Ischemic preconditioning effect in autologous transplantation of rat ovaries

• Grant number: 07/00394-8
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: May 01, 2007
• End date: March 31, 2009
• Field of knowledge: Health Sciences - Medicine - Surgery
• Principal Investigator: Edna Frasson de Souza Montero
• Grantee: Luciana Lamarão Damous
• Host Institution: Departamento de Cirurgia. Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Ischemic lesion and reperfusion is the main cause of organ transplant failure. Numerous experimental studies have shown that ischemic preconditioning has a beneficial effect in ischemic lesions and reperfusion in different organs. In respect to ovarian transplant, women in fertile reproductive ages have been submitted to cytotoxic treatments that are inversely associated with negative effects of fertility. Experimental studies have shown that ischemia that occurs during transplant prior to revascularization is the main factor responsible to the loss of a great quantity of follicular population, about 35 to 50% of primordial follicules. The objective of this research is to study the effect of ischemic preconditioning in autologous transplant of ovaries in rats. Methods: This project has been aproved by the Research and Ethics Committee of the "Escola Paulista de Medicina" under protocol 1327/2006. In this project, 70 adult rats Wistar EPM-1 will be utilized, distributed in seven groups with ten animals in each group: 1) Control Group (CG): unilateral oophorectomy; 2) Oophorectomy Group (OG): bilateral oophorectomy; 3) Fresh Transplant Group (FTG): bilateral oophorectomy followed by unilateral fresh ovarian transplant; 4) Cryopreserved Transplant Group (CTG): bilateral oophorectomy followed by unilateral cryopreserved ovarian transplant; 5) Ischemic preconditioning Group (IPG): ischemic preconditioning; 6) Ischemic preconditioning Group + oophorectomy + transplant of fresh ovary (IPG + TF): ischemic preconditioning followed by bilateral oophorectomy and fresh ovarian transplant; 7) Ischemic preconditioning Group + oophorectomy + transplant of cryopreserved ovary (IPG + TC): ischemic preconditioning followed by bilateral oophorectomy and fresh ovarian transplant. The animals will be anesthetized with intramuscular ketamine and xylazine. The ischemic preconditioning will be done by clamping of the ovarian artery for ten minutes of ischemia followed by ten minutes of reperfusion, as indicated in the group. Fresh ovarian transplant: After oophorectomy, the ovary will be immediately fixed, ortotopically at he level of the right uterine corn. Cryopreserved ovarian transplant: Through the following groups, unilateral oophorectomy will be realized, using previously cryopreserved ovaries 24 hours to transplant, by means of dimethyl sulfoxide as a cryopreservant and placed in a freezer with programmed freezing temperature. In teh groups previous to oophorectomy, ischemic preconditioning will be realized as described anteriorly. After 24 hours, a second laparotomy will be conducted. On the second day after surgery, ovarian functions will be evaluated by means of vaginal smears each two days during the entire experiment, for the analyses of estral cycle, through the obtained material with application of saline solution of 0,9% at the vagina. Follicule stimulant hormonal (FSH) and estradiol dosage will be collected during the experiment in pre stablished periods of time by the pilot protocol. After 21 days of transplant, a new laparotomy will be done on the previous scar and after locating the ovarian graft, which will be dried up and fixed on formol at 10% for the histological studies with hematoxylin-eosine. After collecting the samples, the animals will be subjected to euthanasia by means of lethal dose of medications. Morphological and morphometric analyse, imunohistoquimic by PCNA, myeloperoxidase dosage and apoptosis evaluation through 3-anti caspase clivage antibody and keratin 18 will be done. The datas obtained will the subjected to statistical analyses. (AU)