1 Department of Obstetrics and Gynecology, 2 Department of Pathology and 3 Department of Surgery, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. e-mail: randa.racho@obgyn.gu.se
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Abstract |
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Key words: ischaemia/microsurgery/mouse uterus/preservation/transplantation
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Introduction |
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One crucial aspect in any organ transplantation is the preservation of the graft ex vivo. To slow down the development of ischaemic injuries and lengthen the tolerable ischaemic time between procurement and transplantation, cold ischaemic preservation with a protective buffer is the most common method used. Prolonged cold ischaemia and subsequent reperfusion induces injuries (cold ischaemia/reperfusion injuries; CIRI) which are effectors of delayed graft function and early graft failure (Boom et al., 2000; Nakagawa et al., 2002
). These CIRI are consequences of a complex cascade of events attributed to ischaemia and hypothermia as well as rewarming and reperfusion.
To reduce the injuries caused by cold ischaemia and reperfusion, the vascular system of the explant is flushed with and stored in a preservation solution of intra- or extracellular-like electrolyte compositions. Beside electrolytes, most preservation solutions also contain additives such as colloids, antioxidants, metabolic precursors, chelators of calcium and iron, as well as vasodilators (Muhlbacher et al., 1999; Huddleston et al., 2000
). The most widely used solution for storage of solid organs in clinical transplantation procedure is Belzer University of Wisconsin preservation solution (UW), which has an intracellular-like electrolyte composition. In several experimental and clinical studies UW has proven to be efficient in tissue preservation as exemplified by tests on cultured endothelial cells (Alamanni et al., 2002
), kidney (Faenza et al., 2001
), pancreas (Uhlmann et al., 2002
), liver (Jassem et al., 2000
), lung (Kawahara et al., 1991
) and heart (Masters et al., 1994
; Wildhirt et al., 2000
). No studies exist on cold preservation of the uterus but there are some studies on other genital organs such as the ovary. The complete ovary of the ewe was cryopreserved and replanted with normal ovarian function (Bedaiwy et al., 2003
). Moreover, mouse ovarian tissue was preserved in vitro at 4°C for periods of
48 h (Snow et al., 2001
). The current study aimed to evaluate the tolerance of the murine uterus to cold ischaemia by utilizing our recently developed model for uterine transplantation in the mouse (Racho El-Akouri et al., 2002b
).
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Materials and methods |
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Surgery
The surgical procedures were performed essentially as previously described in detail (Racho El-Akouri et al., 2002b) and are only briefly outlined below. The major difference is that the cervix of the transplanted uterus was attached to a cutaneous stoma and not placed intra-abdominally. The animals were spontaneously cycling and the estrus cycle stage was not determined at surgery. Under isoflurane (2%) anaesthesia, the right uterine horn and the cervix with its feeding and draining vessels were isolated. Several major vessels (inferior mesenteric vessels, inferior epigastric vessels, left common iliac vessels, caudal artery, the external iliac vessels, and the pudendal vessels) were cauterized and cut.
The cervix was then transected below its attachments to the bladder. The lumbar vessels were cauterized and the aorta and vena cava were separated from the point of branching of the renal vessels and 34 mm caudally. A ligature was placed just above the tip of the right uterine horn and the uterus was separated from the oviduct and dissected free from the dorsal peritoneum. The aorta and the vena cava were tied off inferior to the branching of ovarian vessels and the uterus was first flushed with ice-cold 0.154 mol/l NaCl supplemented with heparin sulphate (100 IU/ml; Leo Pharma AB, Sweden) and xylocaine (0.2 mg/ml; Astra Zeneca, Sweden) to empty the organ from blood and then flushed with 1 ml ice-cold UW solution (ViaSpan; DuPont Pharmaceutical, USA). The entire surgical procedure of the donor took 2030 min. The graft was immersed in UW (4°C) solution and the ends of aorta and vena cava were cleared from extravascular tissue, and then stored for either 24 or 48 h before transplantation.
The surgery of the recipient was performed through a mid-line laparotomy after 15 IU of heparin sulphate was given s.c. The aorta and vena cava were mobilized and haemostatic clamps were placed en bloc around the vessels. Aorticaortic and cavalcaval end-to-side anastomosis were performed using continuous 110 nylon suture. The native uterus of the recipient was left in place. The cervix of the grafted uterus was exteriorized and sutured as a stoma to the skin on the right side of the abdomen. The laparotomy was closed and 1 ml of 0.154 mol/l NaCl and 1 ml of glucose (50 mg/ml) were administered (s.c.) to compensate for fluid loss and to counteract hypoglycaemia. The operation of the recipient including the preparation of the specimen took 4050 min. The uterus was kept at room temperature during surgery.
Assessment of morphology
The morphology, after cold ischaemic preservation, was assessed in uteri that had been bisected and separated with one horn preserved in UW and the other horn preserved in NaCl for 24 h (n = 5), for 48 h (n = 4) and 72 h (n = 1). The morphology of uteri transplanted after cold preservation in UW for 24 h (n = 6) and 48 h (n = 2) was assessed 2 weeks after transplantation. All uterine tissues used for morphological studies were immersion fixed in Kidney Biopsy Fixation Solution (Bie & Berntsen A-S, Denmark), dehydrated, embedded in paraffin and cut into 35 µm thick sections. The sections were stained with haematoxylin and eosin or PAS (periodic acidSchiff base) and examined in a light microscope and photographed.
Laser Doppler flowmetry
The laser Doppler flowmetry method (Oberg, 1990) estimates tissue blood perfusion and has been used for studies of blood flow in the reproductive tract of small experimental animals (Zackrisson et al., 2000
). At day 14 after transplantation, the mouse was anaesthetized and a mid-line laparotomy was performed. A miniprobe (Perimed, Sweden) was placed in the lumen of both the graft uterus and the native uterus to record blood flow for periods of 23 min. The Periflux 5000 flowmeter used in this study (Perimed) recorded blood flow using a Perisoft software computer program (Perisoft, Sweden) and blood flow was expressed as arbitrary perfusion units (PU).
Contractility experiments
The ability of preserved uteri to generate spontaneous contractions and respond to prostaglandin F2 (PGF2
) stimulation was examined on fresh samples (control: n = 10), samples preserved for 24 h in either 0.154 mol/l NaCl (n = 5) or UW (n = 5) and samples preserved at for 48 h in NaCl (n = 4) or UW (n = 4).
To harvest a normal uterus, a mid-line laparotomy was performed after induction of anaesthesia. The aorta and vena cava were mobilized and the external iliac vessels and ovarian vessels were ligated on both sides. Two or three lumbar vessels were cauterized and the aorta and vena cava were tied off inferior to the branching of the ovarian vessels. A 27-gauge cannula (B.Braun Melsungen AG, Germany) was inserted into the aorta and the uterus was then flushed with 0.154 mol/l NaCl (4°C) until transparent fluid flowed through an incision of the vena cava. The left horn was then excised and the remaining right horn was flushed with 1 ml of UW. The uterine horns were then kept in their respective solutions at 4°C for either 24 h or 48 h. About 1 h before the start of contractility measurements, an unpreserved fresh uterus was harvested as described above, flushed and kept in 0.154 mol/l NaCl at room temperature to serve as control.
Just prior to measurement of contraction the specimens were prepared as follows: the upper 5 mm of the uterine horn was dissected free from surrounding tissue in chilled KrebsHEPES buffer (122 mmol/l NaCl, 4.7 mmol/l KCl, 1.9 mmol/l KH2PO4, 1.19 mmol/l MgCl2, 5.0 mmol/l HEPES, 2.5 mmol/l CaCl2, pH 7.37). Two silk ligatures (60) were tied 2 mm apart on each specimen and the preparation was mounted in an organ chamber between a metal hook and a force transducer as previously described (Ekerhovd et al., 1997). The organ chamber contained 13 ml KrebsHEPES buffer with 11.5 mmol/l D-glucose maintained at 37°C and was continuously gassed with 100% oxygen. A passive tension force of 5 mN was applied and the preparation was allowed to accommodate for 90 min. Subsequent doses (1, 10, 100 ng/ml) of PGF2
[PGF2
Tris-salt (Sigma Chemicals, USA) dissolved in 70% ethanol] were added in intervals of 50 min with intermediate washings. Isometric contractions were recorded for 10 min before the first addition of PGF2
and 10 min after addition of each PGF2
concentration, on a PC through a Grass polygraph (model 7D; Grass Instruments Ltd, USA) using LabVIEWTM software (National Instruments, USA).
Contractions were measured in terms of start of spontaneous contractions (min after set-up) and area under the curve (AUC) for the 10 min measured, both before and after stimulation. The response to administered PGF2 was expressed as the log-ratio of AUC after dose and AUC for the spontaneous contractions of the same specimen.
Embryo transfer
Mice (n = 6) with transplants (right uterine horn and cervix) that had been preserved in UW for 24 h received embryos 2 weeks after transplantation. To obtain pseudopregnancy, the mice were first caged with vasectomized B6CBAF1 males (day of vaginal mating plug = day 1). As blastocyst donors, 810 week old B6CBAF1 females were used after mating with fertile B6CBAF1 males. Mated females were killed by cervical dislocation on day 4 of pregnancy and the uteri were flushed with Gamete-100 (Vitrolife, Sweden) to obtain the blastocysts. Laparotomy was performed on the pseudopregnant females with uterine transplants at noon on day 3 or 4. Visual inspection confirmed a viable graft and three to six blastocysts were placed inside the lumen of both the graft and the native uterus by transmyometrial puncture as previously described (Hogan et al., 1994). The abdominal wall was closed and 40 mg/kg of cefuroxim was injected i.m.
Assessment of pregnancy rate and growth trajectory of offspring
The transplanted, pregnant mice were taken to term to allow assessment of post-natal development of the offspring. From day 18 of pregnancy the mice were examined every 612 h until spontaneous delivery from the native uterus occurred. Immediately after spontaneous delivery from the native uterus of transplanted mice, the pups were weighed and assigned to a foster-mother. The mother was then anaesthetized and underwent laparotomy with the entire uterine graft, containing the fetuses, being removed. The grafted uterus was cut open and the pups weighed, marked by ear incision and placed together with the other pups and the foster-mother. The laparotomy incision was then closed and the transplanted mouse was placed in the same cage as the pups and foster-mother. Body weights of progeny from the two different groups (native and grafted uteri) were determined at day of delivery and then once every week until 8 weeks of age.
Statistics
The difference between log(AUC)dose and log(AUC)spont was related to log dose using orthogonal linear regression within each specimen. The difference in doseresponse between groups was evaluated using Wilcoxon tests of the slope and intercept parameters from the regression models. The line corresponding to the median slope and intercept was used to characterize the estimated doseresponse for each group graphically.
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Results |
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Out of 25 live-born pups, 20 survived through the first day. Fifteen pups (eight from native and seven from graft uteri) were weighed at regular intervals up to 8 weeks of age. The two groups of animals followed a similar growth trajectory (Figure 7).
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Discussion |
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Early in the history of organ transplantation, it was evident that the length of the ischaemic period had a major impact and significance for the function of the grafted organ (Shoskes et al., 1996). Thus, considerable effort was directed towards characterization of impairment by morphological parameters and also to develop strategies to diminish the ischaemic injuries. These efforts led to the introduction of the concept of cold storage and specially designed preservation solutions.
The signs of morphological degeneration of the preserved uteri of the present study follows essentially the same pattern as described for other organs, with typical vacuolization, nuclear pycnosis, endothelial detachment and finally necrosis (Momii et al., 1990). Light microscopy evaluation in the present study did not reveal any major changes after 24 h of cold preservation and only slight degenerative alterations were seen after 48 h of cold preservation in UW. After 72 h cold preservation, obvious signs of degeneration with pycnotic nuclei were seen, most abundantly in the muscle layer. These findings are consistent with previous studies on the human pancreas that showed only mild injury after 48 h of cold ischaemic preservation in UW (Int Veld et al., 1993
).
It has been convincingly and thoroughly demonstrated by a number of studies that the endothelium, in a time-dependent manner, plays a central and major role in the mechanism of ischaemiareperfusion insult. Expression and activation of various adhesion molecules with increased leukocyte adhesion, interstitial edema compressing the microvascular compartment and direct endothelial lesions (detachment, desquamation) have been identified as major effectors of impaired blood perfusion (no-reflow phenomenon) originating at the capillary level (Massberg et al., 1998; Dindelegan et al., 2003
). If the resulting temporary post-reperfusion ischaemia is further associated with a sustained activity (mandatory in the case of vital organs) with increased metabolic demand, this might result in graft failure ranging from dysfunction to non-function. Our observations of delayed or abolished microvascular filling at initial reperfusion of preserved uteri indicate slight microvascular impairment after 24 h cold ischaemic storage that has become severe after 48 h. However, the normal blood flow and morphology 2 weeks after transplantation of grafts preserved for 24 h indicate that these injuries are reversible.
The complete degeneration found 2 weeks after transplantation in uteri preserved for 48 h was most likely caused by the inadequate perfusion seen at initial reperfusion. This profound microvascular impairment at the time of transplantation wouldif sustainedresult in gradual necrosis similar to the necrobiosis of torsioned leiomyoma or leiomyomas subjected to uterine artery embolization.
There is evidence that the non-pregnant uterus exhibits spontaneous contractions throughout the menstrual cycle. Three typical patterns of uterine contractility have been recognized during the menstrual cycle. During the luteofollicular transition and early follicular phase, the uterine contractility, which involves all myometrial layers, participates in the forward propulsion of the uterine contents (Martinez-Gaudio et al., 1973; de Vries et al., 1990
). The primary function of uterine contractility during the late follicular phase is to transport sperm from the vagina to the distal end of the Fallopian tube (Kunz et al., 1996
). Finally, the uterus reaches a stage of quiescence after ovulation, which characterizes the luteal phase. In this study we used contractile ability as a functional test of viability of preserved uteri, as an indicator of the ionic integrity of the muscle cells as well as the severity of degeneration of structural proteins and enzymes.
The contractile ability was surprisingly intact after 24 h storage in vitro with more pronounced disturbances after 48 h in both storage media. The depolarization of cell membranes caused by inactivation of mainly NaK ATPase is known to be time dependent but the results herein demonstrate that it can be reversed to some extent so that the muscle cells can regain their ability to contract and relax in vitro.
The NaCl preservation for 24 h did not seem to have altered the frequency of contraction/relaxation, but the amplitude and response to PGF2 stimulation was lower than control values. This might be a consequence of alterations in cell-to-cell contacts, damage to actin filaments or other structural proteins involved in contraction. Insult to energy metabolism, protein synthesis and several other mechanisms known to be affected by ischaemiareperfusion are also possible explanations to the altered response.
The UW preservation for 24 h did not alter the amplitude or rhythm of contractions. However, a striking feature was the prolonged contraction induced by PGF2 stimulation, which was also reflected in the AUC. UW is an intracellular preservation solution with a [K+] of 120 mmol/l (Belzer et al., 1988
). During preservation the [K+] in the interstitial space will approach equilibrium with intracellular [K+]. We hypothesize that at repolarization after contraction, the effect of K+-channel opening will therefore be less prominent and the relaxation will be slower.
The relative tolerance of the murine uterus to cold ischaemia is demonstrated by the results of the morphological evaluation as well as the functional studies both before and after reperfusion of preserved uteri. Moreover, 2 weeks after transplantation, the murine uterus was able to resume its reproductive role after extended cold ischaemic preservation. The 2 week period of time was chosen to allow recovery from the transplantation trauma to both graft and recipient animal. It has to be acknowledged that unless the organ plays an immediate life-sustaining function, a period of functional rest at the new site might allow the graft to recover from the transplantation trauma. Such is the case in dialysis after kidney transplantation, which helps in recovery of many initially non-functioning grafts (Hansen et al., 1985) or the pancreatic graft that might resume its function several days after transplantation if during that period the patient has been maintained under insulin therapy (Sutherland et al., 1998
). We assume that the promising results of the present study and the lack of sequelae with regard to the gestational product were due to the lack of functional challenge (pregnancy) during the immediate postoperative period in combination with a relatively high resistance to ischaemic injury of the murine uterus.
In conclusion, both the morphological and functional results reveal that the murine uterus has considerable resistance to ischaemia reperfusion injury and restorative capacity. It has to be emphasized that these findings in the murine uterus do not necessarily fully apply to a human situation, due to apparent differences between these two species in size and anatomy, causing a possible difference of tissue perfusion. Nevertheless, the present study provides valuable observations in an eventual clinical setting, when surgery will probably require elaborate vascular reconstructions in a procedure that takes place in vitro (back-table procedure) and that can take several hours.
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Submitted on April 23, 2003; accepted on June 9, 2003.