Tissue Fixation Research Articles

Comparison of Time-Zero Primary Stability Between a Biodegradable Magnesium Bone Staple and Metal Bone Staples for Knee Ligament Fixation: A Biomechanical Study in a Porcine Model.

Bone staples have been shown previously to be a viable modality for cortical tendon graft fixation in ligament knee surgery. However, soft tissue reactions have been reported, making implant removal necessary. Magnesium alloys are a promising material for biodegradable orthopaedic implants, with mechanical properties closely resembling those of human bone. To compare the primary stability of a biodegradable bone staple prototype made from magnesium to bone staples made from metal in the cortical fixation of tendon grafts during knee surgery. Controlled laboratory study. Primary stability of peripheral tendon graft fixation was assessed in a porcine model of medial collateral ligament reconstruction. Two commercially available metal bone staples (Richards fixation staple with spikes [Me1] and spiked ligament staple [Me2]) were compared with a magnesium bone staple prototype for soft tissue fixation. Primary stability was assessed using a uniaxial materials testing machine. Cyclic loading at 50 and 100 N was applied for 500 cycles each, followed by load-to-failure testing. After 500 cycles at 50 N, elongation was 1.5 ± 0.5 mm in the Me1 group, 1.9 ± 0.5 mm in the Me2 group, and 1.8 ± 0.4 mm in the magnesium group. After 1000 cycles of loading (500 cycles at 50 N and 500 at 100 N), elongation was 3.6 ± 0.9 mm in the Me1 group, 3.5 ± 0.6 mm in the Me2 group, and 4.1 ± 1.0 mm in the magnesium group. No significant differences regarding elongation were found between the groups. Load to failure was 352 ± 115 N in the Me1 group, 373 ± 77 N in the Me2 group, and 449 ± 92 N in the magnesium group, with no significant difference between the groups. In this study, the magnesium bone staples provided appropriate time-zero biomechanical primary stability in comparison with metal bone staples and may therefore be a feasible alternative for cortical fixation of tendon grafts in knee surgery. The biodegradability of magnesium bone staples would eliminate the need for later implant removal.

Rapid Examination of Nonprocessed Renal Cell Carcinoma Using Nonlinear Microscopy.

Histology, the traditional method of examining surgical tissue under a microscope, is a time-consuming process involving the fixation of tissue in formalin, dehydration, embedding in paraffin, and cutting into thin sections for hematoxylin-eosin (H&E) staining. Frozen section analysis is a faster alternative used in surgery to quickly evaluate tissue, but it has limitations, such as the size of the specimens that can be analyzed and difficulties with fatty and bony tissues. To rapidly examine nonprocessed kidney tumors using nonlinear microscopy (NLM), a fluorescence microscopy technique that can rapidly visualize fresh or fixed, rapidly stained, nonprocessed tissue resembling H&E histology. This technology eliminates the need for fixation, embedding, microtome sectioning, or slide preparation. In this study, a total of 190 tissue specimens were collected from 46 patients who underwent partial or radical nephrectomy. Two genitourinary pathologists confirmed that diagnostically important features present in the H&E images could also be identified in the NLM images. The results of this study demonstrated that NLM had a high degree of correspondence with H&E staining for the classical variants of renal cell carcinoma. NLM offers several clinical benefits, such as facilitating rapid renal cell carcinoma diagnosis, assessment of targeted kidney biopsies for both tumor and medical kidney diseases, and collection of fresh renal cell carcinoma tissue for molecular studies.

Virtual histological staining of unlabeled autopsy tissue

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.

Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

Visualization of rat tendon in three dimensions using micro-Computed Tomography

Micro-computed tomography (CT) is an X-ray-based imaging modality that produces three-dimensional (3D), high-resolution images of whole-mount tissues, but is typically limited to dense tissues, such as bone. The X-rays readily pass-through tendons, rendering them transparent. Contrast-enhancing chemical stains have been explored, but their use to improve contrast in different tendon types and across developmental stages for micro-CT imaging has not been systematically evaluated. Therefore, we investigated how phosphotungstic acid (PTA) staining and tissue hydration impacts tendon contrast for micro-CT imaging. We showed that PTA staining increased X-ray absorption of tendon to enhance tissue contrast and obtain 3D micro-CT images of immature (postnatal day 21) and sexually mature (postnatal day 50) rat tendons within the tail and hindlimb. Further, we demonstrated that tissue hydration state following PTA staining significantly impacts soft tissue contrast. Using this method, we also found that tail tendon fascicles appear to cross between fascicle bundles. Ultimately, contrast-enhanced 3D micro-CT imaging will lead to better understanding of tendon structure, and relationships between the bone and soft tissues.•Simple tissue fixation and staining technique enhances soft tissue contrast for tendon visualization using micro-CT.•3D tendon visualization in situ advances understanding of musculoskeletal tissue structure and organization.

Phenolic Determination in Proembryogenic Cell Complexes of Buckwheat Morphogenic Cell Culture with Osmium Tetroxide, Toluidine Blue O Dye, and Iron Chloride.

The study of the localization of secondary metabolites in both plants and the cell cultures on the intravital sections is hampered by the difficulty of obtaining thin, correctly oriented sections. Techniques for fixing tissues in resins allow these difficulties to be overcome. Properly selected tissue fixation techniques allow using different dyes to identify the compound of interest. In addition, some components of tissue fixation can act as fixatives and as a dye for identifying secondary metabolites. For example, osmiumtetroxide, which fixes lipids in tissues, stains phenolic compounds black. This paper describes methods for the detection of phenolic compounds in morphogenic callus culture of buckwheat using osmium tetroxide, ToluidineBlue O dye, and ferric chloride as dyes in epoxy resin-embedded cell culture with double fixation of the material and when material fixed in Karnovsky's fixative.

Histomorphological Comparison of Tissues Fixed in Conventional Formalin and Eco-friendly Jaggery Solution: A Cross-sectional Study

Introduction: Tissue fixation is an essential step in the microscopic preparation of tissues to preserve them by preventing autolysis, bacterial putrefaction, and increasing the tissue's refractive index. The conventional fixative used is 40% formaldehyde. Short-term exposure to formaldehyde can cause irritation to the eyes and respiratory tract, leading to breathlessness and coughing. The International Agency for Research on Cancer (IARC) has classified formaldehyde as a Class 1 human carcinogen capable of potentially causing various neoplasms, including nasopharyngeal carcinoma. Therefore, an innovative approach is being explored to replace formalin with harmless and eco-friendly jaggery. Jaggery possesses cytoprotective, antioxidant, and tissue-preservative properties. At a low pH, the fructose in jaggery breaks down into aldehydes, which cross-link with tissue amino acids, resulting in tissue fixation similar to formaldehyde. Aim: To compare the histological and gross morphological features of tissues fixed in formalin and jaggery. Materials and Methods: This cross-sectional study was conducted in Department of Pathology of a tertiary care centre. Surgical specimens obtained fresh, which were not suspicious of malignancy, were included. A sample size of 23 was selected. Surgical specimens already placed in formalin were excluded. A 30% jaggery solution was prepared by dissolving 300 grams of finely powdered jaggery in 1000 mL of distilled water, which was then filtered using filter paper. A 40% formaldehyde solution was used to prepare a 10% formalin fixative. Tissue bits from each specimen were cut into two halves and placed in formalin and jaggery separately. After 24 hours of fixation, the tissue bits were evaluated for gross morphological features, including tissue shrinkage, consistency, and colour. Tissue shrinkage was classified as mild, moderate, or marked, while consistency ranged from soft to firm to hard. The colour of the specimens varied case by case. Stained slides from jaggeryfixed and formalin-fixed tissues were assessed for histological parameters, such as nuclear details, cytoplasmic details, cellular outline, and overall staining quality. A blinded method was used to compare the stained slides using a microscope. Ratings were assigned to each case on a scale of 1-4. The data were statistically analysed using R software. Results: When comparing the formalin-fixed and jaggery-fixed specimens, no significant differences were observed in gross morphological features. All jaggery-fixed specimens appeared brown grossly. Histological features also showed no significant difference (p-value>0.05) except for cytoplasmic details. Therefore, it was observed that tissue preservation using the jaggery solution was comparable to that of formalin. Conclusion: The tissue-preservative properties of jaggery are on par with formalin. Hence, jaggery solution can be used as an eco-friendly substitute for formalin. Further research with larger sample sizes can pave the way for the effective replacement of hazardous formalin with natural jaggery

Electron Microscopy Techniques for 3D Plant ER Imaging.

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex, whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. Here we present two protocols for 3D EM imaging of the ER across a range of scales. For large-scale ER structure analysis, we describe selective ER staining with ZIO that allows for automated or semi-automated ER segmentation. For smaller regions of ER, we describe high-pressure freezing, which enables almost instantaneous fixation of plant tissues but without organelle specific staining. These fixation and staining techniques are suitable for a range of imaging modalities, including serial sections, array tomography, serial block face-scanning electron microscopy (SBF-SEM), or focused ion beam (FIB) SEM.

Cytochemical and Histochemical Staining with Peptide Antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/absorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

Pilot testing of a simple alternative tissue processing method for immunofluorescence staining

Background: The image quality of tissues obtained with high-resolution imaging systems depends on several factors including the quality of sections prepared by different tissue processing methods. Fresh frozen sections and formalin-fixed paraffinembedded (FFPE) sections are the two routinely employed methods in histopathological examination having their own merits and demerits. Aims and Objectives: We tested the feasibility of a hybrid tissue processing method that could reduce the processing time without compromising the quality of processed and stained tissues. Materials and Methods: Bovine pancreatic tissue was utilized to standardize the process. The steps included fixation of tissue in formalin, followed by embedding in optimal cutting temperature compound and then cryo-sectioning to obtain 50 μm slices for immunostaining. The prepared thick slices were immunostained with islet markers and assessed by confocal imaging. Results: The images revealed that the staining quality was comparable with FFPE sections, though the tissue processing time was significantly curtailed. The modified Leica microsystems scoring system revealed a nearly 80% overall total score for the processed tissue specimens, indicating acceptable levels of quality of the tissues. We also observed that the staining quality of these formalin-fixed cryo-slices was preserved without tissue deterioration for up to 1 month when stored in formalin, indicating its storage potential for future applications. Conclusion: The proof of concept of the hybrid tissue processing method tested in this study will encourage researchers to extrapolate its use to the human pancreas, other solid organs, and other staining techniques for evaluating its general utility.

Telocytes: Detection, Visualization, Tissue Dissociation, and Tamoxifen-Induction of Transgenic Mice.

Telocytes, distinctive interstitial cells, have recently emerged as crucial components of the stem-cell niche in the intestine. Notably, telocytes are distinguished by their extremely long cellular protrusions extending hundreds of microns from the cell body, forming an interconnected network along the intestinal crypt villus axis. Due to these unique cellular characteristics, there is a need for tailored working protocols to effectively characterize and target telocytes. Here, we outline advanced and progressive protocols for tissue fixation, dissociation, visualization, and the use of tamoxifen-induced transgenic mouse models to specifically target telocytes.

Ex vivo assessment of basal cell carcinoma surgical margins in Mohs surgery by autofluorescence‐Raman spectroscopy: A pilot study

AbstractBackgroundAutofluorescence (AF)‐Raman spectroscopy is a technology that can detect tumour tissue in surgically excised skin specimens. The technique does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps within 30 min.ObjectivesTo explore the clinical application of AF‐Raman microscopy to detect residual basal cell carcinoma (BCC) positive margins in ex vivo skin specimens excised during real‐time Mohs surgery. To investigate the ability to analyse skin specimens from different parts of the head‐and‐neck areas and detect nodular, infiltrative and superficial BCC.MethodsFifty Mohs tissue layers (50 patients) were investigated: 27 split samples (two halves) and 23 full‐face samples. The AF‐Raman results were compared to frozen section histology, carried out intraoperatively by the Mohs surgeon and postoperatively by dermatopathologists. The latter was used as the standard of reference.ResultsThe AF‐Raman analysis was completed within the target time of 30 min and was able to detect all subtypes of BCC. For the split specimens, the AF‐Raman analysis covered 97% of the specimen surface area and detected eight out of nine BCC positive layers (similar to Mohs surgeons). For the full‐face specimens, poorer contact between tissue and cassette coverslip led to lower coverage of the specimen surface area (92%), decreasing the detection rate (four out of six positives for BCC).ConclusionsThese preliminary results, in particular for the split specimens, demonstrate the feasibility of AF‐Raman microscopy for rapid assessment of Mohs layers for BCC presence. However, for full‐face specimens, further work is required to improve the contact between the tissue and the coverslip to increase sensitivity.

Quality assessment of histopathological stainings on prolonged formalin fixed thrombus tissues retrieved by mechanical thrombectomy.

Formalin-fixed retrieved clots from mechanical thrombectomy (MT) are now routinely studied using both conventional histopathologic techniques and immunohistochemistry (IHC). However, the effects of prolonged formalin fixation on the histological results of clot analysis remain unknown. The objective of this study was to investigate the effects of prolonged formalin fixation on quality of histopathologic stainings of thrombus tissues retrieved by MT. As part of the multicenter EXCELLENT registry, a total of 80 clots extracted by MT from acute ischemic stroke patients were randomly selected from the tissue database and assigned into four groups according to 10% neutral buffered formalin (NBF) fixation duration (1-30, 30-60, 60-90, and 90+ days, up to 2 years). Samples underwent processing and sectioning. Two serial sections for each case were stained with hematoxylin and eosin (H&E), Martius Scarlet Blue (MSB), and IHC for CD42b (platelet marker). An expert pathologist, who was blinded to tissue fixation duration and patient clinical data, assessed the quality of each stain including stainability, sensitivity, specificity, and consistency of stainings. No significant issues were encountered during tissue processing and sectioning. On H&E stain, 97.5% (78/80) of slides showed good-quality staining, demonstrating clear histological properties of the thrombus tissue as red blood cells (RBC) stained in red, fibrin/platelet stained in pink, and nuclei stained in blue with intranuclear detail. The same histological features were also successfully demonstrated on MSB for all 80 samples. One of the 80 samples (1.2%) showed that RBC lost stainability on H&E due to tissue autolysis. Clear positive signal of platelet staining was expressed in 98.8% of the samples (79/80) with minimal background staining on IHC. There was no significant difference in staining quality across different formalin fixation groups. A good quality of histopathological staining is achievable for the thrombus tissue fixed in 10% neutral buffered formalin for up to 2 years. The findings are limited to the thrombus tissue retrieved by MT and specific fixation and staining protocols used in the study. To apply these results to other tissue or experimental setups, further studies and validations would be necessary. This study was conducted as part of the EXCELLENT study: www.clinicaltrials.gov, unique identifier: NCT03685578.

Does tissue fixation change the mechanical properties of dry ovine bone extracellular matrix?

Tissue fixation is a prevalent method for bone conservation. Bone biopsies are typically fixed in formalin, dehydrated in ethanol, and infiltrated with polymethyl methacrylate (PMMA) Since some experiments can only be performed on fixed bone samples, it is essential to understand how fixation affects the measured material properties. The aim of this study was to quantify the influence of tissue fixation on the mechanical properties of cortical ovine bone at the extracellular matrix (ECM) level with state-of-the-art micromechanical techniques.A small section from the middle of the diaphysis of two ovine tibias (3.5 and 5.5 years old) was cut in the middle and polished on each side, resulting in a pair of mirrored surfaces. For each pair, one specimen underwent a fixation protocol involving immersion in formalin, dehydration with ethanol, and infiltration with PMMA. The other specimen (mirrored) was air-dried. Six osteons were selected in both pairs, which could be identified in both specimens. The influence of fixation on the mechanical properties was first analyzed using micropillar compression tests and nanoindentation in dry condition. Additionally, changes in the degree of mineralization were evaluated with Raman spectroscopy in both fixed and native bone ECM. Finally, micro tensile experiments were conducted in the 3.5-year fixed ovine bone ECM and compared to reported properties of unfixed dry ovine bone ECM.Interestingly, we found that tissue fixation does not alter the mechanical properties of ovine cortical bone ECM compared to experiments in dry state. However, animal age increases the degree of mineralization (p = 0.0159) and compressive yield stress (p = 0.041). Tissue fixation appears therefore as a valid conservation technique for investigating the mechanical properties of dehydrated bone ECM.

Examining Apical or Basolateral Protein Localization inAedes aegyptiTissues: Cross-Section Immunohistochemistry

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues. Here, we describe an IHC protocol for examining apical versus basolateral protein staining through sectioning tissue samples from fixed, embedded tissues (e.g., IHC-paraffin) and adding primary antibodies against a target protein. This IHC protocol provides a guide for tissue fixation, sectioning, and staining of tissue samples.

Gambaran Patologi Anatomi dan Histopatologi Organ Visceral Kambing Kacang (Capra aegagrus hircus) Yang Terinfeksi Sistiserkus

The demand for kacang goat (Capra aegagrus hircus)in Kupang City is very high in the local market, especially before the sacrificial festival. Although the goat population growth is high, there are several reasons that cause low productivity, namely parasitic infection.worms Taenia infect carnivores (as well as humans) as the definitive host and involve an intermediate host which will develop into a cysticercus. The purpose of this study was to determine the anatomical pathology and histopathology of the visceral organs of kacang goats infected with cysticercus. This study used 6 samples of visceral organs of kacang goats consisting of 4 liver organs, 1 rumen organ and 1 colon organ from 6 kacang goats infected with cysticercus, in Pasir Panjang Village, Kupang City. Macroscopic observations, tissue fixation in 10% formalin, tissue processing and HE staining were carried out. The results of the study on liver organs infected with cysticerci showed pathological changes in the form of fibrosis, while in the rumen and colon only cysticerci were formed. Histopathological changes in the liver organs of kacang goat infected with cysticercus in the form of mononuclear and polymorphonuclear inflammatory cell infiltration, necrosis, congestion, hemorrhage, hydropic degeneration, widening of sinusoids and fibrous tissue. Meanwhile, changes that occur in the rumen and colon organs are in the form of infiltration of mild inflammatory cells.

Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Antigen retrieval assisted electrochemiluminescence imaging of surface antigens at single tissue section

Previously, our group has established an electrochemiluminescence (ECL) molecular imaging method to detect carcinoembryonic antigen (CEA) at single tissue sections of carcinoma patients. However, the tissue fixation blocks the binding sites of surface antigens affecting the antigen-antibody interaction and the resultant visualization sensitivity. Herein, we report an antigen retrieval assisted ECL imaging strategy that utilizes heat-induced epitope retrieval (HIER) to re-expose the binding sites of the antigens for more efficient interaction between surface antigen and ruthenium-labeled antibody. With more ruthenium complexes tagged with surface antigens, the ECL intensity at the tissue sections increases significantly which could more accurately reflect the amount and the distribution of surface CEA. Accordingly, the different distribution of CEA at the carcinoma and paracancerous regions of tissue sections from two patients with lung adenocarcinoma (LUAD) is visualized, which could not be easily distinguished without the HIER treatment. Eventually, the combination of antigen retrieval and ECL molecular imaging could improve the visualization sensitivity at single tissue section and minimize the false negative phenomenon, which will greatly advance the development of ECL-based tissue imaging for future application in pathological diagnosis.

Investigating the Role of Metoclopramide and Hyoscine-N-Butyl Bromide in Colon Motility

Metoclopramide is a treatment for gastroenteritis accompanied by vomiting. Hyoscine-n-butyl bromide as an anticholinergic agent causes inhibition of the acetylcholine (Ach) by acting on muscarinic receptors. The study aims to ascertain how metoclopramide affects Ach-induced cortical motility and also investigates the effects of metoclopramide alone and in combination with hyoscine-n-butyl bromide drug effects on colon motility. In this study, 1 cm of colon tissue width was cut, 2 cm long strips were made, and both sides of the tissue were secured with surgical silk at both ends of isolated bath tissues of isolated organs with (1g) tension to the suspended instrument that recorded isometric contractions. Tissue fixation is followed by drug addiction: Ach, metoclopramide, and hyoscine-N-butyl bromide. The tissue was treated with metoclopramide and hyoscine-n-butyl bromide and excess Caine for 10 min. The results show changes in colon frequency, peak-to-peak, and amplitude levels for metoclopramide, hyoscine-N-butyl, and metoclopramide and hyoscine. Apaired T-test statistically analyzes the results. Metoclopramide by itself, as well as in combination with hyoscine-n-butyl bromide, increases colon motility and induces Ach release. In addition, an analysis of the physicochemical characteristics of hyoscine-n-butyl bromide and metoclopramide molecules is conducted. The study includes theoretical calculations of electronic parameters for both protonated and unprotonated forms of these molecules in both gaseous and aqueous environments. These results show the potential use of metoclopramide as a therapeutic option for gastroenteritis with vomiting, warranting additional study, and clinical evaluation. The research also reveals hyoscine-n-butyl bromide and metoclopramide’s molecular features by their physicochemical properties.

Effectiveness of neutral honey as a tissue fixative in histopathology.

Background: In routine histopathology, 10% neutral buffered formalin (NBF) is the choice fixative. However, formalin is a human carcinogen, so there is a necessity for a safer alternative. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological samples. This study determined the effectiveness of neutral buffered honey and other types of fixatives to fix histological tissues. Methods: The study was conducted between July 2019 and August 2020 at Sultan Qaboos University, Oman. Sections from three rat livers, kidneys, and stomach tissues were fixed with 10% NBF, neutral buffered Sumer honey, neutral buffered date honey, formalin, Sumer honey, date honey, alcoholic formalin, alcoholic Sumer honey, and alcoholic date honey for 24 hours. Samples were stained with hematoxylin and eosin (H&E), special stains, and vimentin methods. Three expert biomedical scientists then evaluated the fixed and stained samples for the quality of all sections. The fixation ability of the different honey solutions was then compared to 10% NBF and the utility was determined using nuclear and cytoplasmic criteria, specificity, and intensity. Results: H&E showed adequate staining in all groups compared to 10% NBF. The specificity and intensity of all groups for the Periodic acid-Schiff method were identical to 10% NBF except for Sumer honey and alcoholic date honey. Vimentin showed comparable findings with 10% NBF as there were no significant differences. Conclusions: The findings of this study encourage the use of honey, including neutral, as a possible safe substitute fixative for formalin, however, further experiments on larger specimens should be conducted.