Masson's Trichrome Staining Methods Research Articles

Comparative Evaluation of Masson's Trichrome and Picrosirius Red Staining for Digital Collagen Quantification Using ImageJ in Rabbit Wound Healing Research

The therapeutic potential of Pluronic F127 (PF127) hydrogel loaded with adipose-derived stromal vascular fraction (AdSVF), mesenchymal stem cells (AdMSC), and conditioned media (AdMSC-CM) for repairing full-thickness skin wounds was evaluated using a rabbit model. The rabbits were randomly divided into eight groups with six animals each and treatment was given as per the predetermined protocol (3 doses at one-week interval): Group A (Control), Group B (AdSVF), Group C (AdMSC), Group D (AdMSC-CM), Group E (PF127), Group F (AdSVF + PF127), Group G (AdMSC + PF127), and Group H (AdMSC-CM + PF127). Skin tissue samples were collected from the healing wounds on day 28 for staining and collagen quantification. Collagen density (Area %) was quantified using tissue sections stained with Masson's Trichrome (MT) and Picrosirius Red (PSR) stain using the Colour Deconvolution plugin of ImageJ and RGB stack method, respectively. These techniques function based on separating different colour channels in the stained tissue sections to isolate the collagen fibers and then quantifying them through thresholding and image analysis. Across the treatment groups, both staining methods generally showed a trend of increased collagen density compared to the control group. For most groups, PSR staining consistently indicated slightly lower collagen densities than MT staining. However, the overall trends were similar in both staining. The comparison between PSR and MT staining methods revealed that both techniques effectively assess collagen density in healing wounds. However, there were subtle differences in the absolute values obtained, with PSR staining tending to yield slightly lower collagen density measurements than MT. These differences can be attributed to the distinct mechanisms of these staining methods. Therefore, both staining methods can digitally quantify collagen density in wound healing research.

Effect of methylphenidate at different doses on new bone formation with rapid maxillary expansion: A micro-CT and histomorphometric study in rats.

This study aimed to evaluate the effect of systemically administered methylphenidate hydrochloride (MPH) on new bone formation in premaxillary suture after rapid maxillary expansion (RME). Thirty-three Wistar rats were divided into four groups: Group 1 (high dose, 30/60mg/kg MPH), Group 2 (low dose, 4/10mg/kg MPH), Group 3 (positive control) and Group 4 (negative control). RME was applied on the 70th day of the study. A 5-day RME period was followed by a 12-day retention period. The experiment was terminated on the 87th day. Micro-CT for radiological evaluation, haematoxylin-eosin and Masson's trichrome staining methods were used for histomorphometric evaluation. Among experimental groups with RME, the lowest number of osteoblasts and capillaries in Group 1 (P<.05). New bone formation, fibrous callus formation, distal osteotomy line, proximal osteotomy union and cortex remodelling were observed to be lower in Group 1 and Group 2 than Group 3 (P<.05). There was a statistically significant difference between Group 4 and each of the other groups (P=.000) in the evaluation of the results for bone mineral density, bone volume, bone volume percentage, trabecular thickness and trabecular number. MPH reduces cellular activity for new bone formation in suture in RME groups. Before performing rapid maxillary expansion in patients using MPH, the use of the drug should be postponed after a multidisciplinary decision process or clinical doses should be lowered.

Impact of high salt diets on CHOP-mediated apoptosis and renal fibrosis in a rat model.

Prolonged and excessive salt intake accelerates oxidative stress in kidney tissues, which brings about ER stress. The PERK/ATF4/CHOP/BCL-2 signaling pathway has an essential role in ER stress-induced apoptosis. The present study aimed to investigate the effect of high salt diets on the development of renal fibrosis through CHOP-mediated apoptosis. Twenty-five male Wistar rats were randomly divided into five groups (n = 5 each). Groups 1-5 were treated with 0%, 0.5%, 1%, 1.2%, 1.5% of NaCl dissolved in distilled water, respectively, for 8weeks. To detect the degree of renal tubular damage, urinary KIM-1 was measured. The slides of renal tissues were stained via Masson's Trichrome staining methods for fibrosis detection. The relative gene expression of ATF4, CHOP, and BCl-2 in renal tissues were analyzed using the qRT-PCR method. The results revealed no significantdifference between the urea, creatinine, and urine flow rate of the rats receiving different concentrations of NaCl (groups 2-5) and those of the control group (group 1). The rats treated with 1.5% NaCl (group 5) showed significant elevations in urinary KIM-1 and the mRNA level of CHOP compared to the control group. Mild renal fibrosis was also observed in group 5. Excessive salt intake leads to fibrosis as it induces the PERK/ATF4/CHOP/BCL-2 signaling pathway in renal tissues. KIM-1 is detectable in urine before the impairment of renal function which can be used as a diagnostic marker to prevent the development of progressive renal failure.

Novel hybrid scaffold for improving the wound repair process: evaluation of combined chitosan/eggshell/vitamin D scaffold for wound healing

The current design requirement of cutaneous wound healing is an ideal wound dressing via biodegradable property through which fibroblasts can support, migrate and proliferate. This study aimed to evaluate the effect of the synthesized hybrid scaffold based on chitosan polymer and a natural source of calcium (Ca) and vitamin D on wound healing. To achieve this purpose, three scaffolds include in chitosan (CS), eggshell + chitosan (ES/CS) and chitosan + eggshell + vitamin D (CS/ES/Vit D) were fabricated using the freeze-drying approach. Synthesized scaffolds were characterized by field emission scanning electron microscopy (FESEM), Fourier transformed infrared (FTIR), X-ray diffraction (XRD) and MTT assay. Histological examinations of the wound healing were performed using hematoxylin–eosin (H&E) and Masson's trichrome staining methods on different days in the rat full-thickness skin wound model. Finally, the effect of the synthesized scaffold was evaluated on the TGF-β1 gene expression. The results of the scaffold characterization showed that scaffolds have the homogeneity structure. The MTT assay indicated that the cultured fibroblasts on the ES/CS and Vit D/ES/CS scaffold had viability higher than those cultured on CS. Also, histological studies demonstrated an increased effect on epithelialization and collagen production and accelerated wound healing using designed scaffolds. The TGF-β1 gene expression in all scaffolds was not significantly different between test groups and controls. In general, it can be concluded that the synthesized scaffolds have the potential to accelerate wound healing and can be used as a suitable scaffold in wound management and skin regeneration.

Scoparone alleviates inflammation, apoptosis and fibrosis of non-alcoholic steatohepatitis by suppressing the TLR4/NF-κB signaling pathway in mice

Scoparone, a naturally-occurring, bioactive compound isolated from the Chinese herb Artemisia capillaria, has been shown to ameliorate hepatotoxicity and cholestasis in liver diseases. However, the pharmacological effect of scoparone in non-alcoholic steatohepatitis (NASH) has not been elucidated. In this study, we investigated the protective effects and mechanisms of scoparone in NASH. In vivo, the NASH model was established in mice fed a methionine and choline-deficient (MCD) diet for 4weeks, with or without simultaneous scoparone treatment. In vitro, RAW264.7 cells induced by lipopolysaccharide (LPS) were pretreated with or without different concentrations of scoparone. Hepatic triglycerides and serum AST and ALT levels were examined by biochemical assays. Hepatic histology was assessed by H&E, oil red O and Masson's trichrome staining methods, which were applied to analyze the protective effects of scoparone in NASH. To further explore the underlying mechanism of scoparone, immunohistochemistry, TUNEL, qRT-PCR, and Western blotting assays were applied to liver tissue or LPS-induced RAW264.7 cells. We found that scoparone can effectively improve hepatic steatosis, apoptosis, inflammation, and fibrosis in an MCD diet-induced NASH murine model. Mechanistically, we demonstrated that scoparone treatment alleviates NASH- and lipopolysaccharide (LPS)-induced immune responses in macrophages partly by blocking TLR-4/NF-κB signaling in a dose-dependent manner. Taken together, our results present the potential protective effects and mechanism of scoparone in NASH, suggesting a potentially beneficial drug treatment for NASH.

Acupuncture attenuates renal interstitial fibrosis via the TGF‑β/Smad pathway.

Acupuncture is one of the most useful tools in complimentary medicine, and has demonstrated potential value for treating chronic renal failure (CRF). However, the underlying mechanisms for its therapeutic effect remain unknown. In the present study, the effects of acupuncture on renal interstitial fibrosis (RIF) were explored in a rabbit model of CRF. Rabbits were assigned to the following five groups: sham, model, losartan potassium (Posi), acupuncture (Acup) and acupuncture+inhibitor (Acup+Inhib) groups. The CRF rabbits were administered a drug or/and acupuncture on Shenshu, Mingmen and Pishu. The body weights, urine protein, serum creatinine (SCr) and blood urea nitrogen (BUN) levels of the rabbits were measured. Transforming growth factor β (TGF-β), integrin-linked kinase (ILK) and Smad3 expression were detected by qRT-PCR. Tumor necrosis factor-α (TNF-α) and endothelial nitric oxide synthase (eNOS) expression were analyzed by western blot methods. The concentrations of TGF-β, IL-8, TNF-α and IL-1β in blood serum were detected using ELISA kits. In addition, pathological characteristics of the rabbit tissues were evaluated by H&E and Masson's trichrome staining methods, and TGF-β expression was detected by immunohistochemistry (IHC) assays. Results showing decreased body weights and increased urine protein, SCr and BUN levels confirmed that the CRF model had been successfully constructed. It was also found that acupuncture significantly reduced the levels of TNF-α, Smad3, ILK and TGF-β expression, dramatically decreased the concentrations of TGF-β, IL-8, TNF-α and IL-1β in blood serum, and significantly increased eNOS expression in the CRF model rabbits by affecting the TGF-β/Smad signaling pathway. In addition, it was demonstrated that acupuncture could relieve RIF by affecting the TGF-β/Smad pathway. These observations indicate that acupuncture may be useful for treating CRF, and suggest the TGF-β/Smad pathway as a target for CRF therapy.

Oral Hydrogen water – reduce hepatic inflammatory cells in mouse

Introduction: Nonalcoholic fatty liver disease (NAFLD) is characterized by a wide spectrum of liver damages spanning from steatosis, nonalcoholic steatohepatitis (NASH), cryptogenic liver cirrhosis, even to hepatocellular carcinoma. Oxidative stress is a strong contributor to the progression from simple fatty liver to nonalcoholic steatohepatitis (NASH). In this study we investigated the effect of hydrogen in the prevention of NASH in C57BL6 mouse, known as the NASH-related model. Our aim was to investigate the effects of hydrogen water on liver NASH and the mechanism underling these effects. Method: A methionine choline deficient diet (MCD) was prepared for the mouse models. In our investigation we made two experimental groups as follows: (1) MCD diet + normal water group(n=12); (2) MCD diet + hydrogen water group(n=12). Both groups were fed for three different periods, which are 8 weeks, 12 weeks, and 16 weeks. The livers were histologically examined using H&E, CD68 and Masson's trichrome staining methods. Hydrogen water was produced by placing a metallic magnesium stick into drinking water (final hydrogen concentration; 200-300 ppm). Result: The control group and hydrogen water group average concentration of ROS at 8 weeks (588±24 FORT units vs 164±8; P=0.029), at 12 weeks (587±26 FORT units vs 163±5; P=0.029) and at 16 weeks (439±176 FORT units vs 160±0; P=0.029). From H&E staining the inflammation cells was vicissitudinous in control group and HW group. At 8, 12, and 16 weeks demonstrated that (control group vs HW group; P=0.420)(control group vs HW group; P=0.729)(control group vs HW; P=0.017). Immunohistochemical identification of CD68-positive cells in control and HW group were counted that at 8 weeks P=0.151, at 12 weeks P=0.006 and 16 weeks P=0.000. Conclusion: Daily consumption of hydrogen water reduces nonalcoholic fatty liver disease and may be an effective treatment for NASH by reducing hepatic oxidative stress, apoptosis, and inflammation.

Morphological characteristics of paravertebral muscles in patients with scoliosis caused by primaryprogressive muscular dystrophies

To identify the morphological features of paraspinal muscles in patients with spinal pathology caused by progressive muscular dystrophy. The Traumatologic-and-Orthopedic Department of Axial Skeleton Pathology examined patients with scoliotic spinal deformity due to muscular dystrophy: 1) severe Duchenne X-linked muscular dystrophy (n=7); 2) Erb-Roth's autosomal recessive muscular dystrophy (n=2); 3) Landouzy-Dejerine facioscapulohumeral muscular dystrophy (n=2). For histopathological analysis of paraspinal muscles, an excisional biopsy was performed in the region of the apex of the strain angle (the convex side), and the specimens were fixed with 10% neutral formalin. Paraffin sections were stained with hematoxylin and eosin according to the Van Gieson and Masson trichrome staining methods. The preparations were examined using an AxioScope.A1 stereo microscope and an AxioCam digital camera ('Carl Zeiss MicroImaging GmbH', Germany). Sluggish moderate paraparesis and grade IV progressive neurogenic thoracolumbar scoliosis were predominant in the clinical picture of the disease. The muscle biopsy specimens were established to have muscle fiber profiles with lost polygonality, increased diameter variability, and centrally positioned or numerous internal nuclei (myophagy) and to be characterized by fiber contractures, fatty degeneration fields, interstitial fibrosis, and signs of axonopathy of intramuscular nerve conductors. The arterial blood vessels were spastic with fibrotic t. media and t. adventicia; the venous bed vessels were dilated, thin-walled, full-blooded, which causes blood corpuscle transudation and numerous hemorrhages. The identified morphopathological characteristics of muscle tissue in patients with progressive muscular dystrophy are very similar. However, Duchenne muscular dystrophy is the most severe pathology, in which fatty degeneration and sclerotization of muscle tissue and perimysial vessels are most pronounced. To solve this problem, there is a need for the integration of geneticists, biochemists, molecular biologists, pharmacologists, and histologists.

Baicalin attenuates non-alcoholic steatohepatitis by suppressing key regulators of lipid metabolism, inflammation and fibrosis in mice

AimsBaicalin (BA), an active flavonoid compound originating from the herb of Scutellaria baicalensis Georgi, has been previously shown to exert anti-inflammation and anti-oxidant effects in liver diseases. However, the potential role of BA in the regulation of non-alcoholic steatohepatitis (NASH) remains elusive. In this study, we newly explored the hepatoprotective effects of BA in MCD diet-induced NASH by ameliorating hepatic steatosis, inflammation, fibrosis and apoptosis. Main methodsNASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4weeks. The mice were simultaneously treated with or without BA for 4weeks. Serum liver functional markers and inflammatory indicators were assessed by biochemical and ELISA methods, respectively. The livers were histologically examined using H&E, Oil Red O and Masson's trichrome staining methods. The qRT-PCR, IHC and Western blotting assays were applied to analyze mechanisms underlying BA protection. Key findingsBA treatment significantly attenuated MCD diet-induced hepatic lipid accumulation partly through regulating the expression of SREBP-1c, FASN, PPARα and CPT1a. BA treatment dramatically suppressed MCD diet-induced hepatic inflammation, which was associated with decrease in serum TNF-α, IL-1β and MCP-1 production, macrophage influx and suppression of nuclear factor-κB activation. Additionally, BA was proved to prevent liver fibrosis, which appears to be mediated by inhibition of α-SMA, TGF-β1 and Col1A1. Furthermore, BA markedly inhibited hepatocyte apoptosis and cleaved caspase-3 protein expression in MCD diet-induced mice. SignificanceThese results provide a possible basis of the underlying mechanism for the application of BA in the treatment of NASH.

Histochemical observation of bony reversal lines in bisphosphonate-related osteonecrosis of the jaw

To contrast the pattern of bony reversal linesin bisphosphonate osteonecrosis of the jaw with infected osteomyelitis derived acute osteonecrosis of the jaw. This study investigated the histochemical characteristics of reversal lines in 50 cases of BP-related osteonecrosis of the jaw (BRONJ) compared with non-BP-involved bones in 20 cases of chronic osteomyelitis of the jaws. Necrotic bones were stained by using the toluidine blue, Safranin O, Giemsa, van Gieson, and Masson's trichrome staining methods. All BP-involved bones in BRONJ were distinguishable from non-BP-involved bones in chronic osteomyelitis of the jaws by multiple thick, irregular, reversal lines, which were strongly stained with toluidine blue, Safranin O, and Giemsa solution. The reversal lines of BP-involved bones (average 31.2±10.85μm) were thicker than those of osteomyelitic bones (average 11.1±3.76μm), and they were closely associated with immature bony matrices containing collagenous materials positive for van Gieson and Masson's trichrome staining with statistical significance (P=.0212 in t test statistics). The immature reversal lines of BP-involved bones continuously appeared as thick non-birefringence lines between lamellate structures as observed under a polarizing microscope, whereas the reversal lines of non-BP-involved bones were gradually thinned as their mineralization advanced. BP-involved bones had immature bony matrices outlined by thick reversal lines, which might be crucial to rapid osteonecrosis of BRONJ and also could be hallmarks for the differential diagnosis of BRONJ from chronic osteomyelitis of the jaws.

Negative pressure wound therapy is associated with up-regulation of bFGF and ERK1/2 in human diabetic foot wounds.

Chronic foot wounds are a leading cause of morbidity and hospitalization for patients with diabetes. Negative pressure wound therapy (NPWT) is known to promote healing of diabetic foot wounds, but the underlying molecular mechanisms remain elusive. We propose to gain molecular insights into the wound healing promoting signals underlying the effects of NPWT on diabetic foot wounds in humans. We assessed 30 patients with diabetic foot ulcers. Of these cases, 15 were treated with NPWT, while 15 patients were treated with traditional gauze therapy. Granulated tissue was harvested before and after treatment in both patient groups and histologically analyzed with hematoxylin & eosin as well as Masson's trichrome staining methods. Immunohistochemistry and Western blot analysis was performed to evaluate expression of basic fibroblast growth factor (bFGF) and extracellular signal-regulated kinase (ERK)1/2, previously associated with promoting cellular growth and/or wound healing. Unlike controls, the wounds in the NPWT-treated diabetic patients developed characteristic features of granulated tissue with increased collagen deposition. Immunohistochemical analysis also revealed an increase in bFGF levels in NPWT-treated patients. Western blot analysis further showed a significant up-regulation of bFGF and phosphorylated ERK1/2 protein levels in the NPWT-treated diabetic patients vs. controls. Our studies reveal that NPWT is associated with an up-regulation of bFGF and ERK1/2 signaling, which may be involved in promoting the NPWT-mediated wound healing response.

Efeitos do oleato de etanolamina na parede venosa, de cães

OBJETIVO: Avaliar a resposta biológica que o oleato de etanolamina possa desencadear na parede de veias superficiais normais de cães. MÉTODOS: Utilizados 39 cães, sem raça definida, adultos, machos, com peso variando entre 10 a 18 kg, distribuídos de modo aleatório em três grupos: grupo 1, avaliados após 7 dias, grupo 2, 14 dias e grupo 3, 21 dias. O procedimento foi realizado em duas fases. A primeira constou da injeção de 2 ml do oleato de monoetanolamina a 5%, por punção única na veia cefálica do membro torácico do cão. A segunda, realizada 7, 14 e 21 dias após, constou da retirada da peça operatória, tendo sido executada em três tempos diferentes, conforme o grupo a que pertencia o animal. As veias contralaterais foram extraídas como controle. Para estudo histológico utilizaram-se os métodos de hematoxilina-eosina e tricrômio de Masson. RESULTADOS: A trombose venosa e a organização do trombo ocorreram em todos animais estudados. A recanalização do trombo não foi observada de modo estatisticamente significante, até 21 dias de exame. Encontrou-se lesão de túnica média, que não foi acompanhada de correspondente processo inflamatório. Na túnica adventícia este processo foi visto nos três períodos de tempo estudados. Depósitos de hemossiderina em fagócitos ocorreram aos 14 e 21 dias de experimento. Extravasamento de esclerosante foi observado somente na primeira semana de estudo. Material hialino fibrinóide foi encontrado aos 21 dias de experimento. CONCLUSÕES: O oleato de etanolamina em contato com a parede interna da veia superficial produziu trombose venosa, a qual se organizou em todos os casos, não se observando sua recanalização durante o tempo deste ensaio. Houve lesão da túnica média venosa em todos animais estudados, sem que houvesse processo inflamatório reativo nesse local. Na túnica adventícia venosa surgiu processo inflamatório, além de sinais de extravasamento do esclerosante, da presença de hemossiderina e de material hialino externos à veia.

Fluorescence microscopy of rat embryo sections stained with haematoxylin-eosin and Masson's trichrome method.

The fluorescence pattern induced by haematoxylin-eosin (HE) and Masson's trichrome (MT) staining methods on paraffin sections of rat embryos (from 13 to 18 days old) has been studied. Using optimal excitation (green light, 545 nm), HE- or MT-stained sections showed a selective red emission of the acidophilic tissue components, which was due to eosin Y in the case of HE and to acid fuchsin and/or xylidine ponceau in the case of MT. The fluorescence intensity induced by these anionic dyes was variable and related to the substrate nature and the embryo age. Whereas in young embryos only the immature red blood cells showed a noticeable fluorescence, in the oldest embryos there were also other tissue components that selectively fluoresced with these dyes, in particular fibre lens cells, elastic fibres, zymogen granules and muscle cells. Spectrofluorometric studies on free dyes and densitometric analysis of protein blots confirmed microscopical observations. Our results indicate that the standard HE and MT staining methods can be used in recognizing the appearance of specific protein structures in embryonic tissues by means of fluorescence microscopy.

The Masson Trichrome Staining Methods in Routine Laboratory Use

A resume of Masson's trichrome staining methods is given, with detailed directions for carrying out all of his procedures. The results obtained thru their use in a routine laboratory are discussed ...