Histochemical Techniques Research Articles

Glutaraldehyde-enhanced autofluorescence as a general tool for 3D morphological imaging.

Routine histochemical techniques are capable of producing vast amount of information from diverse samples types, but these techniques are limited in their ability to generate 3D information. Autofluorescence imaging can be used to analyse samples in 3D but it suffers from weak/low signal intensities. Here, we describe a simple chemical treatment with glutaraldehyde to enhance autofluorescence for 3D fluorescence imaging and to generate detailed morphological images on whole-mount samples. This methodology is straightforward and cost-effective to implement, suitable for a wide range of organisms and sample types. Furthermore, it can be readily integrated with standard confocal and fluorescence microscopes for analysis. This approach has the potential to facilitate the analysis of biological 3D structures and research in developmental biology, including studies on model and non-model organisms.

STUDY OF THE ENDOMETRIAL GLANDULAR COMPONENT IN WOMEN WITH FERTILITY AND POST-COVID SYNDROME IN ANAMNESIS

The aim. To increase the effectiveness of the endometrial endometrial glandular component study in women with infertility and post-COVID syndrome. Materials and methods. A study was conducted at the State institution "Carpathian Human Reproduction Centre" of the Ministry of Health of Ukraine in the period from 2022 to the end of the first half-year of 2024. There were included 80 women in the experimental group and 40 women in the control group. The selection criterion for the study group was the presence of documented post-COVID syndrome (COVID-19 symptoms lasted longer than 12 weeks). The selection criterion for the control group was the absence of confirmed SARS-CoV-2 virus in the anamnesis. The following characteristics were common for both groups: diagnosed infertility, age – 25-42 years, written informed consent of the patient to participate in the study. The criteria for exclusion from the study for patients of both groups were: detection of human immunodeficiency virus, viral hepatitis, other sexually transmitted infections, tuberculosis of the genitourinary system, malignant diseases, mental disorders that make communication with person impossible or the awareness of written consent is questioned. General clinical and laboratory examinations and transvaginal sonographic examination of the pelvic organs were performed in the middle stage of proliferation and the middle stage of secretion according to the calendar calculation the menstrual cycle. A visually guided diagnostic office hysteroscopy was performed with the collection of biological material from the most changed areas (biopsies were collected in the middle stage of proliferation and the middle stage of secretion in different menstrual cycles). A routine staining of samples with hematoxylin-eosin, morphometry of the endometrial glandular component, optical determination of the number of glands and measurement of the diameter of the lumen of the studied glands, considering the possible measurement error, glandular-stromal ratio, functional activity of the glands, were performed. A histochemical PAS-reaction to identify the glycoproteins of gland secretions and immunohistochemical staining of the glandular epithelium to express the receptivity of the glands to estrogen and progesterone were carried out. The results. After conducting the PAS-reaction, a higher secretion was noted in the glands than in the stroma of the endometrium during the period of the implantation window in both cohorts. No statistically significant difference between the groups in the amount of glycogen of the middle proliferative phase in the glands (p=0.315) or stroma (p=0.486). Glycogen in this phase was visualized of very small granules and coarse granules, without their formation in clusters. A decrease in secretion was established in 16 (40%) patients of the control group and 49 (61.3%) women of the experimental group in the middle secretory phase, which was statistically significant (p=0.028). A decrease in the expression level of the progesterone receptor in the middle secretory phase was found to be significantly more pronounced in the experimental group than in the control group (р=0.044). Conclusions. In the middle secretory phase, a decrease in the secretion of glycogen by the endometrial glands and a decrease in the level of expression of the progesterone receptor in endometrial biopsies were detected by the routine histological, histochemical techniques and immunohistochemical stude in women with infertility and a history of post-COVID syndrome.

Influence of Physical Exercise on the Rehabilitation of Volumetric Muscle Loss Injury Reconstructed with Autologous Adipose Tissue.

In volumetric muscle loss (VML) injuries, spontaneous muscle regeneration capacity is limited. The implantation of autologous adipose tissue in the affected area is an option to treat these lesions; however, the effectiveness of this therapy alone is insufficient for a complete recovery of the damaged muscle. This study examined the influence of treadmill exercise on the rehabilitation of VML injuries reconstructed with autologous adipose tissue, as a strategy to counteract the limitations of spontaneous regeneration observed in these injuries. Forty adult male Wistar rats were divided into eight groups of five individuals each: normal control (NC), regenerative control (RC), VML control (VML), VML injury reconstructed with fresh autologous adipose tissue (FAT), exercise-rehabilitated control (RNC), exercise-rehabilitated regenerative control (RRC), exercise-rehabilitated VML injury (RVML), and exercise-rehabilitated VML injury reconstructed with fresh autologous adipose tissue (RFAT). Histological and histochemical staining techniques were used for the analysis of structural features and histomorphometric parameters of the tibialis anterior muscle. Grip strength tests were conducted to assess muscle force. Exercise rehabilitation decreased the proportion of disoriented fibers in RFAT vs. FAT group. The percentage of fibrosis was significantly higher in FAT and RFAT groups versus NC and RNC groups but did not vary significantly between FAT and RFAT groups. Overall, muscle grip strength and fiber size increased significantly in the exercise-rehabilitated groups compared to control groups. To conclude, rehabilitation with physical exercise tended to normalize the process of muscle repair in a model of VML injury reconstructed with fresh autologous adipose tissue, but it did not reduce the intense fibrosis associated with these injuries.

Development Postnatal of the Kidney and Liver in Albino Rat Rattus Rattus, Histological and Biochemical Study

Objective: The primary goal of the current study was to look at histological, histochemical, and biochemical aspects of the development structure of rat's kidney and liver. Materials and Method: Seventy eight samples of kidney and liver were obtained from rats at days; 1, 5, 10, 15, 36 and 90 after birth and processed for histological, histochemical and biochemical techniques. Results: Up to the fifth day, new nephron anlages develop in the renal cortex. After ten days, the final anlages form into functional nephrons, and fifteen days after the corticis forms, the cortex seems mature. The collecting ducts and loops of Henle expand longitudinally to form the renal medulla. The immature medulla is structurally similar to the later inner stripe of the outer zone and cannot be split into distinct zones. Within ten days, the inner zone was developed, and within fifteen days, the outside zone's stripe. The kidney tubule's diameter increased with age, the renal medulla was mature at 36 days, the kidney was covered in a moderate amount of collagen fiber, and the henle loops were lengthy at certain ages, but short at adult age. Adults had larger corpuscle and nephron lumens. In contrast, the thick renal capsule at other ages contained less collagen fibers. A tiny renal gap encircling the glomeruli and a notably greater quantity of fibers were visible in adult Bowman's capsules. The liver was encased in a thin capsule on the first day of birth and a thick capsule between 36 and 90 days later. On day 15, the cells surrounding the central veins began to arrange themselves in a regular fashion. The hepatic parenchyma could be seen by symmetrical hepatic cords, which emerged from the central veins as radiating columns. After five days, Von Kuffer cells began to line the sinusoids next to the endothelial cells. On the tenth day, the hepatic sinusoids took on a more regular appearance, stretching between the hepatic cords and joining the central veins. The hepatic lobules with their central vein were visible at the age of 15 days. The capsule then thickened and elastic fibers started to show up. The reticular fibers also thickened and spread, and in 36 days, collagen fibers started to show up in the portal areas. Based on histochemical data, every PAS, AB, and PAS-AB stain was highly reacted with brush in renal and hepatic cells. In conclusion: The kidney and liver developed differently after birth and reached their mature, typical structures in 36 days.

COVID-19-Induced Changes in the Fibrin Network of Pulmonary and Renal Microthrombi

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection often causes coagulation disorders that affect highly vascularized organs, such as the lungs and kidneys. Objective The objective of this study was to report the histopathological findings of variations in the fibrin pattern of pulmonary and renal microthrombi in patients who died from SARS-CoV-2 infection. Methods Minimally invasive autopsies were performed on 40 patients to collect lung (n=40) and kidney (n=16) tissue samples. Histochemical and immunohistochemical staining techniques were used for histopathological analyses. Premortem laboratory data were obtained from the patients' electronic medical records. Results The lung tissue showed a patchy pattern, characterized by areas of both minimal and severe damage. The most significant histopathological finding was the detection of thrombi with fibrin structures organized into discrete star-shaped units, which were more frequently observed in areas with severe lung injury than in those with minimal lung injury (p = 0.012). Star-shaped fibrin structures were also observed in the renal glomerular capillaries. Immunohistochemical staining revealed the presence of platelets and the procoagulant proteins von Willebrand factor (VWF) and Factor VIII within the star-shaped fibrin thrombi. Patients with star-shaped fibrin thrombi had higher levels of the systemic inflammatory indicators C-reactive protein (CRP) and neutrophil-to-lymphocyte ratio (NLR). Conclusion Our observations suggest that the inflammatory microenvironment resulting from SARS-CoV-2 infection may contribute to the formation of star-shaped fibrin units in the pulmonary and renal microthrombi.

Comparison of histopathological and triphenyl tetrazolium chloride test in diagnosing myocardial infarction: An autopsy study

Background Sudden and unexpected deaths are increasing drastically. The main cause of sudden death is cardiovascular disease, out of which coronary artery disease predominates forming 80% of the cases. Most of the time, detecting early changes in myocardial infarction during the autopsy is challenging since gross infarct changes do not appear until after 24 to 48 hours of myocardial ischemia injury. So, the aim of this study was to compare two test to detect early changes of Myocardial Infarction one by using Triphenyl Tetrazolium Chloride (TTC) staining of the myocardial tissue, during autopsy and other by histopathological examination. Methods The sample size of 60 hearts taken from all the sudden deaths cases brought to Mortuary with suspected cause of death due to cardiac origin. The heart was obtained from the deceased by standard post-mortem technique. Serial full-thickness transverse sections of the heart were taken at 2 cm intervals from the apex to the atrioventricular groove. All the serial slices of heart are taken for histochemical staining and TTC staining. Results In histopathological examination 34 hearts were diagnosed with myocardial infarction and 26 hearts reported non myocardial infarction. With TTC 40 hearts remained unstained suggestive of myocardial infarction and 20 hearts were stained suggestive of non-infarcted hearts. TTC staining in our study shows an accuracy of 88.33%. Conclusion The result of this study shows that the Triphenyl Tetrazolium Chloride test, a histochemical staining technique of heart, is reliable approach for forensic pathologists to diagnose early myocardial infarction during the post-mortem examination.

Low ozone concentrations do not exert cytoprotective effects on tamoxifen-treated breast cancer cells in vitro.

Medical treatment with low ozone concentrations proved to exert therapeutic effects in various diseases by inducing a cytoprotective antioxidant response through the nuclear factor erythroid derived-like 2 (Nrf2) transcription factor pathway. Low ozone doses are increasingly administered to oncological patients as a complementary treatment to mitigate some adverse side-effects of antitumor treatments. However, a widespread concern exists about the possibility that the cytoprotective effect of Nrf2 activation may confer drug resistance to cancer cells or at least reduce the efficacy of antitumor agents. In this study, the effect of low ozone concentrations on tamoxifen-treated MCF7 human breast cancer cells has been investigated in vitro by histochemical and molecular techniques. Results demonstrated that cell viability, proliferation and migration were generally similar in tamoxifen-treated cells as in cells concomitantly treated with tamoxifen and ozone. Notably, low ozone concentrations were unable to overstimulate the antioxidant response through the Nfr2 pathway, thus excluding a possible ozone-driven cytoprotective effect that would lead to increased tumor cell survival during the antineoplastic treatment. These findings, though obtained in an in vitro model, support the hypothesis that low ozone concentrations do not interfere with the tamoxifen-induced effects on breast cancer cells.

Histological methods for plant tissues

ABSTRACT Although many of the structures and organelles of vegetative cells are comparable to those of animal tissues, significant differences between the two kingdoms require modifications in histological techniques for both tissue processing steps and histochemical staining techniques. The authors investigated the challenges of working with plant tissues by collecting various flora to represent the four main plant organs: leaf, stem, root, and flower/fruit. Triplicate samples for each specimen were placed into formalin for paraffin embedding, placed into formalin for later frozen sections, and used fresh to undergo immediate frozen sectioning. Frozen sections of plant tissues were more difficult to obtain than formalin-fixed paraffin-embedded (FFPE) sections, exhibited tissue loss during staining, and were inferior morphologically to FFPE sections. Although, historically, plant tissue fixation and processing has employed several different reagents compared with those used in animal tissue processing and took significantly longer times, the current investigation determined reagents and protocols from a modern histology laboratory which processes mammalian tissues can be applied to plant tissue processing with only slight modifications in respect to reagent timing. Additionally, staining techniques were compared and while it is well known that plant cell walls stain well with safranin O, the current investigation determined the uptake of safranin O can be accelerated by incubating at 60°C.

The Contribution of Mast Cells to the Regulation of Elastic Fiber Tensometry in the Skin Dermis of Children with Marfan Syndrome.

Marfan syndrome (MFS) is a hereditary condition accompanied by disorders in the structural and regulatory properties of connective tissue, including elastic fibers, due to a mutation in the gene encodes for fibrillin-1 protein (FBN1 gene) and the synthesis of abnormal fibrillin-1 glycoprotein. Despite the high potential of mast cells (MCs) to remodel the extracellular matrix (ECM), their pathogenetic significance in MFS has not been considered yet. The group of patients with Marfan syndrome included two mothers and five children (three girls aged 4, 11, and 11 and two boys aged 12 and 13). Normal skin was examined in two children aged 11 and 12. Histochemical, monoplex, and multiplex immunohistochemical techniques; combined protocols of simultaneous histochemical and immunohistochemical staining (the results of staining were assessed using light, epifluorescence, and confocal microscopy); and bioinformatics algorithms for the quantitative analysis of detected targets were used to evaluate mast cells and their relationship with other cells from extracellular structures in the skin dermis. Analysis of the skin MC population in children with Marfan syndrome revealed a considerably increased number of intra-organic populations with the preservation of the specific Tryptase+Chymase+CPA3+ protease profile typical of the skin. The features of the MC histotopography phenotype in MFS consisted of closer colocalization with elastic fibers, smooth muscle cells, and fibroblasts. MCs formed many intradermal clusters that synchronized the activity of cell functions in the stromal landscape of the tissue microenvironment with the help of spatial architectonics, including the formation of cell chains and the creation of fibrous niches. In MCs, the expression of specific proteases, TGF-β, and heparin increased, with targeted secretion of biologically active substances relative to the dermal elastic fibers, which had specific structural features in MFS, including abnormal variability in thickness along their entire length, alternating thickened and thinned areas, and uneven surface topography. This paper discusses the potential role of MCs in strain analysis (tensometry) of the tissue microenvironment in MFS. Thus, the quantitative and qualitative rearrangements of the skin MC population in MFS are aimed at altering the stromal landscape of the connective tissue. The results obtained should be taken into account when managing clinical signs of MFS manifested in other pathogenetically critical structures of internal organs, including the aorta, tendons, cartilage, and parenchymal organs.

Lithium-induced apoptotic cell death is not accompanied by a noticeable inflammatory response in the kidney.

Lithium (Li+) therapy is a valuable tool in psychiatric practice that remains underutilized due to safety concerns. Excessive plasma Li+ levels are nephrotoxic and can trigger a local immune response. Our understanding of the immunomodulatory effects of Li+ in the kidney is fragmentary. Here, we studied how immune mechanisms contribute to the development of Li+-induced adverse effects in the kidneys of C57BL/6NJ mice placed on a 0.3% lithium carbonate diet for 28days. We combined histochemical techniques, immunoblotting, flow cytometry, qPCR and proteome profiler arrays to characterize renal tissue damage, infiltrating immune cells and cytokine markers, activation of pyroptotic and apoptotic cascades in the kidneys of mice receiving Li+-containing and regular diets. We found that biomarkers of tubular damage, kidney injury marker, KIM-1, and neutrophil gelatinase-associated lipocalin, NGAL, were elevated in the renal tissue of Li+-treated mice when compared to controls. This correlated with increased interstitial fibrosis in Li+-treated mice. Administration of Li+ did not activate the pro-inflammatory NLRP3 inflammasome cascade but promoted apoptosis in the renal tissue. The TUNEL-positive signal and levels of pro-apoptotic proteins, Bax, cleaved caspase-3, and caspase-8, were elevated in the kidneys of Li+-treated mice. We observed a significantly higher abundance of CD93, CCL21, and fractalkine, accumulation of F4.80+ macrophages with reduced M1/M2 polarization ratio and decreased CD4+ levels in the renal tissue of Li+-treated mice when compared to controls. Therefore, after 28days of treatment, Li+-induced insult to the kidney manifests in facilitated apoptotic cell death without an evident pro-inflammatory response.

Dysregulated zinc homeostasis and microadenomas in the anterior pituitary: pathological insights into suicide risk.

Suicide is a significant public health problem influenced by various risk factors, including dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis. Zinc (Zn), essential for pituitary function in hormone synthesis and release, has been linked to suicide, with studies noting reduced serum levels and altered brain transport mechanisms. Despite Zn's crucial role in pituitary function and its involvement in suicidal behavior, information on pituitary Zn in suicide is scarce. Tumor cells modify Zn dynamics in tissues, and a previous report suggests microadenomas in the anterior pituitary as a risk factor for suicide. Histopathological analysis with hematoxylin-eosin stain and histochemical techniques to assess Zn homeostasis were carried out on anterior pituitary postmortem samples from 14 suicide completers and 9 non-suicidal cases. Pituitary microadenomas were identified in 35% of suicide cases and none in the non-suicidal cases. Furthermore, compartmentalized Zn (detected via dithizone reactivity), but not free Zn levels (detected via zinquin reactivity), was lower in the suicide cases compared to the non-suicidal group. This is the first report of a potential association between disrupted Zn homeostasis and microadenomas in the anterior pituitary as a feature in suicide and provides critical insights for future neuroendocrine Zn-related research.

New insight in secretory structures and secretion composition in Rhus typhina L. – Anatomical, histochemical, and ultrastructural studies

Rhus typhina is a valuable plant used in the pharmaceutical, cosmetic, and food industries due to the presence of biologically active substances accumulated in its organs, especially in secretory structures, i.e. trichomes and secretory ducts. Light microscopy, scanning electron microscopy, and transmission electron microscopy were used to examine the structure of glandular and non-glandular trichomes, as well as secretory ducts present in inflorescence peduncles of R. typhina. The chemical composition of the secretion produced by trichomes and ducts was assessed using histochemical techniques, including observations under brightfield and fluorescence microscopes. Two types of capitate glandular trichomes producing secretions with a similar composition and non-glandular trichomes exhibiting secretory activity were identified. The secretion of glandular trichomes was dominated by acidic and neutral lipids, essential oil, sesquiterpenes, and steroid-containing terpenes. The schizogenic secretory ducts located in the phloem produced a viscous milky substance with acidic polysaccharides, acidic lipids, phenolic compounds, and proteins. The secretion was released into the duct lumen through notches in the walls of the secretory epithelial cell facing the duct lumen. The location, type, and traits of the non-glandular trichomes and secretory structures, as well as the composition of the secreted products are considered important taxonomic features in the family Anacardiaceae and the Rhus genus. Additionally, these characters are important diagnostic markers for the pharmacobotanical identification of the species in medicinal and cosmetic raw materials. The various compounds present in the secretory structures of R. typhina may contribute to plant protection against pathogens or herbivory and probably play a role as attractants for pollinators and seed dispersers.

Histochemistry for Molecular Imaging in Nanomedicine.

All the nanotechnological devices designed for medical purposes have to deal with the common requirement of facing the complexity of a living organism. Therefore, the development of these nanoconstructs must involve the study of their structural and functional interactions and the effects on cells, tissues, and organs, to ensure both effectiveness and safety. To this aim, imaging techniques proved to be extremely valuable not only to visualize the nanoparticles in the biological environment but also to detect the morphological and molecular modifications they have induced. In particular, histochemistry is a long-established science able to provide molecular information on cell and tissue components in situ, bringing together the potential of biomolecular analysis and imaging. The present review article aims at offering an overview of the various histochemical techniques used to explore the impact of novel nanoproducts as therapeutic, reconstructive and diagnostic tools on biological systems. It is evident that histochemistry has been playing a leading role in nanomedical research, being largely applied to single cells, tissue slices and even living animals.

Modified histological staining for the identification of arterial and venous segments of brain microvessels

BackgroundThis study aimed to develop a modified histochemical staining technique to successfully identify arterial and venous segments of brain microvessels. New methodGelatin/red ink-alkaline phosphatase-oil red O (GIAO) staining was developed from the traditional gelatin–ink perfusion method. Oil red Chinese ink for brush writing and painting mixed with gelatin was used to label cerebral vascular lumens. Subsequently, alkaline phosphatase staining was used to label endothelial cells on the arterial segments of cerebral microvessels. Thereafter, the red ink color in vessel lumens was highlighted with oil red O staining. ResultsThe arterial segments of the brain microvessels exhibited red lumens surrounded by dark blue walls, while the venous segments were bright red following GIAO staining. Meanwhile, the nerve fiber bundles were stained brownish-yellow, and the nuclei appeared light green under light microscope. After cerebral infarction, we used GIAO staining to determine angiogenesis features and detected notable vein proliferation inside the infarct core. Moreover, GIAO staining in conjunction with hematoxylin staining was performed to assess the infiltration of foamy macrophages. Comparison with existing methodRed Chinese ink enabled subsequent multiple color staining on brain section. Oil red O was introduced to improved the resolution and contrast between arterial and venous segments of microvessels. ConclusionWith excellent resolution, GIAO staining effectively distinguished arterial and venous segments of microvessels in both normal and ischemic brain tissue. GIAO staining, as described in the present study, will be useful for histological investigations of microvascular bed alterations in a variety of brain disorders.

Direct Immunofluorescence in Cutaneous Vesiculobullous Lesions: A Cross-sectional Study in a Tertiary Care Center

Introduction: Cutaneous noninfectious autoimmune vesiculobullous diseases are a wide variety of lesion. They can show clinical and histopathological overlap, posing diagnostic challenges. Clinically, vesiculobullous lesions might not present with typical morphology and distribution in all cases. The specific histomorphological changes are observed only in an early and intact bulla. Immunofluorescence (IF) is a histochemical staining technique which detects antibodies in the tissue and body fluids. IF an adjuvant to histopathology is considered as gold standard in the diagnosis of vesiculobullous diseases. Methodology: A two-year retrospective cross-sectional descriptive study was conducted in the department of pathology, in a tertiary care center from January 2018 to December 2019. Paraffin embedded hematoxylin and eosin slides were retrieved from the archives and analyzed. Biopsies received in Michel’s medium were utilized for immunofluorescence study. Results: A total of 67 cases with Vesiculobullous lesions were analysed. The most common lesion diagnosed was Bullous Pemphigoid (BP) (29 cases -46.7%), followed by Pemphigus Vulgaris (PV) (19 cases- 30.6%). The highest number of cases were noted in the 7th decade, with a slight female preponderance. Sixteen out of 19 cases of pemphigus vulgaris and 28 out of 29 cases of bullous pemphigoid showed positive histomorphological correlation with IF. The PPV for pemphigus vulgaris was 100% and sensitivity was 84.2%, and in bullous pemphigoid, both the PPV and sensitivity was 96.5%. Higher concordance was noted between Histopathology and IF diagnosis in cases of BP in comparison to PV. Conclusion: Vesiculobullous lesions are heterogeneous in nature and can have clinical and histopathological overlap, posing diagnostic challenges. IF is considered gold standard and is necessary for the diagnosis in every case. A combined clinical, histopathological and IF examination is the best approach to accurate diagnosis. Keywords Bullous, Pemphigus, Vesiculobullous, Direct Immunofluorescence

Understanding the efficiency of Shodhana, a classical drug detoxification technique that may increase safety in using three varieties of Abrus precatorius L. seeds-a toxic Schedule E1 drug used in various herbal formulations

BackgroundAbrus precatorius L. is a noxious vein native to south east asia and popularly called as Gunja in Ayurveda. The seeds of this plant are considered as a potent raw drug used in various herbal formulations such as Gunjadi tailam, Gunjagarbha rasa etc. and used to treat various ailments such as dermal disorders, cysts, snake poisoning and may other conditions. Before utilizing for the herbal formulations, the seeds are classically processed due to the presence of well reported toxic protalbumin named Abrin with the human fatal dose of 0.1–1 µg/kg. MethodsIn this work three varieties of Abrus precatorius L. seeds (Red, White and Black) were collected from the southern part of India and subjected for the detoxification process in order to analyze the efficiency of technique on different varieties of seeds. For processing the seeds, the classically fermented supernatant of rice gruel named kanjika was prepared and the pH was analyzed. Before and after processing, the seeds were analyzed for phytoconstituents with histochemistry technique and the hydroalcoholic extracts were prepared and qualitatively estimated for phytochemicals. The detoxification efficiency was analysed using HPTLC method by qualitative and quantitative estimation of Abrin levels by comparing with standard marker. Following that, cytotoxicity study was conducted against Vero (Healthy kidney) cell lines to ensure reduced toxicity in in-vitro conditions. ResultsThe process of Shodhana has changed the phytochemical levels in the processed seeds in comparison with unprocessed seeds in all three varieties. In connection to that, the extracts prepared with processed seeds have expressed lesser cytotoxicity and can be used as a drug with lesser side effects in whatever indications applicable. ConclusionIt is concluded that Shodhana with kanjika is efficient in processing all three varieties of Abrus precatorius seeds and is more effective in processing white seeds than the red and black varieties.

Automated Whole Slide Imaging for Label-Free Histology Using Photon Absorption Remote Sensing Microscopy.

Pathologists rely on histochemical stains to impart contrast in thin translucent tissue samples, revealing tissue features necessary for identifying pathological conditions. However, the chemical labeling process is destructive and often irreversible or challenging to undo, imposing practical limits on the number of stains that can be applied to the same tissue section. Here we present an automated label-free whole slide scanner using a PARS microscope designed for imaging thin, transmissible samples. Peak SNR and in-focus acquisitions are achieved across entire tissue sections using the scattering signal from the PARS detection beam to measure the optimal focal plane. Whole slide images (WSI) are seamlessly stitched together using a custom contrast leveling algorithm. Identical tissue sections are subsequently H&E stained and brightfield imaged. The one-to-one WSIs from both modalities are visually and quantitatively compared. PARS WSIs are presented at standard 40x magnification in malignant human breast and skin samples. We show correspondence of subcellular diagnostic details in both PARS and H&E WSIs and demonstrate virtual H&E staining of an entire PARS WSI. The one-to-one WSI from both modalities show quantitative similarity in nuclear features and structural information. PARS WSIs are compatible with existing digital pathology tools, and samples remain suitable for histochemical, immunohistochemical, and other staining techniques. This work is a critical advance for integrating label-free optical methods into standard histopathology workflows.

Heterogeneous distribution of mitochondria and succinate dehydrogenase activity in human airway smooth muscle cells

AbstractSuccinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal‐based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

Percutaneous Fluoroscopic-Guided Celiac Plexus Approach: Results in a Pig Cadaveric Model.

Celiac plexus block (CPB) and neurolysis (CPN) are used for pain management in people suffering from abdominal tumours or chronic pancreatitis. The fluoroscopically guided approach common in human medicine has not been described in veterinary settings. The aim of this study was to describe a fluoroscopic approach to the celiac plexus (CP) in fresh pig cadavers. Twelve animals were included in the procedure. Cadavers were positioned in sternal position and, under fluoroscopic guidance, a Chiba needle was inserted parasagittal at 6 cm from the spinal midline at the level of the last thoracic vertebra. From the left side, the needle was directed medio-ventrally with a 45° angle towards the T15 vertebral body; once the vertebral body was contacted, the needle was advanced 1 cm ventrally towards the midline. Iodinated contrast was injected to confirm the location. Following this, 2 mL of dye (China ink) was injected. A laparotomy was performed, and dyed tissue was dissected and prepared for both histochemical and immunohistochemical techniques. In 10 out of 12 samples submitted for histological evaluation, nervous tissue belonging to CP was observed. Fluoroscopy guidance allows for feasible access to the CP in swine cadavers in this study. Further studies are warranted to determine the efficacy of this technique in swine and other veterinary species.

Comparative morphoanatomic and histochemical characterization of Cucurbita pepo L. specimens

Cucurbita pepo L. is popularly known as pumpkin or zucchini. Compared to the others of the genus, it presents the greatest genetic variability. Thus, given its nutritional and medicinal benefits, it results in many widely cultivated and economically important specimens. Due to this, differentiation between specimens becomes necessary, mainly pumpkin and zucchini which have different characteristics, however they receive the same scientific nomenclature (C. pepo). It is noteworthy that the anatomical knowledge of the specimens helps with taxonomy. Furthermore, due to the economic value, when it comes to vegetative propagation, the identification of structural aspects is important for the success of propagation, which depends on the regeneration of plant tissues. This study aimed to characterize and differentiate two specimens of C. pepo through morphoanatomy and histochemistry. For anatomical study, usual methods in plant anatomy were used to prepare and analyze, under an optical microscope, semi-permanent slides containing transverse sections of the roots, stems, leaves, and paradermic sections of the leaf blades of C. pepo specimens. Histochemical tests were performed on cross-sections of the specimens' leaf blades to locate chemical compounds. The optical microscopic evaluation enabled the anatomical characterization, revealing distinct characters among the specimens. For example, the zucchini shows crystals in the root. Meanwhile, the pumpkin shows sclerenchymatic pericycle in the stem and secretory ducts in the stem and leaves. Histochemical techniques showed the presence of different compounds, differentiating in the presence of starch in the zucchini and triterpenes and steroids in the pumpkin. Correct characterization provides important information for quality control of the plant drug and taxonomic differentiation of the specimens since diagnostic characters vary.