Eur. surg. Res. 7: 34-42 (1975)
Surgical Aspects of Spleen Grafting in Rats H. B itter -S uermann 1 Department of Surgery. University of Cambridge, Cambridge
Key Words. Immunologically competent organ graft • Inbred rat system • Heter otopic transplantation, spleen Abstract. Grafting of the spleen as an organ of full immunological competence in inbred rats was found to be a useful tool for studies of immunological parame ters, which would be difficult to evaluate in outbred and larger animals. Early re sults indicate that the spleen is under certain circumstances less susceptible to rejec tion than other organs in the rat and knowledge about principles, underlying this phenomenon, might have application for grafting of organs in other species. Experi ences gained in grafting more than 400 rat spleens heterotopically were described together with the technique employed.
Perhaps because the spleen is regarded as non-vital, transplant sur geons have widely neglected this organ since in 1910 C arrel [5] reported the first successful spleen grafts in dogs. Renewed interest was shown by E yquem and O udot [8], who performed spleen grafting in goats, and Simonsen et al. [19J in 1953 who did this operation in dogs to analyse his tological changes in the grafts and the hosts' own spleens. It had been hoped that the grafted spleen would be useful in the treatment of haemo philia in dogs [17, 18], and man [12], in treating congenital hypogammaglobulinaemia and malignant diseases [15, 20], and metabolic defects in Gaucher’s disease [11]. The results were disappointing, partly because little was known about functions of spleen allografts since all knowledge was gathered in outbred dogs, where rejection made long-term graft ob servations impossible [7, 10, 13, 16]. When rejection was delayed by ad-
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Receivcd: August 12, 1974; accepted: December 2, 1974.
B itter -S uermann
35
ministration of immunosuppressive drugs, desirable functions of the graft ed spleens were interfered with, thus abrogating the effects the grafts were hoped to exert on their hosts. A technique of transplanting rat spleens heterotopically was described by L ee and O rloff [14] and by C oburn [6]. The obvious advantages in the use of the inbred rat system initiated experiments which led to observ ations on graft-versus-host (GVH) and host-versus-graft (HVG) reactivi ties and their possible role in processes resulting in spleen graft accept ance, on the induction of spleen donor-specific unresponsiveness in recip ient rats, on transfer of immunity after pretreatment of spleen donors with recipient cells, and on survival of unmodified spleen allografts [1-4]. In this communication, technical experiences of grafting rat spleens in more than 400 experiments are described. Material and Methods Experimental Animals Rats of three inbred strains with strong histocompatibility differences [9] were used: Wistar, AGUS and PVG rats. Animals included in one experiment were of approximately the same age and weight, since disparities of factors such as body weight and age can readily disturb results after spleen grafting because of differing strength of GVH and HVG reactivities. Animals of either sex served as donors and recipients. Instruments, Sutures and Sterility A binocular loupe (magnification X 4) was used. The abdominal wall was re tracted by a palmar retractor. The anastomoses were accomplished by running overand-over sutures of 8/0 Ethilon, vessels were tied with 5/0 Mersilk. The technique was unsterile. The abdomen was shaved and washed with weak iodine solution. Recipients received a single dose of 30,000-50,000 IU benzyl peni cillin and 30 mg streptomycin i.m. at the end of surgery. With this regime, infec tions have been rare. Haemostasis and Replacement of Blood Loss All vessels to be cut were divided between ligatures. Only lumbar vessels were cauterised with a heated diathermy point. Before releasing the snares round aorta and vena cava, the anastomosis lines were covered with blood-absorbing ‘Sterispon-. After removal of this haemostatic cover, persistent bleeding points were stitched. No rat was closed unless complete haemostasis had been achieved. Shed blood was replaced by intravenous injections of Hartmann's solution into the infrahepatic vena cava.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Anaesthesia The rats were induced with halothane and anaesthesia was maintained by a sin gle injection of 9-12 mg Nembutal i.p. according to body weight. If the animals
36
B itt er -S uermann
Fig. 1. Start of donor operation, the stomach is retracted upwards and the gastrosplenic ligaments are divided, and access to the spleen is now easy.
Donor Operation The abdomen was entered by a midline full-length incision and the bowel was displaced to the right. The stomach was retracted upwards and the gastrosplenic lig aments were divided, which brought the spleen into view (fig. 1). All arteries coming off the coeliac axis were divided except the splenic artery. The pancreas was dissect ed from the spleen as completely as possible. In order to obtain a better view of the portal vein the duodenum was divided and retracted (fig. 2). Then the superior mes enteric artery was divided and the superior mesenteric vein was tied and divided just
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
were too light a swab, soaked with halothane, was placed near the nose. This was found preferable to further doses of Nembutal which frequently caused anaesthetic deaths.
Surgical Aspects of Spleen Grafting in Rats
37
distal to the confluence of the splenic vein. The gastroduodenal vein was divided and the portal vein was cleaned (fig. 3). Both renal arteries and the left renal vein were divided, the aorta was cleaned and tied distal to the isolated coeliac axis. At this stage, 200 units of heparin were injected intravenously and the portal vein and aorta were ready for division. Up to this point the splenic circulation had not been interrupted. The portal vein was divided at the porta hepatis and the first stitch of 8/0 Ethilon was inserted into the anterior wall of the vessel, its lumen being easy to identify because of the heavy bleeding (fig. 4). This stitch was useful for orientation in the recipient operation for avoiding twisting of the vein. Finally the aorta was cut cranial to the coeliac axis and the spleen was stored in Hartmann's solution at 4 °C until transplantation.
Fig. 2. Donor operation, the pancreas has been dissected close to the splenic ves sels, the duodenum is divided which brings the superior mesenteric and portal vein into view.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Recipient Operation The abdomen was opened as in the donor and the infrarenal aorta and vena cava were exposed down to the iliac vessels. 3/0 silk ties were passed around vena cava and aorta caudal to the left kidney and cranial to the iliac vessels and threaded through rubber tubing to form snares, leaving about 2 cm of vessel for anastomosis (fig. 5). The portal vein was anastomosed to the vena cava end-to-side. the back wall was sewn from inside and the front wall from outside. Then the cranial end of the aortic cuff was joined to the aorta some 3-4 mm distal to the venous anastomosis in a similar way (fig. 6). After the snares had been released, an immediate pulsation in the splenic hilum was observed, though it took some 5 min before the spleen was well perfused. This could be ascertained by compression of the draining vein, which
38
B itter -S uermann
Fig. 3. Donor operation, the superior mesenteric artery is divided and held by a tie, the portal vein is now draining splenic blood only after the superior mesenteric vein has been divided. Just cranial to the superior mesenteric artery the coeliac axis is visible which is supplying the spleen alone after ligature of all other arteries. Fig. 4. Donor operation, the aorta is divided just caudal to the coeliac axis and tied, the first 8/0 stitch of Ethilon is inserted into the lumen of the portal vein, the spleen is still circulated at this stage. was a mandatory procedure and made the spleen swell, indicating properly func tioning anastomoses. Both poles of the spleen were stitched to the left psoas muscle to prevent rotation about the long splenic pedicle (fig. 7). The abdomen was closed in layers. Pre- and Post-Operative Care The rats were not starved pre-operatively and recipients were, after surgery, care fully warmed up in their cages and had immediate access to water and food. Their daily observation within a month of operation was desirable in order to monitor signs of GVH disease.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Determination of Graft Acceptance or Rejection Since there is no other reliable way of assessing the viability of a grafted rat spleen, each recipient underwent several laparotomies after spleen grafting and the visible circulation in the splenic pedicle, the colour, consistency, and size of the graft were monitored together with the strength of bleeding at the biopsy site. A slice of tissue of approximately 2X3 mm size was excised for histological examina tion. The capsule was sewn by a mattress suture. Such laparotomies could be re-
Surgical Aspects of Spleen Grafting in Rats
39
Fig. 5. Recipient operation, the infrarenal vena cava and aorta are exposed and ready to be clamped by silk snares. The dotted lines on the walls of the vessels mark the places for venous (top left) and arterial (below right) anastomoses.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Fig. 6. Recipient operation with completed venous (right) and arterial (left) an astomoses. The snares have not yet been released. Note that the anastomoses arc some millimeters apart from each other which makes control of haemostasis easier and will avoid kinking of the vessels after the spleen has been attached to the left psoas muscle (towards the reader).
40
BlTTER-SUERMANN
Fig. 7. Recipient operation before the abdomen is closed, the descendent colon is covering the venous anastomosis (above), whereas the aortic cuff with coeliac axis (below) is visible. The poles of the spleen are fixed to the left psoas muscle and ro tation of the spleen about its long pedicle will be impossible. peated at weekly and shorter intervals, if serial biopsies were required. Rejected spleens were enlarged up to six times normal size after about 2-3 weeks, became hard and patchy, then white, soft, and necrotic, and were finally absorbed complete ly. Accepted spleen allografts grew' to about three times normal size after 2-3 weeks and then showed a tendency to reduce slowly in size. They were fairly soft but al ways had a thickened capsule. Spleen isografts remained macroscopically un changed. Histological findings were variable in accordance to different experimental set-ups and are reported elsewhere.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Mortality Rats which had an unsatisfactory circulation in the graft or were in poor condi tion because of extensive haemorrhage were sacrificed at the end of operation. Such rats were not included in any statistical analysis. The overall mortality within 1
Surgical Aspects of Spleen Grafting in Rats
41
week of surgery due to technical errors, but disregarding immunologically caused deaths (GVH disease), was 19°/o in the first hundred experiments, 9°/o in the second hundred, 4% in the third hundred, and 6% in the fourth hundred.
Discussion The spleen is a lymphoid organ and part of the reticuloendothelial sys tem. Its surgical removal after traumatic lesions and when indicated in cases of disorders of the haemopoietic system combined with splenomega ly is a standard procedure in daily general surgery. The fact that patients do well after splenectomy might have lent little support to the concept of experimentally grafting, rather than removing, this organ. This is reflected by the poor response to a description of a technique of grafting rat spleens in 1969 [6, 14]. Splenic transplantation in inbred rats provides for the study of immu nologically competent allografts under strictly controlled conditions. With the technique employed it becomes possible to perform several such oper ations daily. The lack of knowledge of any functions which could be con tributed solely to the spleen, makes the follow-up of spleen-grafted rats rather difficult when compared with the follow-up of rats carrying grafts of vital organs. Thus the only reliable means of assessing viability, rather than function, of spleen grafts was histological examination of biopsies. Daily observation of operated animals within 2-3 weeks of surgery was necessary to distinguish between deaths caused by technical errors and such induced by GVH disease [3]. The above described technique of grafting rat spleens might encourage further research in this field where surgery and immunology are close each other. A cknowledgements 1 am grateful to Mrs. M. F. A llen , Harston, Cambridge, for the illustrations, to Dr. A. S. M acD onald, University of Halifax, Nova Scotia, Canada, for technical help at the start of this work, and to Professor R. Y. C alne, Department of Surgery, University of Cambridge, for guidance and encouragement. This study was supported by a grant from ‘Deutsche Forschungsgemeinschaft’, Bonn-Bad Godesberg, FRG.
References
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
1 Bitter -Suermann, H.: Prolonged survival of auxiliary spleen allografts in rats inducing acceptance of skin allografts. Transplantation 17: 75 (1974). 2 B r r i E R - S u E R M A N N , H.: Survival of unmodified spleen allografts in rats. Nature 247: 465 (1974).
42
BllTER-SUERMANN
3 BrrTER-SuERMANN, H.: Induction of lethal graft versus host disease in rats by spleen grafting. J. surg. Res. 17: 352 (1974). 4 Bitter -Suermann. H.: Different ways of using GVH reactivities to achieve ac ceptance of rat spleen allografts in the same donor-recipient pairing. Transplan tation (in press, 1974). 5 C arrel, A.: Remote results of replantation of the kidney and the spleen. J. exp. Med. 12: 146 (1910). 6 C oburn , R. J.: Spleen transplantation in the rat. Transplantation 8: 86 (1969). 7 D ammin, G. J.; W heeler , H. B.; M ontague, A. C. W.; D ealy. J. B.: G reen berg , J. B., and M oore , F. D.: The splenic homograft: its course in the unmodi fied and modified canine recipient. Ann. N.Y. Acad. Sei. 99: 861 (1962), 8 E yquem , A. et O udot , J.: Observations biologiques chez des chèvres ayant subi des iso-transplantations de rate. C. R. Acad. Sei. 217: 275 (1953). 9 F esting , M. and Staats, J.: Standardized nomenclature for inbred strains of rats, fourth listing. Transplantation 16: 221 (1973). 10 F isher , B. and F isher , E. R.: Observations concerning spleen homotransplan tation in normal and irrediated animais. Surg. Gyr.ec. Obstet. 112: 455 (1961). 11 G roth , C. G.; H agenfeldt , L.; D reborg , S.; L öfström , B.; Ö ckerman, P. A.; Samuelsson, K.; Svennerholm , L.; W erner , B., and W estberg , G.: Splenic transplantation in a case of Gaucher’s disease. Lancet /: 1260 (1971). 12 H athaway, W. E.; M ull, M. M.; G ithens , J. H.; G roth , C. G.; M archioro, T. L., and Starzl , T. E.: Attempted spleen transplantation in classical hemophilia. Transplantation 7: 73 (1969). 13 J ordan, G. K.; Fiscus, W. G.; T rentin , J. J.; F oster, R. P.; C ollins , V. P„ and Barton, H. L.: Splenic transplantation in the dog. Ann. N.Y. Acad. Sei. 120: 612 (1964). 14 L ee , S. and O rloff , M. J.: A technique for splenic transplantation in the rat. Surgery 65: 436 (1969). 15 M archioro, T. L.; Rowlands , D. T.; R ifkind , D.; W addell, W. R.; Starzl , T. E., and F udenberg , H.: Splenic homotransplantation. Ann. N.Y. Acad. Sei. 120:
H. Bitter -S uermann, Department of Surgery I, University of Göteborg, Sahlgrenska Sjukhuset, S-4I3 45 Göteborg (Sweden)
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626 (1964). 16 M oore , F. D.: W heeler , H. B.; D emissianos, H. V.; Sm ith , L. L.; Bai.ankura, O.; A ree , K.; G reenberg , J. B., and D ammin, G. J.: Experimental whole-organ transplantation of the liver and of the spleen. Ann. Surg. 152: 374 (1960). 17 N orman, J. C.; C ovelli, V. H., and Sise , H. S.: Transplantation o f the spleen: experimental cure o f hemophilia. Surgery 64: 1 (1968). 18 P eacock, E. E.; W ebster , W. P.; P enick , G. D.; M adden , J. W., and H utchin , P. : Transplantation of the spleen. Transplant. Proc. 1: 239 (1969). 19 Simonsen , M.; Buemann, J.; G ammeltoft , A.; J ensen , F., and J örgensen , K.: Biological incompatibility in kidney transplantation in dogs. I. Experimental and morphological investigations. Acta path, microbiol. scand. 32: 1 (1953). 20 Starzl, T. E.; M archioro, T. L.; R ifkind , D.; R owlands, D. T., and W addell, W. R.: Clinical experience with organ transplantation. Sth med. J. 58: 131 (1965).
Surgical Aspects of Spleen Grafting in Rats H. B itter -S uermann 1 Department of Surgery. University of Cambridge, Cambridge
Key Words. Immunologically competent organ graft • Inbred rat system • Heter otopic transplantation, spleen Abstract. Grafting of the spleen as an organ of full immunological competence in inbred rats was found to be a useful tool for studies of immunological parame ters, which would be difficult to evaluate in outbred and larger animals. Early re sults indicate that the spleen is under certain circumstances less susceptible to rejec tion than other organs in the rat and knowledge about principles, underlying this phenomenon, might have application for grafting of organs in other species. Experi ences gained in grafting more than 400 rat spleens heterotopically were described together with the technique employed.
Perhaps because the spleen is regarded as non-vital, transplant sur geons have widely neglected this organ since in 1910 C arrel [5] reported the first successful spleen grafts in dogs. Renewed interest was shown by E yquem and O udot [8], who performed spleen grafting in goats, and Simonsen et al. [19J in 1953 who did this operation in dogs to analyse his tological changes in the grafts and the hosts' own spleens. It had been hoped that the grafted spleen would be useful in the treatment of haemo philia in dogs [17, 18], and man [12], in treating congenital hypogammaglobulinaemia and malignant diseases [15, 20], and metabolic defects in Gaucher’s disease [11]. The results were disappointing, partly because little was known about functions of spleen allografts since all knowledge was gathered in outbred dogs, where rejection made long-term graft ob servations impossible [7, 10, 13, 16]. When rejection was delayed by ad-
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Receivcd: August 12, 1974; accepted: December 2, 1974.
B itter -S uermann
35
ministration of immunosuppressive drugs, desirable functions of the graft ed spleens were interfered with, thus abrogating the effects the grafts were hoped to exert on their hosts. A technique of transplanting rat spleens heterotopically was described by L ee and O rloff [14] and by C oburn [6]. The obvious advantages in the use of the inbred rat system initiated experiments which led to observ ations on graft-versus-host (GVH) and host-versus-graft (HVG) reactivi ties and their possible role in processes resulting in spleen graft accept ance, on the induction of spleen donor-specific unresponsiveness in recip ient rats, on transfer of immunity after pretreatment of spleen donors with recipient cells, and on survival of unmodified spleen allografts [1-4]. In this communication, technical experiences of grafting rat spleens in more than 400 experiments are described. Material and Methods Experimental Animals Rats of three inbred strains with strong histocompatibility differences [9] were used: Wistar, AGUS and PVG rats. Animals included in one experiment were of approximately the same age and weight, since disparities of factors such as body weight and age can readily disturb results after spleen grafting because of differing strength of GVH and HVG reactivities. Animals of either sex served as donors and recipients. Instruments, Sutures and Sterility A binocular loupe (magnification X 4) was used. The abdominal wall was re tracted by a palmar retractor. The anastomoses were accomplished by running overand-over sutures of 8/0 Ethilon, vessels were tied with 5/0 Mersilk. The technique was unsterile. The abdomen was shaved and washed with weak iodine solution. Recipients received a single dose of 30,000-50,000 IU benzyl peni cillin and 30 mg streptomycin i.m. at the end of surgery. With this regime, infec tions have been rare. Haemostasis and Replacement of Blood Loss All vessels to be cut were divided between ligatures. Only lumbar vessels were cauterised with a heated diathermy point. Before releasing the snares round aorta and vena cava, the anastomosis lines were covered with blood-absorbing ‘Sterispon-. After removal of this haemostatic cover, persistent bleeding points were stitched. No rat was closed unless complete haemostasis had been achieved. Shed blood was replaced by intravenous injections of Hartmann's solution into the infrahepatic vena cava.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Anaesthesia The rats were induced with halothane and anaesthesia was maintained by a sin gle injection of 9-12 mg Nembutal i.p. according to body weight. If the animals
36
B itt er -S uermann
Fig. 1. Start of donor operation, the stomach is retracted upwards and the gastrosplenic ligaments are divided, and access to the spleen is now easy.
Donor Operation The abdomen was entered by a midline full-length incision and the bowel was displaced to the right. The stomach was retracted upwards and the gastrosplenic lig aments were divided, which brought the spleen into view (fig. 1). All arteries coming off the coeliac axis were divided except the splenic artery. The pancreas was dissect ed from the spleen as completely as possible. In order to obtain a better view of the portal vein the duodenum was divided and retracted (fig. 2). Then the superior mes enteric artery was divided and the superior mesenteric vein was tied and divided just
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
were too light a swab, soaked with halothane, was placed near the nose. This was found preferable to further doses of Nembutal which frequently caused anaesthetic deaths.
Surgical Aspects of Spleen Grafting in Rats
37
distal to the confluence of the splenic vein. The gastroduodenal vein was divided and the portal vein was cleaned (fig. 3). Both renal arteries and the left renal vein were divided, the aorta was cleaned and tied distal to the isolated coeliac axis. At this stage, 200 units of heparin were injected intravenously and the portal vein and aorta were ready for division. Up to this point the splenic circulation had not been interrupted. The portal vein was divided at the porta hepatis and the first stitch of 8/0 Ethilon was inserted into the anterior wall of the vessel, its lumen being easy to identify because of the heavy bleeding (fig. 4). This stitch was useful for orientation in the recipient operation for avoiding twisting of the vein. Finally the aorta was cut cranial to the coeliac axis and the spleen was stored in Hartmann's solution at 4 °C until transplantation.
Fig. 2. Donor operation, the pancreas has been dissected close to the splenic ves sels, the duodenum is divided which brings the superior mesenteric and portal vein into view.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Recipient Operation The abdomen was opened as in the donor and the infrarenal aorta and vena cava were exposed down to the iliac vessels. 3/0 silk ties were passed around vena cava and aorta caudal to the left kidney and cranial to the iliac vessels and threaded through rubber tubing to form snares, leaving about 2 cm of vessel for anastomosis (fig. 5). The portal vein was anastomosed to the vena cava end-to-side. the back wall was sewn from inside and the front wall from outside. Then the cranial end of the aortic cuff was joined to the aorta some 3-4 mm distal to the venous anastomosis in a similar way (fig. 6). After the snares had been released, an immediate pulsation in the splenic hilum was observed, though it took some 5 min before the spleen was well perfused. This could be ascertained by compression of the draining vein, which
38
B itter -S uermann
Fig. 3. Donor operation, the superior mesenteric artery is divided and held by a tie, the portal vein is now draining splenic blood only after the superior mesenteric vein has been divided. Just cranial to the superior mesenteric artery the coeliac axis is visible which is supplying the spleen alone after ligature of all other arteries. Fig. 4. Donor operation, the aorta is divided just caudal to the coeliac axis and tied, the first 8/0 stitch of Ethilon is inserted into the lumen of the portal vein, the spleen is still circulated at this stage. was a mandatory procedure and made the spleen swell, indicating properly func tioning anastomoses. Both poles of the spleen were stitched to the left psoas muscle to prevent rotation about the long splenic pedicle (fig. 7). The abdomen was closed in layers. Pre- and Post-Operative Care The rats were not starved pre-operatively and recipients were, after surgery, care fully warmed up in their cages and had immediate access to water and food. Their daily observation within a month of operation was desirable in order to monitor signs of GVH disease.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Determination of Graft Acceptance or Rejection Since there is no other reliable way of assessing the viability of a grafted rat spleen, each recipient underwent several laparotomies after spleen grafting and the visible circulation in the splenic pedicle, the colour, consistency, and size of the graft were monitored together with the strength of bleeding at the biopsy site. A slice of tissue of approximately 2X3 mm size was excised for histological examina tion. The capsule was sewn by a mattress suture. Such laparotomies could be re-
Surgical Aspects of Spleen Grafting in Rats
39
Fig. 5. Recipient operation, the infrarenal vena cava and aorta are exposed and ready to be clamped by silk snares. The dotted lines on the walls of the vessels mark the places for venous (top left) and arterial (below right) anastomoses.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Fig. 6. Recipient operation with completed venous (right) and arterial (left) an astomoses. The snares have not yet been released. Note that the anastomoses arc some millimeters apart from each other which makes control of haemostasis easier and will avoid kinking of the vessels after the spleen has been attached to the left psoas muscle (towards the reader).
40
BlTTER-SUERMANN
Fig. 7. Recipient operation before the abdomen is closed, the descendent colon is covering the venous anastomosis (above), whereas the aortic cuff with coeliac axis (below) is visible. The poles of the spleen are fixed to the left psoas muscle and ro tation of the spleen about its long pedicle will be impossible. peated at weekly and shorter intervals, if serial biopsies were required. Rejected spleens were enlarged up to six times normal size after about 2-3 weeks, became hard and patchy, then white, soft, and necrotic, and were finally absorbed complete ly. Accepted spleen allografts grew' to about three times normal size after 2-3 weeks and then showed a tendency to reduce slowly in size. They were fairly soft but al ways had a thickened capsule. Spleen isografts remained macroscopically un changed. Histological findings were variable in accordance to different experimental set-ups and are reported elsewhere.
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
Mortality Rats which had an unsatisfactory circulation in the graft or were in poor condi tion because of extensive haemorrhage were sacrificed at the end of operation. Such rats were not included in any statistical analysis. The overall mortality within 1
Surgical Aspects of Spleen Grafting in Rats
41
week of surgery due to technical errors, but disregarding immunologically caused deaths (GVH disease), was 19°/o in the first hundred experiments, 9°/o in the second hundred, 4% in the third hundred, and 6% in the fourth hundred.
Discussion The spleen is a lymphoid organ and part of the reticuloendothelial sys tem. Its surgical removal after traumatic lesions and when indicated in cases of disorders of the haemopoietic system combined with splenomega ly is a standard procedure in daily general surgery. The fact that patients do well after splenectomy might have lent little support to the concept of experimentally grafting, rather than removing, this organ. This is reflected by the poor response to a description of a technique of grafting rat spleens in 1969 [6, 14]. Splenic transplantation in inbred rats provides for the study of immu nologically competent allografts under strictly controlled conditions. With the technique employed it becomes possible to perform several such oper ations daily. The lack of knowledge of any functions which could be con tributed solely to the spleen, makes the follow-up of spleen-grafted rats rather difficult when compared with the follow-up of rats carrying grafts of vital organs. Thus the only reliable means of assessing viability, rather than function, of spleen grafts was histological examination of biopsies. Daily observation of operated animals within 2-3 weeks of surgery was necessary to distinguish between deaths caused by technical errors and such induced by GVH disease [3]. The above described technique of grafting rat spleens might encourage further research in this field where surgery and immunology are close each other. A cknowledgements 1 am grateful to Mrs. M. F. A llen , Harston, Cambridge, for the illustrations, to Dr. A. S. M acD onald, University of Halifax, Nova Scotia, Canada, for technical help at the start of this work, and to Professor R. Y. C alne, Department of Surgery, University of Cambridge, for guidance and encouragement. This study was supported by a grant from ‘Deutsche Forschungsgemeinschaft’, Bonn-Bad Godesberg, FRG.
References
Downloaded by: Nagoya University 133.6.82.173 - 1/10/2019 10:23:08 PM
1 Bitter -Suermann, H.: Prolonged survival of auxiliary spleen allografts in rats inducing acceptance of skin allografts. Transplantation 17: 75 (1974). 2 B r r i E R - S u E R M A N N , H.: Survival of unmodified spleen allografts in rats. Nature 247: 465 (1974).
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3 BrrTER-SuERMANN, H.: Induction of lethal graft versus host disease in rats by spleen grafting. J. surg. Res. 17: 352 (1974). 4 Bitter -Suermann. H.: Different ways of using GVH reactivities to achieve ac ceptance of rat spleen allografts in the same donor-recipient pairing. Transplan tation (in press, 1974). 5 C arrel, A.: Remote results of replantation of the kidney and the spleen. J. exp. Med. 12: 146 (1910). 6 C oburn , R. J.: Spleen transplantation in the rat. Transplantation 8: 86 (1969). 7 D ammin, G. J.; W heeler , H. B.; M ontague, A. C. W.; D ealy. J. B.: G reen berg , J. B., and M oore , F. D.: The splenic homograft: its course in the unmodi fied and modified canine recipient. Ann. N.Y. Acad. Sei. 99: 861 (1962), 8 E yquem , A. et O udot , J.: Observations biologiques chez des chèvres ayant subi des iso-transplantations de rate. C. R. Acad. Sei. 217: 275 (1953). 9 F esting , M. and Staats, J.: Standardized nomenclature for inbred strains of rats, fourth listing. Transplantation 16: 221 (1973). 10 F isher , B. and F isher , E. R.: Observations concerning spleen homotransplan tation in normal and irrediated animais. Surg. Gyr.ec. Obstet. 112: 455 (1961). 11 G roth , C. G.; H agenfeldt , L.; D reborg , S.; L öfström , B.; Ö ckerman, P. A.; Samuelsson, K.; Svennerholm , L.; W erner , B., and W estberg , G.: Splenic transplantation in a case of Gaucher’s disease. Lancet /: 1260 (1971). 12 H athaway, W. E.; M ull, M. M.; G ithens , J. H.; G roth , C. G.; M archioro, T. L., and Starzl , T. E.: Attempted spleen transplantation in classical hemophilia. Transplantation 7: 73 (1969). 13 J ordan, G. K.; Fiscus, W. G.; T rentin , J. J.; F oster, R. P.; C ollins , V. P„ and Barton, H. L.: Splenic transplantation in the dog. Ann. N.Y. Acad. Sei. 120: 612 (1964). 14 L ee , S. and O rloff , M. J.: A technique for splenic transplantation in the rat. Surgery 65: 436 (1969). 15 M archioro, T. L.; Rowlands , D. T.; R ifkind , D.; W addell, W. R.; Starzl , T. E., and F udenberg , H.: Splenic homotransplantation. Ann. N.Y. Acad. Sei. 120:
H. Bitter -S uermann, Department of Surgery I, University of Göteborg, Sahlgrenska Sjukhuset, S-4I3 45 Göteborg (Sweden)
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626 (1964). 16 M oore , F. D.: W heeler , H. B.; D emissianos, H. V.; Sm ith , L. L.; Bai.ankura, O.; A ree , K.; G reenberg , J. B., and D ammin, G. J.: Experimental whole-organ transplantation of the liver and of the spleen. Ann. Surg. 152: 374 (1960). 17 N orman, J. C.; C ovelli, V. H., and Sise , H. S.: Transplantation o f the spleen: experimental cure o f hemophilia. Surgery 64: 1 (1968). 18 P eacock, E. E.; W ebster , W. P.; P enick , G. D.; M adden , J. W., and H utchin , P. : Transplantation of the spleen. Transplant. Proc. 1: 239 (1969). 19 Simonsen , M.; Buemann, J.; G ammeltoft , A.; J ensen , F., and J örgensen , K.: Biological incompatibility in kidney transplantation in dogs. I. Experimental and morphological investigations. Acta path, microbiol. scand. 32: 1 (1953). 20 Starzl, T. E.; M archioro, T. L.; R ifkind , D.; R owlands, D. T., and W addell, W. R.: Clinical experience with organ transplantation. Sth med. J. 58: 131 (1965).
