Alcian Blue Research Articles

Pain and Inflammation Profile of a Degenerated Intervertebral Disc

Degenerative disc disease (DDD) is characterised by progressive intervertebral disc (IVD) inflammation that ultimately causes lower back pain. The available therapeutic approach for DDD is the management of pain and spinal infusion. In the present investigation, the rat needle punctured disc degeneration model was established under a fluoroscopically guided system, followed by human umbilical cord mesenchymal stem cells (hUC-MSCs) transplantation. The IVD degeneration was examined and confirmed by radiography, histology, and pain and inflammatory marker evaluation. A rat tail disc degeneration model was developed with three successive coccyx IVDs. The needle was inserted into the IVD under fluoroscopic guidance, and its tip was inserted through the lateral annulus fibrosus into the nucleus pulposus (NP) region, twisted 360o twice, and held for 30 seconds. To assess the IVD height from radiography images 2 weeks after disc puncture and MSC transplantation to evaluate the degree of IVD degeneration and regeneration, The pain and inflammatory molecular markers were quantified using qPCR. To assess the severity of IVD degeneration and regeneration, histology was performed using haematoxylin and eosin (H & E) and alcian blue staining. MSCs were successfully isolated from human umbilical cord tissue and exhibited fibroblast-like morphology. Radiographic imaging and histological analyses showed significant IVD degeneration after 2 weeks of injury, and the hUC-MSC group exhibited the restored NP region and significant presence of proteoglycans in contrast to the degenerated group. When compared to healthy IVDs, the molecular expression of pain and inflammatory genes was significantly increased on days 2 and 5, resulting in an immunomodulatory response and a marked IVD degeneration. Conversely, the hUC-MSC group at 2 and 5 days showed significantly downregulated expression of C-JUN, C-FOS, AKT, OPG, RANK, TLR4, and IL-1β compared to the degenerated IVDD group. Moreover, the hUC-MSCs-treated group indicated significant NP hydration. The preclinical transplantation of hUC-MSCs can potentially reduce the pain and inflammation caused by disc degeneration. Therefore, the therapy can ideally deal with all the manifestations that occur due to such debilitating DDD.

Alcian blue as a kidney staining in diabetic mice

One of the complications of Diabetes Mellitus (DM) is damage to the kidneys. Using L. plantarum, it can reduce glucose levels so that it is also possible to repair damaged kidney structures. The process of repairing the kidney structure by L plantarum can be seen microscopically in the glomerulus with Alcian Blue staining (AB). The purpose of the study was to determine the differences in the microscopic results of DM rat kidneys after treatment of L. plantarum with AB staining. The study sample used the kidneys of DM mice. The kidney of DM mice that were given L. plantarum at a dose of 1.0 ml/mice in each treatment, T1 (dose 1×1 times a day), T2 (dose 1×2 times a day), T3 (dose 1×3 times a day), then performed AB staining. The results of cytoplasmic cells in the glomerulus in negative controls/sick mice are stained dark blue/concentrated (strong intensity) with a score of 4, normal controls/healthy mice are not stained blue (negative) with a score of 1. T1 is a stained light blue score of 3, T2 is a stained light blue score of 3, and T3 is a colored pale blue score of 2. Based on these results, it was found that the color intensity of the AB kidney treatment of DM rats was the best in reducing glucose residues.

DYNAMICS OF PATHOMORPHOLOGICAL CHANGES OF THE MYOCARDIUM IN CHRONIC EXPERIMENTAL ENDOTOXICOSIS

Relevance. Modeling the conditions of endotoxicosis by introducing bacterial lipopolysaccharide against the background of toxic liver damage is due to the fact that under such conditions the situation that accompanies almost every chronic pathology, including cardiovascular, is reproduced. The release of substances that play a role in the formation of endogenous intoxication causes heart damage in the absence of cardiac pathology, and in its presence worsens the course and prognosis. However, it should be found out whether these changes are the result of only toxic damage to cardiomyocytes or have another morphological basis and what their dynamics are.
 Objective: to characterize the morphological changes of the myocardium in experimental chronic endotoxicosis.
 Materials and methods. The research was carried out by modeling endotoxicosis in 18 laboratory animals for 90 days by daily intragastric injection of tetrachloromethane at the rate of 3-5 ml/kg of body weight and every 6th day by intraperitoneally adding bacterial lipopolysaccharide at a dose of 0.2 mg/kg of body weight. Animals were removed from the experiment by decapitation at 30, 60, and 90 days. Animals were removed from the experiment by decapitation after anesthesia with intraperitoneal administration of sodium thiopental at a dose of 50 mg/kg. The control group consisted of 6 intact animals. For histological examination, myocardial tissue was condensed in paraffin, and deparaffinized sections were stained with hematoxylin and eosin, examined for the detection of neutral glycosaminoglycans and acidic mucopolysaccharides, stained with Alcian blue and PAS-reaction according to standard methods, and examined under a Nikon Eclipse Ci-E light microscope.
 Results. During researching the myocardium at the light-optical level of experimental animals, after 30 days of chronic endogenous intoxication caused by injection of tetrachloromethane and bacterial LPS, it undergoes changes, first of all, it concerned cardiomyocytes: we observe thinned cells around the vessels and unchanged ones at a distance from the vessels. In some of them, hyperchromia of the nuclei and phenomena of contractile damage and fiber fragmentation were noted. Manifestations from the CMC increased during the next period of the experiment: after 60 days, expressed wave-like deformation of fibers with atrophic changes in muscle cells was detected in the myocardium. Hemodynamic changes were characterized by hyperemia, the number of perivascular hemorrhages increased. On the 90th day of the experiment, in addition to inflammatory manifestations and dystrophic-necrotic changes, cardiomyocyte atrophy in combination with expressed interstitial edema was detected in the myocardial tissue of animals. In the stroma, in addition to small foci of perivascular cardiosclerosis, foci of adipocyte growth were determined. Cardiomyocyte defibrillation and hemorrhages were observed in some areas
 Conclusion. The intensity of structural changes in cardiomyocytes and hemodynamic disorders in chronic endotoxicosis depends on the duration of intoxication and the character of structural changes. The interstitium of the myocardium of rats under experimental endotoxicosis conditions is dystrophic-sclerotic and is manifested by a different combination of edematous and sclerotic manifestations at different stages of exposure to toxicants.

Comparative Anatomical Studies on the Hand of Human and Two Mammalian Species

Background: Morphologically there is a great similarity in the musculoskeletal pattern in different species of the primates especially the development of hands and digits for better functional adaptation to their environment. The factors involved in evolution of modern hand are muscle-tendon architecture, shape, size of bones and articular surfaces in joints. Human hand is functionally highly evolved with manipulative potential for fast and fine skilled activity especially in thumb and index finger as compared with other species.
 Aim: Present study was undertaken to compare the hand anatomy of the Baboon and Squirrel with the Human.
 Methods: Two commonly found mammals in Saudi Arabia i.e. Baboon and Squirrel were selected to reflect the different environmental adaptations. The human hands were provided by the Department of Anatomy. The animals were anesthetized, sacrificed at Anesthesia Department. Fixation done by 9% formaldehyde solution, hands dissected under Zeiss dissecting microscope, Muscle groups identified using 5% iodine stain, Bones and cartilages stained by Alizarin red and Alcian blue stain respectively and photographed (Nikon D3200 digital camera). The hand divided into four parts: Wrist, dorsum of hand, palm and fingers.
 Results: Human Hand: Wrist is wide, bones arranged in two rows gliding over each other for free movements at hand. Dorsum has thin fatty layer, more wrinkles and fewer hairs. Palm has thick skin without hair and well-developed muscles are arranged in groups (Thenar, Hypothenar, Lumbricals and Interossei). Digits have extensor and flexor tendons up to middle and distal phalanx. Thumb is rotated providing opposition and multiaxial movements.
 Baboon Hand: Wrist is short, bones arranged in two rows but fewer movements at hand. Dorsum has fewer wrinkles, more hairs. Palm skin has very thick layer, especially at friction pad located below fingers; muscles are arranged in groups but not fully developed as compared to human.
 Squirrel Hand: Small and close dimensions, five short fingers, pointed, every finger ends with a claw. The skin is thin, covered with hair. Palm is thin but completely free of hair with digital pad. Phalanges are short and all fingers have two phalanges. Thumb has only one phalanx.
 Conclusions: Human hand structure is evolved for better grip and fine movements of hand providing better functional and artistic capabilities.

Abaloparatide and teriparatide enhance mandibular growth in adolescent rats with site-specific and mechano-related effects.

Teriparatide (TPTD) and abaloparatide (ABL) are two osteoanabolic drugs targeting parathyroid hormone (PTH)1R signalling. This study aimed to investigate the effects of TPTD and ABL on the adolescent mandibular growth. In total, 70 4-week-old male Sprague-Dawley rats were randomly divided into 14 groups, treated with intermittent TPDT or ABL at various doses, accompanied by mandibular advancement (MA) or not. 3D printing was used to fabricate an innovative splint for MA. After a 4-week treatment, morphological measurement, histological and immunohistochemical analysis were performed. Mandibular condylar chondrocytes (MCCs) were treated with TPTD or ABL, followed by CCK-8 assay, alcian blue staining, real time-PCR and immunofluorescent staining. In vivo, TPTD or ABL alone increased the condylar length and cartilage thickness, with up-regulated SOX9 and COL II, whilst down-regulated COL X; however, when combined with MA, the promotive effects were attenuated. TPTD or ABL alone increased the mandibular body height and mandibular angle width, whilst increased the mandibular body length and alveolar bone width when combined with MA. In vitro, TPTD or ABL enhanced the MCC proliferation, glycosaminoglycan synthesis, COL II and SOX9 expression, whilst down-regulated COL X, Ihh and PTH1R expression. Both ABL and TPTD enhance mandibular growth in adolescent rats with site-specific and mechano-related effects, including propelling chondrogenesis at the condylar cartilage and promoting bone apposition at other mechano-responsive sites. They behave as promising drugs for mandibular growth modification, and in general ABL seems more potent than TPTD in this context.

Syringol, a wildfire residual methoxyphenol causes cytotoxicity and teratogenicity in zebrafish model

Natural toxicants, particularly methoxy phenols (MPs) generated by wildfire lignin, can accumulate in the environment, and cause serious health hazards in living organisms. Although the toxicity of MPs such as guaiacol and catechol has recently been described, there is minimal evidence of ecotoxicological effects of syringol. As a result, this study focuses on determining the toxicity by evaluating the cytotoxic and teratogenic effects of syringol in vitro and in vivo in human embryonic kidney (HEK-293) cells and zebrafish embryos, respectively. The ecotoxicity of syringol was predicted to be 63.8 mg/L using the ECOSAR (ECOlogical Structure Activity Relationship) prediction tool, and molecular docking analysis was used to determine the interaction and binding affinities of syringol with human apoptotic proteins in silico. In HEK-293 cells, exposure of syringol (0.5–2 mg/L) has induced cytotoxicity in a concentration-dependent manner. In zebrafish larvae, exposure of syringol (0.5–2 mg/L) has induced dose-dependent embryo toxic effects (or growth abnormalities such as yolk sac edema, pericardial edema, skeletal abnormality, and hyperemia), and changes in growth morphometrics (head height, eye, yolk sac, and pericardial area, heart rate) in particular, the heart rate of larvae was found to be significantly decreased (p<0.001). After a 4-day experimental trial, the accumulated concentration of syringol in zebrafish larvae was confirmed both qualitatively (HPLC-MS - High Performance Liquid Chromatography-Mass spectrometry) and quantitatively (LC-QTOF-HRMS - Liquid Chromatography-Quadrupolar Time of Flight-High Resolution Mass spectrometry). The craniofacial abnormalities induced by syringol exposure (0.5–2 mg/L) were detected as anomalies in cartilaginous development and locomotor deficits using alcian blue staining and locomotor analyses, respectively. Significant increase in oxidative stress parameters (including reactive oxygen species generation, lipid peroxidation, superoxide dismutase, catalase, lactate dehydrogenase and nitric oxide production) (p<0.001) and substantial decrease in glutathione levels were observed (p<0.05) in syringol exposed zebrafish larvae through enzymatic analysis. Additionally, through acridine orange staining and gene expression analyses, syringol (2 mg/L) was found to activate apoptosis in zebrafish larvae. Considering the cytotoxic, embryotoxic (teratogenicity), and oxidative stress-related apoptotic effects of syringol in the zebrafish model, syringol has the potential to emerge as a potent environmental toxicant posing serious health hazards in many living systems; however, further research on its toxicological effects on the actual ecosystem and in higher animal models is required to confirm its consequences.

Histological and Histochemical Study of Pyloric Region in Abomasum of Adult Local Buffalos (Bubalus Bubalis)

The current study was conducted to demonstrate the histological feature of the pyloric region of the abomasum in local buffalos and to focus on the distribution of different types of carbohydrates in the pylorus wall, where digestion has a great impact on animal production and economical outcome. Six samples of abomasum were collected from adult healthy local buffalos from the Mosul abattoir. then the pylorus region samples were sectioned from the pylorus, histologically processed, and stained with hematoxylin &amp; eosin stain, Masson’s trichrome, periodic acid Schiff and alcian blue. The histological findings of the pyloric region revealed that the mucosal wall of pylorus has depressions called gastric pits, these pits occupied the most of lamina propria receiving the mucus secretion of pyloric glands, which appeared short, tortuous, tubular in the anterior part of the pylorus, and became longer, straight in the middle part of the pylorus. The submucosa is characterized by the presence of lymphatic nodules. The histochemical findings revealed the presence of neutral and acidic (mixed) glycoprotein in the pyloric region, where acidic glycoprotein was found in terminal pyloric glands, while neutral glycoprotein was present mainly in gastric pits. The mid gland of the anterior part is distinguished by neutral glycoprotein, whereas the acidic glycoprotein was evident in the mid gland of the middle part of the pylorus. The surface epithelium is characterized by the presence of both neutral and acidic types of glycoprotein. Furthermore, glycogen is present in both gastric pits in the caudal part and terminal pyloric glands of the anterior part. Glycosaminoglycans were predominant at the terminal part of pyloric glands and present in little amounts in the middle part and absent in gastric pits. In conclusion presence of acidic glycoprotein in pyloric glands confirm the presence of mucus neck cells due to their acidic secretion, while the presence of neutral glycoprotein mainly in gastric pits indicates the formation of the mucosal gastric barrier.

Microenvironment influence on the development of epithelial-mesenchymal transformation in lung cancer

Relevance. Epithelial-mesenchymal transformation (EMT) largely determines the biological behavior and prognosis of cancers of various localizations. It is known that the determining role in the control and implementation of the transition of the epithelial phenotype to the mesenchymal belongs to the microenvironment. At the same time, the histochemical and microscopic characteristics of stromal elements remain unclear; therefore, the aim of our study was to establish the morphological features of the stroma that affect the development of EMT in lung cancer. Materials and Methods. We studied 32 cases of lung cancer with hematoxylin and eosin staining of sections, Alcian blue at pH 1.0 and 2.5, PAS reaction, as well as immunohistochemical studies with monoclonal antibodies to HMW, AE 1 / AE 3, cytokeratin 18, c-erb B 2, vimentin E-cadherin, alpha-smooth muscle actin, GFAP and chromogranin A, Ki-67. In 16 cases, there was widespread epithelial-mesenchymal transformation (EMT), in 14 cases - focal and in 2 cases EMT was absent. In half of the cases of widespread EMT, it was complete; among the cases of focal EMT, it was not complete. The transition from the epithelial to the mesenchymal phenotype was facilitated by the activation of the stroma, namely, the presence of myofibroblasts and alcianophilia of the extracellular matrix, inflammatory cell infiltration, expression of the epidermal growth factor receptor (c-erb B 2) in stromal cells, proliferation and neurogenic differentiation of stromal cells of stromal cells. Results and Discussion. The activated stroma correlates with the frequency of EMT. Thus, large areas of activated stroma with the presence of myofibroblasts and alcianophilia of the extracellular matrix are more common in cases of widespread EMT (14 out of 16-87.5 %) than in cases of focal EMT (4 out of 14-28.6 %). The differences are statistically significant, p 0.01. Inflammatory cell infiltration, which is the source of a signal for transformation, expression of the epidermal growth factor receptor (c-erb B 2) in stromal cells, proliferation, and neurogenic differentiation of stromal cells also correlated with the frequency of EMT. In all cases, the differences are statistically significant, p0.01. Conclusions. The data obtained indicate the undoubted influence of signals from the activated stroma on the development of epithelial-mesenchymal transformation of tumor cells.

Morphological features of epithelial-mesenchymal transformation and its effect on tumor progression of breast cancer

Introduction. Breast cancer is in the first place in the structure of morbidity among all malignant neoplasms in women. The prognosis of the disease depends on the tumor degree, including the presence of epithelial-mesenchymal transformation (EMT), the degree of invasion, the proliferative index, the preservation or absence of estrogen, progesterone, and epidermal growth factor receptors.Aim. To study the immunohistochemical and morphological characteristics of the epithelial-mesenchymal transformation of breast cancer.Material and Methods. Immunohistochemical study with antibodies to AE1/AE3, HMW, CK18, Snail, HER2/neu, E-cadherin, Vimentin, α-SMA, CD34, Ki-67 and p63 was performed in 60 patients of different age with breast cancer. Native preparations were stained with picrofuchsin according to van Gieson Alcian blue. Inflammatory infiltrate cells were examined for antibodies CD45, CD3, CD4, CD8, CD20, CD68.Results. In ductal carcinoma, positive expression for estrogen and progesterone was found in 82.7% and 86.3%, respectively, the proliferation index ranged before 66,6 %, and p-53 was positive in 97%. In lobular cancer, positive expression to estrogen and progesterone was observed in 83.4% and 66.6%, respectively, the index of proliferative activity at the level of 50 %, and p-53 was positive in 66.6%. Positive moderate expression of HER-2/neu was determined in 47% of ductal and 50% of lobular cancers. Estrogen plays an important role in the development of invasive breast cancer, leads to tumor progression and contributes to EMT. EMT, in turn, leads to the expression of E-cadherin associated with a worse survival prognosis. EMT indirectly leads to the intensification of angiogenesis, and the presence of a large number of newly formed vessels increases the risk of metastasis. Histochemical methods were used to determine the growth of fibrous tissue around invasively growing cancer complexes. Cells located perifocally looked like fibroblasts, immunohistochemically moderately expressed Vimentin and weakly expressed pancytokeratin, which proved the tumor nature of the cells and the acquisition of mesenchymal features by them. The inflammatory infiltrate along the periphery of the tumor growth consisted mainly of T- and B-lymphocytes, and around the cancer complexes - of B-lymphocytes and macrophages.Conclusion. The study of the immunohistochemical tumor phenotype will make it possible to prescribe adequate polychemotherapy and determine the prognosis of the course of the disease.

Aspirin reverses inflammatory suppression of chondrogenesis by stabilizing YAP.

Bone marrow mesenchymal stem cells (BMMSCs) transplantation methods are promising candidates for osteoarthritis (OA) treatment. However, inflammatory factors (such as TNF-α) that occur at cell transplantation sites are critical factors that impair the effectiveness of the treatment. Previous studies have shown that aspirin (AS) had a regulatory role in stem cell differentiation. However, little is known about the role of AS on the chondrogenesis of BMMSCs. The purpose of this study is to explore the protective role of AS against the negative effects of TNF-α on BMMSC chondrogenesis. In this study, we investigated the effects of AS and TNF-α on BMMSCs chondrogenesis by performing the Alcian Blue staining, safranin O-fast green staining, haematoxylin and eosin staining, and immunohistochemical staining, as well as real-time RT-PCR and western blot assays. Our results demonstrated that TNF-α inhibited chondrogenic differentiation of BMMSCs by disrupting the balance of cartilage metabolism and promoting oxidative stress in BMMSCs, while AS treatment attenuated these effects. Furthermore, a detailed molecular mechanistic analysis indicated that Yes-associated protein (YAP) played a critical regulatory role in this process. In addition, AS treatment mitigated the progression of cartilage degeneration in a mouse destabilization of the medial meniscus (DMM) model. AS alleviated the inhibitory effect of TNF-α on chondrogenesis of BMMSCs by stabilizing YAP, which may provide new therapeutic strategies for OA treatment.

Etanercept embedded silk fibroin/pullulan hydrogel enhance cartilage repair in bone marrow stimulation.

Background: Bone marrow stimulation (BMS) is the most used operative treatment in repairing cartilage defect clinically, but always results in fibrocartilage formation, which is easily worn out and needs second therapy. In this study, we prepared an Etanercept (Ept) embedded silk fibroin/pullulan hydrogel to enhance the therapeutic efficacy of BMS. Methods: Ept was dissolved in silk fibroin (SF)-tyramine substituted carboxymethylated pullulan (PL) solution and enzyme crosslinked to obtain the Ept contained SF/PL hydrogel. The synergistical effect of SF/PL hydrogel and Ept was verified by rabbit osteochondral defect model. The mechanism of Ept in promoting articular cartilage repair was studied on human osteoarthritic chondrocytes (hOACs) and human bone marrow mesenchymal stromal cells (hBMSCs) in vitro, respectively. Results: At 4 and 8weeks after implanting the hydrogel into the osteochondral defect of rabbit, histological analysis revealed that the regenerated tissue in Ept + group had higher cellular density with better texture, and the newly formed hyaline cartilage tissue was seamlessly integrated with adjacent native tissue in the Ept + group. In cellular experiments, Ept treatment significantly promoted both gene and protein expression of type II collagen in hOACs, while decreased the protein levels of metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs 5 (ADAMTS5); alcian blue staining, type II collagen and aggrecan stainings showed that addition of Ept significantly reversed the chondrogenesis inhibition effect of tumor necrosis factor alpha (TNF-α) on hBMSCs. Conclusion: BMS could be augmented by Ept embedded hydrogel, potentially by regulating the catabolic and anabolic dynamics in adjacent chondrocytes and enhancement of BMSCs chondrogenesis.

Anatomical, scanning electron microscopy, histological and histochemical studies of the orbital glands of the Egyptian agama Trapelus mutabilis

BackgroundThe orbital gland of the group agama (Agamidae) is not well-studied. Here is a first description of anatomical and histological studies on the orbital gland of the Egyptian agama Trapelus mutabilis.ResultsThe Harderian gland (HG) of the Egyptian agama is well-developed, with a tongue shape, appearing whitish in color, and reaching approximately 1.47 mm in length. The gland can be divided into head and tail regions and lies at the medial corner of the orbit ventral to the ventralis oblique muscle and dorsal to the medialis rectus muscle. Secretory product of the HG is released into the conjunctival fornix, which is deep into the nictitating membrane. The HG is a compound tubuloacinar type, consisting of columnar and pyramidal glandular cells with rounded nuclei and lumina that are variable in size and containing pigments in the connective tissue capsule that envelops the tail portion of this gland. The glandular cells of the HG react positively to periodic acid-Schiff (PAS) and alcian blue staining at pH 2.5. In contrast, lacrimal gland (LG) of Egyptian agama is a reduced mucous gland that appears whitish in color, opens on the surface by numerous orifices at the base of nictitating membrane, and reaches approximately 0.1 mm in length. The lacrimal gland is composed of a simple tubulo-acini of columnar cells with narrow lumen and basal oval-shaped nuclei and exhibits a weak response to PAS but a strong response to alcian blue at pH 2.5.ConclusionsMorphology of LG and HG isn't impacted by features of the orbit, such as the existence or absence of nictitating membrane and/or fixed or movable eyelid. The small size of the LG of the Egyptian agama does not affect the performance of its main function in lubricating the surface of the cornea. The pigmentation envelope of the HG of the Egyptian agama allows heat to be absorbed in order to increase its secretion activity, thereby increasing the protection of the eyes against mechanical damage. Finally, both orbital glands may play a secondary function in digestion via indirect connection with the oral cavity.

Histochemical study of proventriculus in pre-hatch and post-hatch days in northern bobwhite quail (Colinus virginianus)

The present study was designed to investigate the histochemical developmental changes of the proventriculus at pre-hatching and post-hatching in the northern bobwhite quail at periods 11- and 15-days pre-hatching in addition to 14- and 21-days post-hatching. The proventriculus composed of four tunics is tunica mucosa, tunica sub-mucosa, tunica muscularis, and tunica serosa. The tunica mucosa was lined with simple columnar epithelium and the height of epithelium increases with the age also the thickness of the mucosa, the tunica submucosa filled with proventricular glands at 14- and 21-day post-hatch and the gland was height in diameters while in 11- and 15-day pre-hatch the gland less in diameter and the sub mucosa does not fill with the gland. Tunica muscularis was thicker in post-hatch than in pre-hatch, 11- and 15- day pre-hatch composed of a thin inner circular layer and thick outer longitudinal layer but in 14- and 21-day post-hatch the tunica muscularis was composed of a thin inner and outer circular layer and thick middle longitudinal layer. The mucosa and proventriculus glands give a positive reaction for Alcian blue stain and a weak to moderate reaction for periodic acid Schiff stain at pre-hatch, while at post-hatch the mucosa gives a positive reaction for Alcian blue stain and a negative reaction for periodic acid Schiff stain and the proventricular glands give a positive reaction for Alcian blue and periodic acid Schiff stains.

Adenosquamous carcinoma in the palate of a dog: case report

Adenosquamous carcinoma is characterized by the simultaneous presence of squamous and glandular components. This article reports a case of adenosquamous carcinoma in the oral mucosa in a dog. Microscopically, the tumour revealed an infiltrative epithelial proliferation with a tubular pattern and areas of squamous differentiation. In the lumina of the tubules, there were eosinophilic material (mucin). The alcian blue staining showed positive intraluminal material and the cytoplasm of neoplastic cells were immunoreactive for cytokeratin, supporting the diagnosis of adenosquamous carcinoma. The cells exhibited reduced or negative immunoreactivity for E-cadherin, which could be associated with more aggressive tumour behaviour and worse prognosis.

Quantitative Analysis of Mucin Expression Using Combined Alcian Blue-Periodic Acid Schiff (AB-PAS) Stain and Combined High Iron Diamine-Alcian Blue (HID-AB) Stain and the Correlation With Histomorphological Score in Chronic Calculous Cholecystitis.

Introduction Chronic calculous cholecystitis is the most common and important contributing factor for cholecystectomy across the country, with a prevalence of 2-29%. Cholesterol supersaturated bile plays a major role in stone formation. It is very essential to identify the pathogenesis of stone formation in order to prevent its formation. This study is aimed to evaluate histomorphological features of chronic calculous cholecystitis and to quantitatively evaluate alteration in mucin expression using the combined Alcian blue-periodic acid Schiff (AB-PAS) stain and the combined high iron diamine Alcian blue (HID-AB) stain, to correlate with each other and also with biochemical features of gall stones. Methods A cross-sectional study of 64 chronic calculous cholecystitis were taken for histomorphological assessment and grading using Hematoxylin and Eosin (H&E) stainand Masson's trichrome stains.Expression of the type of mucins wasanalyzed using histochemical stains by a standardized scoring system. Results A significant positive correlation was observed between an increase in grades of inflammation and fibrosis with an increase in the quantity of sialomucin and neutral mucin in the deep layers of epithelium, and a significant negative correlation was observed between an increase in grades with a decrease in acidic mucin and sulfomucin of both superficial and deep epithelium except sulfomucin in fibrosis. No significant correlation was obtained with muscle thickness, adipose tissue deposition, and epithelial hyperplasia. A higher frequency of mixed-type stones was associated with severe inflammation. Conclusion Inflammation and fibrosis were strongly correlated with quantitative alteration and reversal of mucin composition in chronic cholecystitis; hence we conclude that these features play a significant role in the pathogenesis of stone formation. Using Combined AB(2.5pH)-PAS stain and Combined HID-AB(2.5 pH) stain to detect mucin hypersecretion and composition of altered mucin is relatively accurate and cost-effective rather than performing costly immunohistochemical (IHC) markers.

Toothpastes for children and their detergent contents affect molecular mechanisms of odontogenesis in zebrafish embryos

We aimed to evaluate how different types of toothpaste (TP) for children affected molecular mechanisms of odontogenesis in zebrafish embryos. Commercially available TPs were selected according to their detergent contents as the cocamidopropyl betaine (CAPB) containing TP (TP1) and sodium lauryl sulfate (SLS) containing TP (TP2). TP3 contained no detergent. Effects of SLS, and CAPB alone were also examined. TP and detergent concentrations affecting development were determined as 750 mg/L and 4 mg/L, respectively. Embryos were exposed to TP1, TP2, TP3, SLS, CAPB, and embryo medium (control) for 72 h post fertilization. Acetylcholinesterase (AChE) activity and oxidant–antioxidant parameters were analyzed spectrophotometrically. Expressions of tooth development genes were evaluated by reverse transcription PCR (RT-PCR). Intraocular distance, lower jaw, and ceratohyal cartilage length were displayed using Alcian Blue staining. axin2 and wnt10a expressions increased in SLS and TP2 groups. igf2a and eve1 expressions decreased in all groups except TP3. nrOb1 expression decreased in TP1, SLS, and CAPB groups. Oxidant–antioxidant balance was disturbed in all groups except TP3, evidenced by increased lipid peroxidation, nitric oxide. SLS, and CAPB groups were more affected in terms of AChE, glutathione-S-transferase, and superoxide dismutase; perturbations were observed in cartilage structures. Altered expression of tooth development gene axin2 correlated with wnt10a, and with changes in cartilage structures in SLS and TP2 groups. TP3 group presented no disruptions in oxidant–antioxidant balance. Our study shows the availability of externally developing zebrafish embryos in examining the effects of TP’ contents on embryogenesis.

A novel method for making cell blocks with higher cellular yield.

Cytopathology is an important part of pathology that is used to diagnose disease on the cellular level. The application of the cell block (CB) technique plays a vital role in cytological diagnosis, as blocks and slides can be further used for special stains, immunohistochemistry (IHC), and molecular pathological analysis. Several methods for making CBs have been reported, but their procedures and cellular yield are still deemed unsatisfactory. In this article, we used gellan gum (GG) as an adjuvant for CBs, which resulted in higher cellular yield with simpler procedures. CBs were prepared by using GG, copper sulfate, plasma/thrombin, or pregelatinized starch methods. The procedures of each of these four methods were then compared. CB sections were stained with hematoxylin and eosin (H&E), and the background and morphological features seen by H&E staining were compared. A preliminary IHC and fluorescence in situ hybridization (FISH) study was performed using cytology specimens from elevenand five cases, respectively. The expression of immunocomplex by IHC and the molecular signals detected by FISH were compared in CB sections made by the four methods and a section derived from the biopsy specimen block from the same patient. Feulgen staining, Alcian blue staining, and Masson trichrome staining were performed on the CB sections from 3 cases of pleural fluid. The cellular yield of CB sections from 83 cases according to the four methods was compared using NDP analysis software. The results demonstrated that sections derived from CBs made with GG had a clear background and good morphological features by H&E staining. The expression of immunocomplex by IHC and the molecular signals of FISH detection in the sections from CBs made by GG were accurately located just as those in biopsy sections from the same patient. The DNA, acidic mucus, and fibrin could be clearly identified through special stains in the CB sections. The procedures involved in the GG method were easily controllable and the coagulated gel increased the ease by which the CB was embedded and sectioned. Specifically, sections from CBs made by the GG method contained higher cellular yield because cells could be concentrated on the bottom of the gel after centrifugation. This novel method for making CBs is a practical, simple method that can result in higher cellular yield. This method is therefore worth promoting in clinical applications.

Hedgehog Signalling Contributes to Trauma-Induced Tendon Heterotopic Ossification and Regulates Osteogenesis through Antioxidant Pathway in Tendon-Derived Stem Cells.

Heterotopic ossification (HO) is defined as the generation of pathological ectopic bony structures in soft tissues, but the molecular mechanisms of tendon HO are not fully revealed. Hedgehog (Hh) signalling is reportedly critical in hereditary HO. Our study focuses on the role of Hh signalling in the formation of trauma-induced tendon ossification. In this study, samples of healthy tendons and injured tendons from C57BL/6J female mice at 1, 4, 7, and 10 weeks after Achilles tenotomy were collected for quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical analysis (IHC). At 1, 4, 7, and 10 weeks postinjury, tendon samples from the mice administered with vehicle, GANT58 (a GLI antagonist), or SAG (a smoothened agonist) were harvested for micro-CT, histological staining, qRT-PCR, and IHC. Rat tendon-derived stem cells (TDSCs) treated with vehicle, GANT58, or SAG were used to induce osteogenic and chondrogenic differentiation in vitro for qRT-PCR, alkaline phosphatase staining, Alcian blue staining, and reactive oxygen species (ROS) levels measurement. We found that Hh signalling is remarkably activated during the formation of trauma-induced tendon ossification in the model of Achilles tenotomy. The in vitro and in vivo assays both confirm that downregulation of Hh signalling significantly suppresses osteogenesis and chondrogenesis to inhibit tendon ossification, while upregulation of Hh signalling promotes this process. Under osteogenic induction, Hh signalling regulates antioxidant pathway and affects ROS generation of TDSCs. Collectively, Hh signalling contributes to trauma-induced tendon ossification and affects ROS generation through antioxidant pathway in osteogenic differentiation of TDSCs, indicating that targeting Hh signalling by GANT58 may be a potential treatment for trauma-induced tendon ossification.

Smooth Muscle Cell Relaxation Worsens Aortic Dilatation and Clinical Presentation in a BAPN/Angiotensin II-Induced Aortic Dissection Model in Rats

Introduction: Beta-aminopropionitrile (BAPN) administration is a chemically induced model for preclinical aortic pathologies research. Angiotensin II (AngII) has been widely used to promotes aortic dissections in mice. Here, we provide insight on a modified aortic dissection model in rats. The effect of smooth muscle cell (SMC) relaxation with vasodilators is studied in this model. Methods: Forty Sprague-Dawley rats were divided in 4 groups: control, isosorbide dinitrate (ISDN, 30 mg/kg/day) in the drinking water, BAPN (0.02%) in the food, BAPN + ISDN (same doses). Thoracic and abdominal aortic diameters were evaluated through transthoracic ultrasound echography. After 6 weeks, all rats were infused with AngII (1 mg/kg/day) subcutaneously. Survival and type of aortic events were numbered. Histological and histochemical analyses of aorta were performed. Results: Initial telesystolic ascending aorta diameters were equal in all groups and became significantly larger in the BAPN + ISDN group compared to the BAPN group (control: 3.37 ± 0.17 mm, ISDN: 3.49 ± 0.16 mm, BAPN: 3.53 ± 0.13 mm, BAPN + ISDN: 3.61 ± 0.16 mm, analysis of variance p < 0.0001). BAPN followed by AngII infusion showed a significant lower survival rate (p = 0.029) and produced a large panel of aortic events. Association of ISDN and BAPN significantly reduces survival (p = 0.001) and provides more aortic events compared to BAPN alone (p = 0.031). In both BAPN-treated groups, orcein staining revealed split and dissected elastic fibers in the media, alcian blue staining showed mucoid degeneration of the aortic wall, and Perls-diaminobenzidine staining revealed an accumulation of Fe²⁺. Conclusion: SMC relaxation with ISDN increases aortic dilatation, worsens aortic prognosis, and reproduces human histological findings in a low-dose BAPN/AngII-induced aortic dissection model in rats.

Gegen Qinlian decoction activates AhR/IL-22 to repair intestinal barrier by modulating gut microbiota-related tryptophan metabolism in ulcerative colitis mice

Ethnopharmacological relevanceGegen Qinlian decoction (GQD) is a traditional Chinese medicine derived from Treatise on febrile diseases and is clinically used for the treatment of acute ulcerative colitis (UC). However, the potential mechanism of GQD treatment for UC remains elusive. Aim of studyIn this study, we aimed to explore the involvement of gut microbiota-related tryptophan metabolism in mediating protective effects of GQD against intestinal barrier damage. Materials and methodsMice with colitis were treated with 3% dextran sulfate sodium (DSS) for 6 days. The therapeutic effects of GQD in UC mice were examined based on body weight, disease activity index (DAI), organ index, length and pathological changes in the colon. The distribution of fluorescein isothiocyanate dextran (FITC-dextran) in the intestinal tract was observed using small animal imaging, while concentration of FITC-dextran in serum was detected using a fluorescein microplate analyser. Bacterial infiltration in colon tissues was observed by fluorescence in situ hybridisation (FISH), and the bacterial load in mesenteric lymph nodes (MLNs) was further examined through bacterial culture. Subsequently, colonic goblet cells were detected using Alcian blue staining. The tight junctions of the colonic epithelium were observed using transmission electron microscopy, and the expression of tight junction proteins was detected by immunofluorescence (IF) and western blot. In addition, flow cytometry was used to analyse the proportion of interleukin-22-positive (IL-22+) ILC3 cells in lamina propria lymphocytes, and the content of IL-22 in colon homogenates was determined using an ELISA kit. In addition, targeted tryptophan metabolomics was used to detect the concentration of indole derivatives produced by tryptophan metabolism in faeces, and 16S rDNA was used to investigate the composition and abundance of gut microbiota-related tryptophan metabolism. ResultsAdministration of GQD significantly alleviated the pathological symptoms, including weight loss, increased DAI score, changes in organ index, colon shortening, and colon pathological injury in UC mice. In addition, GQD reduced the diffusion of FITC-dextran in the intestinal tract, the content of FITC-dextran in serum, and bacterial infiltration in MLNs and colon tissues. Additionally, GQD significantly increased the number of colonic goblet cells, repaired the structure of epithelial tight junctions and increased the expression of tight junction proteins. Furthermore, GQD significantly increased the proportion of IL-22+ ILC3 in the lamina propria, the expression of CYP1A1 protein in colon tissue, and the level of IL-22 in colon homogenates. However, the above protective effects of GQD were inhibited by co-administration of GQD and aryl hydrocarbon receptor (AhR) antagonist. Additionally, GQD restored the content of indole derivatives generated by tryptophan metabolism, regulated the diversity of the gut microbiota, and significantly increased the abundance of genes related to tryptophan metabolism. ConclusionOur results confirmed that GQD repaired the damaged intestinal barrier in UC mice by regulating gut microbiota-related tryptophan metabolism and restoring the generation of indole derivatives to activate AhR-mediated IL-22 production.