Smoth Muscle Actin and Collagen Immunohistochemical evaluation in the endometrium of mares treated with bone narrow stem cells

FUMUSO, E.; CARMO, MT.; HERRERA, MF.; CANTATORE, SE.; LANDIM, FC.; LOMBARDO, DM.; FELIPE, AE.

Hamiltton

Simposio; XITH INTERNATIONAL SYMPOSIUM ON EQUINE REPRODUCTION; 2014

ISERC

Stem cells have been used to treat chronic degenerative diseases in horses mainly in Currently, therapies like mesenchymal stem cells have been used to improve acute and chronic inflammatory conditions in tendons, joints and lower airways with good results[1]. Recently bone narrow stem cells (BNSC) therapy was used in the treatment of mares with chronic degenerative endometrial disease. After SC treatment improvement in endometrial activity, distribution and glandular density was detected [2]. Alpha-smooth muscle actin (Alpha-sm-actin) is a functional marker for fibroblast subtypes that remodels the extracellular matrix. The aim of this study was to evaluate the effect of therapy with BNSC over collagen and Alpha-sm-actin in endometrial tissue of mares with chronic degenerative disease. Eighteen endometrial biopsies were taken from nine mares aged 14-23 years, with chronic degenerative endometrial changes. Nine biopsies where obtained on day 0 (D0) before administration of autologous SC treatment. A total of 8 x 106 cells in a volume of 0,5 ml were injected in each point into 20 different points of approximately 1 cm from each other, following a horizontal line from the tip of one uterine horn to the other. Injections were performed by hysteroscopy with a needle designed for colonoscopic procedures. Sixty days later (D60) a second biopsy was obtained from each mare. Biopsies were fixed in Bouin solution, and then in ethanol 70 % until their inclussion in paraffin. Serial 4 micron thick sections were mounted on silane treated slides and stained with H&E. For measurement of total collagen, additional sections were incubated with a1% picrosirius red saturated picric acid solution, for 60? at room temperature. For Alpha-sm-actin immunohistochemistry, sections were incubated with an anti-human smooth muscle actin Clone 1A4 antibody (DAKO) diluted 1/100, at 4°C overnight. The Dako LSAB?+/HRP kit, was used for visualization before the slides were counterstained with Mayer?s hematoxylin, and mounted with CytosealTM60. Images were captured at 20x and 40x with a Leica ICC50HD camera, using LASEZ ® photographic (Leica Application Suite EZ) incorporated to a Leica DM500 microscope. Eight images per mare with 40x1,25 magnification were analyzed for picrosirius red using a RGB, densitometric profile, Q-Win Plus Leica 2003 Intensity of collagen stain. Five to eight images with 40x1, 25 magnification for Alpha-sm-actin image analysis were captured. The immune marked cumulative % area per field/image was expressed in square microns. Paired t test, from Graph-pad InStat was used for data analysis. After treatment both variables resulted increased at D60. Intensity of collagen stain with picrosirius red was on D0: 151,09; D60: 179,01 (p