Intervertebral disc degeneration in warmblood horses: Histological and biochemical characterization

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

Collagen and Microvascularization in Placentas From Young and Older Mares

In older mares, increasing collagen fibers (fibrosis) in the endometrium and oviduct predisposes to sub-fertility and infertility. In this study, (i) gene transcription of collagen (qPCR: COL1A1, COL1A2, COL3A1, COL5A1); (ii) total collagen protein (hydroxyproline); (iii) collagen distribution (Picrosirius red staining; polarized light microscopy); and (iv) microvascular density (Periodic acid-Schiff staining), were evaluated in mares' placenta, and related to mares age, and placenta and neonate weights. Samples were collected from the gravid horn, non-gravid horn, and body of the placenta from younger (n = 7), and older mares (n = 9) of different breeds. Transcripts of COL1A1, COL3A1 and COL5A1, total collagen protein, chorionic plate connective tissue thickness, and microvascularization increased in the gravid horn of older mares' placentas, compared to the youngest (P < 0.05). Although in other species placenta fibrosis may indicate placental insufficiency and reduced neonate weight, this was not observed here. It appears that older fertile mares, with more parities, may develop a heavier, more vascularized functional placenta with more collagen, throughout a longer gestation, which enables the delivery of heavier foals. Thus, these features might represent morphological and physiological adaptations of older fertile mares' placentas to provide the appropriate nutrition to the equine fetus.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-β1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-β1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-β1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 μg/100 μL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 μg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-β1 Treatment on Collagen Deposition and Biomarkers

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-β1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-β1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-β1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-β1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-β1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

Modulation of Extracellular Matrix by Scrophularia striata Extract in Vitro: A Potential Antiscarring Agent

Background: Hypertrophic scars are the consequences of the aberration of normal wound healing. To date, therapeutic strategies for abnormal scarring have been unsuccessful. ‎‏The abnormal extracellular‎ matrix is one of the most important contributing factors to ‎hypertrophic scars. ‏Scrophularia striata has been used in Iranian folk medicine for the treatment of burn wounds. ‏The plant extract accelerates wound healing and attenuates scar formation. Objectives: The study was performed to investigate the effects of Scrophularia striata hydroalcoholic extract (SSE) on MMP1, MMP8, fibronectin, collagen type I, and total collagen produced by human skin fibroblasts in the culture medium. Methods: The effects of SSE on the expression of MMP1, MMP8, fibronectin, and collagen type I in human skin fibroblast (HSF) were evaluated using Q-PCR and Western blotting methods. In addition, the effect of SSE on the total collagen content was measured in cultured HSF using Red Sirius Kit. Results: SSE significantly induced the expression of MMP1 and suppressed the production of fibronectin at the mRNA and protein levels. The total collagen content was significantly lower in SSE-treated cells than in untreated cells. SSE did not have any significant effect on MMP8 and collagen type I expression. Conclusions: The results of this study revealed that SSE could modulate the extracellular matrix turnover and had the potential for the prevention and treatment of hypertrophic scars.

The Impact of a Polyphenol-Rich Extract from the Berries of Aronia melanocarpa L. on Collagen Metabolism in the Liver: A Study in an In Vivo Model of Human Environmental Exposure to Cadmium

This study examined whether a polyphenol-rich extract from the berries of Aronia melanocarpa L. (AE; chokeberries) may protect from the impact of cadmium (Cd) on the metabolism of collagen in the liver. The study was conducted in an experimental model (rats that were fed a diet containing 1 or 5 mg Cd/kg for 3–24 months) of human exposure to this xenobiotic during a lifetime. The concentration of total collagen and the expression of collagen types I and III at the mRNA and protein levels, as well as the concentrations of matrix metalloproteinases (MMP-1 and MMP-2) and their tissue inhibitors (TIMP-1 and TIMP-2), were assayed. The administration of Cd and/or AE had only a slight and temporary impact on the concentration of total collagen in the liver. The supplementation with AE significantly prevented Cd-mediated changes in the expression of collagen types I and III at the mRNA and protein levels and their ratio (collagen III/collagen I), as well as a rise in the concentrations of MMPs and TIMPs in this organ. The results allow the conclusion that the intake of chokeberry products in the case of Cd intoxication may be effective in prevention from this xenobiotic-induced disturbance in collagen homeostasis in the liver.

Development of a novel in vitro insulin resistance model in primary human tenocytes for diabetic tendinopathy research

Background Type 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-α) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy. Methods hTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis. Results Immunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF–α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h. Conclusion At0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.

Portuguese Local Pig Breeds: Genotype Effects on Meat and Fat Quality Traits

This work investigated the contribution of cross-breeding between two local Portuguese pig breeds to the conservation of animal biodiversity and income of local pig producers. Quality traits of semimembranosus (SM), gluteus medius (GM) and dorsal subcutaneous fat (DSF) were studied in Alentejano (AL), Bísaro (BI), AL × BI, and BI × AL (Ribatejano—RI) castrated male pigs. Pigs were reared outdoors, fed ad libitum, and slaughtered at ~65 (trial 1) and 150 kg BW (trial 2). In trial 1, AL pigs showed higher SM intramuscular fat, lower total collagen, and higher soluble collagen than BI pigs, while AL × BI and BI × AL pigs showed intermediate (NS) values. AL, AL × BI, and BI × AL pigs showed higher SM myoglobin content, and AL a more intense red colour than BI pigs. Finally, AL, AL × BI, and BI × AL showed higher total lipids in DSF than BI pigs. In trial 2, SM and DSF results were similar to those obtained in trial 1. In GM, AL and BI × AL showed higher intramuscular fat than BI and AL × BI pigs, while AL, AL × BI and BI × AL showed lower total collagen content than BI pigs. In conclusion, these results suggest that RI crosses are a productive alternative, with overall muscle and DSF traits statistically not different between AL × BI and BI × AL, and similar to those observed in AL pigs.

Effects of Breed Type, Residual Feed Intake and Post-Mortem Aging on Physio-Chemical Properties of Triceps Brachii Muscle and Their Relationships With Beef Toughness

ObjectivesThe influence of breed type, residual feed intake (RFI) and post-mortem aging on meat and carcass quality attributes and intramuscular connective tissue characteristics were examined in the bovine Triceps brachii, a high connective tissue muscle from the chuck. The hypothesis that selection for low RFI in beef cattle increases beef toughness, increases collagen content and reduces collagen heat solubility of the Triceps brachii was tested.Materials and MethodsSeventy-one beef steers from Angus (n = 23), Charolais (n = 24) and Angus crossbred (n = 24)) genetics were used. Each breed had high RFI and low RFI steers (n = 12). Muscles collected were aged for 3- and 13-days post-mortem (dpm). Final animal live weight, grade marbling, intramuscular pH, water holding capacity (WHC), intramuscular fat, cooking loss, drip loss, protein, temperature, moisture, color, RFI, and Warner Bratzler shear force (WBSF) data were collected for carcass and meat quality measurements. Total collagen, collagen heat solubility, and collagen cross-link Ehrlich’s chromogen (EC) of the isolated perimysium were quantified. Data were analyzed using a split-plot experimental design procedure (R software 3.4.1) with breed and RFI as main effects in the whole plot and postmortem aging included at the subplot level.ResultsFinal weight was significantly greater for Charolais (683 ± 9.58 kg) than Angus (554 ± 9.65 kg) and Angus crossbred (568 kg ± 9.58 kg) steers (P = 0.017), and grade marbling score was higher for high RFI (421 ± 19.85) than for low RFI steer carcasses (385 ± 19.82) (P = 0.001). No significant effects of breed type and RFI (P > 0.05) were observed on meat quality attributes. WBSF value at 3 dpm (3.72 kg) was significantly higher than at 13dpm (3.21 kg) (P < 0.005). Collagen solubility was significantly higher at 13 dpm (25.88%) than at 3 dpm (18.03%) (P < 0.005). Total collagen and wet endomysium were positively correlated (r = 0.44) as were total collagen and EC in raw muscle (r = 0.76), EC and wet perimysium (r = 0.42) and WBSF and EC at 13 dpm (r = 0.27) (P < 0.005). Total collagen and collagen solubility at 3dpm (r = –0.36) and 13 dpm (r = –0.63) were negatively correlated, as were EC and solubility at 3 dpm (r = –0.38) (P < 0.005).ConclusionIncreasing postmortem aging periods reduced WBSF and increased collagen heat solubility of the Triceps brachii muscle. With no effect of RFI on meat quality measurements, the production cost can be reduced by selecting for low RFI animals without sacrificing product quality.Table 7.Least square means (± standard error of the mean) for WBSF, soluble collagen, and collagen solubility of Triceps brachii muscles at 3 and 13 d post-mortem aging (dpm)