Remote Ischemic Preconditioning on Neovascularization and Follicle Viability on Ovary Autotransplantation in Rats L.L. Damous, S.M. Silva, R.S. Simões, R.J. Morello, A.P.F. Carbonel, M.J. Simões, and E.F.S. Montero ABSTRACT Purpose. Verify the optimum remote vascular occlusion time to reduce ovarian injury in autologous transplants in rats. Methods. Twenty-four adult female rats were assigned to four groups: GC (control group): bilateral oophorectomy followed by ovary transplant; GIPC (ischemic preconditioning group): remote ischemic preconditioning at the iliac artery for 5, 10, and 15 minutes (GIPC-5, GIPC-20, and GIPC-15) previous to bilateral oophorectomy and ovarian transplantation. The right ovary was fixed in the retroperitoneum. Euthanasia was performed 4 days after the surgical procedure. The follicles were counted and classified as developing versus atretic. The immunohistochemical assay identified vascular factor of endothelial growth (VEGF) in the ovarian stroma and assessed the proliferation capacity by means of the Ki-67 in the ovarian follicles. Results. Every group showed an inflammatory infiltrate, luteous body, and ovarian follicles in several phases of development. The ischemic preconditioning groups displayed greater amounts of viable ovarian follicles and increased vascularization and vasodilatation than the control group. GIPC-15 showed the highest amount of viable follicles compared to the others (P ⬍ .05 GIPC-15 vs GC; GIPC-15 vs GIPC-5). More VEGF-labeled cells were observed in GIPC-10 than the control group (P ⬍ .05, GIPC-10 vs GC). The proliferation index assessed by Ki-67 marking showed GC: 80%; GIPC-5: 76%; GIPC-10: 67%; and GIPC-15: 64% (P ⬎ .05). Conclusions. The PCI-15 cohort seem to be the most adequate timing to achieve functional support and preservation of a greater number of viable ovarian follicles.
D
UE TO A REPERCUSSION on the fertility and induction of a early ovarian failure,1,2 auto transplantation of the ovary is a resource employed for women in their reproductive age undergoing cytotoxic treatments, whether by radio or chemotherapy for treatment of several malignant or immunosuppressive diseases. These treatments are as aggressive as they are effective, causing a significant increase in the patient survival, But their quality of life after the transplant has been an important point of discussion. It has been reported that 34% of patients with cancer who received conventional chemotherapy show ovarian failure. This finding has also been observed in 92% of patients who received radio- or chemotherapy print to a bone marrow transplant.3 In contrast to the transplantation of other organs in which the surgically performed vascular anastomosis is a major factor for functional preservation, the transplanta© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 861– 864 (2008)
tion of the ovary depends mainly on the early development of a rich vascular web in an early posttransplant stage.4 – 8 Today, it is known that before neovascularization, the graft is vulnerable to the ischemia and reperfusion (I/R) lesion which is the major factor that determines its viability. Studies in sheep6 and in human4 transplantations have shown that 35% to 50% of the ovarian follicles are lost due
From the Department of Surgery (L.L.D., S.M.S., R.J.M., E.F.d.S.M.); and the Department of Morphology (A.P.F.C., M.J.S.), Federal University of São Paulo; and the Department of Gynecology (R.S.S.), University of São Paulo, São Paulo, Brazil. Grant support: The State of São Paulo Research Foundation, Process numbers 2007/00394-8 and 2007/00107-9. Address reprint requests to Edna Frasson de Souza Montero, Al Espada, 134 –Res. Onze, Alphaville 06540-395, Santana de Parnaı´ba, São Paulo, Brazil. E-mail:
[email protected] 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.02.065 861
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to cryopreservation. Thus, appropriate and early vascularization is essential for the functional support of the ovary. Also mandatory is the development of reproducible measures that attenuate the damage caused by initial I/R to the organ. Dissen et al9 showed that ovary transplantations in rats become richly vascularized at 48 hours after transplantation. The process is accompanied by increased expression of genes that codify angiogenic factors, such as vascular factor of endothelial growth (VEGF). During the revascularization period, an intense cellular lesion occurs mainly due to the production of reactive oxygen species due to I/R. Nugent et al10 studied the protective effect of vitamin E on the damage caused by I/R in ovarian transplantations. They observed that previous antioxidant treatment increased the follicles survival. In addition to the use of substances, new surgical techniques have been investigated: graft implant in areas rich in granulation tissue11 and creation of a peritoneal window as the transplant site.12 Ischemic preconditioning (IPC), which was first described by Murry et al13 in an experimental study of the myocardium of dogs, is defined as the beneficial effects of a short period of ischemia followed by reperfusion upon subsequent prolonged or maintained ischemia. Both experimental and clinical studies have confirmed that IPC produces a protective response to I/R lesions in several organs and tissues with consequent increase in organ tolerance to hypoxia.14 –18 IPC involves two protective mechanisms: one in the acute phase, which is independent of protein synthesis, and one in the later phase, which is characterized by the synthesis of antioxidant enzymes.19 The beneficial effects may be related to other factors, such as the glycolytic cascade, in which nitric oxide inhibits the activity of a series of enzymes, resulting in a decreased production of lactate, causing what is called the protective mechanism.20 Afterward, Liauw et al21 introduced the remote IPC (R-IPC) concept, based upon the observation that the ischemia of a muscular group protected the contralateral member from an I/R lesion. Subsequent studies confirmed the existence of protection in different organs, distant from the protection of the organ subject of the study.22,23 Little is known about the mechanism of action of R-IPC, but it is believed that there is a suppression of the inflammatory response, similar to what happens with IPC, among other yet unknown mechanisms. The R-IPC strategy in ovarian transplantation has became a viable alternative, since it has a vascular pedicle of reduced caliber that has the possibility to undergo spasm with consequent sustained ischemia and loss of the protective IPC effect. Due to the facts that organs have different sensitivities to I/R and that there are no previous R-IPC papers on ovaries, we developed this work to verify the optimum remote vascular occlusion time to achieve ovarian protection in autologous transplants in rats.
DAMOUS, SILVA, SIMÕES ET AL
METHODS The experimental procedures were performed according to the ethics principles of the Brazilian Bureau of Animal Experimentation after approval by our Ethics Committee under protocol number 1327/2006. The 24 female EPM-1 virgin and adult Wistar rats were 3 to 4 months old. they were allocated to four groups containing six animals each: GC (control group): animals undergoing bilateral oophorectomy followed by ovary transplant; GIPC (ischemic preconditioning group): animals submitted to R-IPC at the common iliac artery for a period of 5, 10, and 15 minutes prior to bilateral oophorectomy and ovarian transplantation (GIPC-5, GIPC-20, and GIPC-15). All animals were weighed and received anesthesia by intramuscular injection of ketamine (80 mg · kg⫺1) and xylazine (5 mg · kg⫺1) and were kept under spontaneous breathing in environmental air during the surgery. After the trichotomy and antisepsis with polyvinylpyrrolidone-iodine solution, a median laparotomy was performed with inventory of the abdominal cavity.
Ischemic Preconditioning The R-IPC was performed by clamping the common right iliac artery using two vascular microclamps for a preset period, according to the subgroup: 5, 10, or 15 minutes for GIPC-5, GIPC-10, GIPC-15, respectively, before undergoing the other procedures (oophorectomy and transplantation).
Oophorectomy Following ovaries and their blood supply identification, the mesoovary was sectioned between 8-0 nylon suture threads.
Autologous Transplantation of the Ovary After oophorectomy and washing with 0.9% saline solution, the right ovary was fixed onto the retroperitoneum using 8-0 prolene with no vascular anastomosis, under a 16⫻ microsurgical microscope. The abdominal wall was closed in two planes: peritoneumaponeurotic-muscle and skin, both with 6-0 monofilamentary nylon thread. Euthanasia was performed 4 days after transplantation. Ovarian graft was removed surgically from the retroperitoneum, fixed in formalin solution for 12 hours, and stained by hematoxylin-eosin. The follicles were counted as developing follicles (regardless of the phase) versus atretic follicles. The silconized slides were submitted to immunohistochemistry to identify VEGF in the ovarian stroma and to assess the proliferative capacity by means of Ki-67 in ovarian follicles. VEGF expression was classified following the color intensity in the field: absent or 0 (⬍10%), poor or 1 (10% to 25%), moderate or 2 (25% to 50%), and strong or 3 (⬎50%). The proliferative capacity was quantitatively assessed through counting the amount of cells marked in the ovarian follicle by 1000 cells. The proliferation index was calculated as the amount of dead cells by the Ki-67 per 1000 cells. The data were submitted to statistical analysis, using the KruskalWallis test for the follicular counting and the Bonferroni for the immunohistochemistry.
RESULTS Morphology
In all studied groups we observed an inflammatory infiltrate, luteous body in degeneration, and ovarian follicles in
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several phases of development. GIPC-5 and IPC-10 showed more intense inflammatory infiltrates than GC. GIPC-15 had a less intense inflammatory infiltrate compared to the other groups. Compared with the control group, the IPC groups displayed greater amounts of viable ovarian follicles and increased vascularization (neovascularization) and vasodilatation. The GIPC-15 had the greatest number of viable follicles compared with the other groups; the follicle quantity was increased with the timing of the ischemic preconditioning (Table 1). Similar to the results in Table 1, we observed increased VEGF with the time of ischemia. The immunohistochemistry for VEGF, performed by analyzing the cells per field, showed that group GIP-10 had greater staining (3), followed by GIP-15 (2.5), GIP-5 (2.25), and GC (2.0) (P ⬍ .05, GIP-10 vs GC). The proliferation index obtained by Ki-67 staining showed proliferation activity in every group: GC: 80%; GIPC-5: 76%; GIPC-10: 67%; and GIPC-15: 64%. However, there was no significant difference (P ⬎ .05). DISCUSSION
The I/R syndrome observed in transplants or after a surgical trauma generates free radicals, which induce membrane damage resulting in cellular death and necrosis. Various strategies have been proposed to protect tissues from these injuries, such as hypothermia, antioxidants, inflammatory mediators, adhesion molecules, ischemic preconditioning, or drugs.24 –27 Although there are several experimental transplant models described in the literature to maintain ovarian function, follicular loss during ischemia is remarkable, reaching 50%, with improvement through the use of antioxidant treatments10 or new surgical techniques for graft implantation.11 In those cases, neovascularization is a major event to restore appropriate perfusion, due to the absence of a vascular anastomosis and any kind of treatment for better preservation of the follicular population to prolong the function of the transplanted tissue. Within 48 hours, an ovarian transplant shows a noticeable increase in the gonadotrophin secretion that decreases over 6 days after transplantation, thus reflecting the recovery of ovarian steroidal activity.28 Thus, this study elected to perform euthanasia on the fourth day after the transplantation, since the revascularization process had already been Table 1. Number of Viable Follicles With the Time of Ischemia Group
Viable
Atresic
Total
% viable
Control 5 min 10 min 15 min
9 11 16 24
23 24 29 36
32 35 45 60
28 31 36 40
Kruskal-Wallis test, P ⬍ .05; GC vs GIPC-15, and GIPC-5 vs GIPC-15 (for both viable and atresic follicles). Chi-square test, P ⫽ .6759; to compare the percentage of viable follicles among studied groups.
initiated, coincident with the peak of gonadotrophins that occured after 48 hours, without the decline period of that level. IPC is an important mechanism by which tissues protect themself from an imminent lesion. It is believed that in hypoxic conditions, the expression of the VEGF gene produces an increase in the amount of progenitor endothelial cells. When IPC induces a hypoxic period, it increases endothelial function, increasing such cells and contributing to neoangiogenesis.29 Due to the possibility of ovarian artery spasm along with IPC, thus impeding reperfusion after the initial period of ischemia, we chose to perform R-IPC in the common iliac artery just below its origin at the aorta, always on the right. The presence of ovarian follicles in several developing stages after transplantation was consistent with the literature, reinforcing the fact that the viability of the tissue as well as the proportion of the follicular loss did not interfere with several techniques.4,30 –32 In this study, the group that showed the highest percentage of viable ovarian follicles was GIPC-15; it also revealed the greatest amount of viable follicles in absolute figures, although this result did not show significance (P ⫽ .6759). Likewise, IPC did not show a significant difference among groups; all of them had high proliferation rates, and GC showed higher proliferation activity. These findings reflected the functional support of the transplanted organ, as well as the absence of the deleterious effect of IPC in the early phase. However, further studies may better clarify a this finding. Upon the assessment of the mean values of VEGF expression in each group, GIPC-10 reveales the highest value. However, it does not seem convenient to analyze these data separately to indicate that this is an optimum IPC timing in ovarian transplantation among others, which were studied. These data only showed that it was possible to induce an increase in the neovascularization, thus unchaining better protection without damaging the organ with no major benefits compared to the remaining IPC groups (5 and 10 minutes), which did not show any significant difference. When the data were jointly analyzed, it was noticed that GIPC-15 was the group in which there was a greater amount of viable follicles, with high rates of cellular proliferation in the Ki-67 assays, and early stimulation of neoangiogenesis in the VEGF study, which possibly means that from every timing studied, they attained the most satisfactory results as to the functional preservation of the graft. However, later studies assessing a greater follow-up after transplant, as well as viability in relation to the regularity of the estral cycle and reproductive parameters, may further contribute to corroborate these results. In Conclusion, R-IPC causes repercussions in ovarian transplantation, resulting in increased vasodilatation, neoangiogenesis, and preservation of viable ovarian follicles. The last metric was confirmed both by the morphological analysis and by the proliferation index of Ki-67. All data
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together showed GIPC-15 to be the most adequate timing for functional support and preservation of viable ovarian follicles. REFERENCES 1. Donnez J, Squifflet J, Dolmans MM, et al: Orthotopic transplantation of fresh ovarian cortex: a report of two cases. Fertil Steril 84:1018, 2005 2. Mhatre P, Mhatre J, Magotra R: Ovarian transplant: a new frontier. Transplant Proc 37:1396, 2005 3. Meirow D, Fasouliotis SJ, Nugent D, et al: A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertil Steril 71:948, 1999 4. Newton H, Aubard Y, Rutherford A, et al: Low temperature storage and grafting of human ovarian tissue. Hum Reprod 11: 1487, 1996 5. Aubard Y, Piver P, Cogni Y, et al: Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 14:2149, 1999 6. Baird DT, Webb R, Campbell BF, et al: Long-term ovarian function in sheep after ovariectomy and transplantation autografts stored at ⫺196°C. Endocrinology 140:462, 1999 7. Oktay K, Newton H, Gosden R: Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril 73:599, 2000 8. Liu J, Van der Elst J, Van den Broecke R, et al: Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 17:605, 2002 9. Dissen GA, Lara HE, Fahrenbach WH, et al: Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression. Endocrinology 134:1146, 1994 10. Nugent D, Newton H, Gallivan L, et al: Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil 114:341, 1998 11. Israely T, Nevo N, Harmelin A, et al: Reducing ischaemic damage in rodent ovarian xenografts transplanted into granulation tissue. Hum Reprod 21:1368, 2006 12. Donnez J, Dolmans MM, Jadoul P, et al: Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 364:1405, 2004 13. Murry CE, Jennings RB, Reimer KA: Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124, 1986 14. Clavien PA, Selzner M, Rudiger HA: A propective randomized study in 100 consecuive patients undergoing major liver resection with versus without ischemic preconditioning. Ann Surg 238:843, 2003 15. Selzner N, Rudiger H, Graf R: Protective strategies against ischemic injury of the liver. Gastroenterology 125:917, 2003
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