The effect of ovarian steroid deficiency on regeneration of oviductal mucosa following reconstructive surgery

In patients with distal tubal occlusion a microsurgical oviductal reconstruction is, apart from the in vitro fertilization, the only treatment option. Unfortunately, the results of reconstructive surgery are often unsatisfactory. The effects of sex steroids on the regeneration process after reconstructive surgery have not been well investigated. This study was aimed to evaluate the effect of decreased concentrations of ovarian sex steroids (castration) on regeneration of the oviduct mucosa after the reconstructive surgery of distally occluded oviducts. The study was performed on 32 female rabbits that underwent unilateral oviduct ligature and resection of fimbriae. The occlusion lasted six (group I) or twelve weeks (group II). After this time the animals were re-operated, and allocated into 4 groups: castration with reconstructive surgery (IA, IIA), reconstructive surgery only (IB, IIB). After next six or twelve weeks the fallopian tubes were examined under light, scanning and transmission electron microscopes. An immunohistochemical reaction for Ki-67 proliferative antigen was also performed. Ovarian steroid levels were evaluated by radioimmunoassays. The castrated animals had significantly lower levels of estradiol, progesterone and 17-hydroxyprogesterone than the control groups. Long lasting tubal occlusion caused pronounced histological changes of tubal mucous membrane (group II). In the rabbits with preserved ovaries and twelve-week long oviductal occlusion (group IIB), the regeneration of the distal end and restoration of fimbria were not complete twelve weeks after microsurgical reconstruction. In castrated animals with long-lasting occlusion (group IIA) the destructive changes, found in the mucosa of tubal ampullas of occluded oviducts before reconstruction, were still present and even intensified twelve weeks following reconstructive surgery. The castration hampered proliferation of the mucosa cells, thus no fimbriae were restored. Low levels of ovarian steroids were found to have adverse effect on fallopian tube regeneration following reconstructive surgery. The effect was noted even in cases with minor preoperative fallopian tube damage. Therefore, the treatment of concomitant endometriosis or uterine fibroids with GnRH analogues should not be recommended simultaneously with microsurgical tubal reconstruction.     

Rat ear reattachment as an animal model

The external ear of the rat is an excellent model for practicing microsurgical dissection and for the refinement of microvascular anastomoses, techniques that are crucial for microvascular en bloc tissue transfer and replantation. Preparation of the rat ear for replantation requires familiarity with the vascular anatomy and gentle tissue handling with atraumatic dissection of arterial and venous pedicles, steps similarly crucial in raising free flaps for microvascular transfer. The strategy of performing accurate reduction and stabilization of the tubal cartilage prior to vessel repairs, anastomosing the more deeply seated external carotid artery prior to the more superficial posterior facial vein, is as critical to rat ear replantation as for digital reattachment. In addition, the rat ear as compared to other animal models such as the rabbit ear or canine hindlimbs is much less expensive. Compared to the rat hindlimb model, rat ears are much easier to observe, which is a distinct advantage when used as a model for long-term study of replantation, revascularization, or transplantation.     

Evaluation of the hormonal function and histological features of heterotopic isogenic ovarian transplantation in rats

The purpose of the study was to determine the feasibility of preserving ovarian function after heterotopic transplantation by means of microvascular anastomosis of the transplanted vascular pedicles to a set of preselected vessels. Six groups of 10 Sprague-Dawley inbred rats were used in this study. Group I underwent bilateral ovariectomy operation and served as the ovariectomy control. Group II underwent bilateral ovariectomy followed by heterotopic isogenic ovarian implantation. Group III underwent bilateral ovariectomy and isogenic heterotopic ovarian transplantation by means of microvascular anastomosis. Group IV served as the laparotomy sham-operated control. Group V served as the ovarian donor for group II. Group VI served as the donor of the ovarian-kidney vascular pedicle complex for group III. Postoperative ovarian estradiol levels were measured, and histological characteristics were elucidated in groups I, II, III, and IV. The results demonstrated that the estradiol level of the transplantation group was comparable to that of the sham operation group and was significantly higher than that of the implantation group. Histologically normal ovarian architecture was observed in the sham group (IV) and also in the transplantation group (III). Altered architecture was observed in the implantation group (II). These findings indicate that extraabdominal heterotopic ovarian transplantation with microvascular anastomosis led to normal ovarian hormonal function and was effective in preserving oocyte production capacity.     

Time course of angiogenesis in solid pineal autografts

Angiogenesis and reperfusion of blood vessels were analysed qualitatively, at the light- and electron-microscopical levels, in solid pineal autografts placed intracerebrally in adult rats (post-transplantation survival times: 1, 3, 7, 10, 14 and 28 days). Reperfusion of blood vessels was studied in sections from immersion-fixed brains incubated to demonstrate the endogenous peroxidase activity of erythrocytes within the lumen of blood vessels. The possible presence of the blood-brain barrier (BBB) within the grafts was also investigated by injecting native horseradish peroxidase (HRP) intravenously into the rats. Angiogenesis, the morphological and functional properties of blood vessels vascularizing the grafts and the survival of pineal tissue were analysed ultrastructurally following transplantation. Revascularization of pineal autografts placed into the adult host central nervous system occurred very slowly, requiring 7–10 days to establish anastomoses between graft and host blood vessels. During this process, signs of angiogenesis in pineal and cerebral capillaries were evident, suggesting that both contributed to graft revascularization. Morphological and functional studies with HRP revealed that, following transplantation, blood vessels at the graft-host interface or within pineal autografts maintained their morphological and functional properties: they were fenestrated and did not present a BBB to blood-borne peroxidase. Thus, after grafting, the presence or absence of the BBB is graft-determined. Revascularized pineal tissue showed good survival and pinealocytes revealed structural features of active secretory cells.     

Combined transplantation of free tissues

Combined transplantation of free tissues is a new microsurgical technique by which, with only one set of vessels supplying blood, two or more free tissues can be transplanted simultaneously. Very large soft-tissue defects that are not amenable to conventional transplantation, or defects of two or more tissues, either similar or different in nature, can be repaired in a one-stage operation. It is accomplished by vascular combination; i.e., by means of anastomosing the corresponding vessels of their pedicle, the free tissues to be transplanted are reconstituted into an assembly with only one common vascular pedicle which is then rejoined to the vessels selected to supply blood to the grafts in the recipient site. From December of 1983 to July of 1985, the author has performed 17 combined free-tissue transplantations of seven different clinical types for microsurgical repair and reconstruction of extremities. All the transplanted parts survived, and the extremities regained very good function. Seven patients are reported individually in the paper, each representing a definite clinical type. The concepts and operative technique introduced and the indications and advantages of the newly designed procedure are discussed.     

Latent antithrombin does not affect physiological angiogenesis: an in vivo study on vascularization of grafted ovarian follicles

Latent antithrombin (L-AT), a heat-denatured form of native antithrombin (AT), is a potent inhibitor of pathological tumor angiogenesis. In the present study, we have investigated whether L-AT has comparable antiangiogenic effects on physiological angiogenesis of ovarian tissue. For this purpose, preovulatory follicles of Syrian golden hamsters were mechanically isolated and transplanted into dorsal skinfold chambers chronically implanted in L-AT- or AT-treated hamsters. Non-treated animals served as controls. Over 14 days after transplantation neovascularization of the follicular grafts was assessed in vivo by quantitative analysis of the newly developed microvascular network, its microvessel density, the diameter of the microvessels, their red blood cell velocity and volumetric blood flow as well as leukocyte-endothelial cell interaction using fluorescence microscopic techniques. In each group, all of the grafted follicles were able to induce angiogenesis. At day 3 after transplantation, sinusoidal sacculations and capillary sprouts could be observed, finally developing complete glomerulum-like microvascular networks within 5 to 7 days. Overall revascularization of grafted follicles did not differ between the groups studied. Interestingly, follicular grafts in L-AT- and AT-treated hamsters presented with higher values of microvessel diameters and volumetric blood flow, when compared to non-treated controls, which may be best interpreted as a reactive response to an increased release of vasoactive mediators. In conclusion, the present study demonstrates, that L-AT has no adverse effects on physiological angiogenesis of freely transplanted ovarian follicles. Thus, L-AT may be an effective drug in tumor therapy, which blocks tumor growth by selective suppression of tumor vascularization without affecting new vessel formation in the female reproductive system.     

Revascularized periosteal grafts--a new method to produce functional new bone without bone grafting

Rib periosteum was transplanted to the groins of 9 dogs. In half of the periosteal grafts, no microvascular anastomoses were done (free grafts); at 6 weeks after grafting they had become resorbed. The other periosteal grafts were revascularized by microvascular anastomoses of the intercostal vessels to local muscular vessels; at 6 weeks those with confirmed vascular patency had all formed substantial amounts of new bone. Five cm, full-thickness defects were created in the tibias of 10 dogs. The control animals (without grafting) did not heal in two months. However, the experimental dogs, with vascularized periosteal grafts in the defects regenerated their tibias with healthy new bone by 6 weeks--and were walking on them then.     

Factors involved in the testicular development from fetal mouse ovaries following transplantation

We have previously shown that fetal mouse ovaries develop testicular structures after transplantation into adult male mice. The mechanisms of gonadal sex reversal is poorly understood. In the present study, we examined how a host environment is involved in the induction of testicular development in ovarian grafts. Fetal ovaries on the twelfth day of gestation were microencapsulated with semipermeable membranes, transplanted beneath the kidney capsules of adult male mice, and fixed for histological examinations between the sixteenth and twenty-second day after transplantation. Fifteen of forty-seven ovarian grafts were found to be completely enclosed in microcapsules, whereas the microcapsule membranes of other grafts were partly broken or had been lost. These differences of microencapsulation conditions made it possible to study the role of host factors in gonadal sex reversal. All ovarian grafts surrounded by microcapsule membranes developed ovarian structures. In contrast, most ovarian grafts which had lost the microcapsules developed testicular structures in addition to ovarian structures. When ovarian grafts were partially enclosed in microcapsule membranes, testicular structures developed only in the area in contract with the host kidney. These results suggest that direct interaction between the ovarian graft and cells or large macromolecules from the host is involved in the development of testicular structures in ovarian grafts.     

Changes of the dermal-fascial autograft after transplantation on the vascular-nervous connections using a microsurgical technique

In 56 dogs 86 microsurgical operations on transplantation of free dermal-facial autografts from the internal knee surface have been performed on the hidden vascular-nervous bundle. The animals have been observed for 1 day up to 1 year. The implanted grafts (63) have been studied, using a complex of anatomical, histological and roentgenological methods. During early time (up to 7 days) after the operation in the flap signs of edema, dystrophy and inflammatory infiltration of tissues predominate. The graft gets blood at the expense of the restored main artery and has no vascular connections with the surrounding tissues. Its nervous conductors are fragmented. During 2 weeks--1 month epidermis completely regenerates along the line of the dermal suture. In the flap bed mature granulations result in vascular connections with its surrounding tissues. These connections become stable by the end of the first month, this means that the graft has implanted. Its nervous fibers are also restored. Long-term observations demonstrate that the adaptive changes of the flap and its vascular bed are near to completion. By the end of the 1st year restoration of the main innervational connections of the graft takes place. According to the data obtained, the nervous conductors grow into it along the sewed hidden nerve and along the course of paravasal nerve plexuses. Across the scar from the surrounding tissues the dermal-fascial autograft does not reinnervate.     

Comparison of lymphatic and venous interpositional autografts in experimental microsurgery of the canine lymphatics

In mongrel dogs, 56 autologous lymphatic and vein grafts were interpositioned to bridge a defect in the femoral collecting lymphatics. In one group, 26 lymphatic autografts were interpositioned with good results. No obstruction was observed over 6 months. In another group, 20 venous autografts were interpositioned after irrigation with heparinized saline and another 10 autografts were interpositioned without irrigation. After 1 week, four irrigated grafts were partially occluded with a red thrombus; after 6 months, all grafts were totally occluded. In a third group, 15 lymphaticolymphatic anastomoses were enveloped by a silicone sheet to provoke prolonged devascularization. None of the vessels was patent. Anastomotic patency was inspected in vivo postoperatively. The specimens were studied with light microscopy and scanning and transmission electron microscopy. Prolonged devascularization damaged the endothelial cells. The results show that the lymphatic vessel autograft is the best choice for an interpositional autografting to bridge a defect in lymphatic vessels.     

Arterial repair after microvascular anastomosis. Scanning and transmission electron microscopy study

In order to study the morphological aspects of endothelial regeneration and vascular wall reaction after microvascular anastomosis, rat femoral arteries were sectioned and successively sutured (end-to-end anastomosis) with microsurgical techniques. Control arteries and anastomosed vessels (recovered after 1, 4, 7, 14, 21, 30, 60, 120, 180 and 360 days) were studied by means of scanning (SEM) and transmission electron microscopy (TEM). The reendothelialization phenomena started after 7 days and were mainly evident at 21 days. Areas of subendothelial connective tissue with fibrin deposition remained exposed to the blood stream up to 21-30 days. Thrombus formations or post-anastomotic stenosis have been occasionally observed. Regenerating endothelium showed evident morphological differences from the control. These changes mainly consisted of shortened cell length, absence of pinocytotic vesicles, presence of cytoplasmic prolongations, and microvillous proliferations. The arterial wall showed subintimal thickening. The anastomotic site appeared completely covered by new endothelium after 30-60 days. Subintimal vascular wall changes (thickening of the media) as well as slight alterations of endothelial cells (shortened length, reduced number of pinocytotic vesicles) were evident in 60-day vessels. Lumen reduction, due to the protruding of endothelial-covered sutures, was occasionally observed in 60- to 120-day arteries. Endothelial cell morphology normalized after 60-120 days. However, thickening of the media and occasional lumen reduction were observed also after 180-360 days. Although the endothelial regeneration phenomena were clearly evident after 2 weeks, nevertheless the reestablishment of arterial wall took longer time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biology of micro-vascularized cartilaginous transplantation in the growing dog

Epiphyseal bone transplantations including growth area have been extensively studied experimentally in different animals and applied in children for several decades. Results were unpredictable and the amount of growth obtained with the graft could get 50 per cent of the normal. Development of microsurgical techniques has determined new possibilities of epiphyseal transfer with the respect of the epiphyseal vascularization. Growth plate germinal cell division is directly related to the epiphyseal vascular respect. In a large multifacetted program we could successively determine the vascular patterns of the donor site areas and then utilized different grafts in some experimental situations reproducing the clinical pathology in children, like hip dysplasia or epiphyseal defects after resection for tumor or infection. Results demonstrate that the main problem, in epiphyseal transplantation, is not vascular but mechanical. Further studies have to be done in this direction.     

Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts

Cancer patients, treated by either chemo- or radiotherapy, frequently suffer from ovarian failure and infertility. One of the new emerging techniques to preserve reproductive potential of such patients is cryopreservation of ovarian fragments prior to treatment and their retransplantation after healing. A major obstacle in survival of the ovarian implants is vascular failure, which leads to tissue necrosis. In order to investigate the role of angiogenesis in implant preservation, we used a xenograft model in which rat ovaries were transplanted into immunodeficient mice. Graft reception and maintenance were monitored by magnetic resonance imaging (MRI) and histology. Two transplantation sites were explored, i.e., subcutaneous and intramuscular. Comparison between these two transplantation sites revealed the importance of vascular smooth muscle cells and pericytes in sustaining vascular and tissue integrity. Histological examination of the grafts, at different time points and sizes, revealed that loss of perivascular cells preceded damage to endothelial cells and was closely correlated with loss of follicular and oocyte integrity. Intramuscular implantation provided better maintenance of implant perivascular cells relative to subcutaneous implantation. Accordingly, follicular integrity was superior in the intramuscular implants and the number of damaged follicles was significantly lower compared with the subcutaneous transplantation site. These results suggest that improving ovarian implant maintenance should be directed toward preservation of perivascular support.     

Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs - amputated digits - xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts

Ovarian cortical tissue cryopreservation with subsequent autografting is a potential strategy for the preservation of fertility in patients undergoing systemic chemotherapy and pelvic radiotherapy. Non-vascular implants are first subjected to a period of ischaemia before revascularization and are, therefore, vulnerable to ischaemia-reperfusion injury from reactive oxygen species. Ischaemia-reperfusion injury was investigated during the first week after surgery in murine ovarian grafts and human ovarian xenografts in mice with severe combined immune deficiency (SCID) by measuring total lipid peroxides and malondialdehyde concentrations with a colorometric assay. The effects of administering an antioxidant, vitamin E, on these concentrations were also tested. Products of lipid peroxidation were higher in non-supplemented murine autografts compared with control ovaries (P < 0.05), and were significantly reduced on day 3 by vitamin E administration (P < 0.05). Similarly, in human xenografts, there was a significant reduction in lipid peroxidation with vitamin E administration. These results correspond to a significantly greater total follicle survival in the murine grafts of the supplemented group (45 versus 72%; P < 0.05). They suggest that antioxidant treatment improves the survival of follicles in ovarian grafts by reducing ischaemia-reperfusion injury.     

Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs—amputated digits—xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Utility and diagnostic accuracy of fallopian tube touch imprint cytology

OBJECTIVE: To determine the diagnostic accuracy of cytology smears in distinguishing between tube and non-tube structures. METHODS: One hundred cytology smears of fallopian tube and non-tube structures (vessels, round and ovarian ligaments) were prepared from surgically removed uterus and fallopian tube specimens and stained by the Papanicolaou method. The slides were reviewed blindly by pathologists and interpreted as tube or non-tube structures. The results were compared to the histological examination of the same specimens. FINDINGS: Results indicated an overall accuracy of 97% with a specificity of 98% and sensitivity of 96% for cytology smears, taking histology as the gold standard. Positive and negative predictive values were 96.1% and 97.9%, respectively. CONCLUSION: Cytology smears are a convenient and cost effective tool for laboratory confirmation of tubal sterilization. This method can reduce the costs of laboratory examination, especially in developing countries, where tubal sterilizations are done in large cohorts. However, histological slides remain the gold standard in cases of medicolegal problems.     

Microsurgery: review of the literature and discussion of microtechniques

This review of microsurgery considers the current status of both experimental and clinical uses of microsurgery in the fields of otolaryngology, ophthalmology, gynecology, the central and peripheral nervous systems, the vascular system and transplantation. A report of studies on the microcirculation of blood vessels and nerves demonstrated the usefulness of mignification for research studies and emphasized certain factors which could be significant in selecting the best techniques for repairing nerves and blood vessels. Techniques of repair are included, for they have been changed on the basis of experimental studies. Consideration has been given to the advantages of magnification in identification and removal of tumors and vascular anomalies. A report on the use of microtechniques for repairing fallopian tubes is included. It provides a basis for reviewing the problems associated with the repair of small tubes and ducts. The need for microtechniques should increase as medical progress calls for greater precision for the fields of reparative, reimplantation and transplantation surgery.     

Angiogenesis after sintered bone implantation in rat parietal bone

We studied the effect of bone substitutes on revascularization and the restart of blood supply after sintered bone implantation in comparison with synthetic hydroxyapatite implantation and fresh autogenous bone transplantation (control) in rat parietal bones. Methods for the study included the microvascular corrosion cast method and immunohistochemical techniques were also used. The revascularization of the control group was the same as that for usual wound healing in the observations of the microvascular corrosion casts. The sintered bone implantation group was quite similar to that of the control group. In the synthetic hydroxyapatite group, immature newly-formed blood vessels existed even on the 21st day after implantation and the physiological process of angiogenesis was interrupted. Immunohistochemically, vascular endothelial growth factor (VEGF), which activates angiogenesis, appeared at the early stages of both the control group and the sintered bone implantation group. VEGF reduced parallel with the appearance of the transforming growth factor factor-beta-1 (TGF-beta-1), which obstructs angiogenesis, and the angiogenesis passed gradually into the mature stage. In the hydroxyapatite implantation group, TGF-beta-1 appeared at the early stage of the implants. The appearance of VEGF lagged and it existed around the pores of hydroxyapatite even on the 21st day of the implantation. Proliferation and wandering of endothelial cells continued without any maturing of the vessels. These findings suggest that the structure and the components of the implant material affect angiogenesis after implantation as well as new bone formation.