Pulmonary Endothelial Cell Modifications After Storage in Solid Organ Preservation Solutions

The Journal of International Medical Research 1995; 23: 200-206 Pulmonary Endothelial Cell Modifications After Storage in Solid Organ Preservation Solutions P CARBOGNANI, L SPAGGIARI, M RusCA, L CAITELANI, P SOLLI, A F ALESSANDRINI, P DELL'ABATE, M VALENTEANDP BOBBIO ROMANI, Department of General Thoracic and Vascular Surgery, University of Parma, Parma, Italy During lung preservation, the vascular endothelium is probably the first site of damage and these lesions are considered the main limiting factor in solid-organ preservation. In the present study, the ultrastructural changes in the ofhuman pulmonary artery hypothermically stored (at 4 C) for 6 and 12 h in Euro Collins, University of Wisconsin and Ringer-lactate solutions were compared. The arteries obtained from three patients who underwent pneumonectomy were divided into 20 segments and preserved in the three solutions mentioned. The specimens, which were fixed in osmic acid, were examined using transmission electron microscopy. Transmission electron microscopy indicated that the cells stored in the University ofwisconsin solution either for 6 or 12 h were the best preserved, while the most severely damaged cells were those stored in Euro-Collins solution, even after just 6 h. The cells stored in Ringer-lactate showed an intermediate level of damage. The data from an ultrastructural grading scale, which quantified the damage to the cytoplasm, mitochondria and nucleus, were in broad agreement with the general transmission electron microscopy observations. Analysis of variance of the grading scale data showed that there were statistically significant differences between the groups after both 6 and 12 h storage (P < 0.05). 200

P Carbognani, L Spaggiari, M Rusca et al. KEY WORDS: EURO-COLLINS SOLUTION; UNIVERSITY OF WISCONSIN SOLUTION; RINGER-LACTATE SOLUTION; LUNG TRANSPLANTATION; LUNG STORAGE; PULMONARY ARTERY; ENDOTHELIAL CELLS INTRODUCTION In the last few years lung transplantation has become a therapeutic reality. There have been improvements in surgical technique, post-operative care and immunosuppression, but the problem of preserving the lung for long periods remains unsolved.':" Research is continuing in an effort to find a method of preservation that will permit the storage of the organ for mote than 10 h, with a minimum of cytotoxic effects on the lung cells." The vascular endothelium is probably the first site of damage during preservation and lesions of its cells are considered the major limiting factor in solid-organ preservatiou.':" The prevention of endothelial damage should, thus, be considered the principal function of a preservation solution. The aim of the present work was to compare the time-related ultrastructural changes in the of human pulmonary arteries when stored in Euro-Collins and University of Wisconsin solutions, and in a simple extracellular solution, in this case, Ringer-lactate solution. MATERIALS AND METHODS Human pulmonary arteries were obtained from three patients, all males, aged 60-66 years, who underwent pneumonectomy. In order to reduce endothelial damage, the vessels were removed using the no-touch method." The lumen was washed with a precooled physiological saline solution containing heparin (10000 U/SOO ml). The arteries were divided into 20 segments: five segments were fixed immediately and used as a control, five were stored in Euro-Collins solution, five in University of Wisconsin solution and five in Ringer-lactate solution. The stored segments were examined after 6 and 12 h of storage at 4 C. The fresh specimens, measuring 3-4 rnrn, were fixed in osmic acid and 1% veronal buffer for 2 h (ph 7.3) and, after dehydration in acetone, were embedded in Durcupan (ACM Fluka, Switzerland). Semithin sections were stained with lead citrate. 7 After fixation, 60 pieces were obtained from each specimen and were cut into ultrathin sections. Approximately 20 specimens for each solution were examined using transmission electron microscopy, EM Philips 300. The influence of the solutions was evaluated using an ultrastructural grading scale with a score ranging from 0 to 3, where 0 means no damage and 3, complete disruption (Table 1). Analysis of variance was used to determine the significance of the differences between the three means of storage. F values greater than the critical ratio were considered to be statistically significant. RESULTS Ultrastructural evaluation of the human pulmonary artery by transmission electron microscopy demonstrated that the tissue fixed at time 0 (control) was normal. After 6 h of cold storage in Euro-Collins solution, the ultrastructure of the pulmonary 201

/' (;Ur/HI.!-:IIUlli. I, Spu,r.:,r.:iul'i.,\1 /{IlS('U et a I. Stora,~p solutions lor pulmonary endothefial r.ells TABLE 1 Ultrastructural grading scale used to quantify the effects ofstoring human pulmonary artery endothelialcells In thrae differentsolutions Grade Cytoplasm MItochondrIa Nucleus 0 Normal Normal Normal 1 Slight intracellular Oval Slight chromatin oedema aggregation 2 Rarefaction Cristae degeneration Diffuse chromatin aggregation 3 Complete disruption Complete disruption Complete disruption artery was severely damaged (Fig. 1). Intracellular and extracellular oedema were extensive. leading to rarefaction of the cytoplasm and to vacuolization caused both by the swelling of the endoplasmic reticulum and by the loss of organellar structure; further. the mitochondria were rounded and the cristae were degenerate. Ribosomes were rarely recognizable and nuclear chromatin was frequently aggregated. After 12 h, the ultrastructural damage was extensive, the cytoplasm was electron transparent and the cellular structure was hardly recognizable. Numerous lysosomes and phagolysosomes were present in the peripheral cellular extension. In the pulmonary. preserved for 6 and 12 h at 4 C in Ringer-lactate solution (Fig. 2). intracellular oedema was present but was less extensive than in the cells stored in Euro-Collins solution; vacuolization of the endoplasmic reticulum was also less extensive, and mitochondrial fine structure was clearer. The nuclear chromatin and the nuclear membranes were well preserved. and intracellular junctions were easily recognizable and intact. The best preserved were.'-'~::;r.:~;<. FIGURE 1 *... -,'.... -' "..'\.- -.....( ""'-. ~..).~ ". " -;;:;.r:., ~.'-..-.~'-:::...J'" '- ".1"',~, ;~loi::"""~.qi<~"jiiitli'if~. -..:. Transmission electron micrograph showing the ultrastructura of of human pulmonary artery preserved for 6 h In Euro-Colllns solution; the cytoplasm Is electron transparent and highly vacuolated and the nuclei and the cellular organelles are degenerated. e; endothelium;.., lumen; v, vacuoles. Magnification: x 13400. 202

I' Carbognuni. I. Spll,r.:gilJri. M /{US('II PI al. Storagl~ solutions Iur pulmonary FIGURE 2 ",:,.,,-'-~--- --~.~~~): -.:... -...r._~ a Electron micrographs of human pulmonary artery preserved for (a) 6 h and (b) 12 h In Ringer-lactate solution; the cytoplasm Is slightly vacuolated and oedematous, the ribosomes are rare, but the cellular organelles are moderately preserved; the Intrecellular Junctions (Fig. 2a) and the nuclear membranes (Fig. 2b, a"ow) are Intact. e, endothelium;., lumen; v, vacuoles; m, mitochondria; g, Goigi complex. Magnification: (a) x 16600; (b) x 26 200. 203

I' C(JrI)//,~lI(JlIi.t. SI)(l,~,~i(Jri, M Runcu et ai. endnthuliul t:l~lis FIGURE 3 Electron micrograph of human pulmonary artary preserved for 12 h In University of Wisconsin solution: the cytoplasm retains Its e/ectron-denslty; the cellular organelles are mostly well preserved; numerous dense bodies are present In the cytoplasm (big arrow); and the Intracellular Junctions are Intact (small arrows). e, endothelium;.., lumen; m, mitochondria. Magnification: x 26 200. those stored for 6 and 12 h in University of Wisconsin solution (Fig. 3). Intracellular oedema was the least evident and organellar ultrastructure was largely intact; the mitochondria were mostly oval in shape and the cristae were generally visible. Irregular dense bodies were present in the cytoplasm and the intercellular junctions were again preserved. The analysis of variance of the data obtained from the ultrastructural grading scale confirmed that there were statistically significant differences (P < 0.05) between the three means of storage after both 6 and 12 h (Tables 2 and 3). DISCUSSION The difficulty of preserving the lung for just 10 h is the most important limitation in TABLE 2 SCores Indicating extent of ultrastructural'damage to human pulmonary artery endothelial cells after storage for 6 or 12 h In three different storage solutions Storage solution Ultrastructural score (mean ± SO) after storage for Ringer-lactate Euro-Collins University of Wisconsin Meansarefor threesamples in eachsolution. 3.33 ± 0.58 5.67±0.58 2.33 ± 0.58 4±1 7±1 2.33±0.58 204

P Carbognani, L Spaggiari, M Rusca et al. transplantation. The ideal storage solution has not yet been identified since the pathophysiology of lung preservation is not yet clearly understood. The response of the vascular endothelium to different storage fluids was evaluated because of the primary importance of this 'simple membrane' in organ preservation; during pulmonary artery flush-out and lung storage it is in direct contact with the solutions.v" The ultrastructural changes of the endothelial cells of the pulmonary artery were studied after hypothermic storage (4 C) for 6 and 12 h in Euro-Collins, University of Wisconsin and Ringer-lactate solutions. The Euro-Collins and University of Wisconsin solutions were selected as the most widely used and tested intracellular types of solution, and Ringer-lactate was chosen as a simple extracellular solution.b.l0 Transmission electron microscopy indicated that the cells stored in the University of Wisconsin solution either for 6 or 12 h were the best preserved. The most severely damaged cells were those incubated in Euro- Collins solution even after just 6 h. The cells stored in Ringer-lactate solution showed less cytotoxic effects than those stored in Euro Collins solution after 6 and 12 h, but appeared to be more damaged than those stored in University of Wisconsin solution either after 6 or 12 h. When an ultrastructural grading scale was used to quantify the damage to the cytoplasm, mitochondria and nucleus, the results confirmed the general transmission electron microscopy observations (Table 2); statistical analysis of the data indicated that there were significant differences between the solutions (P < 0.05; Table 3). In conclusion, after 12 h the endothelial cells were best preserved when hypothermically stored in University of Wisconsin solution. Specific constituents of this intracellular-type solution are probably important, particularly the oncotic substances that prevent cellular swelling and the antioxidants that reduce the amount of oxygen freeradical injury during the ischaemic period.":" TABLE 3 Analysis of variance for the data on ultrastructural damage to human pulmonary artery endothelialcells after storage for 6 or 12 h in three storage solutions (Table 2) Sum of squares Variance estimate Fvalue Degrees of Source of variation 6h 12 h freedom 6h 12 h 6h 12 h Between means 17.56 33.56 2 8.78 16.78 26.33 21.57 Within means 2.00 4.67 6 0.33 0.78 Total 19.56 38.22 8 The F values after both 6 and 12 h storage exceed the critical ratio of 19.33 at P < 0.05. 205

P Carbognani, L Spaggiari, M Basco et ai. REFERENCES 1 Novik RJ, Menkis AH, McKenzie FN: New trend in lung preservation: a collective review. J Heart Lung Transplant 1992; 11: 337-392. 2 Belzer FO, Southard JH: Principles of solid-organ preservation by cold storage. Transplantation 1988; 45: 673-676. 3 Christie NA, Waddel TK: Lung preservation. In: Chest Surgery Clinics of North America: Lung Transplantation (Patterson GA, Cooper JD, eds). Philadelphia: Saunders, 1993; pp. 29-47. 4 Killinger [r WA, Dorofi DB, Keagy BA, et al: Endothelial cell preservation using organ storage solutions. Ann Thome Surg 1992; 53: 979-982. 5 Von Oppel UO, Pfeiffer S, Preiss P, et a1: Endothelial cell toxicity of solid-organ preservation solutions. Ann Thome Surg 1990; 50: 902-910. 6 Gottlob R, Stockinger L, Gestring GF: Conservation of vein with preservation of viable endothelium. J Cardiovase Surg 1982; 23: 109-116. 7 Reynolds ES: The use of lead citrate at high ph as an electronpaque stain in electron microscopy. J Cell Biol1963; 17: 208-212. 8 Whalers T, Haverickh A, Fieguth HG, et a1: Flush perfusion using Euro-Collins solution versus cooling by means of extracorporeal circulation in heart-lung preservation. J Heart Transplant 1986; 5: 89-98. 9 Caldwell-Kenkel JC, Currin TC, Tanaka Y, et al: Kuppfer cell activation and endothelial cell damage after storage of rat livers: effect of reperfusion. Hepatology 1991; 13: 83-95. 10 Hardesty RL, Aeba R, Armitage JM, et al: A clinical trial of University of Wisconsin solution for pulmonary preservation. J Thorae Cardiovase Surg 1991; 105: 660 666. 11 Wahlberg JA, Southard JH, Belzer FO: Development of a cold storage solution for pancreas preservation. Cryobiology 1986; 23: 477-482. 12 Detterbeck FC, Keagy BA, Paull DE, et al: Oxygen free radical scavengers decrease reperfusion injury in lung transplantation. Ann Thorae Siug 1990; 50: 204 210. P Carbognani, L Spaggiari, M Rusca, L Cattelani, P Solli, A Romani, P Dell'Abate, M Valente and P Bobbio Pulmonary Endothelial Cell Modifications After Storage in Solid-Organ Preservation Solutions The Journal ofinternational Medical Research 1995; 23: 200-206 Received for publication 25 November 1994 Accepted 12 December 1994 Copyright 1995 Cambridge Medical Publications Address for correspondence DR P CARBOGNANI Department of General Thoracic and Vascular Surgery, University of Parma, 14 Gramsci Street, 43100 Parma, Italy. 206