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Cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted mice
Transplant Immunology 16 (2006) 99 – 104 www.elsevier.com/locate/trim
Cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted mice Sándor Sipka Jr. a,⁎, Endre Bráth a , Ferenc F. Tóth b , Magdolna Aleksza c , Andrea Kulcsár c , Ákos Fábián d , Sándor Baráth c , Péter Balogh e , Sándor Sipka c , István Furka a , Irén Mikó a a
c
Department of Operative Techniques and Surgical Research, Medical and Health Science Center, University of Debrecen, Hungary b Department of Radiology, Medical and Health Science Center, University of Debrecen, Hungary 3rd Department of Internal Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei blv. 98, 4012, Debrecen, Hungary d Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Hungary e Department of Immunology and Biotechnology, University of Pécs, Hungary Received 24 November 2005; received in revised form 20 March 2006; accepted 28 March 2006
Abstract Background: Our department worked out a modified surgical form of spleen autotransplantation earlier, named “spleen apron method” introduced already into the clinical practice. Recently we tested the immunological changes in a group of patients autotransplanted with about 10–15% of their spleen, what was the at least always implantable amount after the severe splenic injuries. In the current work we aimed at measuring some cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted inbred mice two and eight months after the operations in order to get more unambiguous results than that we could obtain in our patients with this technique. Materials and methods: We divided 96 two months old Balb/c female mice into eight groups (n = 12/group). The group of controls, sham operated, splenectomized and autotransplanted animals with two and eight months of survival time after the operations. During the autotransplantation we inserted the same amount of spleen, five slices, “chips,” about 10–15% of total mass of spleen, into the greater omentum similarly as it was used in the patients. The concentration of serum proteins were measured by laser nephelometry. The lymphocyte subsets were analyzed by flow cytometry. Results: We found that two months after the operations the number of CD 19+ B-cells increased in the splenectomized but decreased in the autotransplanted animals. Eight months after the operations the number of both CD3+ T and CD19+ B lymphocytes decreased both in the splenectomized and autotransplanted animals compared to the controls and sham operated mice. However, the numbers of T and B cells were slightly but not significantly higher in the autotransplanted than in the splenectomized mice. The serum level of IgM was also decreased in the splenectomized and autotransplanted mice at both time points, however, eight months after the operations the concentration of IgM was significantly higher in the autotransplanted group than in the splenectomized animals. Conclusion: The effects of autotransplanted “chips” were different at the various ages of the animals. Additionally, they showed some immunological benefit being quantitatively in accordance to the amount of the transplanted spleen. The elevated level of serum IgM what we found in the autotransplanted mice even with this amount of transplanted spleen eight months after the operations, however, might have the potentially greatest importance compared to splenectomy. These experiments can prove that the attempts for autotransplantation may have real perspectives but their efficacy depends on the amount of the successfully transplanted (saved) mass of spleen. © 2006 Published by Elsevier B.V. Keywords: Splenectomy; Spleen autotransplantation; IgM
1. Introduction
⁎ Corresponding author. Tel.: +36 52 411 600; fax: +36 52 411 4357. E-mail address: [email protected] (S. Sipka). 0966-3274/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.trim.2006.03.013
Physiologically the spleen has a fundamental role in the filtration of erythrocytes and platelets, in the production of T and B lymphocytes and immunoglobulins. It responses actively to
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S. Sipka Jr. et al. / Transplant Immunology 16 (2006) 99–104
the blood-born antigens, the more so if particulate. In blunt abdominal trauma the spleen is often injured. The treatment depends on the grade of the injury. At grade I (small subcapsular hematoma/capsular tear) conservative therapy, careful follow-up and coagulation, at grade II (1–3 cm depth parenchymal rupture, 10–50% subcapsular hematoma) careful follow-up, suture or bioplasts should be used. At grade III (large or expanding rupture or hematoma) suture or resection should be carried out. In the case of partial (grade IV) or complete devascularization, or when the spleen is completely shattered (grade V) it should be removed [1,2]. The removal of spleen after traumatic injuries, however, has numerous negative consequences. The most severe problem is the appearance of “overwhelming postsplenectomy infection (OPSI) syndrome” what can cause a mortality rate higher than 75%, and what can be observed in 2.1–6.3% after splenectomy [3–5]. Therefore, the spleen saving surgical techniques are really needed [6–8]. The technical aspects of heterotopical efficient splenic autotransplantation with various amounts of the healthy spleen tissue have been widely reported from several research groups [9–23]. Since decades the greater omentum has been recognized already as a potential place for transplantation [9]. Using the greater omentum, a “spleen apron method” was earlier developed by Furka and coworkers on dogs in our institute [24,25]. According this method, the slices of spleen (“chips”) were inserted into the two layers of greater omentum (forming an “apron”), preventing the formation of adhesions. This method was adapted also for mice by a microsurgical technique [26,27]. We proved earlier
histologically that the “spleen chips” representing 10–15% of total spleen mass could excellently survive there for several months. By the end of the second month, the splenic chips were already histologically regenerated in mice after a central necrosis taking place in the first days after the operation [26,27]. This technique has been adopted also into the clinical practice in several places in Hungary. However, it is a great problem that after a high grade (IV–V) spleen injury (e.g. in the case of a shattered spleen) only a very limited part (about 10–15%) of the damaged spleen can be used for autotransplantation preserving the original tissue architecture. Recently we tested some immunological changes in patients autotransplanted with about 10–15% of the spleen but the intergroup changes were not significant [2]. Therefore, in the current work we applied an autotransplantation model with 10–15% of implanted spleen in inbred mice in order to get unambiguous changes and comparable data among the various experimental groups. We tested the changes in the distribution of subsets of peripheral lymphocytes and in the levels of serum immunoglobulins and complement components in the splenectomized and spleen autotransplanted inbred mice two and eight months after the operations. Although, we were aware of those results that about 25–30% of replaced spleen could be needed to greatly restore the lost splenic functions. 2. Objective We suppose that use of inbred mice is suitable to find and demonstrate significant changes in the immunological parameters
Fig. 1. Changes in the number of CD3+ T lymphocytes in the peripheral blood of mice two and eight months after the operations.
S. Sipka Jr. et al. / Transplant Immunology 16 (2006) 99–104
of splenectomized, autotransplanted (even with 10–15% of spleen), sham operated and control mice. 3. Materials and methods 3.1. Animals Our experiments were approved by the Committee of Animal Research of the University of Debrecen. Two months old female Balb/c mice (body weight: 23–26 g, n = 96) were used for the study. The animals were housed under conventional circumstances in standard size cages, they were fed by standard food, and they had free access of water to drink. The animals were operated in two rounds, and the laboratory measurements were carried out in all groups of animals simultaneously. All mice were two months old at the time of the operations. There was a difference of sixth months between the two rounds of the microsurgical interventions and the ages of the animals at the time of the laboratory measurements in the two sets of groups. Animals with eight months old operations were ten months old mice in the groups A (SE-8; n = 12), B (AU8; n = 12), C (SH-8; n = 12), D (C-8; n = 12). The animals with two months old operations were four months old mice in the groups E (SE-2; n = 12), F (AU-2; n = 12), G (SH-2; n = 12), H (C-8; n = 12).
3.2. Surgical operations The animals were anesthetized with an intraperitoneal injection of pentobarbital (35 mg/kg, Phylaxia Sanofi Ltd., Hungary) and were operated using an operating microscope in clean but not sterile conditions. The mice with accessorial spleen were excluded from the study. In the first series of experiments, eight months before the measurements: in group A the splenectomy (SE-8) was performed after a midline incision; in group B spleen autotransplantation (AU-8) was carried out after splenectomy using the microsurgical adaptation of Furka's spleen chip technique, described by Miko previously [26,27].
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Briefly, after removing the spleen, we cut 5 small pieces of the splenic tissue – called “chips” – with the size of 2 × 2 × 2 mm and we placed them in physiological saline solution at room temperature. The greater omentum was raised and after forming 5 omental nests by opening the first layer of the greater omentum, we placed the chips inside. The nests were signed and fixed with 7/0 Prolene stitches. In group C a sham operation (SH-8) was performed (lifting out and reposition of spleen after a midline incision); in group D, as a control, /C-8/ no surgical intervention was made. In the second series of experiments, two months before the laboratory measurements: in group E /SE-2/ the splenectomy was performed; in group F /AU-2/ spleen autotransplantation was carried out after splenectomy; in group G /SH-2/ a sham operation was performed; in group H /C-2/ no surgical intervention was made. Before closing of the abdominal cavity, 1.0–1.2 ml of sterile physiological saline solution was injected to each animal intraperitoneally. The abdominal wall was closed in two layers (peritoneal–aponeurotic plane and skin). After the operation, the animals were kept under a heating lamp for 6 h. Mice usually recovered from anesthesia within 1 h, and then regular food and water were given them ad libitum. There was no antibiotic administration. All animals recovered excellently and survived for the postoperative period.
3.3. Laboratory measurements Blood was taken under pentobarbital anesthesia (35 mg/kg) by an intracardial puncture after thoracotomy. After taking blood and histological samples the mice were sacrificed immediately. The subsets of lymphocytes were measured by a flow cytometer (FACSCalibur, Becton-Dickinson, USA) using the following antibodies: PE anti-mouse CD3; FITC anti-mouse CD 19; PE anti-mouse NK1.1 (CD-56) (Pharmingen, USA). Immunoglobulin and complement components were determined by a laser nephelometer (Behring, Germany), using the following reagents: IgM, IgA, C3, C4 (DAKO, Denmark), and IgG (Behring, Germany) from sera stored at −20 °C.
Fig. 2. Changes in the number of CD19+ B lymphocytes in the peripheral blood of mice two and eight months after the operations.
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Fig. 3. Changes in the serum levels of immunoglobulin IgM in mice two and eight months after the operations. The quantitative and qualitative cell count determinations were carried out by a microcell counter (DiaTerm Abacus, Hungary) from heparinized blood (7 U/ml).
3.4. Statistic analysis All results are expressed as means and standard deviations (mean ± S.D.). When the distribution of data was normal, the one-way ANOVA test was used for the inter-group comparisons. If the result of ANOVA test was significant, the Dunett test was used to compare the means in the pairs. When the distribution was not normal, the Kruskal–Wallis test was applied. P values smaller than 0.05 were considered statistically significant.
4. Results 4.1. Changes in the number of CD3+ T lymphocytes in the peripheral blood of mice two and eight months after the operations Two months after the operations there was still no difference in the number of circulating CD3+ T cell in the peripheral blood. On the other hand, eight months after the operations the number of T cells was significantly decreased in both groups of operated (splenectomized SE8 and autotransplanted AU-8) animals compared to the two groups of controls (sham operated SH-8 and time matched untreated mice C-8). (SE-8: 0.49 ± 0.10 vs. C-8: 1.05 ± 0.16, p b 0.001; SH-8: 1.25 ± 0.14 p b 0.001 G/l; and AU-8: 0.64 ± 0.06 vs. C-8: 1.05 ± 0.16; SH-8: 1.25 ± 0.14; p b 0.001 G/l) (Fig. 1). 4.2. Changes in the number of CD19+ B lymphocytes in the peripheral blood of mice two and eight months after the operations Two months after the operations the number of circulating CD19+ B cells increased significantly in the splenectomized (SE-2) and decreased in the autotransplanted (AU-2) animals compared to the two
groups of controls (C-2 and SH-2). (SE-2: 0.72 ± 0.15 vs. C-2: 0.44 ± 0.05, G/l, p b 0.01, and SH-2: 0.42 ± 0.07 G/l p b 0.01; and AU-2: 0.25 ± 0.06 vs. C-2: 0.44 ± 0.05 G/l, P b 0.05, SH-2: 0.42 ± 0.07 G/l p b 0.05). Eight months after the operations, however, in both groups of operated animals (SE-8 and AU-8) the number of B cells was diminished compared to the two groups of controls (C-8 and SH-8) but only the fall measured in the splenectomized mice was significant (SE-8: 0.55 ± 0.09 vs. C-8: 0.81 ± 0.11 G/l p b 0.01, SH-8: 0.92 ± 0.16 G/l p b 0.01;) The value of AU-8: 0.71 ± 0.12 was not significant compared to the three other groups, however, it was higher than that measured in the splenectomized animals (0.55 ± 0.09) (Fig. 2). 4.3. Changes in the serum levels of immunoglobulin IgM in mice two and eight months after the operations The level of serum IgM was significantly decreased in the two operated (splenectomized and autotransplanted) groups compared to the controls both two and eight months after the operations (two months group: SE-2: 68.67 ± 8.43 vs. C-2: 88.25 ± 10.72 G/l p b 0.05; SH-2: 104.67 G/l, p b 0.01; and AU-2: 65.05 ± 6.41 vs. C-2: 88.25 ± 10.72 G/l, p b 0.01; SH-2: 104.67 ± 18.25 G/l, p b 0.01; eight months group: SE-8: 45.24 ± 7.03 vs. C8: 84.67 ± 9.16 G/l, p b 0.001, SH-8: 102.34 ± 10.74 G/l, p b 0.001; and AU-8: 63.08 ± 8.98 vs. C-8: 84.67 G/l, p b 0.001, SH-8:102.34 ± 10.74 G/l, p b 0.001). The level of serum IgM, however, was significantly higher in the autotransplanted (AU-8) than in the splenectomized (SE-8) group of animals (63.08 ± 8.96 vs. 45.24 ± 7.03 G/l, p b 0.05) (Fig. 3).
5. Discussion In the current work we present some significant changes in the peripheral distribution of CD3+ T and CD19+ B cells and in
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the level of serum IgM of autotransplanted mice compared to the splenectomized animals suggesting that a surgical attempt for the partial restoration of spleen after splenectomy may have some immunological benefits. The immune competence of the spleen is crucially dependent on the tissue architecture and the recirculation efficiency of lymphoid cells to and from the spleen. In our histological analyses performed eight months after the autotransplantation the lymphoid tissue structure was clearly compartmentalized into red pulp and lymphocyte-rich white pulp, latter with a slight Bcell dominance compared to controls (manifested as approximately 30% increase of B-cell area, and an approximately 50% reduction of T-cell zone, respectively). In preliminary adoptive cell-transfer studies in autotransplanted mice the majority of injected lymphocytes 90 min after the injection tended to pass through the red pulp compartment, with only a minority moving towards the white pulp, similarly to control mice (manuscript in preparation). However, the main point of this present work is that some functional and immunological activity of the grafted spleen slices can be observed in the inbred mice. (The series of the not significant changes found e.g. in the number of CD56+ NK cells, in the serum levels of IgG, IgA and the complement components, C3 and C4 are not presented). The first conclusion drawn from the data of significant alterations is that there are time dependent differences in the changes of immune system of mice, especially in the peripheral distribution (in the number) of circulating B cells. In addition, the alterations caused by splenectomy or autotransplantation also can be different two or eight months after the operations. It was surprising that the absolute number of CD3+ T cells was almost the same in the four groups two months after the operations. (But there were differences in the percent of cells. These data are not presented). On the other hand, a striking fall of these cells was observed in the two operated groups eight months after the interventions. In addition, it was also not negligible that the number of T cells was slightly (although not significantly) higher in the autotransplanted than in the splenectomized animals. In the case of B cells the directions of changes were different in the animals of different ages. Whereas the number of B lymphocytes was significantly elevated in the splenectomized mice compared to the controls two months after the operations, there was a rather great fall in the number of these cells in the autotransplanted mice. In this period of time, we found that the number of lymphocytes in the peripheral blood of splenectomized mice was 2.32 ± 0.65 G/l compared to the 1.81 ± 0.67 G/ l value of autotransplanted animals showing a slight but not significant elevation [28]. The increased bone marrow activity of splenectomized mice could be even better reflected by the remarkable neutrophil granulocytosis observed simultaneously [28]. We think that the reason(s) of the rather great differences in the number of circulating B and T lymphocytes in the autotransplanted animals can be related to the special and time dependent change in the “homing” circumstances for them in the “chips” as it was demonstrated in our in vivo cell transfer experiments mentioned above. It has to be stressed, however,
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that though eight months after the interventions, the number of circulating B cells was decreased in both splenectomized groups of animals, in the autotransplanted mice, this fall was less than in the splenectomized animals, showing a slightly better capacity for the production of immunoglobulins. The production of IgM is crucial after splenectomy from the point of view of “overwhelming postsplenectomy infection” (OPSI) in the surgical practice [3–8]. Therefore, it could be regarded to be the most important finding in this work that in the autotransplanted animals we measured a significantly higher level of serum IgM than in the splenectomized mice eight months after the operations. This change could be in parallel with the slightly elevated number of B cells in these mice helping possibly a better antimicrobial defense [29,30] than that of splenectomized animals, where the impaired production of IgM was reflected both two and eight months after the interventions in spite of the significantly high number of peripheral B cells in the group of two months old grafting. It was common in the present experiments that in the autotransplanted animals the values of T and B cell number and the serum IgM level were higher than in the splenectomized mice eight months after the operations reflecting the functional activity of replanted spleen tissue, however, it represented only the 10–15% of original mass. The fact that these elevations were found significant only in the cases of IgM measurements could possibly explained by the greater sensitivity of laser nephelometry than that of cytofluorimetry. This approach allows us to declare that possibly a direct and quantitative linkage can exist between the successfully implanted mass of spleen and its positive influence on the number of T and B cells and circulating IgM compared to splenectomy. Supposedly, the larger the mass of successfully transplanted spleen, the better is the clinical effect. The results of this work were observed in inbred mice but they showed a series of similarities to those that we got in the patients treated by the same surgical technique [2]. All the main cellular and serological parameters of splenectomy measured in human were the same what we found in these animals. The serum IgM elevating effect of autotransplantation was also demonstrated there but only the positive tendency because the differences did not reach the level of statistical significance. In conclusion, we think that the various surgical trials, not only the “ours,” for the partial restoration of the immunological function of spleen after splenectomy even with 10–15% part of the removed organ may have some real practical benefit. To prove these beneficial effects, however, our present work on inbred mice could give a more unambiguous experimental evidence than that we obtained in the patients. Therefore, these results can encourage the continuation of works on spleen autotransplantation also with the acceptance of the limitations related to it. Acknowledgments This work was supported by OTKA T034211. The authors express their thanks for technical assistance to Erika Somlyai and Katalin Éles.
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References [1] Moore EE, Cogbill TH, Jurkovich GJ, Shackford SR, Malagoni MA, Champion HR. Organ injury scaling: spleen and liver (1994 revision). J Trauma 1995;3:323–4. [2] Acs G, Furka F, Miko I, Szendroi T, Hajdu Z, Sipka Jr S, et al. Splenectomisalt es lep autotranszplantalt betegek osszehasonlito haematologiai es immunologiai vizsgalatai (Comparative hematological and immunological studies of patients with splenectomy and spleen autotransplantation). Magy Seb (Hung J Surg) 2005;58(2):74–9 [in Hungarian]. [3] King H, Shumacker HB. Susceptibility to infection after splenectomy performed in infancy. Ann Surg 1952;136:239–42. [4] Hiatt JR, Phillips EH, Morgenstern L, editors. Surgical diseases of the spleen. Berlin Heidelberg: Springer-Verlag; 1997. [5] Waghorn DJ. Overwhelming infection in asplenic patients: current best practice preventive measures are not being followed. J Clin Pathol 2001;54: 214–8. [6] Carlstedt A, Tholin B. Infectious complications after splenectomy. Acta Chir Scand 1984;150:607–10. [7] Cooney DR, Dearth JC, Swanson SE, Dewanjee MK, Telander RL. Relative merits of partial splenectomy, splenic reimplantation and immunisation in preventing postsplenectomy infection. J Pediatr Surg 1979;86:561–9. [8] McKinnon WM, Sanders HS, Zamora LF, Marion L. Splenectomy: indications, results and complication in 406 patients. Am Surg 1973;39:72–4. [9] Tavassoli M, Ratzan RJ, Crosby WH. Studies on regeneration of heterotopic splenic autotransplants. Blood 1973;41(5):701–9. [10] Pabst R, Reilmann H. Regeneration of heterotopically transplanted autologous splenic tissue. Cell Tissue Res 1980;209(1):137–43. [11] Patel JM, Williams JS, Shrivel B, Hingham JR. Preservation of splenic function by autotransplantation of traumatized spleen in man. Surgery 1981;90(10):683–8. [12] Nielsen JL, Sakso P, Sorensen FH, Hansen HH. Demonstration of splenic functions following splenectomy and autologous spleen implantation. Acta Chir Scand 1984;150(4):469–73. [13] Pabst R, Kamran D. Autotransplantation of splenic tissue. J Pediatr Surg 1986;21(2):120–4. [14] Thalhamer J, Pimpl W, Pattermann M. The role of the spleen and splenic autotransplants in clearing experimental bacteremia caused by the gramnegative bacterium E. coli. Res Exp Med 1986;186(3):229–38. [15] Büyükünal C, Danismend N, Yeker D. Spleen-saving procedures in pediatric splenic trauma. Br J Surg 1987;74(5):350–2. [16] Iinuma H, Okinaga K, Sato S, Tomioka M, Matsumoto K. Optimal site and amount of splenic tissue for autotransplantation. J Surg Res 1992;53 (2):109–16.
[17] Timens W, Leemans R. Splenic autotransplantation and the immune system. Adequate testing required for evaluation of effect. Ann Surg 1992;215(3):256–60. [18] Petroianu A, Simal CJR, Barbosa AJ. Assessment of phagocytic function in remnants of subtotal splenectomy and in autologous spleen implantation. Med Sci Res 1993;21(10):715–7. [19] Weber T, Hanisch E, Baum RP, Seufert RM. Late results of heterotopic autotransplantation of splenic tissue into the greater omentum. World J Surg 1998;22(8):883–9. [20] Leemans R, Manson W, Snijder JA, Smit JW, Klasen HJ, The TH, et al. Immune response capacity after human splenic autotransplantation. Ann Surg 1999;229(2):279–85. [21] Marques RG, Petroianu A, Coelho JM, Portela MC. Regeneration of splenic autotransplants. Ann Hematol 2002;81(11):622–6. [22] Marques RG, Petroianu A, de Oliveira MB, Bernardo-Filho M, Boasquevisque EM, Portela MC. Bacterial clearance after total splenectomy and splenic autotransplantation in rats. Appl Radiat Isotopes 2002;57 (6):767–71. [23] Marques RG, Petroianu A, Coelho JMC de O. Bacterial phagocytosis by macrophage of autogenous splenic implant. Braz J Biol 2003;63(3): 492–495. [24] Furka I, Miko I, Tarsoly E. Heterotopic autotransplantation of the spleen in animal experiments. Khirurgija 1989;9:125–7. [25] Furka I, Miko I, Serfozo J, Frendl I, Hauck M. Autotransplantation of the spleen. Second World Week of Professional Updating in Surgery and in Surgical and Oncological Disciplines of the University of Milan. Lecture BookBologna: Monduzzi Editore; 1990. p. 767–9. [26] Miko I, Brath E, Furka I, Kovacs J, Kelvin D, Zhong R. Spleen autotransplantation in mice: a novel experimental model for immunology study. Microsurgery 2001;21(4):140–2. [27] Miko I, Brath E, Nemeth N, Toth F, Sipka S, Kovács J, et al. Hematological, hemorheological, immunological, and morphological studies of spleen autotransplantation in mice: preliminary results. Microsurgery 2003;23 (5):483–8. [28] Sipka Jr S, Brath E, Toth F, Fabian Á, Krizsán Cs, Baráth S, et al. Distribution of peripheral blood cells in mice after splenectomy or autotransplantation. Microsurgery 2006;26(1):43–9. [29] Lucas HA. IgM memory B cells: origin, mutation, and mobilisation by polysaccharide vaccination. Blood 2004;104:3417–8. [30] Leemans R, Harms G, Rijkers GT, Timens W. Spleen autotransplantation provides restoration of functional splenic lymphoid compartments and improves the humoral immune response to pneumococcal polysaccharide vaccine. Clin Exp Immunol 1999;117(3):596–604.
Cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted mice Sándor Sipka Jr. a,⁎, Endre Bráth a , Ferenc F. Tóth b , Magdolna Aleksza c , Andrea Kulcsár c , Ákos Fábián d , Sándor Baráth c , Péter Balogh e , Sándor Sipka c , István Furka a , Irén Mikó a a
c
Department of Operative Techniques and Surgical Research, Medical and Health Science Center, University of Debrecen, Hungary b Department of Radiology, Medical and Health Science Center, University of Debrecen, Hungary 3rd Department of Internal Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei blv. 98, 4012, Debrecen, Hungary d Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Hungary e Department of Immunology and Biotechnology, University of Pécs, Hungary Received 24 November 2005; received in revised form 20 March 2006; accepted 28 March 2006
Abstract Background: Our department worked out a modified surgical form of spleen autotransplantation earlier, named “spleen apron method” introduced already into the clinical practice. Recently we tested the immunological changes in a group of patients autotransplanted with about 10–15% of their spleen, what was the at least always implantable amount after the severe splenic injuries. In the current work we aimed at measuring some cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted inbred mice two and eight months after the operations in order to get more unambiguous results than that we could obtain in our patients with this technique. Materials and methods: We divided 96 two months old Balb/c female mice into eight groups (n = 12/group). The group of controls, sham operated, splenectomized and autotransplanted animals with two and eight months of survival time after the operations. During the autotransplantation we inserted the same amount of spleen, five slices, “chips,” about 10–15% of total mass of spleen, into the greater omentum similarly as it was used in the patients. The concentration of serum proteins were measured by laser nephelometry. The lymphocyte subsets were analyzed by flow cytometry. Results: We found that two months after the operations the number of CD 19+ B-cells increased in the splenectomized but decreased in the autotransplanted animals. Eight months after the operations the number of both CD3+ T and CD19+ B lymphocytes decreased both in the splenectomized and autotransplanted animals compared to the controls and sham operated mice. However, the numbers of T and B cells were slightly but not significantly higher in the autotransplanted than in the splenectomized mice. The serum level of IgM was also decreased in the splenectomized and autotransplanted mice at both time points, however, eight months after the operations the concentration of IgM was significantly higher in the autotransplanted group than in the splenectomized animals. Conclusion: The effects of autotransplanted “chips” were different at the various ages of the animals. Additionally, they showed some immunological benefit being quantitatively in accordance to the amount of the transplanted spleen. The elevated level of serum IgM what we found in the autotransplanted mice even with this amount of transplanted spleen eight months after the operations, however, might have the potentially greatest importance compared to splenectomy. These experiments can prove that the attempts for autotransplantation may have real perspectives but their efficacy depends on the amount of the successfully transplanted (saved) mass of spleen. © 2006 Published by Elsevier B.V. Keywords: Splenectomy; Spleen autotransplantation; IgM
1. Introduction
⁎ Corresponding author. Tel.: +36 52 411 600; fax: +36 52 411 4357. E-mail address: [email protected] (S. Sipka). 0966-3274/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.trim.2006.03.013
Physiologically the spleen has a fundamental role in the filtration of erythrocytes and platelets, in the production of T and B lymphocytes and immunoglobulins. It responses actively to
100
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the blood-born antigens, the more so if particulate. In blunt abdominal trauma the spleen is often injured. The treatment depends on the grade of the injury. At grade I (small subcapsular hematoma/capsular tear) conservative therapy, careful follow-up and coagulation, at grade II (1–3 cm depth parenchymal rupture, 10–50% subcapsular hematoma) careful follow-up, suture or bioplasts should be used. At grade III (large or expanding rupture or hematoma) suture or resection should be carried out. In the case of partial (grade IV) or complete devascularization, or when the spleen is completely shattered (grade V) it should be removed [1,2]. The removal of spleen after traumatic injuries, however, has numerous negative consequences. The most severe problem is the appearance of “overwhelming postsplenectomy infection (OPSI) syndrome” what can cause a mortality rate higher than 75%, and what can be observed in 2.1–6.3% after splenectomy [3–5]. Therefore, the spleen saving surgical techniques are really needed [6–8]. The technical aspects of heterotopical efficient splenic autotransplantation with various amounts of the healthy spleen tissue have been widely reported from several research groups [9–23]. Since decades the greater omentum has been recognized already as a potential place for transplantation [9]. Using the greater omentum, a “spleen apron method” was earlier developed by Furka and coworkers on dogs in our institute [24,25]. According this method, the slices of spleen (“chips”) were inserted into the two layers of greater omentum (forming an “apron”), preventing the formation of adhesions. This method was adapted also for mice by a microsurgical technique [26,27]. We proved earlier
histologically that the “spleen chips” representing 10–15% of total spleen mass could excellently survive there for several months. By the end of the second month, the splenic chips were already histologically regenerated in mice after a central necrosis taking place in the first days after the operation [26,27]. This technique has been adopted also into the clinical practice in several places in Hungary. However, it is a great problem that after a high grade (IV–V) spleen injury (e.g. in the case of a shattered spleen) only a very limited part (about 10–15%) of the damaged spleen can be used for autotransplantation preserving the original tissue architecture. Recently we tested some immunological changes in patients autotransplanted with about 10–15% of the spleen but the intergroup changes were not significant [2]. Therefore, in the current work we applied an autotransplantation model with 10–15% of implanted spleen in inbred mice in order to get unambiguous changes and comparable data among the various experimental groups. We tested the changes in the distribution of subsets of peripheral lymphocytes and in the levels of serum immunoglobulins and complement components in the splenectomized and spleen autotransplanted inbred mice two and eight months after the operations. Although, we were aware of those results that about 25–30% of replaced spleen could be needed to greatly restore the lost splenic functions. 2. Objective We suppose that use of inbred mice is suitable to find and demonstrate significant changes in the immunological parameters
Fig. 1. Changes in the number of CD3+ T lymphocytes in the peripheral blood of mice two and eight months after the operations.
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of splenectomized, autotransplanted (even with 10–15% of spleen), sham operated and control mice. 3. Materials and methods 3.1. Animals Our experiments were approved by the Committee of Animal Research of the University of Debrecen. Two months old female Balb/c mice (body weight: 23–26 g, n = 96) were used for the study. The animals were housed under conventional circumstances in standard size cages, they were fed by standard food, and they had free access of water to drink. The animals were operated in two rounds, and the laboratory measurements were carried out in all groups of animals simultaneously. All mice were two months old at the time of the operations. There was a difference of sixth months between the two rounds of the microsurgical interventions and the ages of the animals at the time of the laboratory measurements in the two sets of groups. Animals with eight months old operations were ten months old mice in the groups A (SE-8; n = 12), B (AU8; n = 12), C (SH-8; n = 12), D (C-8; n = 12). The animals with two months old operations were four months old mice in the groups E (SE-2; n = 12), F (AU-2; n = 12), G (SH-2; n = 12), H (C-8; n = 12).
3.2. Surgical operations The animals were anesthetized with an intraperitoneal injection of pentobarbital (35 mg/kg, Phylaxia Sanofi Ltd., Hungary) and were operated using an operating microscope in clean but not sterile conditions. The mice with accessorial spleen were excluded from the study. In the first series of experiments, eight months before the measurements: in group A the splenectomy (SE-8) was performed after a midline incision; in group B spleen autotransplantation (AU-8) was carried out after splenectomy using the microsurgical adaptation of Furka's spleen chip technique, described by Miko previously [26,27].
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Briefly, after removing the spleen, we cut 5 small pieces of the splenic tissue – called “chips” – with the size of 2 × 2 × 2 mm and we placed them in physiological saline solution at room temperature. The greater omentum was raised and after forming 5 omental nests by opening the first layer of the greater omentum, we placed the chips inside. The nests were signed and fixed with 7/0 Prolene stitches. In group C a sham operation (SH-8) was performed (lifting out and reposition of spleen after a midline incision); in group D, as a control, /C-8/ no surgical intervention was made. In the second series of experiments, two months before the laboratory measurements: in group E /SE-2/ the splenectomy was performed; in group F /AU-2/ spleen autotransplantation was carried out after splenectomy; in group G /SH-2/ a sham operation was performed; in group H /C-2/ no surgical intervention was made. Before closing of the abdominal cavity, 1.0–1.2 ml of sterile physiological saline solution was injected to each animal intraperitoneally. The abdominal wall was closed in two layers (peritoneal–aponeurotic plane and skin). After the operation, the animals were kept under a heating lamp for 6 h. Mice usually recovered from anesthesia within 1 h, and then regular food and water were given them ad libitum. There was no antibiotic administration. All animals recovered excellently and survived for the postoperative period.
3.3. Laboratory measurements Blood was taken under pentobarbital anesthesia (35 mg/kg) by an intracardial puncture after thoracotomy. After taking blood and histological samples the mice were sacrificed immediately. The subsets of lymphocytes were measured by a flow cytometer (FACSCalibur, Becton-Dickinson, USA) using the following antibodies: PE anti-mouse CD3; FITC anti-mouse CD 19; PE anti-mouse NK1.1 (CD-56) (Pharmingen, USA). Immunoglobulin and complement components were determined by a laser nephelometer (Behring, Germany), using the following reagents: IgM, IgA, C3, C4 (DAKO, Denmark), and IgG (Behring, Germany) from sera stored at −20 °C.
Fig. 2. Changes in the number of CD19+ B lymphocytes in the peripheral blood of mice two and eight months after the operations.
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Fig. 3. Changes in the serum levels of immunoglobulin IgM in mice two and eight months after the operations. The quantitative and qualitative cell count determinations were carried out by a microcell counter (DiaTerm Abacus, Hungary) from heparinized blood (7 U/ml).
3.4. Statistic analysis All results are expressed as means and standard deviations (mean ± S.D.). When the distribution of data was normal, the one-way ANOVA test was used for the inter-group comparisons. If the result of ANOVA test was significant, the Dunett test was used to compare the means in the pairs. When the distribution was not normal, the Kruskal–Wallis test was applied. P values smaller than 0.05 were considered statistically significant.
4. Results 4.1. Changes in the number of CD3+ T lymphocytes in the peripheral blood of mice two and eight months after the operations Two months after the operations there was still no difference in the number of circulating CD3+ T cell in the peripheral blood. On the other hand, eight months after the operations the number of T cells was significantly decreased in both groups of operated (splenectomized SE8 and autotransplanted AU-8) animals compared to the two groups of controls (sham operated SH-8 and time matched untreated mice C-8). (SE-8: 0.49 ± 0.10 vs. C-8: 1.05 ± 0.16, p b 0.001; SH-8: 1.25 ± 0.14 p b 0.001 G/l; and AU-8: 0.64 ± 0.06 vs. C-8: 1.05 ± 0.16; SH-8: 1.25 ± 0.14; p b 0.001 G/l) (Fig. 1). 4.2. Changes in the number of CD19+ B lymphocytes in the peripheral blood of mice two and eight months after the operations Two months after the operations the number of circulating CD19+ B cells increased significantly in the splenectomized (SE-2) and decreased in the autotransplanted (AU-2) animals compared to the two
groups of controls (C-2 and SH-2). (SE-2: 0.72 ± 0.15 vs. C-2: 0.44 ± 0.05, G/l, p b 0.01, and SH-2: 0.42 ± 0.07 G/l p b 0.01; and AU-2: 0.25 ± 0.06 vs. C-2: 0.44 ± 0.05 G/l, P b 0.05, SH-2: 0.42 ± 0.07 G/l p b 0.05). Eight months after the operations, however, in both groups of operated animals (SE-8 and AU-8) the number of B cells was diminished compared to the two groups of controls (C-8 and SH-8) but only the fall measured in the splenectomized mice was significant (SE-8: 0.55 ± 0.09 vs. C-8: 0.81 ± 0.11 G/l p b 0.01, SH-8: 0.92 ± 0.16 G/l p b 0.01;) The value of AU-8: 0.71 ± 0.12 was not significant compared to the three other groups, however, it was higher than that measured in the splenectomized animals (0.55 ± 0.09) (Fig. 2). 4.3. Changes in the serum levels of immunoglobulin IgM in mice two and eight months after the operations The level of serum IgM was significantly decreased in the two operated (splenectomized and autotransplanted) groups compared to the controls both two and eight months after the operations (two months group: SE-2: 68.67 ± 8.43 vs. C-2: 88.25 ± 10.72 G/l p b 0.05; SH-2: 104.67 G/l, p b 0.01; and AU-2: 65.05 ± 6.41 vs. C-2: 88.25 ± 10.72 G/l, p b 0.01; SH-2: 104.67 ± 18.25 G/l, p b 0.01; eight months group: SE-8: 45.24 ± 7.03 vs. C8: 84.67 ± 9.16 G/l, p b 0.001, SH-8: 102.34 ± 10.74 G/l, p b 0.001; and AU-8: 63.08 ± 8.98 vs. C-8: 84.67 G/l, p b 0.001, SH-8:102.34 ± 10.74 G/l, p b 0.001). The level of serum IgM, however, was significantly higher in the autotransplanted (AU-8) than in the splenectomized (SE-8) group of animals (63.08 ± 8.96 vs. 45.24 ± 7.03 G/l, p b 0.05) (Fig. 3).
5. Discussion In the current work we present some significant changes in the peripheral distribution of CD3+ T and CD19+ B cells and in
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the level of serum IgM of autotransplanted mice compared to the splenectomized animals suggesting that a surgical attempt for the partial restoration of spleen after splenectomy may have some immunological benefits. The immune competence of the spleen is crucially dependent on the tissue architecture and the recirculation efficiency of lymphoid cells to and from the spleen. In our histological analyses performed eight months after the autotransplantation the lymphoid tissue structure was clearly compartmentalized into red pulp and lymphocyte-rich white pulp, latter with a slight Bcell dominance compared to controls (manifested as approximately 30% increase of B-cell area, and an approximately 50% reduction of T-cell zone, respectively). In preliminary adoptive cell-transfer studies in autotransplanted mice the majority of injected lymphocytes 90 min after the injection tended to pass through the red pulp compartment, with only a minority moving towards the white pulp, similarly to control mice (manuscript in preparation). However, the main point of this present work is that some functional and immunological activity of the grafted spleen slices can be observed in the inbred mice. (The series of the not significant changes found e.g. in the number of CD56+ NK cells, in the serum levels of IgG, IgA and the complement components, C3 and C4 are not presented). The first conclusion drawn from the data of significant alterations is that there are time dependent differences in the changes of immune system of mice, especially in the peripheral distribution (in the number) of circulating B cells. In addition, the alterations caused by splenectomy or autotransplantation also can be different two or eight months after the operations. It was surprising that the absolute number of CD3+ T cells was almost the same in the four groups two months after the operations. (But there were differences in the percent of cells. These data are not presented). On the other hand, a striking fall of these cells was observed in the two operated groups eight months after the interventions. In addition, it was also not negligible that the number of T cells was slightly (although not significantly) higher in the autotransplanted than in the splenectomized animals. In the case of B cells the directions of changes were different in the animals of different ages. Whereas the number of B lymphocytes was significantly elevated in the splenectomized mice compared to the controls two months after the operations, there was a rather great fall in the number of these cells in the autotransplanted mice. In this period of time, we found that the number of lymphocytes in the peripheral blood of splenectomized mice was 2.32 ± 0.65 G/l compared to the 1.81 ± 0.67 G/ l value of autotransplanted animals showing a slight but not significant elevation [28]. The increased bone marrow activity of splenectomized mice could be even better reflected by the remarkable neutrophil granulocytosis observed simultaneously [28]. We think that the reason(s) of the rather great differences in the number of circulating B and T lymphocytes in the autotransplanted animals can be related to the special and time dependent change in the “homing” circumstances for them in the “chips” as it was demonstrated in our in vivo cell transfer experiments mentioned above. It has to be stressed, however,
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that though eight months after the interventions, the number of circulating B cells was decreased in both splenectomized groups of animals, in the autotransplanted mice, this fall was less than in the splenectomized animals, showing a slightly better capacity for the production of immunoglobulins. The production of IgM is crucial after splenectomy from the point of view of “overwhelming postsplenectomy infection” (OPSI) in the surgical practice [3–8]. Therefore, it could be regarded to be the most important finding in this work that in the autotransplanted animals we measured a significantly higher level of serum IgM than in the splenectomized mice eight months after the operations. This change could be in parallel with the slightly elevated number of B cells in these mice helping possibly a better antimicrobial defense [29,30] than that of splenectomized animals, where the impaired production of IgM was reflected both two and eight months after the interventions in spite of the significantly high number of peripheral B cells in the group of two months old grafting. It was common in the present experiments that in the autotransplanted animals the values of T and B cell number and the serum IgM level were higher than in the splenectomized mice eight months after the operations reflecting the functional activity of replanted spleen tissue, however, it represented only the 10–15% of original mass. The fact that these elevations were found significant only in the cases of IgM measurements could possibly explained by the greater sensitivity of laser nephelometry than that of cytofluorimetry. This approach allows us to declare that possibly a direct and quantitative linkage can exist between the successfully implanted mass of spleen and its positive influence on the number of T and B cells and circulating IgM compared to splenectomy. Supposedly, the larger the mass of successfully transplanted spleen, the better is the clinical effect. The results of this work were observed in inbred mice but they showed a series of similarities to those that we got in the patients treated by the same surgical technique [2]. All the main cellular and serological parameters of splenectomy measured in human were the same what we found in these animals. The serum IgM elevating effect of autotransplantation was also demonstrated there but only the positive tendency because the differences did not reach the level of statistical significance. In conclusion, we think that the various surgical trials, not only the “ours,” for the partial restoration of the immunological function of spleen after splenectomy even with 10–15% part of the removed organ may have some real practical benefit. To prove these beneficial effects, however, our present work on inbred mice could give a more unambiguous experimental evidence than that we obtained in the patients. Therefore, these results can encourage the continuation of works on spleen autotransplantation also with the acceptance of the limitations related to it. Acknowledgments This work was supported by OTKA T034211. The authors express their thanks for technical assistance to Erika Somlyai and Katalin Éles.
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