1 Department of Obstetrics and Gynaecology, 2 Department of Pathology, 3 Department of Cardio-Thoracic Surgery and 4 Department of Radiology, University of Bonn, Faculty of Medicine, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
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Abstract |
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Key words: extracorporeal perfusion/human uterus/physiological recirculation system
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Introduction |
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The purpose of the present study was to establish a system for extracorporeal perfusion of a human uterus with recirculation of the perfusate and regeneration by interval exchange to guarantee tissue vitality of the perfused organ for up to 24 h.
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Materials and methods |
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The surgical specimens were carefully prepared, avoiding traction and laceration in order to get vascular stumps suitable for catheterization. Organ weights were always measured immediately before and after perfusion. After cannulation of both uterine arteries with 14 G bulb-headed cannulas, the uterus was flushed bilaterally with a modified heparinized KrebsHenseleit bicarbonate buffer at 37°C to remove blood products and cell detritus caused by the operation procedures. The composition of the perfusion medium was as follows: pH 7.40, NaCl 6.89 g/l, KCl 0.37 g/l, MgSO47H2O 0.25 g/l, CaCl22H2O 0.37 g/l, KH2PO4 0.14 g/l, NaHCO3 2.35 g/l, D(+)-glucose 1.50 g/l, saccharose 0.70 g/l, glutatione 0.005 g/l, 1,4-dithiothreitol 0.10 g/l, 100 IE insulin bolus at the beginning of perfusion, refobacin 40 mg/l perfusate, heparin 150 IU/ml. Following this flush-perfusion the uterus was connected with the recirculating perfusion system and placed in a cabinet (baby incubator, Dräger, Köln, Germany) at 37°C with humidity values of ~97% on wire mesh above a cylindrical teflon receptacle for perfusate collection under sterile conditions. Oxygenation of the perfusion medium was accomplished by a membrane oxygenator (Jostra, Hirrlingen, Germany) flushed with 95% O2 and 5% CO2 and monitored by gas flowmeters. The perfusate was moved through silicone rubber tubing by separate roller pumps (Storz, Tuttlingen, Germany) monitoring flow and pressure rates throughout the perfusion period.
The recirculation was achieved by pumping the perfusion medium from the teflon receptacle back to the perfusate reservoir with another separate roller pump. In order to coordinate the effluate volume and the recirculation flow rate, a regulating element triggered the recirculation roller pump by measuring the filling level of the perfusate in the collecting receptacle. For the description of the complete perfusion system see Figure 1.
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Samples of pH, pO2 and pCO2 were analysed with an ABL 505 Blood Gas Analyzer (Radiometer, Copenhagen, Denmark); all other biochemical parameters were measured using standard procedures (Vitros-Analyzer, Johnson & Johnson, Rochester, NY, USA).
Several myometrium and endometrium biopsies for light and electron microscopy were taken from the corpus uteri and the cervix uteri with a biopsy needle at the beginning, during (at 6, 12 and 18 h of perfusion) and at the end of perfusion.
For light microscopy the samples were fixed in 4% paraformaldehyde and then embedded in paraffin blocks. From these specimens several sections of 4 µm thickness were stained with haematoxylin and eosin (HE). For the electron microscopical investigations samples were fixed in 2.2% glutaraldehyde and PBS buffer. Cubes of about 1 mm3 in size were cut from each specimen and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and were examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands).
To evaluate the best interval for regeneration of the perfusate in this recirculating perfusion model, isovolumetric exchange of the perfusion medium after 1 (group 1), 2 (group 2), 4 (group 3) and 6 (group 4) h was compared to a non-recirculating system (group 0). In each group five uterine perfusions were performed. After each experiment we performed X-ray studies with opaque contrast medium using digital subtraction angiography (DSA) and perfused each uterus with methylene blue to show the distribution of the perfusion medium throughout the organ. For statistical evaluation we used the KruskalWallis one-way analysis of variance for non-parametric data.
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Results |
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Perfusions were considered to be appropriate when constant flow rates of 1535 ml/min through each artery could be maintained at pressure rates ranging from 70 to 130 mmHg. The venous effluate was collected in a cylindrical teflon reservoir from which the perfusate was recirculated by a separate roller pump to the reservoir for the perfusion buffer (Figure 1).
The biochemical parameters were initially elevated in all groups due to preparation and cannulation time. Within the first hours of perfusion, arterio-venous gradients of pH, pO2, and pCO2 decreased significantly in groups 0, 1, 2 and 3 and remained stable in the further course of perfusion as a sign of physiological oxygen consumption of the perfused organ (Figure 2ac).
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The injected X-ray contrast fluid through both arteries showed good distribution of the perfusate throughout the myometrium. The uterus is well supplied with arcuate and radial arteries crossing vertically and horizontally to form a network of small anastomoses which results in perfusion of the entire organ (Figure 5a,b). Application of methylene blue demonstrated a homogeneous distribution of the perfusion medium throughout the corpus uteri including the endometrium (not shown).
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Discussion |
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In our modified experimental model, perfusions under physiological flow and pressure conditions with modified oxygenated KrebsRinger bicarbonate buffer could be maintained at 37°C for the entire perfusion period at a steady rate of oxygen consumption, low output of lactate, LDH and CK activity as markers for hypoxia and cytolytic processes and preservation of tissue integrity. In contrast to current clinical methods for extracorporeal preservation of organs by hypothermia, our purpose was to obtain an experimental model suitable for metabolic studies. Cooling causes modifications of physiochemical functions and metabolic processes during prolonged hypothermic storages: in particular, temperatures close to freezing inactivate the sodiumpotassium cell pump with consecutive loss of cellular potassium and magnesium and a corresponding sodium influx into the cell with cellular swelling.
In this study it was our intention to simulate the in-vivo situation as close as possible. Therefore, human specimens were used and uterine perfusion was performed at body temperature with the organ placed in an automatically thermo- and humidity-controlled incubator.
With the use of autoregulated perfusion pumps, with adjustable minimum and maximum levels for perfusion flow and perfusion pressure rates according to physiological conditions, microvascular damages such as capillary destruction could be avoided.
In a perfusion system with recirculation of the perfusion medium, the composition of the perfusate is of particular importance. From the biological point of view the use of undiluted heparinized blood appears to be preferable to synthetic media. However, in-vitro perfusions using blood can cause problems in mid-term and long-term perfusion experiments regarding its physiological instability, the probable interaction with experimental pharmacological substances and the large volume required for perfusion.
Currently various preservation solutions, for example EuroCollins solution, UWBelzer solution, Bretschneider HTK solution and KrebsHenseleit bicarbonate buffer solution are used experimentally and clinically for cold storage preservation of organs in transplantation medicine or investigations in oncology (Marinelli et al., 1991; Bresticker et al., 1992
; Collins and Wicomb, 1992
; Erhard et al., 1994
; Blech et al., 1997
; Collins, 1997
). Based on the experiences of Bulletti et al. (1988b, 1993) we used a modified KrebsHenseleit bicarbonate buffer with saccharose, glutathione, 1,4-dithiothreitol, insulin and refobacin and demonstrated that exchanging the perfusate every 4 h seems to be sufficient to maintain organ architecture and tissue vitality under optimal conditions of oxygenation and nutrition, thus avoiding infection problems.
Under these circumstances the necessary volume of the expensive perfusion medium could be reduced significantly.
Furthermore, observing uterine tissue fragments by light and electron microscopy, which show good preservation of the endometrial and myometrial intracellular structures as well as the absence of inter- and intracellular oedema up to 24 h of perfusion time, confirm the results of our investigations. Performing X-ray studies with the DSA technique and perfusions with methylene blue demonstrated the homogeneous distribution of the perfusion medium throughout the entire organ.
By avoiding the degeneration of the organ by thrombus formation, problems with organ and perfusate temperature (hypothermia), increased capillary resistance or capillary destruction, unphysiological perfusion flow and perfusion pressure, insufficient gas exchange and relatively activated anaerobic metabolism and bacterial infection, we could maintain the viability and function of the organ.
Previous investigations by Bulletti et al. (1988a,b, 1993a,b, 1997) have shown the feasibility of the extracorporeal perfusion of the human uterus for various purposes. In conclusion, our model for the extracorporeal perfusion of the human uterus with recirculation of the perfusate by isovolumetric interval exchange every 4 h represents a reproducible experimental system which offers, especially in mid-term and long-term perfusion experiments, a physiological and economical scientific approach for further observations on the endometrium and myometrium in reproductive medicine.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 14, 1999; accepted on March 1, 2000.