Tissue Preparations Research Articles

Study of nerve cell regeneration on nanofibers containing cerium oxide nanoparticles in a spinal cord injury model in rats

Since the CNS is unable to repair itself via neuronal regeneration in adult mammals, alternative therapies need to be found. The use of cerium oxide nanoparticles to repair nerve damage could be a promising approach for spinal cord reconstruction. In this study, we constructed a scaffold containing cerium oxide nanoparticles (Scaffold-CeO2) and investigated the rate of nerve cell regeneration in a rat model of spinal cord injury. The scaffold of gelatin and polycaprolactone was synthesized, and a gelatin solution containing cerium oxide nanoparticles was attached to the scaffold. For the animal study, 40 male Wistar rats were randomly divided into 4 groups (n = 10): (a) Control; (b) Spinal cord injury (SCI); (c) Scaffold (SCI + scaffold without CeO2 nanoparticles); (d) Scaffold-CeO2 (SCI + scaffold containing CeO2 nanoparticles). After creation of a hemisection SCI, scaffolds were placed at the site of injury in groups c and d, and after 7 weeks the rats were subjected to behavioral tests and then sacrificed for preparation of the spinal cord tissue to measure the expression of G-CSF, Tau and Mag proteins by Western blotting and Iba-1 protein by immunohistochemistry. The result of behavioral tests confirmed motor improvement and pain reduction in the Scaffold-CeO2 group compared to the SCI group. Decreased expression of Iba-1 and higher expression of Tau and Mag in the Scaffold-CeO2 group compared to the SCI group could be the result of nerve regeneration caused by the scaffold containing CeONPs as well as relief of pain symptoms.Graphical

Negative Chronotropic Effects of Class I Antiarrhythmic Drugs on Guinea Pig Right Atria: Correlation with L-Type Ca2+ Channel Blockade

The negative chronotropic effects of eight Vaughan Williams Class I antiarrhythmic drugs were examined in guinea pig right atrial tissue preparations. The drugs decreased the spontaneous beating rate at concentrations overlapping with their therapeutic blood levels. Cibenzoline, aprindine, flecainide, and propafenone showed stronger effects; 10 µM of each drug decreased the beating rate to about 75% of initial values. Disopyramide, mexiletine, pilsicainide, and ranolazine showed weaker effects; 10 µM of each drug decreased the beating rate to about 90% of initial values. The potency of drugs correlated with the reported IC50 values to block the L-type Ca2+ channel current rather than the Na+ and K+ channel currents. The reported IC50 values for the blockade of the hyperpolarization-activated inward current (If) and the Na+-Ca2+ exchanger current were much higher than those for the blockade of the L-type Ca2+ channel current. These results indicate that the negative chronotropic effects of Class I antiarrhythmic drugs can be largely explained by their blockade of the L-type Ca2+ channel.

Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix during in vitro culture.

How does in vitro culture alter the human ovarian cortical extracellular matrix (ECM) network structure? The ECM composition and architecture vary in the different layers of the ovarian cortex and are remodelled during in vitro culture. The ovarian ECM is the scaffold within which follicles and stromal cells are organized. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Tissue preparation for culture of primordial follicles within the native ECM involves mechanical loosening; this induces undefined modifications in the ECM network and alters cell-cell contact, leading to spontaneous follicle activation. Fresh ovarian cortical biopsies were obtained from six women aged 28-38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1 × 1 mm and cultured for 0, 2, 4, or 6 days (D). Primordial follicle activation, stromal cell density, and ECM-related protein (collagen, elastin, fibronectin, laminin) positive area in the entire cortex were quantified at each time point using histological and immunohistological analysis. Collagen and elastin content, collagen fibre characteristics, and follicle distribution within the tissue were further quantified within each layer of the human ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex-medulla junction regions. Primordial follicle activation occurred concomitantly with a loosening of the ovarian cortex during culture, characterized by an early decrease in stromal cell density from 3.6 ± 0.2 × 106 at day 0 (D0) to 2.8 ± 0.1 × 106 cells/mm3 at D2 (P = 0.033) and a dynamic remodelling of the ECM. Notably, collagen content gradually fell from 55.5 ± 1.7% positive area at D0 to 42.3 ± 1.1% at D6 (P = 0.001), while elastin increased from 1.1 ± 0.2% at D0 to 1.9 ± 0.1% at D6 (P = 0.001). Fibronectin and laminin content remained stable. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Analysis at the sub-region level showed that collagen deposition was maximal in the outer cortex and the lowest in the mid-cortex (69.4 ± 1.2% versus 53.8 ± 0.8% positive area, respectively, P < 0.0001), and cortical collagen staining overall decreased from D0 to D2 (65.2 ± 2.4% versus 60.6 ± 1.8%, P = 0.033) then stabilized. Elastin showed the converse distribution, being most concentrated at the cortex-medulla junction (3.7 ± 0.6% versus 0.9 ± 0.2% in the outer cortex, P < 0.0001), and cortical elastin peaked at D6 compared to D0 (3.1 ± 0.5% versus 1.3 ± 0.2%, P < 0.0001). This was corroborated by a specific signature of the collagen fibre type across the cortex, indicating a distinct phenotype of the ovarian cortical ECM depending on region and culture period that might be responsible for the spatio-temporal and developmental pattern of follicular distribution observed within the cortex. N/A. Ovarian cortical biopsies were obtained from women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution and structure of non-pregnant women. Clarifying the composition and architecture signature of the human ovarian cortical ECM provides a foundation for further exploration of ovarian microenvironments. It is also critical for understanding the ECM-follicle interactions regulating follicle quiescence and awakening, leading to improvements in both in vitro activation and in vitro growth techniques. Medical Research Council grant MR/R003246/1 and Wellcome Trust Collaborative Award in Science: 215625/Z/19/Z. The authors have no conflicts to declare. N/A.

6-OHDA-Induced Changes in Colonic Segment Contractility in the Rat Model of Parkinson's Disease.

Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model. Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K+ Krebs-induced contractility and spontaneous contractions. This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation.

Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila melanogaster after Rapid Cryofixation.

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.

Prevention of subclinical forms of ketosis and hypocalcemia in dairy cows

Relevance. Against the background of stress and high physiological load, cows experience a lack of energy, the need for which increases many times over in comparison with the dry period, in connection with this, their body is forced to use the reserves of adipose tissue. It is important to timely prevent and monitor the status of cows during the transition period.Methods. The methodology of the work was to study the effectiveness of the use of immunostimulating drugs PS-2, Prevention-N-E and PDE + E-selen. For scientific research, 4 groups of down-calving cows of the Holsteinized Black-and-White breed were selected, 10 animals each. Dry cows of the 1st and 2nd experimental groups received PS-2 and Prevention-N-E intramuscularly at a dose of 10.0 ml three times (40, 20 and 10 days before calving), animals of the 3rd experimental group were injected subcutaneously with tissue preparation PDE (denatured emulsified placenta) at a dose of 20.0 ml and intramuscularly — with a complex mineral and vitamin preparation E-selen at a dose of 10.0 ml 20 days before calving. Biological preparations were not used in animals of the control group.Results. An analysis was made of the prevalence of postpartum metabolic disorders: clinically pronounced hypocalcemia was observed in 5.9% of the newly-calved cows, ketosis — in 11.6%, subclinical hypocalcemia — in 17.7%, and a latent form of ketosis — in 22.7%. Biological preparations PS-2 and Prevention-N-E have a corrective effect on the synthesis of aminotransferases, mineral and carbohydrate metabolism, digestibility of macronutrients, against which background the number of newly-calved cows with subclinical forms of ketosis and hypocalcemia is reduced by 2 times.

The Impact of Tissue Preparation on Salivary Gland Tumors Investigated by Fourier-Transform Infrared Microspectroscopy.

Due to the wide variety of benign and malignant salivary gland tumors, classification and malignant behavior determination based on histomorphological criteria can be difficult and sometimes impossible. Spectroscopical procedures can acquire molecular biological information without destroying the tissue within the measurement processes. Since several tissue preparation procedures exist, our study investigated the impact of these preparations on the chemical composition of healthy and tumorous salivary gland tissue by Fourier-transform infrared (FTIR) microspectroscopy. Sequential tissue cross-sections were prepared from native, formalin-fixed and formalin-fixed paraffin-embedded (FFPE) tissue and analyzed. The FFPE cross-sections were dewaxed and remeasured. By using principal component analysis (PCA) combined with a discriminant analysis (DA), robust models for the distinction of sample preparations were built individually for each parotid tissue type. As a result, the PCA-DA model evaluation showed a high similarity between native and formalin-fixed tissues based on their chemical composition. Thus, formalin-fixed tissues are highly representative of the native samples and facilitate a transfer from scientific laboratory analysis into the clinical routine due to their robust nature. Furthermore, the dewaxing of the cross-sections entails the loss of molecular information. Our study successfully demonstrated how FTIR microspectroscopy can be used as a powerful tool within existing clinical workflows.

Single-cell and spatial sequencing application in pathology.

Traditionally, diagnostic pathology uses histology representing structural alterations in a disease's cells and tissues. In many cases, however, it is supplemented by other morphology-based methods such as immunohistochemistry and fluorescent in situ hybridization. Single-cell RNA sequencing (scRNA-seq) is one of the strategies that may help tackle the heterogeneous cells in a disease, but it does not usually provide histologic information. Spatial sequencing is designed to assign cell types, subtypes, or states according to the mRNA expression on a histological section by RNA sequencing. It can provide mRNA expressions not only of diseased cells, such as cancer cells but also of stromal cells, such as immune cells, fibroblasts, and vascular cells. In this review, we studied current methods of spatial transcriptome sequencing based on their technical backgrounds, tissue preparation, and analytic procedures. With the pathology examples, useful recommendations for pathologists who are just getting started to use spatial sequencing analysis in research are provided here. In addition, leveraging spatial sequencing by integration with scRNA-seq is reviewed. With the advantages of simultaneous histologic and single-cell information, spatial sequencing may give a molecular basis for pathological diagnosis, improve our understanding of diseases, and have potential clinical applications in prognostics and diagnostic pathology.

Modulation of post-pacing action potential duration and contractile responses on ventricular arrhythmogenesis in chloroquine-induced long QT syndrome

BackgroundExcitation-contraction (E-C) coupling, the interaction of action potential duration (APD) and contractility, plays an essential role in arrhythmogenesis. We aimed to investigate the arrhythmogenic role of E-C coupling in the right ventricular outflow tract (RVOT) in the chloroquine-induced long QT syndrome. MethodsConventional microelectrodes were used to record electrical and mechanical activity simultaneously under electrical pacing (cycle lengths from 1000–100 ms) in rabbit RVOT tissue preparations before and after chloroquine with and without azithromycin. KB-R7943 (a Na+-Ca2+ exchanger [NCX] inhibitor), ranolazine (a late sodium current inhibitor), or MgSO4 were used to assess their pharmacological responses in the chloroquine-induced long QT syndrome. ResultsSequential infusion of chloroquine and chloroquine plus azithromycin triggered ventricular tachycardia (VT) (33.7%) after rapid pacing compared to baseline (6.7%, p = 0.004). There were greater post-pacing increases of the first occurrence of contractility (ΔContractility) in the VT group (VT vs. non-VT: 521.2 ± 50.5% vs. 306.5 ± 26.8%, p < 0.001). There was no difference in the first occurrence of action potential at 90% repolarization (ΔAPD90) (VT vs. non-VT: 49.7 ± 7.4 ms vs. 51.8 ± 13.1 ms, p = 0.914). Pacing-induced VT could be suppressed to baseline levels by KB-R7943 or MgSO4. Ranolazine did not suppress pacing-induced VT in chloroquine-treated RVOT. ΔContractility was reduced by KB-R7943 and MgSO4, but not by ranolazine. ConclusionΔContractility (but not ΔAPD) played a crucial role in the genesis of pacing-induced VT in the long QT tissue model, which can be modulated by NCX (but not late sodium current) inhibition or MgSO4.

Monitoring TGFβ signaling in irradiated tumors.

Transforming growth factor β (TGFβ) is exquisitely regulated under physiological conditions but its activity is highly dysregulated in cancer. All cells make TGFβ and have receptors for the ligand, which is sequestered in the extracellular matrix in a latent form. Ionizing radiation elicits rapid release of TGFβ from these stores, so-called activation, over a wide range of doses and exposures, including low dose (<1Gy) whole-body irradiation, creating an extraordinarily potent signal in the irradiated tissue or tumor. Hence, accurate evaluation of TGFβ activity is complicated because of its ubiquitous distribution as a latent complex. Here we describe conditions for assays that reveal TGFβ activity in situ using either tissue preparations or functional imaging.

A methodology to evaluate different histological preparations of soft tissues: Intervertebral disc tissues study.

A tissue preparation method will inevitably alter the tissue content. This study aims to evaluate how different common sample preparation methods will affect the tissue morphology, biomechanical properties, and chemical composition of samples. The study focuses on intervertebral disc (IVD) tissue; however, it can be applied to other soft tissues. Raman spectroscopy synchronized with nanoindentation instrumentation was employed to investigate the compositional changes of IVD, specifically, nucleus pulposus (NP) and annulus fibrosus (AF), together with their biomechanical properties of IVD. These properties were examined through the following histological specimen types: fresh cryosection (control), fixed cryosection, and paraffin-embedded. The IVD tissue could be located using an optical microscope under three different preparation methods. Paraffin-embedded samples showed the most explicit details where the lamellae structure of AF could be identified. In terms of biomechanical properties, there was no significant difference between the fresh and fixed cryosection (p > 0.05). In contrast, the fresh cryosection and paraffin-embedded samples showed a significant difference (p < 0.05). It was also found that the tissue preparations affected the chemical content of the tissues and structure of the tissue, which are expected to contribute to biomechanical properties changes. Fresh cryosection and fixed cryosection samples are more promising to work with for biomechanical assessment in histological tissues. The findings fill essential gaps in the literature by providing valuable insight into the characteristics of IVD at the microscale. This study can also become a reference for a better approach to assessing the mechanical properties and chemical content of soft tissues at the microscale.

FEATURES OF PREPARATION OF SUPPORTING TEETH BEFORE PROSTHETICS

The tactics of providing prostheses in the front teeth of dentitions under sparing preparation of the hard tissue of supporting teeth was presented in the article. The peculiar features of prosthodontics in patients with higher esthetic demands and its advantages were determined.

Primary total knee replacement without drain: A good and safe practice to inculcate

BACKGROUND: The use of drain in primary total knee replacement (TKR) started with the belief that it prevents hematoma formation and infection. The fear of infection made this practice an essential step that enjoyed a long journey. However, in recent years, majority of evidence-based studies have not only been failed to provide substantial benefits of the drain but also have proven this a counterproductive step.OBJECTIVE: The purpose of our study is to assess the outcome of performing primary TKR without the use of a drain.METHODOLOGY: After meeting inclusion and exclusion criteria, a prospective observational study was conducted on 186 patients (191 knees) who underwent primary cemented total knee arthroplasty without the use of drain from September 2018 to March 2020 and were followed up for one year. Surgery was performed under tourniquet, bleeders were electro cauterized, and injection tranexamic acid was used to control bleeding. A good preoperative screening for foci of infection, tight glycemic control for diabetics, part preparation, prophylactic antibiotics, and gentle handling of soft tissue was ensured to control infection. Deep vein thrombosis prophylaxis was instituted after risk stratification. Ambulation and physiotherapy were started as per protocol. The clinical parameters such as pain score and range of motion (ROM) were measured by a physiotherapist. Postoperative hemoglobin (Post Op Hb) and requirement of dressing change/reinforcement were monitored by the ward nurse. Aspiration of the knee if required was done by the treating surgeon.RESULTS: Post Op Hb drop was insignificant and none of the patients required blood transfusion. Tense arthrocoele, requiring rescue analgesia, were found in 5(2.6%) patients which responded well with knee aspiration. There was significant improvement (P < 00001) in pain score and the targeted ROM were achieved in 3 weeks' postoperative. Although ecchymosis was a common finding (20.4%), it did not require special attention. Blisters developed in six patients which were managed by antibiotic-impregnated paraffin dressing. Wound healing was not delayed in any patient. None of the patients acquired infection.CONCLUSION: Performing primary TKR without drain is a safe practice to exercise as it neither increases the risk of infection nor poses a significant threat of blood loss and blood transfusion. The presence of arthrocoele in postoperative period does not compromise short term clinical and functional outcomes. The incidence of developing tense hemarthrosis is very less and if aspirated, relieves patient's discomfort and aborts the surgeon's fear of infection. Moreover, not only the cost of drain and utilization of workforce is saved but the question of caring for the drain and its complications also ends.

EXPERIMENTAL ASSESSMENT OF THE BONE CAVITY HEATING AT DIFFERENT SPEEDS DURING PREPARATION FOR DENTAL IMPLANTATION

The objective of the study was to evaluate the level of bone tissue heating experimentally during various high-speed and slow-speed protocols for the preparation of the bone cavity for dental implantation. Materials and Methods: 8 samples of skeletonized veal ribs (length 10.00–12.00 cm and thickness 1.50–2.00 cm) were dissected, four cuts were made according to different speed protocols at a distance of 1.00–1.50 cm from each other. The duration of each manipulation was 15 s. The Lindemann guide drill with a diameter of 2.20 mm (apex diameter – 1.70 mm) and length of 35.00 mm (with a limiter) was used for work without any cooling of the operating field. The following preparation parameters were applied: 1000 rpm, 300 rpm, 100 rpm, and 50 rpm; in all cases, the torque of the surgical drill was 35 N×cm. Bone tissue temperature was measured with an electronic contact thermometer before and immediately after manipulation. Results: Measurements showed that heating of bone tissue was observed in almost all applied protocols of bone tissue preparation. This fact can represent a danger to the patient by increasing the risk of developing localized osteomyelitis of the alveolar ridge due to trauma. This, in turn, can increase the risk of the installed dental implant disintegration or the development of fiber integration. During 15 seconds of preparation at a speed of 1000 rpm, the bone tissue heated up the most; the average temperature increase was 5.64 ± 2.23 °С (М = 4.60 °С), the value – from 3.00 to 10.30 °С. At the speed of 300 rpm, heating was less – by 3.79 ± 1.76 °C (M = 3.80 °C), the values were in the range of 1.10–6.40 °C. At the speed of 100 rpm, the heating of the bone canal was 2.99 ± 1.36 °С (М = 2.65 °С), in the range of values within 1.10–5.20 °С. During preparation at the speed of 50 rpm, the heating of bone tissue was minimal – 2.06 ± 0.66 °С (М = 1.95 °С), the value was recorded in the range of 1.00–3.20 °С. Considering that in most experimental studies, the time of preparation of bone tissue (or its model) was longer – 1 min, while in our study such prolongation of interaction with bone tissue was impractical (the bone cavity was fully prepared in 15 s) and the interaction of the cutter with the surrounding tissue was minimized, a decision was made to create a mathematical regression model of the thermal reaction of bone tissue during such treatment. The application of the created regression mathematical model of bone tissue heating during preparation without cooling for 1 min showed that the use of surgical drills at the speed of 1000 rpm and torque of 35 N×cm could lead to critical heating in 62.50% of cases, at the speed of 300 rpm min and a torque of 35 N×cm – in 50.00% of cases, at a speed of 100 rpm and torque of 35 N×cm – in 25.00%. The use of the speed of 50 rpm and torque of 35 N×cm without water cooling did not impose the risk of critical heating of bone tissue. Conclusions: the experimental data on the bone tissue heating when using different speeds during preparation without cooling the operating field were obtained. The safest method used was the scheme of 50 rpm with a torque of 35 N×cm.

Uji Ketahanan Pewarna Alami Kain Tenun Ikat Kecamatan Noemuti Timur Pada Preparat Basah Jaringan Tumbuhan

This study aims to determine the resistance of natural dyes and to determine the length of time of resistance of natural dyes of ikat woven fabrics in wet preparations of plant tissues. This study used the method of description and direct experiment. The design used a complete random design (RAL) with 2 treatment factors, namely plant dye and length of coloring time. Coloring plants are turmeric (Curcuma longa L) and mahogany (Swietenia mahagoni (L) Jack.,, and the length of coloring time is 0 minutes, 60 minutes, 120 minutes, and 180 minutes. Data analysis in this study is descriptive qualitative and descriptive quantitative. The results showed that the natural dye of ikat woven cloth in East Noemuti District consisted of yellow color extracted from turmeric rhizomes (Curcuma longa L), maroon color extracted from turmeric rhizomes (Curcuma longa L) added lime and detergent, red color extracted from mahogany bark (Swietenia mahagoni (L). Jack) can survive wet preparations of spinach (Amaranthus tricolor) and corn (Zea mays) plant tissues and the length of time of dye resistance to wet preparations of spinach (Amaranthus tricolor) and corn (Zea mays) plant tissues which shows the best results is 180 minutes.

A Histomorphometric Analysis of Interganglionic Distance in the Myenteric Plexus of the Entire Large Intestine of Aborted Human Foetuses: A Cross-sectional Study

Introduction: The Enteric Nervous System (ENS) contributes to the innervation of the gut and is considered the second brain in the gut. It is a part of the peripheral nervous system and the ganglionated plexus (GP) of enteric neurons, present in the layers of the digestive tract, is more complex than any other structure of the peripheral nervous system. The establishment of a valid standard method for quantitative analysis of the myenteric plexus has been a topic of debate among enteric neuropathologists and is essential for better characterisation of patients with gut dysmotility. Aim: To determine the histomorphometric analysis of the interganglion distance of the myenteric plexus in the entire large intestine of aborted human foetuses. Materials and Methods: A cross-sectional descriptive study was carried out in the Department of Anatomy at Pondicherry Institute of Medical Sciences, Pondicherry, India. The study duration was eight years and seven months, from October 2014 to May 2022. A total of 50 aborted human foetus samples were collected from Rajiv Gandhi Government Women and Children’s Hospital, Puducherry, India, from the Department of Obstetrics and Gynaecology. The age of the aborted human foetuses was determined based on Biparietal Diameter (BPD) and Crown Rump Length (CRL) and correlated with clinical history. The aborted human foetuses were assigned to two groups: group A (n=29), ≤20 Weeks of Gestation (WG), and group B (n=21), ≥20 WG. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) diaphorase histochemistry, an enzyme histochemistry technique, was used to study the tissue preparations. Statistical significance was determined using Statistical Package for Social Sciences (SPSS) version 20.0, International Business Machines (IBM) corporation. The independent sample t-test was conducted for parametric analysis. Results: The interganglion distance was measured from the caecum (A1 segment) to the anal canal (A7 segment) in aborted foetuses of less than 20 WG. The mean values ranged from 78.86±38.09 μm to 126.94±23.87 μm in foetuses of less than 20 WG. The mean values ranged from 66.41±8.05 μm to 107.67±30.17 μm in aborted human foetuses of more than 20 WG. Conclusion: The anal canal showed a long interganglion distance. The average reference values were found to be helpful as a diagnostic tool for identifying disorders related to gut innervation and motility.

The immunology of corneal limbal stem cells: the up-to-date approach to stem cell transplantation.

Limbal epithelial stem cells (LSC, LESC) are multipotent cells used as regenerative treatment of the cornea in patients with limbal epithelial stem cell deficiency (LSCD, LESCD). There are different types of stem cell grafting including cultivated limbal epithelial transplantation (CET) and simple limbal epithelial transplantation (SLET). The outcomes of the techniques have been assessed as similar, with differences in the sample size required during the procedures. The most important culture components for stem cell cultivation include 3T3 murine fibroblasts, human amniotic membrane (HAM), fibrin gel, and culture medium. The culture medium may be enriched with serum or not; however, xenobiotic-free materials are preferred because of the low risk of pathogen transmission. Multiple studies have defined molecules important for maintaining the function of LSC including C/EBP δ, Bmi-1, p63 α, interleukins (IL-6), epithelial structural proteins - keratins, and antibodies against epidermal growth factor receptor (EGFR). The cell phenotype of LSC has been described with factors of transplantation success rate such as a high percentage of p63 positive cells. The article emphasizes the role of recipient tissue preparation, modern cultivation techniques and pathophysiological processes in LSC transplantation effectiveness.

Использование поляризационно-интерференционной микроскопии для определения оптических свойств фетальных эритроцитов в тканях

Objective: to evaluate the possibility of using polarization interference microscopy for morphological diagnostics of fetal erythrocytes in tissues. Materials and methods. Blood smears used during the diagnostic and treatment process and histological sections prepared from autopsy material were used. During the diagnosis, a polarization-interference microscope PZO BIOLAR PI with a microphotometric nozzle UPI was used to determine the optical density and the measure of light transmission in erythrocytes. The method of polarization-interference microscopy contributed to the study of optical properties in individual erythrocytes. The optical properties of erythrocytes containing fetal-type hemoglobin and erythrocytes with adult-type hemoglobin were compared. The difference in the composition of the structure of erythrocytes determined the difference in light transmission and refraction. Based on the results of light transmission, the optical density of fetal and adult erythrocytes was calculated. In the conducted study of clinical blood smears, in neonatal patients, 60 measurements of the light transmission coefficient and 60 similar studies were performed in patients of the comparison group - persons older than 18 years. In the study of autopsy material, 120 measurements of the erythrocyte light transmission coefficient were carried out in placental tissue samples (chorionic villi and interstitial space). Results. The optical properties of erythrocytes were determined both in blood smears and in placental tissue preparations. Erythrocytes in blood smears with HbF had an increased light transmission coefficient in relation to the optical properties of erythrocytes with HbA in the control group. During the study of placental tissues, it was revealed that the indicators of the optical density of maternal erythrocytes with HbA in the erythrocytes of the interstitial space differed in higher values of indicators compared with the optical density of fetal erythrocytes with HbF in the vessels of chorionic villi. Conclusion. The determination of differences in the physico-optical properties of erythrocytes with fetal and adult hemoglobins makes it possible to determine the affiliation of erythrocytes both in blood smears and in the interstitial space of the placenta.

Morphological characteristics and notes of the skin in preclinical toxicity assessment.

In this review, the histological structures of the skin are summarized for fundamental knowledge for toxicological assessment. The skin is composed of epidermis, dermis, subcutaneous tissue, and associated adnexa. In the epidermis, keratinocytes comprise four layers, and three other cell types, besides keratinocytes, play various roles. Epidermal thickness varies with species and body site. In addition, it can be affected by tissue preparation procedures, which can render toxicity assessments difficult. Bulge stem cells are the origin of sebaceous glands, epidermal basal layer, and hair follicle formation, and they play an important role in the maintenance of the basic structure of the skin. Stem cells and appendages formed from stem cells sometimes become toxic targets, and it is useful to study the origins of the hair follicle/hair cycle to interpret their toxicity. Irritant contact dermatitis and allergic contact dermatitis are the main adverse reactions in topical application studies. The mechanism involves direct chemical irritation of the skin, and histologically, epidermal necrosis and accompanying inflammatory cell infiltration. In allergic contact dermatitis, an inflammatory reaction and intercellular or intracellular edema, histologically represented by lymphocytic infiltration of the epidermis and dermis, are observed. Regional and species differences exist in the dermal absorption of compounds, and differences in the thickness of the stratum corneum substantially contribute to these differences. Learning the basic structures, functions, and possible artifacts will contribute to the evaluation of skin toxicity by topical and systemic applications.

Negative Inotropic Effects of Class I Antiarrhythmics on Guinea Pig Ventricular Myocardium: Correlation with L-Type Ca&lt;sup&gt;2+&lt;/sup&gt; Channel Blockade

The negative inotropic effects of nine Vaughan Williams class I antiarrhythmic drugs were examined in guinea pig ventricular tissue preparations. The drugs decreased the contractile force of papillary muscles with different potencies: the potency order was propafenone > aprindine > cibenzoline > flecainide > ranolazine > disopyramide > pilsicainide > mexiletine > GS-458967. The potency of drugs correlated with the reported IC50 values to block the L-type Ca2+ channel rather than the Na+ channel. The effects of drugs were roughly the same when examined under a high extracellular K+ solution, which inactivates the Na+ channel. Furthermore, the attenuation of the extracellular Ca2+-induced positive inotropy was strong with propafenone, moderate with cibenzoline, and weak with pilsicainide. These results indicate that the negative inotropic effects of class I antiarrhythmic drugs can be largely explained by their blockade of the L-type Ca2+ channel.