17ß-estradiol protects nucleus pulposus cells from serum deprivation-induced apoptosis and regulates expression of MMP-3 and MMP-13 through promotion of autophagy.

Serum deprivation is a likely contributor to intervertebral disc (IVD) degeneration (IVDD).17ß-estradiol (E2) have been noted to protect nucleus pulposus cells (NPCs) against apoptosis. Autophagy and apoptosis play a paramount role in maintaining the homeostasis of IVD. So far, little research has been published on whether autophagy plays a role for the E2 mediated protection of NPCs. The aim of this study is to understand whether autophagy is involved in the protective effect of E2 against serum deprivation-induced cell apoptosis and expression of matrix metalloproteinase (MMP)-3 and MMP-13. mCherry-GFP-LC3-adenovirus transfection is used to monitor autophagy detection. The expression levels of autophagy-related proteins were measured by Western blotting, Apoptosis and MMPs were detected by flow cytometry and Western blotting. Accordingly, Autophagy and apoptosis was detected in NP cells under serum deprivation conditions, the autophagy incidence began to reached a peak value at 48 h, the apoptosis and MMPs incidence began reached a minimum value treat with E2 (10-7 M). Whereas the combined use of E2 and 3-MA led to a dramatic decrease in autophagy, while aberrantly elevated expression levels of apoptotic and MMPs. These data suggest that serum deprivation-induced apoptosis and MMP-3, MMP-13, which was efficiently suppressed by the E2 through promoting autophagy in rat NPCs.

Autophagy Protects Advanced Glycation End Product-Induced Apoptosis and Expression of MMP-3 and MMP-13 in Rat Chondrocytes.

Aging is one of the most prominent risk factors for the pathological progression of osteoarthritis (OA). One feature of age-related changes in OA is advanced glycation end products (AGEs) accumulation in articular cartilage. Autophagy plays a cellular housekeeping role by removing dysfunctional cellular organelles and proteins. However, the relationship between autophagy and AGE-associated OA is unknown. The aim of this study is to determine whether autophagy participates in the pathology of AGE-treated chondrocytes and to investigate the exact role of autophagy in AGE-induced cell apoptosis and expression of matrix metalloproteinase- (MMP-) 3 and MMP-13. AGEs induced notable apoptosis that was detected by Annexin V/PI double-staining, and the upregulation of MMP-3 and MMP-13 was confirmed by Western blotting. Autophagy-related proteins were also determined by Western blotting, and chondrocytes were transfected with mCherry-GFP-LC3B-adenovirus to monitor autophagic flux. As a result, autophagy significantly increased in chondrocytes and peaked at 6 h. Furthermore, rapamycin (RA) attenuated AGE-induced apoptosis and expression of MMP-3 and MMP-13 by autophagy activation. In contrast, pretreatment with autophagy inhibitor 3-methyladenine (3-MA) enhanced the abovementioned effects of AGEs. We therefore demonstrated that autophagy is linked with AGE-related pathology in rat chondrocytes and plays a protective role in AGE-induced apoptosis and expression of MMP-3 and MMP-13.

Visual method for measuring the roughness of a grinding piece based on color indices.

The existing machine-vision surface roughness measurement technique extracts relevant evaluation indices from grayscale images without using the strong sensitivity of color information. In addition, most of these measurements use a micro-vision imaging method to measure a small area and cannot make an overall assessment of the workpiece's surface. To address these issues, a method of measuring surface roughness that uses an ordinary light source and a macro-vision perspective to generate a red and green color index for each pixel is proposed in the present study. A comparison test is conducted on a set of test samples before and after surface contamination using the color index and gray-level algebraic averaging, the square of the main component of the Fourier transform in the frequency domain, and the entropy. A strong correlation between the color index and the surface roughness is established; this correlation is not only higher than that of other indices but also present despite contamination and very robust. Verification using a regression model based on a support vector machine proves that the proposed method not only has a simple apparatus and makes measurement easy but also provides high precision and is suitable over a wide measurement range. The impact of the red and green color blocks, the lighting, and the direction of the surface texture on the correlation between the color index and the roughness are also assessed and discussed in this paper.

Antiproliferative effects of low molecular weight heparin.

AIM: To investigate the effect of low molecular weight heparins (LMWH) on the inhibition of intimal hyperplasia (IH) developing in prosthetic vascular patch graft implanted into sheep carotid artery. METHODS: A gelatin sealed Dacron patch graft was implanted into the common carotid artery of sheep, which were then allocated to a control group (n = 10) or to one of four treatment groups (each group n = 10) receiving either a low dose (LD) or high dose (HD) of one of two LMWH (enoxaparin 1 or 2 mg/kg/day, dalteparin 100 or 200 units/kg/day) administered subcutaneously for 4 weeks. Anti-activated factor X and activated partial thromboplastin time were assayed from blood collected prior to and at 1 and 2 h after LMWH administration on days 3, 7, 14, 21 and 28. Animals were killed on day 28 after taking blood samples prior to, then at 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h following the last injection. Grafts were collected for analysis and measurements of intimal thickness obtained under light microscopy from eight transverse sections of each grafted artery aided by computer image analysis. An IH index was calculated by dividing the area of IH (mm2) by the width of the graft (mm). RESULTS: Intimal hyperplasia index measurements (mean +/- SD) were: controls 0.574 +/- 0.077, LD enoxaparin 0.471 +/- 0.056, LD dalteparin 0.404 +/- 0.025, HD enoxaparin 0.398 +/- 0.068, HD dalteparin 0.332 +/- 0.048. The reductions in IH index compared to controls were significant (P < 0.05) for both LD and HD dalteparin and for HD enoxaparin. CONCLUSION: Both LMWH dalteparin and enoxaparin reduced the amount of IH formation with dalteparin showing a greater effect in the present animal study. The possibility that different LMWH might exert differing antiproliferative effects requires further investigation.

Vascular closure staples reduce intimal hyperplasia in prosthesis implantation.

BACKGROUND: Vascular surgery, like the various other surgical specialities, has seen an increasing demand toward faster and more minimally invasive procedures. One such need is to create a reliable vascular anastomosis that is faster, easier and less damaging to the tissue. The vascular closure staples (VCS*) device provides such characteristics but, to date, no studies have investigated its effectiveness in reducing intimal hyperplasia when used for vascular prosthesis implantation. The present study evaluated its effectiveness compared with suturing of a graft in vascular prosthesis implantation. METHODS: Twelve female Merino sheep underwent gelatin sealed Dacron patch graft implantation into the left and right common carotid artery. Grafts were randomly allocated so that one carotid artery and graft was anastomosed using sutures and the other with VCS*. The two techniques were compared for operation time, clip/suture numbers and blood loss during the implantation procedure. After a 4-week period, the sheep were killed and the grafts were harvested for intimal hyperplasia (IH) assessment. RESULTS: There was a significant reduction in the amount of IH seen in the VCS* group (mean +/- SD: 0.278 +/- 0.079 mm2/mm) when compared with the sutured group (0.575 +/- 0.331 mm2/mm) (P < 0.05). There was also significant reduction in anastomosis time (mean +/- SD: 14 +/- 4.4 min) and fewer points of contact (23 +/- 1.4) using the VCS* compared with suturing (22 +/- 3.2 min, P < 0.01; 27 +/- 3.3, P < 0.05, respectively). CONCLUSIONS: In this model, the VCS* shows several distinct advantages over suturing with significant time saving at operation and, most importantly, the reduction of IH seen at 1 month.

New methodology for assessment of intimal hyperplasia of vascular prostheses.

BACKGROUND: When investigating the degree of formation of intimal hyperplasia (IH) quantification of the extent of the lesion is crucial to its assessment and analysis. The aim of the present study was to establish a new methodology (IH index) for estimating the degree of IH associated with vascular prosthesis implantation. METHODS: Ten female Merino sheep underwent a standard gelatin sealed Dacron (GSD) patch grafting procedure in the left common carotid artery. The grafts were harvested 4 weeks following implantation and processed for assessment of IH by two methodologies - mean intimal thickness (MIT) and IH index. The advantages and disadvantages of the two methods for expressing the degree of IH were compared. RESULTS: The IH index is less labour intensive but is as accurate as the MIT method in quantifying the IH lesion, statistical analysis showing high correlation and measurement agreement between the two methods. CONCLUSION: The IH index is a labour saving standardized methodology for quantification of IH in the current animal model.

Perigraft adventitia and intima remodeling after synthetic patch implantation in sheep carotid artery: role of apoptosis and proliferation.

BACKGROUND: The mechanisms of neointima formation after synthetic vascular grafting are not clear. The aim of this study was to investigate the intima and perigraft adventitia remodeling process in terms of cell apoptosis versus proliferation after synthetic patch implantation. METHODS: Female Merino sheep were randomized equally into two groups and underwent implantion with a patch of gelatin sealed Dacron graft into the left common carotid artery. At 1 and 6 months, grafted vessels were harvested, processed, and assessed. Intimal area and lumen sizes were measured with histologic assessment of eight segments from each animal assisted with image analysis. Immunohistochemical labeling of alpha-actin and D33 desmin was performed on tissue sections of perigraft adventitia, graft matrix, and intima. Cell proliferation and cell phenotype were determined with double immunohistochemical staining with anti-proliferating cell nuclear antigen and anti-alpha-actin or antimacrophage antibodies (HAM 56) in perigraft adventitia, graft matrix, and intima. Apoptosis was detected with in situ terminal deoxynucleiotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-fluorescence nick end labeling (TUNEL) in perigraft adventitia, graft matrix, and intima. RESULTS: The carotid artery lumen size at 6 months was significantly larger than at 1 month (P