Ultrathin Sections Research Articles

Assessment of Rat Spleen Structure under an Isolated and Combined Action of Selenium and Ethyl Carbamat

The immune system, having a complex structural organization, performs the main functions in providing an anti-inflammatory and antitumor immune response. The spleen, as a peripheral organ of the immune system, plays an important role in realization of these functions and contains a significant number of immune cells. Ethylcarbamate, being a group 2A carcinogen, can provoke the development of neoplastic processes through activation of Kras gene mutation and stimulation of oncogenic signals. Alongside with that, selenium affects the immune response, reducing the proliferation of tumor cells and producing an anti-inflammatory effect. Despite the existence of several studies on the effect of ethyl carbamate and selenium on the animal body, there is no data on the combined effect of these substances on the rat spleen under experimental conditions in the scientific literature. The aim of the study was to evaluate morphological and morphometric changes in the structure of the spleen in male rats with oral administration of selenium into their body and intraperitoneal injection of ethyl carbamate. Materials and methods of the study. The body mass of male rats and their spleen were studied, morphological and morphometric parameters of the spleen were evaluated, the lymphoid coefficient and the Kernoghan index were calculated. The animals were divided into four groups: N1 – control group; N2 – animals receiving selenium with drinking water; N3 – animals injected intraperitoneally with ethyl carbamate; N4 – animals receiving selenium with drinking water followed by injection of ethyl carbamate. Results. It was found that in the group 2, the area of lymphoid nodules was reduced, the Kernoghan index was reduced and the lymphoid coefficient was increased, while the body weight of rats and their spleen remained unchanged. In a month after an isolated intake of ethyl carbamate, both the body weight and the spleen weight in rats were significantly decreased. At this, in group 3, the area and the diameter of the germinal center, the thickness of the periarterial lymphoid sheath and the Kernoghan index were increased, and the lymphoid coefficient was reduced. At the ultramicroscopic level, an increase in the number of plasma cells was detected in group N3, as well as the number of swollen mitochondria in lymphocytes. In group 4, an increase in the area and the diameter of lymphoid nodules, the germinal center, as well as the lymphoid coefficient was determined, and the Kernoghan index was unchanged. When studying ultrathin sections in group 4 lymphocytes of various transitional forms were revealed. Conclusions. It was found that against the background of selenium intake into the body of male rats, a morphological rearrangement of the white pulp of the spleen occurred, probably associated with the reaction of T-lymphocytes. Ethyl carbamate injection resulted in a predominant effect on the B-cell link of the immune response of the spleen. In combined intake of selenium and ethyl carbamate, hyperplasia of the white pulp was revealed, consisting in an increase in its structures.

Ultrastructure of adrenal glands endocrinocytes after thermal skin injury under conditions of correction with cryo-lyophilized xenograft skin substrate

According to WHO studies, burn injuries are one of the most common injuries in the world, and their number has increased significantly due to the military actions in Ukraine. The adrenal glands, which regulates metabolism and participates in the body's defense reactions, is sensitive to burn injuries, which leads to morphological changes in the organ. The aim of the study was to investigate the ultrastructural changes in endocrinocytes of the adrenal glands after experimental thermal skin injury under conditions of correction with cryo-lyophilized xenograft skin substrate. A second-degree burn was modeled by applying heated copper plates to the skin of the back of laboratory male rats, which accounted for 18-20 % of the body surface. For electron microscopic analysis on days 7th, 14th, and 21st of the experiment, pieces of the adrenal glands were taken. They were fixed in a glutaraldehyde solution, post-fixed in osmium tetraoxide, and processed according to standard procedures. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and submicroscopic changes were studied using a PEM-125K electron microscope. On the 7th day after the burn with correction (using a cryo-lyophilized xenograft skin substrate), moderate changes in the ultrastructure of the nuclei and organelles of the endocrinocytes were found. Mitochondrial hyperplasia, changes in the shape of the nuclei and the presence of small clumps of heterochromatin in the karyoplasm were observed. On the 14th day, under the conditions of correction, there were signs of restoration of the cell ultrastructure, in particular, the structure of the nucleus and the membrane of the zona glomerulosa cells was restored, ribosomes and hypertrophied mitochondria with an electronically bright matrix appeared, as well as numerous lipid droplets. After 21 days, the ultrastructure of the cells of the zona glomerulosa was restored, and lipid droplets were evenly distributed in the cytoplasm. In the zona reticularis and adrenal medulla, changes in the submicroscopic organization of endocrinocytes were insignificant. We observed a uniform distribution of secretory granules in the cytoplasm of chromaffinocytes, mitochondrial recovery and unchanged ultrastructural organization of the organelles of the synthetic apparatus. Thus, the results of the study confirmed that the cryo-lyophilized xenograft skin substrate effectively promotes the restoration of the ultrastructure of adrenal endocrinocytes, reducing the degree of damage to cytoplasmic membranes, organelles of the synthetic apparatus of cells, endocrinocyte nuclei, especially activating the regeneration processes in the late stages after thermal skin injury.

Advancing yeast cell analysis: A cryomethod for serial block-face scanning electron microscopy imaging in mitochondrial morphology studies.

Conventional Transmission Electron Microscopy analysis of biological samples often provides limited insights due to its inherent two-dimensional (2D) nature. This limitation hampers a comprehensive understanding of the three-dimensional (3D) complexity of cellular structures, occasionally leading to misinterpretations. Serial block-face scanning electron microscopy emerges as a powerful method for acquiring high-resolution 3D images of cellular volumes. By iteratively removing ultrathin sample sections and capturing images of each newly exposed surface, Serial block-face scanning electron microscopy allows for the meticulous reconstruction of a comprehensive 3D volume. In this study, we investigate the 3D architecture of altered mitochondrial morphologies in Saccharomyces cerevisiae using Serial block-face scanning electron microscopy imaging. We have developed a novel cryomethod based on plunge freezing and a dedicated freeze-substitution protocol. This protocol enhances ultrastructural preservation enabling a more accurate understanding of mitochondrial defects observed in 2D electron microscopy. Our findings underscore the utility of Serial block-face scanning electron microscopy coupled with optimized sample preparation techniques in elucidating complex cellular structures in 3D.

Cryopreservation of Human Adult Ventricular Tissue for the Preparation of Viable Myocardial Slices.

Living myocardial slices (LMS) are ultrathin sections of adult myocardium that can be maintained in culture. These slices provide a unique platform for studying interactions between cardiomyocytes (CMs), other cardiac cell types, and the extracellular matrix while maintaining the cytoarchitecture and electrical phenotype of CMs over extended periods. Despite their advantages over other cardiac models, LMS have limitations, particularly their reliance on slice quality. The primary factor influencing the quality of the slices is the method used to process the cardiac tissue block. Current methods typically require immediate slice preparation following the excision of the tissue block, which restricts the timing of experiments. To address this limitation, we developed a simple procedure for cryopreserving human adult myocardium, allowing the preparation of LMS at a later stage. The protocol provides a list of required equipment and reagents, as well as a detailed description of the methodology for processing the myocardium and slice preparation. We present typical results demonstrating that cryopreserved human cardiac tissue retains biomass and structural integrity comparable to freshly obtained myocardium. Furthermore, we assessed the LMS derived from both fresh and cryopreserved samples. Histological analysis confirmed the preservation of viability, normal cytomorphology, and gap junctions between CMs in all LMS after 24 h and up to 5 days of culture in the absence of electrical stimulation. Cryopreservation extends the interval between tissue collection and LMS preparation, facilitating longer-term and more complex experiments. Further research into the impact of cryopreservation on various cardiac cell types will promote better donor organ management and efficient banking of cardiac samples from a multitude of donors and disease states. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and preservation of human adult myocardium Basic Protocol 2: Preparation of adult living myocardial slices from cryopreserved blocks.

Infiltration of salivary proteins into dentin during erosive processes

ObjectiveUltrastructural analyses showed that during erosion under oral cavity conditions, dentin is infiltrated by a substrate morphologically similar to salivary proteins. This in-situ study aimed to investigate the presence of salivary proteins in demineralized dentin. MethodsBovine dentin specimens were attached to individual maxillary splints (n = 1 per subject and condition) and worn intraorally by four subjects for 1 min. During intraoral exposure, the subjects rinsed with 10 ml of either sterile water or 1 % citric acid. Additional specimens were eroded in-vitro as controls (n = 2). The specimens were then fixed, embedded in acrylic resin, and ultrathin sections were prepared. The salivary protein lysozyme was labelled with immunogold staining and examined using transmission electron microscopy. ResultsLysozyme was absent in the in-vitro controls but was detected on the dentin surface after rinsing with water, indicating its role in the initial pellicle formation on dentin. After rinsing with citric acid, lysozyme infiltrated deeper layers of the dentin. ConclusionsThese findings support the initial morphological observations that lysozyme, representing other salivary proteins, is deposited in demineralized dentin during erosion with citric acid. Clinical significanceInfiltration of salivary proteins into dentin during erosive processes can be a factor to consider in remineralization approaches under oral cavity conditions.

Analyzing Extracellular Vesicle-associated DNA Using Transmission Electron Microscopy at the Single EV-level.

Extracellular vesicles (EVs) play an important role in cell-cell communication, carrying bioactive molecules including DNA. EV-associated DNA (EV-DNA) has created enormous interest in the field of biomarkers, particularly related to liquid biopsy. However, its analysis is challenging due to the nanoscale structure of EVs, the low abundance of EV-DNA, and surrounding biogenetic debate. Therefore, novel protocols to enhance the accurate detection of EV-DNA are essential to study its role in normal physiology and disease states. Here, we provide two protocols for confirming the presence of EV-DNA from biological samples. In the first protocol, ultrathin sectioning of EVs is combined with immunogold labeling to detect the presence of double-stranded (ds) DNA within the EV lumen using transmission electron microscopy (TEM). In the second protocol, whole-mount EV immunogold labeling allows detailed morphological analysis of EVs and their surface-associated DNA. Using TEM imaging, we have demonstrated that cancer-cell-derived individual EVs exhibit simultaneous positivity for dsDNA and the EV surface protein tetraspanin 9. We believe that this method can be used to label any proteins of interest inside as well as on the surface of EVs. This can aid in the characterization of single EVs and in the identification and verification of EV-associated biomarkers. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: EV isolation from cell-culture-conditioned medium, EV embedding, ultrathin sectioning, labeling, and imaging Basic Protocol 2: Whole-mount immunolabeling of EV-DNA.

Ultrastructural Perspectives on the Biology and Taphonomy of Tonian Microfossils From the Draken Formation, Spitsbergen.

Silicified peritidal carbonates of the Tonian Draken Formation, Spitsbergen, contain highly diverse and well-preserved microfossil assemblages dominated by filamentous microbial mats, but also including diverse benthic and/or allochthonous (possibly planktonic) microorganisms. Here, we characterize eight morphospecies in focused ion beam (FIB) ultrathin sections using transmission electron microscopy (TEM) and X-ray absorption near-edge structure (XANES) spectromicroscopy. Raman and XANES spectroscopies show the highly aromatic molecular structure of preserved organic matter. Despite this apparently poor molecular preservation, nano-quartz crystallization allowed for the preservation of various ultrastructures distinguished in TEM. In some filamentous microfossils (Siphonophycus) as well as in all cyanobacterial coccoids, extracellular polysaccharide sheaths appear as bands of dispersed organic nanoparticles. Synodophycus microfossils, made up of pluricellular colonies of coccoids, contain organic walls similar to the F-layers of pleurocapsalean cyanobacteria. In some fossils, internal content occurs as particulate organic matter, forming dense networks throughout ghosts of the intracellular space (e.g., in Salome svalbardensis filaments), or scarce granules (in some Chroococcales). In some chroococcalean microfossils (Gloeodiniopsis mikros, and also possibly Polybessurus), we find layered internal contents that are more continuous than nanoparticulate bands defining the sheaths, and with a shape that can be contracted, folded, or invaginated. We interpret these internal layers as the remains of cell envelope substructures and/or photosynthetic membranes thickened by additional cellular material. Some Myxococccoides show a thick (up to ~ 0.9 μm) wall ultrastructure displaying organic pillars that is best reconciled with a eukaryotic affinity. Finally, a large spheroid with ruptured wall, of uncertain affinity, displays a bi-layered envelope. Altogether, our nanoscale investigations provide unprecedented insights into the taphonomy and taxonomy of this well-preserved assemblage, which can help to assess the nature of organic microstructures in older rocks.

Remodeling of the structural components of the capsule and glomerular zone of the adrenal glands cortex of white rats under the influence of a complex of food additives at the late terms of the experimental study

With food additives, we can add flavour to food, extend its shelf life, and make it look good. However, even small amounts of these additives can lead to diseases of various organs and systems. The aim of our work is to establish metrics and morphological changes in the structure of the capsule and the glomerular zone of the cortex of the adrenal glands of rats at the later stages of the experimental study under the conditions of consumption of a complex of food additives: monosodium glutamate, sodium nitrite and Ponceau 4R. The control group of rats consumed oral drinking water and saline. The rats of the experimental groups were orally administered once a day with a 10 % solution of sodium nitrite (E250), sodium glutamate (E621) or Ponceau 4R. The dosages of food additives were two times lower than the permissible norm in food. The rats were withdrawn from the experiment after 8, 12 and 16 weeks. The adrenal glands were removed for the study. Paraffin sections were stained with hematoxylin and eosin, and semi-thin sections with toluidine blue, and examined using a light microscope Biorex with a digital microphoto attachment DSM 900. Ultrathin sections were examined in a TEM-125 K electron microscope. For morphometric analysis, the structure of the capsule and the glomerular zone of the cortex of the adrenal glands of rats at the later stages of the experimental study was studied. The study results were analysed using the software package “InStat”. At the late stages of the experimental study, the adrenal capsule underwent irreversible changes. The average thickness of the adrenal connective tissue capsule decreased by the end of the 16th week of the experimental research. Similarly, the average thickness of the cortex decreased with each subsequent week relative to the control values, indicating significant structural and functional disorders. The average thickness of the glomerular zone of the adrenal cortex periodically changed as a compensatory reaction in response to the administration of a complex of food additives to rats. The phenomena of nucleus pyknosis and sludge syndrome were detected, which indicated compensatory and adaptive processes at the microscopic level. The analysis of electron micrographs revealed destroyed granular endoplasmic reticulum cisternae and mitochondrial remnants. Thus, it has been established that using a complex of food additives (monosodium glutamate, sodium nitrite and Ponceau 4R) directly affects the adrenal glands of rats. This effect causes structural changes in the cortex and medulla, causing alteration and hypertrophy of cells in these areas. Additionally, hyperhydration of the connective tissue stroma and compensatory-restorative reactions aimed at transcription factors and internal mechanisms of nitric oxide formation were recorded.

CLINICOPATHOLOGIC CHANGES OF VITREOMACULAR INTERFACE IN IDIOPATHIC EPIRETINAL MEMBRANE WITH DISORGANIZATION OF RETINAL INNER LAYERS.

To compare the pathological characteristics of the vitreomacular interface of the idiopathic epiretinal membrane with and without disorganization of retinal inner layers (DRIL) and to correlate with clinical data. In this clinicopathologic study, the samples of epiretinal membrane and internal limiting membrane were extracted from DRIL(+) (19 eyes) and DRIL(-) (22 eyes) idiopathic epiretinal membrane eyes. Ultrathin series sectioning for transmission electron microscopy was observed and correlated with surgery status and prognosis. All idiopathic epiretinal membrane eyes presented fibrocellular membranes accompanied by vitreous collagen, glial cells, and myofibroblasts, regardless of association with DRIL. A robust signal indicative of Collagen Type VI was observed in eyes DRIL(-), whereas Collagen Type I was discovered in DRIL eyes. Cell debris and microvascular basement membrane were seen on the retinal side of DRIL eyes and a larger cell count on the vitreous side. These have more intraoperative complications and less surgery benefit. Although internal limiting membrane peeling seems important, the histopathologic findings underscore the potential for retinal injury in DRIL(+) idiopathic epiretinal membrane eyes. This suggests that further research is needed to investigate individual preoperative assessment and to modify surgical procedures.

Giant membrane rings/loops in the cytosol of P. falciparum-infected erythrocytes and their relation to the parasite.

Striking morphological transformations characterize the invasion of a red blood cell by the malaria parasite. Shortly after the infection, parasite-induced membranes appear in the cytosol of the affected host erythrocyte. One intensely investigated membrane type, commonly called Maurer's clefts, has a slit-like morphology and can be arranged in the form of extended three-dimensional membrane stacks or networks. Here we report the three-dimensional reconstruction of a second membrane type, giant or extended membrane rings/loops, that have only occasionally been described on single ultrathin sections, however that have never been systematically examined so far. Serial ultrathin sectioning of P. falciparum-infected red blood cells, subsequent three-dimensional reconstructions, and in addition examination of Giemsa-stained blood films revealed that intraerythrocytic membrane rings/loops are not isolated structures but are locally in contact with the parasite. They consist either of the parasitophorous vacuolar membrane alone or contain the parasitophorous vacuolar membrane including the plasma membrane of the parasite and small amounts of parasite cytoplasm. We demonstrate that membrane rings/loops represent surface extensions of the parasite that maybe involved in ring stage parasite formation and Maurer's cleft generation at least in a subset of infected red blood cells.

Ultraplex microscopy: versatile highly-multiplexed molecular labeling and imaging across scale and resolution.

The molecular organization of cells and tissue is challenging to study due to the inefficiency of multiplexed molecular labeling methods and the limited options for combining microscopy modalities in a single specimen, especially when high spatial resolution is needed. Here we describe ultraplex microscopy, which combines serial multiplexing, ultrathin sectioning, and reversible embedding to circumvent incompatibilities between labeling and imaging techniques, enhance resolution, and expand multiplexing capacity within and across modalities. Samples can be labeled with antibodies, RNA probes, and tissue stains for imaging by brightfield, epifluorescence, super-resolution, and electron microscopy without specialized reagents or materials. We demonstrate applications in brain tissue including molecular profiling of single cells and axonal boutons, high-resolution molecular colocalization, and correlative imaging of fluorescent proteins with confocal and ultraplex microscopy. The power and versatility of ultraplex microscopy will be valuable in addressing currently intractable experimental questions in many systems and contexts.

Comparative effect of ivermectin and amitraz on cellular architecture of ovaries, synganglion and Gené's organ of Rhipicephalus microplus (Acari: Ixodidae).

The present study investigated the effect of ivermectin and amitraz on the cellular architecture of vital organs of Rhipicephalus microplus. Adult female ticks were treated with lethal concentrations (LC95) of ivermectin and amitraz, and the ovaries, synganglion, and Gené's organ were processed 48h post treatment. In both the treatment groups, the ultra-thin sections of ovary exhibited deformed oocytes, irregular plasmic membrane and chorion layer, extensive vacuolation in the cytoplasm mainly at periphery of the cell and oocyte-pedicel junction. Marked vacuolations in the cortex and neuropile region with significant structural disorganization of the neural fibers were common alterations observed in the synganglion of ticks exposed to ivermectin and amitraz. The tissue sections of Gené's organ revealed deformed tubular glands with severe loss of cellular limit of secretory epithelium and cytoplasmic vacuolations in the ivermectin treated ticks whereas, the alterations were comparatively less severe in amitraz exposed ticks. The cellular deformities in these vital organs probably impaired reproductive function, nerve signal transmission and metabolic activities and thus affected fecundity and survivability of the treated ticks. The findings suggested that the action of ivermectin and amitraz are not restricted to the nervous system of ticks, but also on other vital organs, ovary and Gené's organ affecting the oviposition. The study provided insights into the development of targeted interventions for tick control strategies.

The effects and mechanisms of novel antibacterial amyloid peptides derived from Streptococcus mutans proteome

Streptococcus mutans (S. mutans) is one of the primary cariogenic pathogens. Recently the aggregation prone region (APR) has been applied to develop novel antibacterial amyloid peptides due to its ability to target and trigger the aggregation of proteins containing similar APRs. In this study, we modified 63 novel amyloid peptides based on the APRs screened from S. mutans proteome. Three of the peptides exhibited significant antibacterial activity against S. mutans, but without specificity. Transmission electron microscope (TEM), stain of Congo red and ThT, and Fourier transform infrared spectroscopy (FTIR) showed that the antimicrobial amyloid peptides (C9 and C12) aggregated into unmature fibrils, not typical morphology of mature amyloid fibrils. Distortion and perforation of cell membrane were observed by various microscopes. Membrane permeabilization assay and SYTO9/PI staining assay verified that the membrane permeability increased after being treated by C9 and C12. Aggregates and unmature fibrils could be observed inside dead cells in the ultrathin sections by TEM. Transcriptome sequencing (RNA-seq) detected the significant expression changes of genes related to protein aggregates formation (dnaK, groL and groES) and metabolism (gtfB), which was further verified by quantitative real-time PCR (qRT-PCR). In conclusion, we innovatively developed antimicrobial amyloid peptides based on the APRs in S. mutans proteome and found their combination antimicrobial mechanism of damaging the membrane with triggering protein aggregation, which might shed light on novel antibacterial peptides development.

External surface Al-rich ZSM-48 zeolite with high efficient n-dodecane hydroisomerization activity

ZSM-48 zeolites with exterior Al-rich (Z-48-Sx) are synthesized by the recrystallization method. The crystal structure of Z-48-Sx with the Al-rich outer layer is verified by analyzing the crystallization process of Z-48-Sx and the EDS of the ultrathin section. The diffusion properties of Z-48-S3 and Z-48-C are investigated by the zero-length column (ZLC) method, and the synthesized ZSM-48 zeolite with an Al-rich external layer effectively improves the diffusion ability. The effective diffusion constants of Z-48-S3 are 53 %–102 % higher than those of the conventional ZSM-48 (Z-48-C). Meanwhile, the values of turnover frequency (TOF) of Z-48-S3 are increased by 14 %–63 % at similar n-dodecane conversions compared with Z-48-C, which is closely related to the diffusion characteristics. In addition, the depth of hydroisomerization (Iiso) of Z-48-S3 is decreased by 65.3 %, and the isomerization selectivity is increased by 52.5 % at 340 °C compared with that of Z-48-C. Hence, the ZSM-48 molecular sieves synthesized by the recrystallization hydrothermal method are beneficial for controlling the depth of hydroisomerization of the catalyst and reducing the occurrence of the cracking reaction. Meanwhile, the isomer distribution does not change significantly compared with that of the conventional ZSM-48, and the 5-methylundecane is still the main isomerization product.

Array tomography: trails to discovery.

Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.

The morphofunctional state of the hematoalveolar barrier of the lungs in COVID-19 patients compared to pandemic influenza

Background. It is known that viral infections, such as pandemic influenza A(H1N1) and COVID-19, are characterized by acute respiratory distress syndrome, which in some cases is accompanied by high mortality. It has also been established that diffuse alveolar damage (DAD) is common to both influenza A and COVID-19. However, there are some differences between these infections. In contrast with pandemic flu, the most frequent symptom of COVID-19 is thromboembolic complications. The examination of the autopsy material showed that, despite the large number of conducted studies, the morphofunctional state of the hematoalveolar barrier of the lungs has not been sufficiently studied and some mechanisms of the development of pathological reactions of the оrganism in response to the action of influenza viruses A(H1N1) pdm09 and SARS-CoV-2 remain unclear. But the post-mortem picture of the hematoalveolar barrier can allow us to see all previous links of the viral lung damage process. This, in turn, will help to solve the problem of timely diagnosis of the disease severity, prognosis, prevention of further development of the disease and appropriate methods of therapy. The purpose of the work was to study the features of the SARS-CoV-2 virus effect on the morphofunctional state of the hematoalveolar region of the lungs at autopsy, which allows the pathogenesis of COVID-19 to clarified, critical mechanisms, thedevelopment of which leads to fatal consequences, to be established, and a therapy strategy to be proposed. Materials and methods. 8 samples of autopsy material of the lungs of patients with COVID-19 were studied. The comparison groups were patients with pandemic influenza A caused by the H1N1 virus (3 autopsies) and lung samples obtained during surgery from a patient with traumatic pneumothorax at a distance from the main focus. Lung samples were processed according to standard methods of biological material preparation for electron microscopy. From the obtained blocks, semi-thin sections with a thickness of 1 μm were made. They were stained with methylene blue, and studied under a light microscope at a magnification of 500 times. Digital photographs were obtained from the most informative areas. Ultrathin sections were analyzed in electron microscope after being contrasted in uranium and lead salts. The incidence of thrombosis in the studied groups was calculated. Statistical processing was performed using the Fisher’s non-parametric exact test and the Biostat PC software package. Results. The conducted studies showed that patients with pandemic influenza A had a pronounced lesion of the alveolar part of the lungs with damage to the alveolar walls of the lungs and the appearance of hyaline membranes in them. Desquamated pneumocytes, erythrocytes, neutrophilic leukocytes, macrophages and fibrin clots were found in alveoli filled with serous fluid. Among the features of the influenza virus, the preservation of cellular elements in the alveolar walls, the convergence of the alveolar septums and the formation of large areas with a high level of neutrophilic and macrophage invasion should be noted. Macrophages patrol the fields of damaged alveolar septums, separating them from the surrounding tissue. The subsequent fibrotization indicates the replacement of the destroyed lung tissue. The group of patients with COVID-19, in contrast to the pandemic flu, has significantly higher rates of thrombosis of various calibers. This is the result of disseminated intravascular coagulation, which often leads to fatal consequences. Erythrocytes in vascular thrombi have a tendency to agglutination, which is more pronounced in oncological patients. For all studied cases, epithelial membrane damage is characterized by almost complete loss of epithelial cells, which is accompanied by the denudation of capillaries, in which there is an accumulation of erythrocytes that gives them the look of «blood sausages». Sometimes these vessels are attached to hyaline membranes, sometimes they are arranged in chains or groups. No significant neutrophilic invasion or granulomatous inflammation is observed. Conclusions. The conducted studies showed that patients with pandemic influenza A had a pronounced lesion of the alveolar part of the lungs with a high level of neutrophilic and macrophage invasion. The mechanisms of the pathogenesis of COVID-19 are the direct destructive effect of the virus on the epithelial cells of the hemato-alveolar barrier, which obviously leads to the release of inflammatory cytokines that stimulate the systemic reaction of intravascular coagulation. It has been established that for oncology patients with a history of chemotherapy, the processes of thrombosis and destruction of alveolar walls during COVID-19 were one of the most pronounced lesions among the patients. The obtained results suggest that the fatal consequences of COVID-19 can obviously be prevented if therapeutic measures aimed at destroying the virus, blocking the targets for its penetration into cells, breaking the chain of intravascular blood coagulation with the use of corticosteroids, anticoagulants, cytokine inhibitors, etc. are taken immediately after receiving a positive diagnosis for COVID-19 and carrying out a set of laboratory tests to assess the severity of the disease.

Investigation of the ultrastructure of dental pulp odontoblasts in a patient following a COVID-19 viral infection

Relevance. Recent studies on odontoblast ultrastructure have indicated that these cells possess immune functions. Unfortunately, both domestic and international sources lack transmission electron microscopy research data that characterise the ultrastructure of odontoblasts. This gap in the literature limits the expansion of knowledge about these specific cells of the human dental pulp and their practical applications. An analysis of existing literature and our own research suggests that the organs of the oral cavity may become targets for the coronavirus. Odontoblasts, as specialized cells of the dental pulp, perform multiple functions. Their primary role is in dentinogenesis, but equally important is their participation in immune functions, evidenced by the presence of pattern recognition receptors (PRRs) on their surface. These include Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs), some of which can mount an immune response to the entry of SARS-CoV-2 into the cell.Materials and methods. This study involved a comprehensive clinical-laboratory investigation of the pulp from an intact tooth 2.8, extracted for orthodontic reasons from a patient who had recovered from a COVID-19 infection a few weeks prior to the tooth extirpation. The ultrastructure of the dental pulp was examined using transmission electron microscopy.Results. In the ultrathin sections of the pulp specimens from the extirpated tooth, specific odontoblast cells with clearly traceable ultrastructure were identified. The electron micrographs visualized features characteristic of viral entry into the cell: initial apoptotic changes in the nucleus of the odontoblast, the presence of clathrin-coated vesicles in the cytoplasmic membrane of the odontoblast process. Additionally, clathrin-coated invaginations of the cytolemma in the terminal parts of the odontoblasts were observed.Conclusion. The electron micrographs revealed ultrastructural changes indicative of a viral infection, suggesting the involvement of odontoblasts, equipped with the TLR3 and TLR4 system, in the antiviral defence against SARS-CoV-2.

Plastic deformation of the microfibrils in wet polyacrylonitrile fibers induced by the stretching field

AbstractThe plastic deformation of the microfibrils governs the formation and development of orientation, crystallization, and structural defects in polyacrylonitrile (PAN) fibers due to the flow‐fiber coupling effect during stretching process. In this study, we investigated the morphological changes of microfibrils in PAN fibers during wet‐fiber stretching using ultrathin section technology and electron microscopy observation. Their plastic deformation mechanism was also revealed. An interconnected network was formed and separated by the pore/voids during coagulation process. Stretching forces facilitated the plastic deformation of microfibrils, resulted in their elongation and orientation. Moreover, the inter‐fibril distance reduced and pore/void shape and dimensions were changed. The distinct response of microfibril elements under stretching force resulted in skin‐core structures within the fiber. Tensile strength increased from 27 to 382 MPa, while elongation at break decreased from 225% to 28%. Aligned microfibrils exhibited high tensile strength, whereas unoriented microfibrillar networks showed relatively high elongation properties. Additionally, we proposed a relationship between microfibril morphology changes and fiber mechanical properties.

Extensive soma-soma plate-like contact sites (ephapses) connect suprachiasmatic nucleus neurons.

The hypothalamic suprachiasmatic nucleus (SCN) is the central pacemaker for mammalian circadian rhythms. As such, this ensemble of cell-autonomous neuronal oscillators with divergent periods must maintain coordinated oscillations. To investigate ultrastructural features enabling such synchronization, 805 coronal ultrathin sections of mouse SCN tissue were imaged with electron microscopy and aligned into a volumetric stack, from which selected neurons within the SCN core were reconstructed insilico. We found that clustered SCN core neurons were physically connected to each other via multiple large soma-to-soma plate-like contacts. In some cases, a sliver of a glial process was interleaved. These contacts were large, covering on average ∼21% of apposing neuronal somata. It is possible that contacts may be the electrophysiological substrate for synchronization between SCN neurons. Such plate-like contacts may explain why the synchronization of SCN neurons is maintained even when chemical synaptic transmission or electrical synaptic transmission via gap junctions is blocked. Such ephaptic contact-mediated synchronization among nearby neurons may therefore contribute to the wave-like oscillations of circadian core clock genes and calcium signals observed in the SCN.

#2518 The decrease of the filtration slit density, not proteinuria, is the early indication of the filtration barrier dysfunction

Abstract Background and Aims Intact podocyte foot processes (FP) play a crucial role in ensuring proper selective filtration in the kidney. Changes in FP morphology are thought to be the initial trigger for proteinuria, but definitive proof is still missing. As FP structures are below the optical resolution of light microscopes, changes are investigated by electron microscopy (EM) on ultrathin sections. In this study, the correlation between morphology and proteinuria was investigated using the Podocyte Exact Morphology Measurement Procedure (PEMP), a 3D-SIM-based technique. Our investigation was focused on podocyte damage caused by treatment with nephrotoxic serum (NTS) as a model for focal segmental glomerulosclerosis. Method The mice were treated with nephrotoxic serum (NTS), and kidneys were taken at 24, 48, and 72 hours after injection. Kidney sections (3 µm thick) were prepared for 3D-SIM analysis after staining with podocin and integrin and then fixed in formalin and embedded in paraffin. Filtration slit density (FSD) was quantified using the Podocyte Exact Morphology Measurement Procedure (PEMP) as described earlier (Siegerist et al. 2017). In addition, EM was performed on glutaraldehyde-fixed tissue samples using standard protocols. The albumin to creatinine ratio was also determined. Results We observed that NTS-treated mice developed moderate proteinuria after 48 hours. Using the super-resolution microscopy-based 3D-SIM technique to quantify podocyte PF morphology by calculating the filtrations slit density (FSD) revealed a significant reduction of the FSD in NTS-treated mice already after 24 hours. This structural change was confirmed by transmission electron microscopy analysis, showing that the effacement of podocytes FPs is the first sign of a change in the filtration barrier function before proteinuria is measurable. Conclusion PEMP analysis of standard histological sections from NTS-injected and control mice demonstrated that the first event in the functional loss of the size selectivity of the filtration barrier is the effacement of FP morphology, followed by detectable proteinuria. Therefore, the assessment of the FSD is a promising strategy for early and accurate detection of the loss of filtration barrier integrity, providing opportunities for faster and more precise diagnostics.