Serial Paraffin Sections Research Articles

First record of Phoronopsis (Phoronida) from Japan, with a description of Phoronopsiscalifornica Hilton, 1930 from Okinawa.

Phylum Phoronida currently contains two genera, Phoronis Wright, 1856 and Phoronopsis Gilchrist, 1907, with approximately thirteen speices. Phoronida is distributed worldwide, ranging from northern Europe to southern New Zealand and also from intertidal to 400 m depths. From Japanese waters, four species of Phoronis have been reported, viz. Phoronisijimai Oka, 1897, Phoronisaustralis Haswell, 1883, Phoronispsammophila Cori, 1889 and Phoronisemigi Hirose et al., 2014. We describe the morphology of Phoronopsiscalifornica Hilton, 1930, based on five specimens collected at a sandy-mud habitat in Nago Bay and Oura Bay, Okinawa, Japan. We examined the internal anatomy by serial paraffin sections. We also examined the genetic distance of Japanese specimens from the other phoronid sequences in GenBank. This is the first record of Phoronopsis from Japanese waters and the fifth species record of phoronids in addition to the previously recorded four species of Phoronis.

Comparison of the infrapatellar and subcutaneous adipose tissue biopsy material as a source of mesenchymal stromal cells for regenerative medicine in traumatology

Objective. The use of infrapatellar fat pad (IPFP) as the source of mesenchymal stem cells (MSCs), show an age-independent proliferation and differentiation potential. In addition, the pronounced chondrogenic potential of IPFP-ASCs makes them promising candidates for research for use in other methodsof regenerative therapy. Methods. A direct immunohistochemical study was carried out in serial paraffin sections of the biopsy material of Hoffa`s fat pad and subcutaneus fat tissue, using monoclonal antibodies. The minimum criteria established by the International Society for Cell Therapy to ensure the identity of MSCs use CD73 and CD105 as positive markers and CD34, CD31, CD45 as a negative. Results. According to the results of histological, immunohistochemical, morphometric and statistical studies, it was found that in the biopsy of both tissues the relative number of cells with an immunoprofile CD105+, CD73+, CD34–, CD31–, CD45– in the standard field of view (×200) was: 7.25 (5.42; 8.89 ) %, and 11.11 (8.46; 13.45) %, no statistically significant difference was found between comparison groups (р > 0,05). Conclusion. The therapeutic effect of mesenchymal stem cells of subcutaneous adipose tissue has been proven, thus the identification of similar cells in the biopsy material and relatively similar density in the Hoffa body makesit an important source of adipose-derived stem cells that can be used for regenerative engineering tissue.

Comparative characteristics of the stem cells’ number in the stromal vascular fraction of infrapatellar fat pad and subcutaneous fat tissue

ObjectivesThe use of infrapatellar fat pad adipose stem cells (IPFP-ASCs) shows an age-independent proliferation and differentiation potential. In addition, the pronounced chondrogenic potential of IPFP-ASCs makes them promising candidates for research for use in other methods of regenerative therapy. The purpose of this study was to ascertain the presence and compare the relative abundance of cells exhibiting an immunohistochemical profile characteristic of adipose-derived mesenchymal stem cells in selected samples of the stromal vascular fraction (SVF) obtained from the IPFP and subcutaneous fat tissue. MethodsA direct immunohistochemical study was carried out in serial paraffin sections of the SVF of the infrapatellar fat pad (IPFP) and subcutaneous tissue, using monoclonal antibodies. The minimum criteria were established by the International Society for Cell Therapy to ensure the identity of mesenchymal stem cells use CD73, CD90, and CD105 as positive markers and CD34, CD31, and CD45 as a negative. ResultsAccording to the results of histological, immunohistochemical, morphometric, and statistical studies, it was found that in the SVF of IPFP and subcutaneous adipose tissue, the relative number of cells with the profile CD105+, CD73+, CD34+, CD31−, CD45− in the standard field of view (×200), the SVF of IPFP was 1.58%, whereas the SVF of subcutaneous adipose tissue was 6.92 %, which was statistically significantly greater by 4.38 times (p ​< ​0.05). ConclusionThe presence of a sufficient number of mesenchymal stromal cells in IPFP in combination with their topographic relationship with the structures of the joint determines the use of the SVF of the IPFP for the treatment of diseases of the knee joint. Level of evidenceIII.

Ameliorative mechanism of dietary vitamin d and magnesium on newborn’s pulmonary toxicity induced by cadmium

Cadmium (Cd) exposure in mothers can cause respiratory issues in newborns, but the exact toxicity mechanisms are not fully understood. Vitamin D deficiency in Cd-exposed rats is associated with increased cadmium accumulation in tissues. Finding a cost-effective medication that is vital for the body while also reducing the effects of poisoning is crucial in treating poisonings. To investigate the mechanisms of Cd-induced lung toxicity, we examined the impact of prolonged Cd exposure in female rats before pregnancy on newborn lung health, focusing on sera TNF-α level, lung P53, Foxo1 mRNA, and lung VEGF, and BMP-4 protein level. A total of 50 rats were divided into control, Cd, Cd+Vitamin D, Cd+Mg, and Cd + Vitamin D+Mg groups. Cd exposure resulted in higher serum TNF-α levels and a significant rise in P53 mRNA levels. Additionally, the occurrence of hemorrhage, inflammatory cell infiltration, and thickening of alveolar walls decreased following treatment with vitamin D + Mg. Although Cd did not affect the newborns' body weight, it did impair their lung function. These findings suggest that the Cd-induced increase in the P53 gene expression could be alleviated by vitamin D and Mg, along with the elevation of VEGF and BMP-4 proteins and Foxo1 gene expression. The study revealed that environmental toxins can sometimes harm molecules and proteins, leading to damage in critical fetal tissues. However, these issues can be mitigated through essential supplements. Structured AbstractThe increasing role of Cd in the erratic behavior of numerous biological and molecular entities, notably the development of fetal lung tissue, has made it beneficial to investigate the possible adverse effects of Cd exposure in pregnant mothers and fetal organ development, where instinctive molecular events occur. Researchers are encouraged to create new aspects of medications to reduce clinical symptoms and improve the quality of life due to exposure to metal toxins, particularly in industrialized countries. The present study aimed to evaluate histopathological and molecular modifications of fetal lungs caused by maternal Cd toxicosis and evaluate the possible ameliorating effects of vitamin D and Mg alone and in combination with fetal lung developmental abnormalities, followed by maternal toxin induction, which can be generalized to humans.Fifty female Wistar rats were purchased from the Pasteur Institute of Iran. To induce the model, cadmium at a dose of 2 mg/kg body weight was injected intraperitoneally into the female rats over 28 days before mating (5 days after injection in a week). Afterward, the female rats were randomly divided into type IV polycarbonate cages and mated with healthy male rats. The pregnancy was confirmed by observation of the vaginal plaque, which was subsequently observed, and the number of days of embryo formation was calculated. Subsequently, the pregnant rats were assigned to the following groups and received PBS, vitamin D, Mg, or vitamin D + Mg. At the end of the nine-day treatment period (the 6th day of pregnancy to the 14th day), the neonates were born vaginally, and their body weight and mortality were recorded. The P53 and Foxo1 gene expression levels in the left and right lobes of the homogenized lungs of the newborns in each group were assessed. TNF-alpha was detected in the sera collected from the newborns by ELISA. The isolated left and right lung tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer and the superior phase was collected to determine the total protein content by Lowry's method and VEGF and BMP-4 protein levels. The obtained lung samples from newborn rats were fixed in a 10% formalin solution for tissue processing. The fixed samples were embedded in paraffin, and serial paraffin sections were prepared for hematoxylin and eosin staining. This study is the first to examine how maternal Cd exposure affects fetal lung development and to estimate the impact of prescribing Mg and vitamin D during pregnancy. The present study assessed the effects of a repeated dose of Cd for 4 weeks before pregnancy on the lung development of newborn rats born to mothers treated with vitamin D and Mg. The results showed that the P53 gene was overexpressed in the model group, while Foxo1 gene expression was downregulated, negatively impacting the lung structure and developmental indices of the fetuses. Therefore, the intake of vitamin D and Mg may contribute to improving the various stages of Cd-induced lung injury by modulating lung inflammation and mucosal secretion while also positively influencing the number of surviving offspring.

Determination of the embryotoxic effects of propofol injected into eggs on the cerebellum and spinal cord using histologic methods: an animal study.

This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

Potentiality of Bone Marrow-Derived Mesenchymal Stromal Cells (BM-MSCs) to Differentiate into the Osteogenic Lineage in in Vitro Model of Tissue Regeneration

Introduction:The mesenchymal stromal cells (MSCs) have gained considerable popularity owing to the vast possibilities and lack of ethical constraints. MSCs serve as multipotent progenitors for several niche components in the bone marrow (BM) and play an essential role in the quiescence, proliferation, and differentiation of hematopoietic stem cells. Recently, MSCs from BM (BM-MSCs) represent an attractive cell source for tissue engineering, thanks to their immunomodulatory property, self-renewing and high proliferative capability. BM-MSCs have multi-lineages (adipogenesis, chondrogenesis, osteogenesis) potential and in combination with biomaterials, well support cell-attachments and stimulate extracellular matrix synthesis. For these reasons, BM-MSCs have a great potential for regenerative medicine applications. The present research aims to evaluate the ability of BM-MSCs to proliferate and differentiate toward the osteogenic lineage into innovative three-dimensional bioresorbable scaffold based on gelatin-chitosan hydrogel with a poly(lactic acid) lattice structure (PLA-CH) for regenerative medicine applications in the presence of fetal bovine serum (FBS) or human platelet lysate (HPL) with or without the osteogenic medium (OM). Methods: BM-MSCs were expanded to the passages 3 or 4 and seeded into the PLA-CH, in dry state, at a cellular density of 7x10 5 cells/scaffolds in growth medium (GM). For inducing osteogenic differentiation, cells/scaffolds constructs were cultured in 24-well plates for 4 weeks with OM consisting of a high-glucose DMEM supplemented with 10% FBS or 5% HPL, 10 −7M Dex, 25 mg/ml l-ascorbic acid, and 3mM NaH 2PO 4. For histomorphological analysis at optical microscope, scaffolds were embedded in paraffin using an automatic processor Donatello series 2 (Diapath S.p.A., Bergamo, Italy). Serial paraffin sections (5 µm thick) of each sample were cut, deparaffinized, and rehydrated, according to standard procedures and stained with hematoxylin-eosin stain for general morphology and Von Kossa (Bio-Optica, Milan, Italy) for calcium deposition. Immunohistochemistry for the osteogenic marker Osteocalcin (OSC) (Santa Cruz Biotechnologies, USA) was also performed. For Scanning Electron Microscopy (SEM) (EVO LS-10 manufactured by ZEISS) observation, samples have been progressively dehydrated through immersion in alcohol solutions. The local changes in elemental composition were investigated using the Energy Dispersive X-ray (EDX) analyzer. Results: The results showed that BM-MSCs have higher affinity to attachment, migrate and differentiate toward the osteogenic lineage inside the novel scaffold PLA-CH both with HPL and FBS as supplement in the culture media. The use of HPL for cell expansion offers the possibility to obtain a safer cellular products without xenogenic contaminants. Cells develop a large spreading area with elongated fibroblast-like morphology preferentially inside the hydrogel pores, showing great affinity with the scaffold. This is confirmed also by histomorphological analyses at optical microscope, showing homogeneous colonization of cells inside the hydrogel pores (figure 1a). The results showed the presence of calcium deposits in the mash of the scaffold with Von Kossa stain (figure 1b), moreover cells appeared well integrated with a positivity for OSC showing a clear differentiation state as osteoblasts (figure 1c). Calcium and phosphorous were detected with SEM-EDX on the samples incubated in osteogenic medium (figure 1 d,e). Conclusion: MSCs, derived from bone marrow, associated with scaffold PLA-CH proved to be biocompatible and promising for personalized medicine. In fact, BM-MSCs were able to growth, colonize and osteo-differentiate throughout the hydrogel, demonstrating their potential application for bone regenerative medicine. Figure 1. Histomorphological analysis at the optical microscope of PLA-CH with BM-hMSCs in the GM at 100 m m ( a), Von Kossa stain showing calcium deposit distribution on PLA-CH with BM-hMSCs in the OM at 30 m m ( b), immunohistochemistry for OCN with BM-hMSCs differentiated to osteoblast in the OM at 10 m m ( c). Evaluation of calcium (Ca) and phosphorous (P) with SEM-EDX of PLA-CH with BM-hMSCs in OM day 28 ( d,e).

Hepatocellular Ballooning is Due to Highly Pronounced Glycogenosis Potentially Associated with Steatosis and Metabolic Reprogramming.

Hepatocellular ballooning is a common finding in chronic liver disease, mainly characterized by rarefied cytoplasm that often contains Mallory-Denk bodies (MDB). Ballooning has mostly been attributed to degeneration but its striking resemblance to glycogenotic/steatotic changes characterizing preneoplastic hepatocellular lesions in animal models and chronic human liver diseases prompts the question whether ballooned hepatocytes (BH) are damaged cells on the path to death or rather viable cells, possibly involved in neoplastic development. Using specimens from 96 cirrhotic human livers, BH characteristics were assessed for their glycogen/lipid stores, enzyme activities, and proto-oncogenic signaling cascades by enzyme- and immunohistochemical approaches with serial paraffin and cryostat sections. BH were present in 43.8% of cirrhotic livers. Particularly pronounced excess glycogen storage of (glycogenosis) and/or lipids (steatosis) were characteristic, ground glass features and MDB were often observed. Decreased glucose-6-phosphatase, increased glucose-6-phosphate dehydrogenase activity and altered immunoreactivity of enzymes involved in glycolysis, lipid metabolism, and cholesterol biosynthesis were discovered. Furthermore, components of the insulin signaling cascade were upregulated along with insulin dependent glucose transporter glucose transporter 4 and the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin signaling pathway associated with de novo lipogenesis. BH are hallmarked by particularly pronounced glycogenosis with facultative steatosis, many of their features being reminiscent of metabolic aberrations documented in preneoplastic hepatocellular lesions in experimental animals and chronic human liver diseases. Hence, BH are not damaged entities facing death but rather viable cells featuring metabolic reprogramming, indicative of a preneoplastic nature.

Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis.

The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts. The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wolffian duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the difficulty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial paraffin sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Specific regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found first in the specific portion of the MTs near the MT-rete junction at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal regions. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD first at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5. This 3-D analysis suggests that gonadal androgen acts first on the MTs near the MT-rete junction but that estrogen is the first to influence MTs near the WD, while potential PGR activity is delayed and limited to the epithelium.

Three-dimensional analysis of junctions between efferent and epididymal ducts in the human caput epididymis.

Due to the scarcity of studies using human tissues, the limited information is currently available on the gross structure of the caput epididymis in humans, at which efferent ducts connect to the epididymal duct. The present study investigated the three-dimensional structures of efferent and caput epididymal ducts in humans, with a focus on junctions between the former and the latter. We examined three sets of human efferent and caput epididymal ducts in specimens obtained from prostatic carcinoma patients. They were reconstructed from serial paraffin sections using a segmentation model created by a deep learning protocol and high-performance three-dimensional reconstruction software. Serial sections and three-dimensional images of human efferent and caput epididymal ducts were combined to obtain the detailed anatomical information. When a single efferent duct was defined as a duct connecting to both the extra-testicular rete testis and epididymal duct, there were 14.7 efferent ducts with a total length of 3.0m per specimen on average. The cranial portion of the efferent ducts joined to a single duct and terminated at the end of the epididymal duct, whereas other efferent ducts terminated independently on the side of the epididymal duct. These two types of junctions between the efferent and epididymal ducts differed in the patterns of the epithelial-type switch. The epididymal duct consisted of multiple segments, which were separated by a minimal amount of connective tissue septa or even without them. Efferent ducts occupied most of the volume of the caput epididymis. By utilizing deep learning, we reconstructed human efferent and caput epididymal ducts and revealed their precise three-dimensional structures, which differed from those of rodents in several aspects. The present results may be useful for analyzing anatomical abnormalities related to some types of male infertility.

Cyphetrylin effect on somatostatin receptors expression in transplantable Ca-755 mice tumor

Background. Expression evaluation of somatostatin receptors (SSTRs) in tumor cells is necessary for the reasonable use of therapy directed at such receptors.Aim. The affinity determination of the original analogue of somatostatin cyphetrylin for SSTRs of transplanted mice mammary adenocarcinoma Ca-755.Materials and methods. Cyphetrylin was synthesized in the Chemical Synthesis Laboratory of the Research Institute of Experimental Diagnostics and Therapy of Tumors, N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russia. Cyphetrylin in tablet form was administered orally at a therapeutic dose of 10 mg/kg for 7 days to female F1 (C57Bl/6 × DBA/2) tumor-grafted Ca-755 mice. Animals of the control group were not administreted with сyphetrylin. Tumor tissue samples were obtained from animals on the 9th and 14th days after Ca-755 transplantation and sent for immunohistochemical study, which was performed on serial paraffin sections by the immunoperoxidase method using primary antibodies to various types of SSTRs.Results. The high frequency of positive expression of SSTR1, SSTR2, and SSTR5 (in 80, 100 and 100 % of tumor samples, respectively) was shown in tumor samples of the control group animals. As a result of cyphetrylin introduction in tumor samples obtained on the 9th day after Ca-755 inoculation, a change in the tumor receptor status was found towards a decrease in the level of expression of SSTR2 (80 % of samples) and SSTR5 (80 % of samples); SSTR1 expression did not change (80 % of samples). Compared to the controlled, in tumor samples after cyphetrylin administration, obtained on day 14 from Ca-755 transplantation, a decrease in the expression level of SSTR2 (80 % of samples), SSTR1 and SSTR5 (60 % of samples for SSTR each type) was noted, due to cyphetrylin binding to SSTRs of tumor cells. The receptors SSTR3 and SSTR4 did not show a high level of expression in the studied Ca-755 tumor samples.Immunohistochemical staining of Ca-755 cells with antibodies to SSTRs showed a tendency to reduction of antigen-positive cells number from 15–50 % in control to 10–40 % on day 9 after Ca-755 transplantation and 10–30 % on day 14 after Ca-755 transplantation.Conclusion. The data obtained indicates the presence in mice transplanted mammary adenocarcinoma Ca-755 of SSTR1, SSTR2 and SSTR5 high level expression due to the binding to which the direct cyphetrylin antitumor effect is realized.

OP-3 - Immunohistochemical analyses of steroid hormone receptors in the Wolffian remnants of female mice

The developing mesonephros contains the mesonephric duct, mesonephric tubules, and rete cells. In males, they differentiate into the epididymal duct, efferent tubules, and rete testis, respectively. These structures in females cease development and persist as the epoöphoron and rete ovarii. The morphology and function of the female reproductive organs are closely related to steroid hormones. Therefore, we examined the expression of steroid hormone receptors in the epoöphoron and rete ovarii to clarify whether these remnants can react to the steroid hormones in adult female mice. Serial paraffin sections of ovaries collected from adult C57BL/6 J mice were prepared. Androgen receptor (AR), estrogen receptor (ESR1), progesterone receptor (PR), Ad4BP as a marker for the rete ovarii, and Pax2 as a marker for the epoöphoron were detected by immunohistochemistry. Additionally, the rete ovarii and epoöphoron were reconstructed in Amira software from image data of the serial sections. The rete ovarii was positive for Ad4BP at the hilum of ovary, and Pax2 was detected in the epoöphoron within the adipose tissue around the ovary. Pax2 was also found in a part of epithelial cells of the rete ovarii. The Pax2 positive epithelial cells of the epoöphoron and rete ovarii were also positive for AR and ESR1. The PR expression was confirmed in neither the rete ovarii nor epoöphoron. Three-dimensional reconstruction confirmed a convoluting duct, which divided 3-4 branches extending toward the rete ovarii. The Pax2-positive epithelium of the rete ovarii also protruded to the epoöphoron. However, no contact between the rete ovarii and epoöphoron was found in almost all cases. The epithelium of mesonephric tubules, which have connected to the rete ovarii, is suggested to be partially stranded on the rete ovarii during the regression in the female mouse. These epithelial cells left in the postnatal rete ovarii probably retain the sensitivity for androgen and estrogen.

OP-12 - Three-dimensional and immunohistochemical analysis of sex steroid receptors in the developing murine mesonephros

Sex steroids are essential for the development of the reproductive tracts, and their function is mediated by activating their receptors. The expression patterns of the receptors have been investigated in previous studies, but the results differ. One of the reasons is a lack of a method to distinguish each region of the reproductive tracts. To distinguish the cranial and caudal Wolffian duct (WD), which contribute to the common efferent duct and epididymal duct, respectively, it is necessary to check the connection with the mesonephric tubules (MTs) that differentiate into the efferent ductules. In this study, we examined the expression patterns of sex steroid receptors in detail to combine the three-dimensional analysis. Serial paraffin sections of the developing murine mesonephros were prepared, and immunohistochemistry for sex steroid receptors, such as estrogen receptor (ESR1), androgen receptor (AR), and progesterone receptor (PR), was performed. Additionally, the sections in which Collagen Type IV was detected were used to reconstruct the MTs and WD. ESR1 was found in the mesenchymal cells around the WD, and the MTs and cranial WD also became positive from the middle period of the development, but the caudal WD was negative for ESR1. AR was first found at the distal end (gonadal side) of the MTs and weakly in the mesenchymal cells. The epithelial expression became strong in the whole length of the MTs and cranial portion of the WD in the middle period of the development. Finally, the caudal part of the WD also showed strong positive just before birth. PR was weakly positive in the MTs and cranial WD, but the distal end of the MTs where the rete cells were adherent was negative for PR. Androgen secreted from the gonad probably acts on the distal end of the MTs to maintain and develop the efferent tubules. ESR1 and PR may also contribute to the development of the efferent duct. Additionally, estrogen treatment in the neonatal male mice induces epididymal inflammation after puberty, but its mechanisms are still unclear. This study found the ESR1 expression in not epithelial cells but mesenchymal cells around the caudal WD. These results suggest that neonatal estrogen treatment probably affects the mesenchymal cells around the epididymal duct, resulting in the inability to suppress the autoimmune response to spermatozoa after puberty.

Immunohistochemical analysis of caspase expression in the brains of individuals with obesity or overweight.

Mechanisms underlying the negative effects of obesity on the brain are still unknown. Obesity is associated with oxidative stress in the brain and neuroinflammation that promotes neurodegenerative diseases. Chronic low-grade neuroinflammation in obesity could be associated with lower volumes of gray matter and lower neuronal density. If neuroinflammation mediated by the expression of cytokines and chemokines leads to apoptosis, this can be assessed by examining caspase expression. The aim of this study was to compare the expression of caspases in the 16 brains of donors with obesity/overweight (n=8; Body Mass Index [BMI]=31.6±4.35kg/m2; 2 females; Age=52.9±4.76years) and normal weight (n=8; BMI=21.8±1.5kg/m2; 3 females; Age=37.8±19.2years). Sixteen human brain samples were processed. Serial paraffin sections were examined by anti-caspase immunochemistry (caspase-3, caspase-4, caspase-6, caspase-1, caspase-8, and caspase-9 antibodies). Postmortem samples of cerebral cortex tissue were captured as photomicrographs and the images obtained were analyzed using ImageJ software to obtain the percentage of positive caspase expression. Nonparametric Mann-Whitney U tests were performed to compare caspase expression between samples from donors with obesity/overweight and normal weight. Taking into consideration the immunohistochemistry results, the Search Tool for the Retrieval of Interacting Genes was used to model molecular interactions. Results showed that brain samples from individuals with obesity/overweight exhibited significantly greater values of positive expression for Caspase-1 (U=16.5, p=0.05, Cohen d=0.89) and -8 (U=15, p=0.03, Cohen d=0.99) than those from donors with normal weight. This study contributes to the knowledge about the inflammatory effects of obesity/overweight on brain, suggesting theactivation of the alternative inflammasome pathway in which interact caspase-1 and -8.

Age specific features of nNOS immunoreactive neurons in rat neocortex

The aim of the study was to evaluate the morphological features of nNOS-positive (nNOS-IR) neurons in the dorsolateral cortex of the frontal lobe of the cerebral hemispheres in albino rats during 180 days of postnatal development.Material and methods. The study was performed on 40 outbred white Wistar rats of different ages, from 1 to 180 days. The object of the study was an area of the right cerebral hemisphere on the dorsolateral surface near the frontal pole (neocortex). On paraffin serial sections of the frontal lobe, an immunohistochemical reaction was performed with antibodies to nNOS and a detection system with horseradish peroxidase. Neuronal morphometry was performed by microphotographs using the ImageJ-Fiji (NIH) 1.51h program, measuring the sectional area of the neuron body, the area of the nucleus, the nuclear-cytoplasmic ratio, and the intensity of the reaction.The significance of differences was assessed by paired Student's t-test.Results. It was found that in mature rats in the frontal lobe cortex nNOS-IR was detected in large multi-polar cells with high activity of the enzyme located in the supragranular layers, spindle-shaped cells with long positive processes at the border with the white matter (type 1), and two varieties of low-positive neurons – accumulations in the VI layer and single ones in other layers (type 2). Polymorphism of nNOS-IR neurons manifests from the birth, but it was possible to distinguish all subpopulations only from the 21st day. Each subpopulation is distinguished by its own age dynamics of the studied parameters and the nature of the distribution of positivity. In addition, in 3–7 day old rat pups, numerous small neurons at the border of the cortex and white matter have transient immunoreactivity.Conclusion. Thus, the division of nNOS-IR neurons into two morphological types proposed in the works of predecessors does not correspond to the number of subpopulations that could be described in the dorsolateral region of the prefrontal cortex in rats. This diversity of nNOS-IR neurons is consistent with the numerous functions described for nitric oxide. For an objective characterization of various classes of nNOS-IR cortical interneurons, it is necessary to use additional data obtained from transcriptomic, histological, electrophysiological, and functional experiments, taking into account species, topographic, and age features. Only an extended approach will make it possible to selectively influence different types of cells and reasonably interpret the results of experimental studies.

Molar extraction alters gastric mucosa and ghrelin expression in rat stomach: A preliminary study

Objectives: Ghrelin is a key regulator of food intake and is considered a hunger hormone that affects cognition, memory, glucose metabolism, and antidepressant effects. Altered occlusion, such as a loss of molars, has been thought to retard digestive function. However, the association between occlusion and digestive function remains poorly understood. Here, we aimed to explore the effect of bilateral maxillary molar extraction on the gastrointestinal mucosa of growing rats and the expression of ghrelin and its receptor, growth hormone secretagogue receptor (GHSR). Material and Methods: Twenty-four male 5-week-old Wistar rats were divided into control (CON) and experimental (EXP) groups (n = 12/group). The rats in the EXP group underwent extraction of the bilateral maxillary first, second, and third molars under general anesthesia. Rats in the CON group underwent a sham operation. All rats in both the CON and EXP groups were fed a powder diet and water ad libitum. The body weight of all rats was monitored throughout the EXP period. Rats in both the CON and EXP groups were euthanized on days 14 and 28, and the stomachs were isolated and subjected to histological analysis. Paraffin serial sections were prepared using a microtome for hematoxylin-eosin and immunohistochemical staining using anti-ghrelin and anti-GHSR antibodies. The distribution and expression of ghrelin-immunopositive and GHSR cells were detected and observed under a light microscope. Data were statistically analyzed using t-tests (P &lt; 0.05). Results: There were no significant differences in body weight between the CON and EXP groups throughout the EXP period. Histological analysis showed that the area of the submucosa (ASM), and the number of ghrelinimmunopositive cells were significantly decreased in the EXP group compared with the CON group on day 14. Alternatively, there was no significant difference in the ASM and the number of ghrelin-immunopositive cells between the CON and EXP groups on day 28, whereas the number of ghrelin receptors showed no differences across groups. Furthermore, the number of eosinophilic blood cells significantly increased in the EXP group on days 14 and 28. Conclusion: Our findings suggest that bilateral maxillary molar extraction may trigger stomach mucosal changes and alter digestive function through ghrelin expression in rats. This is the first report that occlusal deficiency could alter ghrelin expression in the mucosa of the rat stomach, thus raising concerns about the consequential role of ghrelin.

Comparative morphometric analysis of age-related changes in the pyramidal neurons of the human prefrontal and posterior associative cortex from birth to 7 years

Aim: This article is devoted to the study of age-related changes in the body volume of pyramidal neurons (VP) of the human prefrontal (PFC) and posterior associative cortex (PAC) in children from birth to 7 years of age. Methods: The material consisted of left cerebral hemispheres obtained from 60 male individuals. We studied the areas 8, 45 and 10 in the PFC and area 37 in subareas 37ac, 37a, 37d in the PAC. For morphometry of pyramidal neurons in cortical sublayer III3, we used virtual images of serial frontal paraffin sections of cortex 10 thick stained by Nissl. We determined the mean value of VP (mean VP), the standard error of the mean, and the confidence interval for each age group. Results: The most significant changes in the mean VP in all studied cortical areas occurred during the first 6 months of life. The greatest increase in the VP was detected in the PFC from 3.5 to 7 years, and in the PAC from 1.5 to 3 years of age. There are close positive correlations between agerelated changes in the size of neurons in the PFC and in the PAC. Conclusion: In children, the development of pyramidal neurons of the external pyramidal plate in the PAC occurs earlier and at a more intensive pace compared to the PFC. We suggest that VP is an important quantitative indicator that allows us to assess the timing and rate of age-related structural changes in the cerebral cortex.

Morphogenetic and pathogenetic features of hypertrophic and keloid scars of the head and neck

Evaluate the morphogenetic and pathogenetic features of hypertrophic and keloid scars of the head and neck. The study included 286 patients, among them 176 (61.5%) patients with hypertrophic and 110 (38.5%) with keloid scars aged 18 to 65 years with a disease duration from 1 month to 2 years. Material for histological and immunohistochemical (IHC) studies of scar tissue was fixed in 10% buffered formalin. Serial paraffin sections were stained with H&E, according to Van Gieson and Weigert. IHC was performed using monoclonal mouse antibodies to collagen type I (clone 3G3, Santa Cruz, dilution 1:100), collagen type III (clone B-4, Santa Cruz, dilution 1:50), collagen type IV (clone COL-94, Santa Cruz, dilution 1:50), MMP-1 (clone 3B6, Santa Cruz, dilution 1:100), α-SMA1 (clone 1A4, Dako Agilent, dilution 1:100) and rabbit polyclonal anti-TGFβ antibodies (clone 3C11, Santa Cruz, 1:100 dilution). Pathogenetic, morphological and immunohistochemical differences in hypertrophic and keloid scars were established depending on their degree of maturity. In the formation of hypertrophic scars, the key factor in sclerotic processes is TGF-b on the background of low MMP1 activity. Keloid scars were distinguished not only by the accumulation of hard-to-degrade collagens, but also by the development of an osteoclast-like reaction with a high content of MMP1. Immature scar tissue was characterized by the presence of myofibroblastic α-SMA1 positive focus and center of inflammatory changes. The data obtained allow substantiating new approaches to the treatment of patients with hypertrophic and keloid scars.

CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids.

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.

Structural and molecular markers of aging heart: Sechenov University classification

Abstract Background The process of aging at the level of the cells is connecting with arising of senescence cell, that are characterized by several possible signs, including damage to the mitochondria and contraction of telomeres, epigenetic changes, impaired regulation of protein metabolism, sensitivity to nutrients, mitochondrial dysfunction, stem cell collapse, impaired intercellular communication. Postmitotic cells may also develop senescence and are called amitosenescence cells (AMCs). Goal To investigate structural and molecular characteristic of aging heart with special identification of AMCs and creating classification of aging heart status. Methods Study was performed on 52 biopsies (endomyocardial and operative) of the heart and 48 autopsy cases (38 women and 62 men) with diagnosis of dilated cardiomyopathies, chronic ischemic heart disease, hypertensive cardiomyopathies, rheumatic heart disease and their combination. All patients were divided in two groups according to the age of patients: 1 group included 80 patients (average age 72.9±13.3 years) and 2 control group - 20 patients (average age 42.9±13.3 years). Serial paraffin sections were stained with hematoxylin and eosin, picrofuchsin; p16ink, Apocas, CD68 and CD 45 were detected by immunohistochemistry (DakoCytomation). We have calculated percent of AMC, Apo and RCB in 30 fields of vision on X400 magnification. Results AMCs were found in the myocardium of 77 aging patients and only in 2 young patients (premature aging?). AMCs were characterized by prominent degenerative changes, p16ink expression and accumulation of lipofuscin. Reduction of capillary bad (RCB), increased number of apoptotic bodies and presence of CD68+ macrophages and CD45+ lymphocytes were found in stroma. There were three aging heart status degree: 1 – presence less than 15% of AMCs, 0,1% of Apo and RCB –less than 15% (20 patients); 2 – presence 16–30% of AMCs, 0,2- 1% of Apo and RCB – 16–30% (35); 3 – presence more than 31% of AMCs, 1–2% of Apo and RCB – more than 31% (27). Conclusion Aging heart has increased number of amitosenecence cells that results severity and chronicity of heart diseases in aging patients. Funding Acknowledgement Type of funding sources: None.

Three-dimensional reconstruction of testis cords/seminiferous tubules.

BackgroundDue to the development of novel equipment for the acquisition of two‐dimensional serial images and software capable of displaying three‐dimensional (3D) images from serial images, the accurate 3D reconstruction of organs and tissues has become possible.MethodsBased on published studies, this review summarizes techniques for the 3D reconstruction of the testis cords/seminiferous tubules, with special reference to our method using serial paraffin sections and 3D visualization software.Main findingsThe testes of mice, rats, and hamsters of various ages were 3D reconstructed and species and age differences in the structures of the testis cords/seminiferous tubules were analyzed. Our method is advantageous because conventional paraffin‐embedded normal and pathological specimens may be utilized for the 3D analysis without the need for complicated and expensive equipment.ConclusionBy further decreasing the time and labor required for the procedure and adding information on molecular localization, the technique for 3D reconstruction will contribute to the elucidation of not only the structures, but also the functions of various organs, including the testis.