Conditions for late gadolinium enhancement MRI in myocardial infarction model rats that better reflect microscopic tissue staining.

Late gadolinium enhancement (LGE) is a widely used magnetic resonance imaging method for assessing cardiac disease. However, the relationship between different LGE signal thresholds and microscopic tissue staining images is unclear. In this study, we performed cardiovascular MRI on myocardial infarction (MI) model rats and evaluated the relationship between LGE with different signal thresholding methods and tissue staining images. We prepared 16 rats that underwent MRI 14-18 days following a surgery to create an MI model. We captured cine and LGE images of the cardiac short-axis and longitudinal two- and four-chamber views. The mean ± 2SD, ± 3SD, and ± 5SD of the pixel values in the non-infarcted area were defined as the LGE area. We compared areas of Sirius red staining, determined by the color tone, with their respective LGE areas at end-diastole and end-systole. We observed that the LGE area calculated as the mean ± 2SD of the non-infarcted area at end-diastole demonstrated a significant positive correlation with the area of Sirius red staining (Pearson's correlation coefficient in both: 0.81 [p < 0.01]). Therefore, the LGE area calculated as the mean ± 2SD of the non-infarcted area at end-diastole best reflected the MI area in tissue staining.

Liver fibrosis quantified by image morphometry predicts clinical outcomes in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Liver fibrosis predicts adverse clinical outcomes, such as liver-related death (LRD) and hepatocellular carcinoma (HCC) in patients with non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the accuracy of semi-automated quantification of collagen proportionate area (CPA) as an objective new method for predicting clinical outcomes. METHOD: Liver biopsies from patients with NAFLD underwent computerized image morphometry of Sirius Red staining with CPA quantification performed by ImageScope. Clinical outcomes, including total mortality, LRD, and combined liver outcomes (liver decompensation, HCC, or LRD), were determined by medical records and population-based data-linkage. The accuracy of CPA for predicting outcomes was compared with non-invasive fibrosis tests (Hepascore, FIB-4, APRI). RESULTS: A total of 295 patients (mean age 50 years) were followed for a median (range) of 9 (0.2-25) years totalling 3253 person-years. Patients with CPA ≥ 10% had significantly higher risks for total death [hazard ratio (HR): 5.0 (1.9-13.2)], LRD [19.0 (2.0-182.0)], and combined liver outcomes [15.6 (3.1-78.6)]. CPA and pathologist fibrosis staging (FS) showed similar accuracy (AUROC) for the prediction of total death (0.68 vs. 0.70), LRD (0.72 vs. 0.77) and combined liver outcomes (0.75 vs. 0.78). Non-invasive serum markers Hepascore, APRI, and FIB-4 reached higher AUROC; however, they were not statistically significant compared to that of CPA except for Hepascore in predicting total mortality (0.86 vs. 0.68, p = 0.009). CONCLUSION: Liver fibrosis quantified by CPA analysis was significantly associated with clinical outcomes including total mortality, LRD, and HCC. CPA achieved similar accuracy in predicting outcomes compared to pathologist fibrosis staging and non-invasive serum markers.

[Inhibitory effect of Xinhui citrus fermentation liquor on liver fibrosis in mice].

OBJECTIVE: To investigate the inhibitory effect of Xinhui citrus fermentation liquor on liver fibrosis in mice. OBJECTIVE: Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4 in 105 male C57BL/6 mice, followed by gavage of 0.1 mL 40% CCl4 olive oil 3 times a week (model group, n=49) or daily gavage of citrus liquor at the dose of 0.26 mL (citrus liquor group, n=56) for 8 weeks. Seven mice receiving only olive oil treatment (0.1 mL, 3 times a week) and another 7 treated with citrus liquor served as the control group. Liver tissues and serum samples were collected from 7 mice in the citrus liquor group and model group each week and from the mice in the two control groups at the 8th week for pathological examination of the liver tissues using HE staining and Sirius red staining and for determination of the biochemical indexes of liver function. OBJECTIVE: The mice in the model group showed progressively worsened liver fibrosis with obvious hepatic steatosis, necrosis and inflammatory cell infiltration. These liver pathologies were much ameliorated in citrus liquor group, which showed significantly reduced vacuolation, inflammatory cell infiltration, collagen deposition and the Ishak score of the liver tissue (P < 0.05). Serum levels of cholyglycine, alanine aminotransferase, transglutaminase and alanine aminotransferase were all significantly lower in citrus liquor group than in the model group (P < 0.05). OBJECTIVE: Xinhui citrus fermentation liquor has protective effect on the liver and can significantly ameliorate liver fibrosis in mice.

A model of partial ligation of common carotid artery and neointima / 解剖学报

Objective To discuss a method to induce vascular remodeling (intimal regeneration) after partial ligation of the left common carotid artery. Methods Forty 8-week-old male C57BL / 6 J mice were randomly divided into 6 groups: normal group (WT), sham group (sham), 4 weeks, 8 weeks, 10 weeks and 12 weeks postoperative groups. Only the internal carotid, external carotid and occipital arteries in the four distal branches of the left common carotid artery (LCA) were ligated, and the superior thyroid artery was retained, resultsing in intimal neovascularization and vascular remodeling of the left common carotid artery. The changes of body weight and forage were observed after operation. Morphological changes of blood vessels were observed by HE staining. The aggregation of collagen fibers in blood vessels was observed by sirius red staining. Results In addition to the WT group, the weight and dietary quantity of other groups were reduced 1-2 days after the operation, and began to rise from the third day, while the sham group began to rise from the second day. HE staining showed that no new intima was formed in the left common carotid artery of sham group and WT group, and thickening of intima and media layer and stenosis of the nasal cavity were observed in the 4, 8, 10 and 12 weeks groups after partial ligation. There was no intima thickening in the unligated carotid artery on the right side, but there was an enlargement of the lumen. This sirius red stain demonstrated collagen accumulation in the intima and tunica media. Conclution Partial ligation of the left common carotid artery can establish a mouse model of arterial endothelial injury with obvious intima regeneration.

Quantification of myocardial infarct area based on TRAFFn relaxation time maps - comparison with cardiovascular magnetic resonance late gadolinium enhancement, T1ρ and T2 in vivo.

BACKGROUND: Two days after myocardial infarction (MI), the infarct consists mostly on necrotic tissue, and the myocardium is transformed through granulation tissue to scar in two weeks after the onset of ischemia in mice. In the current work, we determined and optimized cardiovascular magnetic resonance (CMR) methods for the detection of MI size during the scar formation without contrast agents in mice. METHODS: We characterized MI and remote areas with rotating frame relaxation time mapping including relaxation along fictitious field in nth rotating frame (RAFFn), T1ρ and T2 relaxation time mappings at 1, 3, 7, and 21 days after MI. These results were compared to late gadolinium enhancement (LGE) and Sirius Red-stained histology sections, which were obtained at day 21 after MI. RESULTS: All relaxation time maps showed significant differences in relaxation time between the MI and remote area. Areas of increased signal intensities after gadolinium injection and areas with increased TRAFF2 relaxation time were highly correlated with the MI area determined from Sirius Red-stained histology sections (LGE: R2 = 0.92, P < 0.01, TRAFF2: R2 = 0.95, P < 0.001). Infarct area determined based on T1ρ relaxation time correlated highly with Sirius Red histology sections (R2 = 0.97, P < 0.01). The smallest overestimation of the LGE-defined MI area was obtained for TRAFF2 (5.6 ± 4.2%) while for T1ρ overestimation percentage was > 9% depending on T1ρ pulse power. CONCLUSION: T1ρ and TRAFF2 relaxation time maps can be used to determine accurately MI area at various time points in the mouse heart. Determination of MI size based on TRAFF2 relaxation time maps could be performed without contrast agents, unlike LGE, and with lower specific absorption rate compared to on-resonance T1ρ relaxation time mapping.

Comprehensive Assessment of Authenticating Method of Pathological Staining in Cardiac Fibrosis / 医学研究杂志

Objective To compare hematoxylin and eosin(HE) staining,Masson's staining and picric sirius red(PSR) staining in evaluation of cardiac fibrosis.Methods Twenty adult sprague dawley (SD) rats were given subcutaneous injection of isoprenaline for 5mg/(kg · d) for 3 weeks,which induced cardiac fibrosis model successfully.In order to observing the fibrotic areas and collagen deposition,we prepared paraffin sections by using the heart specimen of rat,and carried out HE staining,Masson staining and PSR staining respectivelly for the special staining.Results All staining methods could clearly show the collagen fibers and fibrosis stage.We could clearly distinguish the type Ⅰ and type Ⅲ collagen fibers through PSR staining with immunoflurescent techniques,but not Masson and HE staining.Conclusion The results demonstrated that PSR staining is better than HE and Masson staining to be used for the evaluation of degree and type of proliferation of collagen fibers.It has important signification for treatment and prevention of cardiac fibrosis.

Comprehensive Assessment of Authenticating Method of Pathological Staining in Cardiac Fibrosis / 医学研究杂志

Objective To compare hematoxylin and eosin(HE) staining,Masson's staining and picric sirius red(PSR) staining in evaluation of cardiac fibrosis.Methods Twenty adult sprague dawley (SD) rats were given subcutaneous injection of isoprenaline for 5mg/(kg · d) for 3 weeks,which induced cardiac fibrosis model successfully.In order to observing the fibrotic areas and collagen deposition,we prepared paraffin sections by using the heart specimen of rat,and carried out HE staining,Masson staining and PSR staining respectivelly for the special staining.Results All staining methods could clearly show the collagen fibers and fibrosis stage.We could clearly distinguish the type Ⅰ and type Ⅲ collagen fibers through PSR staining with immunoflurescent techniques,but not Masson and HE staining.Conclusion The results demonstrated that PSR staining is better than HE and Masson staining to be used for the evaluation of degree and type of proliferation of collagen fibers.It has important signification for treatment and prevention of cardiac fibrosis.

A highly reproducible mice model of chronic kidney disease: Evidences of behavioural abnormalities and blood-brain barrier disruption.

AIMS: In the present study, a novel mice model of chronic kidney disease (CKD) was developed, and psycho-motor behavioural abnormalities, blood-brain barrier (BBB) integrity and brain histology were studied. MAIN METHODS: Swiss albino female mice were given high adenine diet (0.3% w/w mixed with feed) for 4weeks. Serum urea and creatinine levels and renal histological studies were performed to validate the model. Psycho-motor behavioural abnormalities and neurological severity were studied. BBB integrity was assessed using Evans blue extravasation method. Nissl staining was performed to see possible morphological aberrations in brain. KEY FINDINGS: There was a significant increase in serum urea and creatinine levels in mice given high adenine diet, and the mice had abnormal kidney morphology. Deposition of adenine and 2,8-dihydroxyadenine crystals, and increased collagen deposits in the renal tissues were found, which validate induction of CKD in the mice. Motor behavioural abnormalities, depression-like and anxiolytic behaviour and increase in neurological severity were prevalent in mice with CKD. Evans Blue dye extravasation was found to occur in the brain, which signifies disruption of BBB. However, Nissl staining did not reveal any morphological aberration in brain tissue. SIGNIFICANCE: The present study puts forward a highly reproducible mice model of CKD validated with serum parameters and renal histopathological changes. The mice showed psycho-motor behavioural abnormalities and BBB disruption. It is a convenient model to study the disease pathology, and understanding the associated disorders, and their therapeutic interventions.

Identification of antrodin B from Antrodia camphorata as a new anti-hepatofibrotic compound using a rapid cell screening method and biological evaluation.

AIM: Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) resulting from chronic liver diseases. Efficient and well-tolerated drugs for its treatment are urgently needed. This study aims to identify the active ingredients of Antrodia camphorata by a bioassay-guided fractionation approach and explore the acting mechanism by using a hepatic stellate cell (HSC) line CFSC-8B stimulated by transforming growth factor-ß1 (TGF-ß1). METHODS: The accumulation of collagens was evaluated using chromogenic precipitation reaction with picro-sirius red (PSR) dye solution and quantified by spectrophotometric analysis of the dissolved stain. MTT assay, cell migration assay, quantitative polymerase chain reaction and western blotting analysis were used to determine the cell viability, cell migration and gene expression. RESULTS: We established a rapid colorimetric assay suitable for screening of anti-hepatofibrotic reagents. Stimulation with 10 ng/mL TGF-ß1 for 48 h and 200 µL PSR dye solution were optimal for the colorimetric assay in CFSC-8B cells. We used SB431542, silybin and another 11 antifibrotic reagents to verify the cellular model. Within the safe doses, they attenuated ECM production induced by TGF-ß1. Bioactivity-guided fractionation led to the identification of antrodin B from A. camphorata. Antrodin B significantly ameliorated cell proliferation, cell migration, suppressed HSC activation marker α-smooth muscle actin expression and ECM components Col1, Col3 and Fn expression, and blocked the phosphorylation of Smad2/3 induced by TGF-ß1 in CFSC-8B cells in a dose-dependent manner. CONCLUSION: We developed a simple assay based on TGF-ß1-induced total collagen accumulation in CFSC-8B cells and identified antrodin B which may serve as a potential candidate for treatment of liver fibrosis.

Biocomposite nanofibrous strategies for the controlled release of biomolecules for skin tissue regeneration.

Nanotechnology and tissue engineering have enabled engineering of nanostructured strategies to meet the current challenges in skin tissue regeneration. Electrospinning technology creates porous nanofibrous scaffolds to mimic extracellular matrix of the native tissues. The present study was performed to gain some insights into the applications of poly(l-lactic acid)-co-poly-(ε-caprolactone) (PLACL)/silk fibroin (SF)/vitamin E (VE)/curcumin (Cur) nanofibrous scaffolds and to assess their potential for being used as substrates for the culture of human dermal fibroblasts for skin tissue engineering. PLACL/SF/VE/Cur nanofibrous scaffolds were fabricated by electrospinning and characterized by fiber morphology, membrane porosity, wettability, mechanical strength, and chemical properties by Fourier transform infrared (FTIR) analysis. Human dermal fibroblasts were cultured on these scaffolds, and the cell scaffold interactions were analyzed by cell proliferation, cell morphology, secretion of collagen, expression of F-actin, and 5-chloromethylfluorescein diacetate (CMFDA) dye. The electrospun nanofiber diameter was obtained between 198±4 nm and 332±13 nm for PLACL, PLACL/SF, PLACL/SF/VE, and PLACL/SF/VE/Cur nanofibrous scaffolds. FTIR analysis showed the presence of the amide groups I, II, and III, and a porosity of up to 92% obtained on these nanofibrous scaffolds. The results showed that the fibroblast proliferation, cell morphology, F-actin, CMFDA dye expression, and secretion of collagen were significantly increased in PLACL/SF/VE/Cur when compared to PLACL nanofibrous scaffolds. The accessibility of human dermal fibroblasts cultured on PLACL/SF/VE/Cur nanofibrous scaffolds proved to be a potential scaffold for skin tissue regeneration.