tetroxide are widely used for transmission electron microscopy (TEM) and lead to adequate preservation of muscle ultrastructure, but do not preserve the molecular features of samples. Methacarn is suggested to be a preferable chemical fixative for light microscopy because it maintains immunohistological features of samples. However, the efficacy of methacarn to preserve ultrastructural features as a primary chemical fixative for TEM is currently unclear. Additionally, cryo-preservation of samples for TEM analysis involves freezing processes such as plunge freezing, slam freezing, or high pressure freezing. High pressure freezing is the considered the gold standard but requires costly equipment and may not be a viable option for many labs collecting tissue samples from remote locations. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant that may allow for better structural preservation of samples by impairing ice damage that occurs during plunge/snap freezing. We aimed to assess the effectiveness of methacarn as a primary chemical fixative and determine the effect of pre-coating samples with DMSO before plunge/snap freezing tissues to be prepared for TEM. The micrographs of the methcarn-fixed samples indicate a loss of Z-disk integrity, intermyofibrillar space, mitochondria structure, and lipids. Ultimately, methacarn is not a viable primary fixative for tissue sample preparation for TEM. Similarly, liquid nitrogen freezing of samples wrapped in aluminum foil produced non-uniform Z-disk alignments that appeared smeared with swollen mitochondria. DMSO coating before freezing appears to lessen the alterations to contractile and mitochondrial morphological structures. DMSO appears to be useful for preserving the ultrastructure of sarcomeres if samples are covered before freezing.
Topics: Acetic Acid; Aluminum; Chloroform; Cryopreservation; Dimethyl Sulfoxide; Fixatives; Glutaral; Ice; Methanol; Microscopy, Electron, Transmission; Muscles; Osmium Tetroxide
PubMed: 35988477
DOI: 10.1016/j.ultramic.2022.113600 -
Endocrinology Oct 2017Obesity during maturation can affect the growing skeleton directly and indirectly, although these effects and the mechanisms behind them are not fully understood. Our...
Obesity during maturation can affect the growing skeleton directly and indirectly, although these effects and the mechanisms behind them are not fully understood. Our objective was to determine how a high-fat diet with or without metformin treatment affects skeletal development. We also sought to characterize changes that occur in white adipose tissue, circulating metabolites, lipids, and gut microbiota. A diet-induced obesity C57BL/6J mouse model was used to test the effects of obesity and metformin on bone using bone histomorphometry and microcomputed tomography. Bone marrow adipose tissue was quantified with osmium tetroxide microcomputed tomography and histology. Dual-energy x-ray absorptiometry was used to analyze body composition. Hematoxylin and eosin staining was used to assess changes in white adipose depots, mass spectrometry was used for circulating lipids and protein metabolite analysis, and ribosomal RNA sequencing was used for gut microbiome analysis. Mice fed a high fat-diet since wean displayed increased medullary areas and decreased osteoblast numbers in the long bones; this phenotype was partially normalized by metformin. Marrow and inguinal adipose expansion was also noted in obese mice, and this was partially normalized by metformin. A drug-by-diet interaction was noted for circulating lipid molecules, protein metabolites, and gut microbiome taxonomical units. Obesity was not detrimental to trabecular bone in growing mice, but bone marrow medullary expansion was observed, likely resulting from inhibition of osteoblastogenesis, and this was partially reversed by metformin treatment.
Topics: Absorptiometry, Photon; Adipose Tissue, White; Adiposity; Animals; Body Composition; Bone Marrow; Cell Count; Chromatography, Liquid; Cortical Bone; Diet, High-Fat; Gastrointestinal Microbiome; Immunohistochemistry; Lipid Metabolism; Male; Mass Spectrometry; Metabolomics; Metformin; Mice; Mice, Inbred C57BL; Obesity; Organ Size; Osteoblasts; Phenotype; RNA, Bacterial; RNA, Ribosomal, 16S; Tandem Mass Spectrometry; X-Ray Microtomography
PubMed: 28977604
DOI: 10.1210/en.2017-00299 -
Neural Regeneration Research Dec 2017Peripheral nerve injury is a serious disease and its repair is challenging. A cable-style autologous graft is the gold standard for repairing long peripheral nerve...
Peripheral nerve injury is a serious disease and its repair is challenging. A cable-style autologous graft is the gold standard for repairing long peripheral nerve defects; however, ensuring that the minimum number of transplanted nerve attains maximum therapeutic effect remains poorly understood. In this study, a rat model of common peroneal nerve defect was established by resecting a 10-mm long right common peroneal nerve. Rats receiving transplantation of the common peroneal nerve in situ were designated as the in situ graft group. Ipsilateral sural nerves (10-30 mm long) were resected to establish the one sural nerve graft group, two sural nerves cable-style nerve graft group and three sural nerves cable-style nerve graft group. Each bundle of the peroneal nerve was 10 mm long. To reduce the barrier effect due to invasion by surrounding tissue and connective-tissue overgrowth between neural stumps, small gap sleeve suture was used in both proximal and distal terminals to allow repair of the injured common peroneal nerve. At three months postoperatively, recovery of nerve function and morphology was observed using osmium tetroxide staining and functional detection. The results showed that the number of regenerated nerve fibers, common peroneal nerve function index, motor nerve conduction velocity, recovery of myodynamia, and wet weight ratios of tibialis anterior muscle were not significantly different among the one sural nerve graft group, two sural nerves cable-style nerve graft group, and three sural nerves cable-style nerve graft group. These data suggest that the repair effect achieved using one sural nerve graft with a lower number of nerve fibers is the same as that achieved using the two sural nerves cable-style nerve graft and three sural nerves cable-style nerve graft. This indicates that according to the 'multiple amplification' phenomenon, one small nerve graft can provide a good therapeutic effect for a large peripheral nerve defect.
PubMed: 29323049
DOI: 10.4103/1673-5374.221167 -
Frontiers in Neuroscience 2018Stable posture and body movement in humans is dictated by the precise functioning of the ampulla organs in the semi-circular canals. Statistical analysis of the...
Stable posture and body movement in humans is dictated by the precise functioning of the ampulla organs in the semi-circular canals. Statistical analysis of the interrelationship between bony and membranous compartments within the semi-circular canals is dependent on the visualization of soft tissue structures. Thirty-one human inner ears were prepared, post-fixed with osmium tetroxide and decalcified for soft tissue contrast enhancement. High resolution X-ray microtomography images at 15 μm voxel-size were manually segmented. This data served as templates for centerline generation and cross-sectional area extraction. Our estimates demonstrate the variability of individual specimens from averaged centerlines of both bony and membranous labyrinth. Centerline lengths and cross-sectional areas along these lines were identified from segmented data. Using centerlines weighted by the inverse squares of the cross-sectional areas, plane angles could be quantified. The fit planes indicate that the bony labyrinth resembles a Cartesian coordinate system more closely than the membranous labyrinth. A widening in the membranous labyrinth of the lateral semi-circular canal was observed in some of the specimens. Likewise, the cross-sectional areas in the perilymphatic spaces of the lateral canal differed from the other canals. For the first time we could precisely describe the geometry of the human membranous labyrinth based on a large sample size. Awareness of the variations in the canal geometry of the membranous and bony labyrinth would be a helpful reference in designing electrodes for future vestibular prosthesis and simulating fluid dynamics more precisely.
PubMed: 29535601
DOI: 10.3389/fnins.2018.00107 -
Viruses Mar 2018Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select...
Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV () in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.
Topics: Animals; Antiviral Agents; Cell Line; Chlorocebus aethiops; Cytopathogenic Effect, Viral; Disinfectants; Ebolavirus; Hemorrhagic Fever, Ebola; Humans; Microbial Sensitivity Tests; Sensitivity and Specificity; Vero Cells; Virus Inactivation
PubMed: 29533988
DOI: 10.3390/v10030126 -
Cancers Jan 2023Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of...
Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.
PubMed: 36672277
DOI: 10.3390/cancers15020328 -
Journal of Visualized Experiments : JoVE Jul 2018Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently...
Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2μm thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.
Topics: Animals; Histological Techniques; Peripheral Nerves; Rats; Staining and Labeling; Tolonium Chloride
PubMed: 30035773
DOI: 10.3791/58031 -
International Journal For Parasitology Sep 2021Parasitic infections can be challenging to study because two dimensional light and electron microscopy are often limited in visualising complex and inaccessible...
Parasitic infections can be challenging to study because two dimensional light and electron microscopy are often limited in visualising complex and inaccessible attachment sites. Exemplifying this, Trichuris spp. inhabit a tunnel of epithelial cells within the host caecum and colon. A significant global burden of this infection persists, partly because available anthelminthics lack efficacy, although the mechanisms underlying this remain unknown. Consequently, there is a need to pioneer new approaches to better characterize the parasite niche within the host and investigate how variation in its morphology and integrity may contribute to resistance to therapeutic intervention. To address these aims, we exploited three-dimensional X-ray micro-computed tomography (microCT) to image the mouse whipworm, Trichuris muris, in caeca of wild-type C57BL/6 and SCID mice ex vivo. Using osmium tetroxide staining to effectively enhance the contrast of worms, we found that a subset exhibited preferential positioning towards the bases of the intestinal crypts. Moreover, in one rare event, we demonstrated whipworm traversal of the lamina propria. This morphological variability contradicts widely accepted conclusions from conventional microscopy of the parasite niche, showing Trichuris in close contact with the host proliferative and immune compartments that may facilitate immunomodulation. Furthermore, by using a skeletonization-based approach we demonstrate considerable variation in tunnel length and integrity. The qualitative and quantitative observations provide a new morphological point of reference for future in vitro study of host-Trichuris interactions, and highlight the potential of microCT to characterise enigmatic host-parasite interactions more accurately.
Topics: Animals; Mice; Mice, Inbred C57BL; Mice, SCID; Mucous Membrane; Trichuriasis; Trichuris; X-Ray Microtomography
PubMed: 34216623
DOI: 10.1016/j.ijpara.2021.04.006 -
Zebrafish Feb 2015Zebrafish has been used as a powerful model system in biological and biomedical studies studying development and diseases. Comparative, functional, and developmental...
Zebrafish has been used as a powerful model system in biological and biomedical studies studying development and diseases. Comparative, functional, and developmental studies on zebrafish morphology require precise visualization of 3D morphological structures. Few methods that can visualize whole-volume of zebrafish tissues are available because optical bio-imaging methods are limited by pigmentation and hard tissues. To overcome these limitations, the 3D microstructures of a hypercholesterolemic zebrafish model are visualized using synchrotron X-ray micro-computed tomography (SR-μCT). The model spatial resolution ranged from sub- to several microns. The microstructures of various zebrafish organs are observed by combining high-contrast staining (osmium tetroxide and uranyl acetate) and embedding a protocol to enhance the image contrast of soft tissues. Furthermore, blood vessels are identified using a barium sulfate injection technique. The internal organs and cells, such as liver, intestine, oocytes, and adipocytes, of a hypercholesterolemic zebrafish are compared with those of normal organs and cells. The SR-μCT is useful for understanding the pathogenesis of circulatory vascular diseases by detecting the modifications in the 3D morphological structures of the whole body of the zebrafish. This bio-imaging technique can be readily used to study other disease models.
Topics: Animals; Female; Hypercholesterolemia; Lipid Metabolism; Organ Specificity; Synchrotrons; X-Ray Microtomography; Zebrafish
PubMed: 25521241
DOI: 10.1089/zeb.2014.1039 -
Journal of Periodontal Research Jun 2019The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a...
The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week-old male C57BL/6 mice (N = 5) were experimentally translated for 2 weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship's resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurred as an attempt to restore PDL-space of the load-bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs provided insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally induced resorption, and naturally occurring mineral resorption to experimentally induced formation at the enthesial sites to permit tooth translation.
Topics: Animals; CD146 Antigen; Dental Cementum; Integrin-Binding Sialoprotein; Male; Mice, Inbred C57BL; Periodontal Ligament; Regeneration; Sp7 Transcription Factor; Tooth Mobility; Tooth Movement Techniques; Ubiquitin Thiolesterase; X-Ray Microtomography
PubMed: 30485431
DOI: 10.1111/jre.12625