Eduardo Malschitzky

A novel strategy of mesenchymal stem cells delivery in the uterus of mares with endometrosis

Mesenchymal stem cells (MSCs), because of their immunomodulation and trophic activities, in addition to their capacity to regenerate damaged tissues, have potential for treatment of many diseases. The success of stem cell therapies depends, in part, on the method of cell delivery, which should provide wide cell distribution and homing in to injured sites. The objective of the present study was to developing a novel strategy for delivery of MSCs into the uterus of mares with endometrosis (degenerative alteration of uterine glands and surrounding stroma). Endometrosis was confirmed in all mares (N = 6) used in this study. To trace multipotent equine adipose tissue-derived MSCs (eAT-MSCs) in endometrial tissue, before transplantation, cells were stained with a fluorescent dye. During a synchronized estrus, the eAT-MSCs (2 × 10(7) diluted in 20 mL of sodium chloride 0.9%) were inoculated into uterus using a simple technique, similar to artificial insemination (AI) in mares. At 7 and 21 days after transplantation, homing of fluorescently labeled eAT-MSCs was observed by confocal microscopy of uterine biopsies collected from the uterine body and in both uterine horns, including glandular and periglandular spaces, in three of four treated mares. Herein, we propose a new method of MSCs delivery in uterus of mares with endometrosis, which was minimally invasive and technically simple.

Changes in Expression Pattern of Selected Endometrial Proteins following Mesenchymal Stem Cells Infusion in Mares with Endometrosis

Mesenchymal stem cells (MSCs) due to their self-renewal potential and differentiation capacity are useful for tissue regeneration. Immunomodulatory and trophic properties of MSCs were demonstrated suggesting their use as medicinal signaling cells able to positively change local environment in injured tissue. Equine endometrosis is a progressive degenerative disease responsible for glandular alterations and endometrial fibrosis which causes infertility in mares. More precisely, this disease is characterized by phenotypic changes in the expression pattern of selected endometrial proteins. Currently, no effective treatment is available for endometrosis. Herein, we aimed at the evaluation of expression pattern of these proteins after allogeneic equine adipose tissue-derived multipotent mesenchymal stem cells (eAT-MSCs) infusion as well as at testing the capacity of these cells to promote endometrial tissue remodeling in mares with endometrosis. eAT-MSC (2×107/animal) were transplanted into mares’ uterus and control animals received only placebo. Uterine biopsies were collected before (day 0) and after (days 7, 21 and 60) cells transplantation. Conventional histopathology as well as expression analysis of such proteins as laminin, vimentin, Ki-67-antigen, α-smooth muscle actin (α-SMA) and cytokeratin 18 (CK18) have been performed before and after eAT-MSCs transplantation. We demonstrated that eAT-MSCs induced early (at day 7) remodeling of endometrial tissue microenvironment through changes observed in intra cellular and intra glandular localization of aforementioned proteins. We demonstrated that eAT-MSCs were able to positively modulate the expression pattern of studied secretory proteins as well as, to promote the induction of glandular epithelial cells proliferation suggesting local benefits to committed endometrial tissue environment after eAT-MSCs transplantation.

Uterine nitric oxide levels and isofluopredone treatment effect in mares susceptible to persistent post-breeding endometritis

Transient endometritis is a normal consequence of breeding and results from uterine contamination with both semen and bacteria. The modulation of the inflammatory response with the use of isoflupredone has been proposed as efficient for the treatment of endometritis by increasing pregnancy rates. The aim of the current study was to determine the effects of isoflupredone on nitric oxide (NO) levels in uterine samples from mares susceptible to persistent postbreeding endometritis, presenting or not the infectious process. Seven consecutive estrous cycles were induced in 11 mares, being the first one used as control (no treatment). All mares were submitted to the following four treatments: treatment 1: control, treatment 2: glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days, treatment 3: infected treatment (intrauterine infusion of 1x109 CFU/ml Streptococcus equi subsp. zooepidemicus), treatment 4: combination of GC + infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected by flushing and NO was determined. After nitrate reduction, total nitrite was determined by spectrophotometer. No significant differences on nitric oxide concentration were verified by analysis of variance in the different experimental groups. It is concluded that the use of isoflupredone did not alter the nitric oxide concentration in uterine flushing’s from susceptible mares 12 h after treatment.