Osmium Tetroxide Research Articles

Quantification of porcine lower thoracic spinal cord morphology with intact dura mater using high-resolution μCT.

Spinal cord stimulation (SCS) is approved by the Food and Drug Administration for treating chronic intractable pain in the back, trunk, or limbs through stimulation of the dorsal column. Numerous studies have used swine as an analog of the human spinal cord to better understand SCS and further improve its efficacy. We performed high-resolution imaging of the porcine spinal cord with intact dura mater using micro-computed tomography (μCT) to construct detailed 3-dimensional (3D) visualizations of the spinal cord and characterize the morphology of the dorsal and ventral rootlets. We obtained spinal cords from Yorkshire/Landrace crossbred swine (N=7), stained samples with osmium tetroxide, and performed μCT imaging of the T12-T15 levels at isotropic voxel resolutions ranging from 3.3 to 50μm. We measured the anatomical morphology using the 3D volumes and compared our results to measurements previously collected from swine and human spinal cords via microdissection techniques in prior literature. While the porcine thoracic-lumbar spinal cord is a popular model for SCS, we highlight multiple notable differences compared to previously published T8-T12 human measurements including rootlet counts (porcine dorsal/ventral: 12.2±2.6, 26.6±3.4; human dorsal/ventral: 5.3±1.3, 4.4±2.4), rootlet angles (porcine ventral-rostral: 161±1°, ventral-caudal: 155±6°, dorsal-rostral: 148±9°, dorsal-caudal: 142±6°; human ventral-rostral: 170±3°, ventral-caudal: 22±10°, dorsal-rostral: 171±3°, dorsal-caudal: 15±7°), and the presence and count of dorsal rootlet bundles. Detailed measurements and highlighted differences between human and porcine spinal cords can inform variations in modeling and electrophysiological experiments between the two species. In contrast to other approaches for measuring the spinal cord and rootlet morphology, our method keeps the dura intact, reducing potential artifacts from dissection.

Streamlined chemical fixation method for morphological investigation of Candida albicans with scanning electron microscopy

This method describes the preparation of Candida albicans specimen for scanning electron microscopy (SEM), starting from specimen collection to obtaining SEM images. Our approach involves preserving cellular structure and preventing cell degradation through chemical fixation and post-fixation without Osmium tetroxide (OsO4), followed by air dried specimen placed onto Si substrate. Additionally, the method outlines the steps for mounting the prepared specimen onto SEM sample holder aluminum stubs, followed by coating with an ultrathin gold layer via thermal evaporation to enhance specimen/substrate/stub conductivity. The specimens are then mounted in the SEM sample holder. This step-by-step procedure demonstrates successful morphological analysis of C. albicans, providing a comprehensive and effective specimen preparation for SEM analysis.•Effective preparation of Candida albicans specimens for SEM analysis, preserving cellular structure.•Step-by-step specimen mounting and gold coating for enhanced detailed SEM imaging.•Chemical fixation of yeast cells for electron microscopy visualization.

Basal vacuolization of renal tubules in hypothermia: A comprehensive pathological study based on 79 forensic autopsy cases.

This study investigated the usefulness of histopathological examination in diagnosing ketoacidosis in hypothermia cases. Out of 79 cases (38 females), 30 showed basal vacuolization (BV), a representative histopathological finding of ketoacidosis, in their hematoxylin and eosin (H&E)-stained specimens. Through the use of both lipid staining with osmium tetroxide postfixation and immunohistochemistry for adipophilin (ADFP-IHC), BV pattern pathology was identified in 43 cases. Serum was available in 75 cases, with 37 having serum β-hydroxybutyrate levels exceeding 1000 μmol/L. Serum β-hydroxybutyrate concentrations showed a significantly negative correlation with age and tended to be lower in outdoor hypothermia cases than in indoor cases. When comparing serum β-hydroxybutyrate levels with histopathological findings, all but one of the BV-positive cases on H&E staining were serologically diagnosed as ketoacidosis. However, when combining multiple staining methods, six cases were histologically positive but serologically negative. Using multiple staining methods increases BV detection sensitivity, but it may also detect findings of unknown pathological significance. Therefore, identifying a distinct BV pattern in H&E-stained specimens is crucial for suspecting ketoacidosis in the usual practice of forensic pathology, and ADFP-IHC is useful for confirming lipid droplet presence. Our results suggest that BV positivity rates are strongly influenced by the case characteristics of the study cohort, particularly the proportion of older adults. Thus, the extent to which BV is complicated by hypothermia should be examined and understood on a country-by-country or region-by-region basis.

The Exploratory Study of the PTEN-AKT/mTOR Signaling Pathway in the Corresponding Dorsal Root Ganglion during Compensatory Repair via Small Gap Amplification in Sciatic Nerve Injury.

Peripheral nerve injury is a challenging orthopedic issue in clinical management that often leads to limb dysfunction or even disability in severe cases. A thorough exploration of the repair process of peripheral nerve injury and the underlying mechanism contributes to formulate more effective therapeutic strategies. In the present study, we established a sciatic nerve transection injury model in Sprague-Dawley (SD) rats. A 12-week compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis was then performed via sleeve jointing the proximal common peroneal nerve to the distal tibial nerve and common peroneal nerve, with a 2 mm interval. Compensatory repair via small gap amplification was observed via gross observation of nerve specimen, osmic acid staining, and electrophysiological stimulation of sciatic nerve branches of the tibial and common peroneal nerve. Rat limbs were observed, and the functional recovery of effector muscles of the gastrocnemius and tibialis anterior muscles was assessed through weighing the muscle wet weight, Hematoxylin and Eosin (H&E) staining, and muscle strength detection. H&E staining, Masson staining, and toluidine blue staining were performed to observe the morphological changes of the dorsal root ganglion. Positive expressions of key proteins involved in the Phosphatase and tensin homologue deleted on chromosome ten (PTEN)-protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, including PTEN, AKT, mTOR, Toll-like receptor 4 (TLR4), and Caspase9 in the dorsal root ganglion during compensatory repair of sciatic nerve after injury via small gap amplification, were detected by immunohistochemical staining. It is found that the compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis via sleeve jointing effectively restored the continuity, number of myelinated nerve fibers, and nerve conduction velocity. It promoted toe abduction recovery, improved muscle fiber morphology and increased the wet weight and muscle strength of the gastrocnemius muscle and tibialis anterior muscle. Moreover, it increased the number of neurons and nerve fibers, and improved their morphology. Downregulated PTEN, TLR4, and Caspase9 in the dorsal root ganglia and upregulated AKT and mTOR were observed after small gap amplification than those of the transection injury group, which were closer to those of the control group. Compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis via sleeve jointing can restore the morphology and function of the sciatic nerve, effector muscles, and corresponding dorsal root ganglia by activating the PTEN-AKT/mTOR signaling pathway in the dorsal root ganglia. Our findings provide novel therapeutic targets for peripheral nerve injuries.

Withaferin A Ameliorated the Bone Marrow Fat Content in Obese Male Mice by Favoring Osteogenesis in Bone Marrow Mesenchymal Stem Cells and Preserving the Bone Mineral Density.

Obesity and osteoporosis are two prevalent conditions that are becoming increasingly common worldwide, primarily due to aging populations, imbalanced energy intake, and sedentary lifestyles. Obesity, characterized by excessive fat accumulation, and osteoporosis, marked by reduced bone density and increased fracture risk, are often interconnected. High-fat diets (HFDs) can exacerbate both conditions by promoting bone marrow adiposity and bone loss. The effect of WFA on the osteogenesis and adipogenesis was studied on the C3H10T1/2 cell line and bone marrow mesenchymal stem cells (BM-MSCs) isolated from mice. We used oil red O and alkaline phosphatase (ALP) staining to observe adipogenesis and osteogenesis, respectively, in MSCs. Real-time PCR and Western blot analyses were used to study the molecular effects of WFA on MSCs. We employed micro-CT to analyze the bone microarchitecture, bone mineral density (BMD), and abdominal fat mass in male mice. We have used osmium tetroxide (OsO4) staining to study the bone marrow fat. WFA induced the C3H10T1/2 cell line and BM-MSCs toward osteogenic lineage as evidenced by the higher ALP activity. WFA also downregulated the lipid droplet formation and adipocyte specific genes in MSCs. In the in vivo study, WFA also suppressed the bone catabolic effects of the HFD and maintained the bone microarchitecture and BMD in WFA-treated animals. The bone marrow adipose tissue was reduced in the tibia of WFA-treated groups in comparison with only HFD-fed animals. Withaferin A was able to improve the bone microarchitecture and BMD by committing BM-MSCs toward osteogenic differentiation and reducing marrow adiposity. The findings of this study could provide valuable insights into the therapeutic potential of Withaferin A for combating bone marrow obesity and osteoporosis, particularly in the context of diet-induced metabolic disturbances.

OsO2 as the Contrast-Generating Chemical Species of Osmium-Stained Biological Tissues in Electron Microscopy.

Electron imaging of biological samples stained with heavy metals has enabled visualization of subcellular structures critical in chemical-, structural-, and neuro-biology. In particular, osmium tetroxide (OsO4) has been widely adopted for selective lipid imaging. Despite the ubiquity of its use, the osmium speciation in lipid membranes and the process for contrast generation in electron microscopy (EM) have continued to be open questions, limiting efforts to improve staining protocols and therefore high-resolution nanoscale imaging of biological samples. Following our recent success using photoemission electron microscopy (PEEM) to image mouse brain tissues with synaptic resolution, we have used PEEM to determine the nanoscale electronic structure of Os-stained biological samples. Os(IV), in the form of OsO2, generates nanoaggregates in lipid membranes, leading to a strong spatial variation in the electronic structure and electron density of states. OsO2 has a metallic electronic structure that drastically increases the electron density of states near the Fermi level. Depositing metallic OsO2 in lipid membranes allows for strongly enhanced EM signals and conductivity of biological materials. The identification of the chemical species and understanding of the membrane contrast mechanism of Os-stained biological specimens provides a new opportunity for the development of staining protocols for high-resolution, high-contrast EM imaging.

P-017 DNA fragmentation Index before and after density gradient centrifugation using flow cytometry, its reflection in sperm ultrastructure and its association with clinical outcome

Abstract Study question Is DNA fragmentation Index (DFI) affected by Density Gradient Centrifugation (DGC), reflected in sperm ultrastructure and associated with clinical outcome? Summary answer DFI was not affected by DGC, nor associated with clinical outcome, but it was negatively correlated with sperm concentration/motility and positively correlated with ultrastructural anomalies. What is known already Oxidative stress induces DNA fragmentation during sperm transit through the male genital tract and it is suggested to be the main mechanism causing DNA breaks after ejaculation during in vitro manipulation and sperm selection for injection/insemination. High levels of sperm DNA fragmentation have been linked to lower fertilization rates, poor embryo quality and delayed cleavage. Limited studies, have investigated in detail the effects of DGC on DFI and the reflection in sperm ultrastructure, while the predictive value of DFI on clinical outcome still remains controversial. Study design, size, duration Semen samples were collected on the day of the female partener’s oocyte collection and analysed before and after DGC for standard characteristics by light microscopy, according to World Health Organization (WHO) criteria (n = 120), for DNA Fragmentation by Flow Cytometry and for ultrastructure by Transmission Electron Microscopy (TEM). The predictive value of DFI for clinical outcome was also assessed. Participants/materials, setting, methods Standard semen analysis and DGC using the Sil-Select 40%/ 80 % layers were performed at an IVF Laboratory of an academic Hospital. Sperm DFI before and after DGC was assessed by Flow Cytometry at the Department of Immunology and Histocompatibility of the same academic Hospital. Ultrastructure analysis by TEM was carried out at an academic Histology/Embryology Laboratory following fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance DGC did not alter DFI significantly (p = 0.052), but lead to a statistically significant increase in Progressive Motility (p < 0.001). DFI was higher in men with a male factor infertility compared to men without a male factor (p = 0.032) and Oligoasthenoteratospermia (OAT) exhibited the highest DFI levels (36.38% ± 5.36). DFI was negatively correlated with sperm concentration (rho = -0.361, p < 0.01) and sperm motility (rho = -0.301, p < 0.05). No significant association was observed between DFI and clinical outcome, but statistically significant differences were observed both in the age of men (37.54±1.33 vs 42.8±1.27 p = 0.017) and the age of women (35.77±1.25 vs 40.17±0.78 p = 0.007) in the group of patients who achieved a pregmamcy and those that did not. TEM showed that DGC did not lead to an increase in ultrastructural anomalies of spermatozoa, but DFI was positively correlated with ultrastructural anomalies. In teratozoospermic men TEM revealed head, neck and tail defects, including elongated forms, axonemal defects and premature initiation of the acrosome reaction. Limitations, reasons for caution The ultrastructure analysis was not possible to be performed in severely oligospermic samples. Wider implications of the findings This study shows that sperm ultrastructure and DFI are not adversely affected by DGC confirming the safety of the preparation method for sperm selection prior to insemination/injection. Larger-scale studies are needed, to elucidate any potential correlations between DFI and the probability of pregnancy. Trial registration number not applicable

Serial block-face scanning electron microscopy of adherent cells on thin plastic substrate

Abstract Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

Deficiency of glucocorticoid receptor in bone marrow adipocytes has mild effects on bone and hematopoiesis but does not influence expansion of marrow adiposity with caloric restriction.

Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids. In this study, we utilized a recently described mouse model (BMAd-Cre) to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. Nr3c1) whilst maintaining GR expression in other adipose depots. Mice lacking GR in BMAds (BMAd-Nr3c1 -/-) and control mice (BMAd-Nr3c1 +/+) were fed ad libitum or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female BMAd-Nr3c1-/- mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and BMAd-Nr3c1-/- mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and μCT revealed no difference in BMAT accumulation between control or BMAd-Nr3c1 -/- mice. Differences in BMAd size were not observed between BMAd-Nr3c1-/- and control mice. Interestingly, BMAd-Nr3c1-/- mice had decreased circulating white blood cell counts 4h into the light cycle. In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.

Exploring contrast-enhancing staining agents for studying adipose tissue through contrast-enhanced computed tomography

Contrast-enhanced computed tomography offers a nondestructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO4) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO4, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using contrast-enhanced computed tomography but fail to provide chemical information. This study introduces isotonic Lugol’s iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae and bovine muscle tissue strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte gray values post-IL staining were associated with chemical distinctions between bovine muscle tissue and murine caudal vertebrae. More specific, a difference in the degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of gray value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.

Separation of Osmium and Ruthenium Metals by Selective Dissolution with Two Phase System Consisted of Sulfuric Acid, Sodium Chlorate, and Tributyl Phosphate

ABSTRACT In general, separating osmium and ruthenium metals via hydrometallurgical methods poses significant challenges. This study presents a novel approach to separate osmium from a metallic mixture with ruthenium through selective dissolution using a two phase system. The system comprises an aqueous phase containing sulfuric acid solution with sodium chlorate and an organic phase consisting of pure tributyl phosphate. Among the several variables investigated in this work, sulfuric acid concentration and stirring speed showed most significant effect on the dissolution of osmium metal. Based on the potential-pH diagram of osmium metal, an oxidation reaction of osmium metal by the sulfuric acid and sodium chlorate mixture was proposed. The role of tributyl phosphate was to prevent the evaporation of the dissolved osmium tetroxide species by extracting into organic phase. The separation of the two metals was accomplished by selective dissolution of osmium at the following conditions: 3 M sulfuric acid, 0.7 M sodium chlorate, pure tributyl phosphate, pulp density of 2 g/L, 20°C, 60 minutes and stirring speed of 200 rpm. Use of a mixture of thiourea and hydrochloric acid solution containing ascorbic acid completely stripped the osmium from the loaded tributyl phosphate. Comparison of dissolution efficiency of osmium metal between two phase system and one phase system of sulfuric acid-sodium chlorate-water indicated that the employment of tributyl phosphate in two phase system was of importance in the recovery of the dissolved osmium species. Our results demonstrated that osmium can be separated from ruthenium by selective dissolution with the two phase system.

Synchrotron-source micro-x-ray computed tomography for examining butterfly eyes.

Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly mapping the microscale anatomies of related species poses a major impediment to using comparative anatomy approaches for identifying evolutionary adaptations. We describe a method using synchrotron source micro-x-ray computed tomography (syn-μXCT) combined with machine learning algorithms for high-throughput imaging of Lepidoptera (i.e., butterfly and moth) eyes. Our pipeline allows for imaging at rates of ~15 min/mm3 at 600 nm3 resolution. Image contrast is generated using standard electron microscopy labeling approaches (e.g., osmium tetroxide) that unbiasedly labels all cellular membranes in a species-independent manner thus removing any barrier to imaging any species of interest. To demonstrate the power of the method, we analyzed the 3D morphologies of butterfly crystalline cones, a part of the visual system associated with acuity and sensitivity and found significant variation within six butterfly individuals. Despite this variation, a classic measure of optimization, the ratio of interommatidial angle to resolving power of ommatidia, largely agrees with early work on eye geometry across species. We show that this method can successfully be used to determine compound eye organization and crystalline cone morphology. Our novel pipeline provides for fast, scalable visualization and analysis of eye anatomies that can be applied to any arthropod species, enabling new questions about evolutionary adaptations of compound eyes and beyond.

Submicroscopic changes of type II alveolocytes in the late development of experimental acute pancreatitis

Background. Today, acute pancreatitis is one of the most common and severe visceral disorders in patients worldwide. The aim of the work was to study the dynamics of ultrastructural changes of type II alveolocytes in the long-term periods of development of experimental acute pancreatitis. Material and methods. The experiments were carried out on 54 white Wistar male rats weighing 180–220 g. The animals were divided into three groups: first — intact, second — control, third — experimental with a model of acute pancreatitis, which was reproduced by intraperitoneal administration of a 20% solution of L-arginine at a total dose of 5 g/kg at one-hour interval. The control group of animals was intraperitoneally injected with an equivalent dose of isotonic sodium chloride solution. All research were performed under sodium thiopental anesthesia at the rate of 60 mg/kg body weight. Lung tissue for electron microscopic examination was collected from the lower lobe of the left lung at 3–5 and 7 days. Pieces of lung tissue measuring 1×1×1 mm were fixed in a 2.5% glutaraldehyde solution, followed by additional fixation in a 1% osmium tetroxide solution. After dehydration, the material was poured into Epon-Araldite. Sections with a thickness of 20–50 nm obtained on “Tesla BS-490” ultramicrotome were studied in a PEM-125K electron microscope. Results. It was found that three days after the start of the study, along with dystrophic-destructive changes, type II alveolocytes in a state of increased functional activity were observed. Continuation of the experiment (5 days) leads to the progression of ultrastructural changes in type II alveolocytes are determined, which are most pronounced on the 7 day of the study. Conclusion. Acute experimental pancreatitis is accompanied by marked changes in the ultrastructural structure of type II alveolocytes. The nature and severity of structural changes of type II alveolocytes depends on the duration of the course of arginine-induced acute pancreatitis.

Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

Anatomical, pathological, and histological features of experimental respiratory infection of birds by biofilm-forming bacteria Staphylococcus aureus.

The pathogenesis of staphylococcal infections is mediated by virulence factors, such as enzymes, toxins, and biofilms, which increase the resistance of microorganisms to host immune system evasion. Testing and searching for standardized multi-level algorithms for the indication and differentiation of biofilms at the early stages of diagnosis will contribute to the development of preventive measures to control the critical points of technology and manage dangerous risk factors for the spread of infectious diseases. This research aimed to study the main stages of Staphylococcus aureus biofilm formation in in vitro experiments and to analyze the dynamics of respiratory syndrome development in chickens infected with these bacteria. Experimental reproduction of the infectious process was performed using laboratory models: 10-day-old White Leghorn chickens (n = 20). Before the experiments, the birds were divided into two groups according to the principle of analogs: Group I (control, n = 10): the birds were intranasally inoculated with 0.5 cm3 of 0.9% NaCl solution; Group II (experiment, n = 10): the birds were intranasally inoculated with a suspension of S. aureus bacteria, 0.5 cm3, concentration 1 billion/cm3. Colonization of individual areas of the substrate under study in vitro occurred gradually from the sedimentation and adhesion of single motile planktonic cells to the attachment stage of microcolony development. Staining preparations with gentian violet due to the "metachromosia" property of this dye are a quick and fairly simple way to differentiate cells and the intercellular matrix of biofilms. Fixation with vapors of glutaraldehyde and osmium tetroxide preserves the natural architecture of biofilms under optical and scanning electron microscopy. Pure cultures of S. aureus microorganisms were isolated from the blood, lungs, small intestine, liver, kidneys, and spleen after 5-10 days during experimental infection of chickens. Clinical signs of respiratory syndrome developed within 5-6 days after infection. Acute and subacute serous-fibrinous airsacculitis, characterized by edema and thickening of the membranes of the air sacs and the presence of turbid, watery, foamy contents in the cavity, was the most characteristic pathomorphological sign. The signs of acute congestive hyperemia and one-sided serous-fibrinous pneumonia developed with significant thickening of fibrinous deposits. In Garder's gland, there was an increase in the number of secretory sections, indicating hypersecretion of the glands. In the lymphoid follicles of Meckel's diverticulum, leukocytes, usually lymphocytes, and pseudoeosinophils were detected. Hydration and heteromorphism of the internal environment of biofilms determine the localization of differentiated cells in a three-dimensional matrix for protection against adverse factors. The most characteristic pathomorphological sign was the development of acute and subacute serous-fibrinous airsacculitis when reproducing the infectious process in susceptible models. There was a significant thickening of fibrinous deposits and signs of acute congestive hyperemia and one or two serous-fibrinous pneumonia developed.

The use of scanning electron microscopy and fixation methods to evaluate the interaction of blood with the surfaces of medical devices

Testing the hemocompatibility of medical devices after their interaction with blood entails the need to evaluate the activation of blood elements and the degree of their coagulation and adhesion to the device surface. One possible way to achieve this is to use scanning electron microscopy (SEM). The aim was to develop a novel SEM-based method to assess the thrombogenic potential of medical devices and their adhesiveness to blood cells. As a part of this task, also find a convenient procedure of efficient and non-destructive sample fixation for SEM while reducing the use of highly toxic substances and shortening the fixation time. A polymeric surgical mesh was exposed to blood so that blood elements adhered to its surface. Such prepared samples were then chemically fixed for a subsequent SEM measurement; a number of fixation procedures were tested to find the optimal one. The fixation results were evaluated from SEM images, and the degree of blood elements’ adhesion was determined from the images using ImageJ software. The best fixation was achieved with the May–Grünwald solution, which is less toxic than chemicals traditionally used. Moreover, manipulation with highly toxic osmium tetroxide can be avoided in the proposed procedure. A convenient methodology for SEM image analysis has been developed too, enabling to quantitatively evaluate the interaction of blood with the surfaces of various medical devices. Our method replaces the subjective assessment of surface coverage with a better-defined procedure, thus offering more precise and reliable results.

Peculiarities of morphological indicators of quail spleen (Coturnix japonica Temminck et Schlegel, 1849) when using protein-vitamin mineral supplements

In most countries of the world, poultry farming is one of the main branches of agriculture, which provides the population with high-quality food products. To date, the positive dynamics of the growth of such a branch of industrial poultry farming as quail breeding has been noted. Quails have a complex of excellent economic and productive advantages compared to other agricultural birds – high body temperature, intensive metabolism in the body, small size, precocity, high egg productivity. The question of increasing the productivity of quail breeding, as a branch of precocious animal husbandry, has always occupied the attention of biologists, morphologists, physiologists, geneticists and technologists. This topical problem has various scientific aspects: morphological, histochemical, physiological, genetic, etc. Special attention in the conducted research is required to study the impact of additives on the body of quails, the absence of negative effects and the production of safe products for humans. We chose the spleen as a biomarker, because the spleen is an important peripheral organ of immune protection, sensitive to the action of various factors, whose morphofunctional indicators determine the magnitude and nature of the impact. The purpose of the research was to determine the effect of modern protein-vitamin mineral supplements, which are advertised and offered by producers in the poultry industry to stimulate the growth and development of the bird, on the quail body. In the studies, the features of the microscopic structure and morphometry of the spleen of control and experimental animals were used as sensitive characteristics of the action of additives. The proposed methods are aimed at researching the impact of protein-vitamin mineral supplements on the relevant industry. So, for 21 days, mineral supplements “Multilife”, “Missy” and “Standard Agro” were added to the main ration of quails. Using macrometric methods, the body weight of quails, the weight of the spleen (absolute and relative) were determined. For histological studies, pieces of the spleen were fixed in a 10 % formalin solution, and after embedding in paraffin, histological sections were prepared from it, which after deparaffinization were stained with hematoxylin and eosin, according to the methods of Brachet and Van Gieson. For electron microscopic examination, the spleen was immediately fixed with a 2.5 % solution of glutaraldehyde in a phosphate buffer with additional fixation in a 1 % solution of osmium tetroxide according to Caulfield. For the first time, the ultrastructural organization of the spleen of Japanese quail was investigated after feeding protein-vitamin mineral supplements, which may indicate the absence of their negative impact on the structural and functional state of the spleen. Important morphological structural indicators of the morphofunctional state of the spleen were determined: the relative area of the white pulp, red pulp, lymphoid nodules, periarterial lymphoid sheaths, and the contractile apparatus. According to the analysis of the morphological results obtained by us, it was investigated that the microscopic structure of the quail spleen is characterized by the presence of a support-contractile apparatus with a relative area of 4.58 ± 0.76 % and single radial trabeculae. The parenchyma is formed by white and red pulp without clear boundaries. Lymphoid nodules without bright centers, periarterial lymphoid sheaths, and ellipsoids were identified as part of the white pulp. The ultrastructural organization of the spleen of quails had peculiarities: the nuclei of leukocytes contain large nucleoli, the endotheliocytes of the central artery of the lymphoid follicle perform a phagocytic function by accumulating autophagosomes in their cytoplasm. The macroscopic, microscopic and ultramicroscopic structure of quails that were added to the main diet with the listed additives did not undergo any changes. As a result, the following supplements led to an increase in the body weight of quails: by 5.78 % when using the Multilife supplement, by 12.51 % when using the Missy supplement, and by 8.12 % when using the Standard Agro supplement, and the weight of the spleen on 7.17 %, 16.04 % and 10.54 %, respectively. The conducted studies showed that the relative weight of the organ practically did not change. At the same time, there is a trend towards an increase in the relative area of the white pulp and the supporting-contractile apparatus. Taking into account the obtained data, it can be concluded that our research confirms the feasibility of using modern protein-vitamin mineral supplements in poultry farming to stimulate the growth and development of poultry. Especially valuable is the absence of a negative effect on the morphofunctional indicators of the spleen, which was used as a biomarker in research.

Development and Comparison of 4-Thiouridine to Cytidine Base Conversion Reaction.

To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be converted into a cytidine analogue. Although several reactions can convert 4sU into a cytidine analogue, few studies have compared the features of these reactions. In this study, we performed three reported base conversion reactions, including osmium tetroxide, iodoacetamide, and sodium periodate treatment, as well as a new reaction using 2,4-dinitrofluorobenzene. We compared the reaction time, conversion efficacy, and effects on reverse transcription. These reactions successfully converted 4sU into a cytidine analogue quantitatively using trinucleotides. However, the conversion efficacy and effect on reverse transcription vary depending on the reaction with the RNA transcript. OsO4 treatment followed by NH4Cl treatment showed the best base-conversion efficiency. Nevertheless, each reaction has its own advantages and disadvantages as a tool for studying the transcriptome. Therefore, it is crucial to select the appropriate reaction for the target of interest.

Ultrastructural organization of alveolar macrophages during late development of experimental acute pancreatitis

Background. Nowadays, acute pancreatitis continues to remain an actual problem both in surgery and in intensive care medicine. The aim of the work was to study the dynamics of ultrastructural changes of alveolar macrophages in the long-term development of experimental acute pancreatitis. Material and methods. The experiments were carried out on 62 white Wistar male rats weighing 180–220 g. The animals were divided into three groups: first — intact, second — control, third — experimental with a model of acute pancreatitis, which was reproduced by intraperitoneal administration of a 20% solution of L-arginine in a total dose of 5 g/kg at one-hour interval. The control group of animals was intraperitoneally injected with an equivalent dose of isotonic sodium chloride solution. All research were performed under sodium thiopental anesthesia at the rate of 60 mg/kg body weight. Lung tissue for electron microscopic examination was collected from the lower lobe of the left lung at 3–5 and 7 days. Pieces of lung tissue measuring 1×1×1 mm were fixed in a 2.5% glutaraldehyde solution, followed by additional fixation in a 1% osmium tetroxide solution. After dehydration, the material was poured into Epon-Araldite. Sections with a thickness of 20–50 nm obtained on “Tesla BS-490” ultramicrotome were studied in a PEM-125K electron microscope. Results. It was found that three days after the start of the study, significant heterogeneity of alveolar macrophages was observed in the alveolar lumen. Next to active phagocytic macrophage elements with dystrophic-destructive changes are determined. Continuation of the experiment (5 days) leads to the progression of submicroscopic changes in alveolar macrophages, which are most pronounced on the7 day of the study. Conclusion. Acute experimental pancreatitis is accompanied by pronounced changes in the ultrastructural structure of alveolar macrophages. The features and severity of structural changes in alveolar macrophages depends on the duration of arginine-induced acute pancreatitis.

(067) How Many Nerve Fibers Innervate the Human Glans Penis: A Histomorphometric Analysis of the Human Dorsal Nerve of the Penis

Abstract Introduction The mainstream media has frequently quoted that the penis has “half as many” nerve fibers as the human clitoris. Recent work has demonstrated that the number of nerve fibers in the dorsal nerve of clitoris was under-estimated, and no study has yet quantified the number of axons in the human dorsal nerve of the penis (DNP). Improving our understanding of penile innervation and sexual response has potential implications for fields of medical and surgical practice. Several nerve transfers have recently emerged targeting the DNP for re-innervation in order to restore penile sensation and/or erectile function, demonstrating variability in outcomes. Understanding the axon counts of the DNP at the anatomic level that these nerve transfers are performed may inform technical refinements and improve our understanding of current outcomes. Comparing DNP axon counts to the recently reported axon counts of the dorsal nerve of the clitoris may also inform techniques in transgender genital surgery, anticipate sensory outcomes in penile allotransplantation and further our understanding of sexual responses across genitalia. Objective The objective of this study is to quantify the number of axons in the human dorsal nerve of the penis. Methods Bilateral DNP samples were obtained from 5 fresh frozen cadavers. Samples were collected at the base of the penis at the level of the pubic symphysis, a location commonly targeted for coaptation in nerve transfers. Specimens were placed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide and serially dehydrated in ethanol and toluene. They were embedded in araldite, sectioned on ultramicrotome into 1 μm cross-sections and counter-stained with 1% toluidine blue. Histomorphometric evaluation was performed at 1000x magnification using Leitz Laborlux S microscope and image analysis software (Clemex Vision Professional, Longueuil, Quebec) to obtain nerve fiber counts from each specimen (Figure). Results 10 DNP samples were analyzed. The mean number of axons in the human dorsal nerve of penis was 3,844 +/- 1,238 (range 1,915 to 5,494). Accounting for the bilaterality of the DNP, the mean total number of axons innervating the human glans penis was 7,688 +/- 1,762. Conclusions This study is the first to report the number of axons in the human dorsal nerve of the penis and approximate the number of axons innervating the human glans penis. At the level of the pubic symphysis, axon counts of the dorsal nerve of penis varied significantly and averaged 3,844. The variation in axon counts at this anatomic level was greater than seen in prior reports on the DNC and may be one factor contributing to variations in outcomes following nerves transfers for penile sensation and erection. This variation could also reflect the anatomic level of sample collection or differences in specimen origin (cadaver vs. live person). DNP axon counts were less than or equal to recently reported axon counts of the human glans clitoris, despite the much larger size of the glans penis. This finding may be important when considering clitoral construction in transgender genital surgery, penile allotransplantation and different in sexual responsiveness across genitalia. Disclosure No.