- Email: [email protected]
A technique for complete thymectomy in adult rats
ELSEVIER
JOURNALOF IMMUNOLO61CAL METHODS Journal of ImmunologicalMethods 171 (1994) 33-36
A technique for complete thymectomy in adult rats Eiji Kobayashi *'a'b, Naoshi Kamada b, Shin Enosawa c, Nobuyuki Toyama a,b, Luc Delriviere h, Shigeru Goto b, Roger Lord b, Steve Stamatiou b, Michio Miyata
a
a Department of Surgery, Omiya Medical Center, Jichi Medical School, Amanuma 1-847, Omiya 330, Japan, b Department of Surgery, Universityof Queensland and QIMI~ Brisbane QLD 4029, Australia, c Department of Experimental Surgery, National Children's Medical Research Center, Tokyo 154, Japan
(Received 23 August 1993; revised received 19 November 1993; accepted 15 December 1993)
Abstract
50 open thymectomies were performed in adult rodents using intubation combined with a fibrin glue able to prevent hemorrhage and pulmonary air leakage. This method had a 100% success rate and lower mortality than the ordinal suction procedure. Although the conventional suction thymectomy has been widely used, the open thymectomy method would permit more complete thymectomies for immunological studies. Key words: Thymectomy; Tracheal intubation; Suction method; Fibrin glue
1. Introduction
It is well known that the thymus is indispensable for T cell maturation and tolerance induction (Kappler et al., 1987; Kisielow et al., 1988; Finkel et al., 1989). In immunological studies of adult rodents suction thymectomy is a widely used procedure. However, there are problems associated with this method, since it has a high mortality rate and it is difficult to ensure that complete ablation has been achieved. In 1979, Barbul et al. introduced a method of open thymectomy using tracheal intubation combined with positive pressure. Unfortunately, although a wide sternotomy gives a full view of the thymus,
* Corresponding author. Tel.: (81-48) 647-2111; Fax: (81-48) 648-5188.
this technique has not proved to be successful in our hands. In particular, we experienced problems of both h e m o r r h a g e and p n e u m o t h o r a x possibly attributable to our lack of experience with this technique. Accordingly, we have developed a modification of this procedure using a fibrin glue which controls bleeding and pulmonary air leaks (Borst et al., 1982). Here, we describe in detail a satisfactory method of complete thymectomy which gives a high success rate, and we compare it to the ordinal suction method.
2. Materials and methods 2.1. Rats
Male and female P V G ( R T 1 c) and ( L E W x BN) F 1 hybrid (RT1 l/n) rats (6-12 weeks old, weighing 125-330 g) were used.
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00016-P
34
E. Kobayashi et al. /Journal of lmmunological Methods 171 (1994) 33-36
2.2. Surgical procedure
E t h e r anesthesia was used because the depth of anesthesia could easily controlled. Sodium pentobarbitorate may also be used in place of ether for anesthesia in laboratories where the use of ether is restricted or unavailable. However, rats should be anesthetized slowly, since rapid induction increases the production of secretions. After controlled anesthesia, a 4 - 5 cm middle neck incision was carried out. Two surgical procedures were used. (1) The ordinal suction method as described by Ford (1987) was performed in 20 rats. (2) The tracheal intubation procedure was performed in 50 rats using a modification of the method of Barbul et al. (1979). The pretracheal muscles were separated until the trachea was exposed. The separated muscles were ligated with 4-0 Dexson (American Cyanamid, Pearl River, NY, USA) to permit traction in operation time and final rapid approximation. After traction of the ligated muscles, the trachea was separated from its surroundings and a 2-0 silk suture was passed around it. The trachea was punctured with a fine steel wire between two intratracheal rings and the wire was passed retrograde through the mouth. Occasionally there was some difficulty in passing the wire, but the process was unusually assisted by the traction of the rat's tongue. A 4 F G polyethylene tube (Portex, UK) was then introduced into the trachea using the wire as a guide (Fig. 1). The intubated tube was tied to the lower jaw of the rat in order to fix it in place. A 1.5 cm (approximately) midline sternotomy was carried out to expose the thymus (Fig. 2). The u p p e r poles of the thymus were then gently teased until free using iris forceps. The thymus was then removed by separating it from the pleural linings. Care was taken not to injure the superior vena cava or internal carotid artery, when cauterizing the tissue (Fig. 3a). It was essential to remove the thymus completely and without trauma (Fig. 4). When the pleural cavity was open, respiration was controlled by positive pressure. Pressure was applied to evacuate air from the pleural cavity, and fibrin glue (Beriplast P, Hoechst Japan, Tokyo) was then applied to the separated muscle tissue (Fig. 3b). The pin hole at
(a)
(b)
Fig. 1. Illustration of the incubation method and use of the guide wire. a: exposure of trachea; b: passing a fine steel wire through the mouth; c: intubation using the wire as a guide.
the punctured trachea was also closed using this glue. A few minutes were sufficient to allow tight closure. Only one ligation was required between the muscle using the tensioned suture. The skin was then closed with a running 4-0 Dexson suture.
Fig. 2. Full view of the thymus after median sternotomy.
35
E. Kobayashi et al. /Journal of Immunological Methods 171 (1994) 33-36
hl,iI LII,,, ,' ' ii
,
!
', ,/1
(a)
(b) Fig. 3. Removal of the thymus and closure using fibrin glue. a: complete thymectomywithout bleeding; b: quick closure and sealing of air leakage using fibrin glue.
2.3. A s s e s s m e n t
The success of the thymectomy was confirmed 1-10 weeks later both macroscopically and histologically.
3. Results A n open thymectomy procedure was performed in 50 rats by the same operator (E.K.). The results obtained are shown in Table 1. In the case of three rats in the intubation method group, the trachea was damaged, nasal bleeding was observed and eventually the rats died of bleeding and air leakage from the punctured trachea. We believe, therefore, that in the event of injury or bleeding due to such complications the animals should be killed. However, there were no cases of damage to the trachea in another series of 50 rats
Fig. 4. Resected thymus. Complete thymuswithout injury.
undergoing thymectomy using this method. The operation time required was 14 + 2 min in the surviving 47 cases. 94% of rats survived and complete thymectomy was confirmed macroscopically. Histological examination confirmed the absence of thymic tissue and revealed that a number of mononuclear cells had infiltrated the tissue surrounding the fibrin glue 7 - 1 4 days after the operation. As previously reported with mice, no glue was observed 3 weeks postoperatively (Kobayashi et al., 1991). 20 thymectomies were carried out using the suction method and four of these animals died post-operatively. The causes of death were hemorrhage from the branch of the superior vena cava in two cases and pneumothorax in the other two rats. The total operating time for the suction procedure was 5 min. 16 rats survived and in five cases (31%) remnants of the thymic organ, including epithelial tissue, remained in situ.
Table 1 Comparison of suction and intubation methods of thymectomy Procedure n Mortality
Complete rate
Operation time
Suction method Intubation method
69% (11/16) 100% (47/47)
within 5 min 11-17 min
20 50
20% (4*/20) 6% (3**/50)
* Fatal hemmorrhage (2 cases) and pneumothorax (2 cases). ** Damage to the trachea caused by difficulties in passing the guide wire. *'** There was no statistically significant difference between the groups (Fisher's exact test, t = 0.0967).
36
E. Kobayashi et al. /Journal of Immunological Methods 171 (1994) 33-36
4. Discussion Tolerance induction by neonatal thymectomy has been well documented (Miller, 1961), and the effect of thymectomy in adults has also been reported (Miller, 1962; Metcalf, 1965). The surgical technique of suctionthymectomy is the conventional method in general use and has been well standardized (Ford, 1978). However, this procedure is performed 'blind' and carries a high mortality rate in adult rodents. In particular, there is a danger of animals developing a pneumothorax because the thymic capsule is intimately attached to the pleural lining (Haimov et al., 1971). To overcome these problems, open-thymectomy was introduced by Barbul et al. in 1979. This technique offers a full view of the thymus using tracheal intubation. However, there are still some problems with the technique and air leakage can occur from the opened pleura and from the hole punctured by the guide wire. The survival of the rat requires the maintenance of positive pressure and this can be achieved either manually or with a respirator. Fibrin sealant made from concentrated fibrinogen has been introduced as a multicomponent biological adhesive. It can be used to establish hemostasis and also as an adhesive in wound repair. The sealing effect of fibrin glue has also been advocated in cardiovascular (Borst et al., 1982) and digestive operations (Ham et al., 1992) to prevent leakage. We have shown that open thymectomy using fibrin glue protects the rat from bleeding and air leakage after each operation. Comparing the intubation method and the ordinal suction technique of thymectomy showed that the suction method took less time and was easier to perform, but the thymus was not always completely removed. Although the intubation method appears difficult to perform, complete thymectomy using this technique was made easier because it en-
sured a full view of the organ. Open thymectomy was associated with a lower mortality rate and, in our hands, was always confirmed histologically.
Acknowledgements The authors thank Mrs. M. Saul for her excellent illustration and Mrs. H. Matthews for her precise photography.
References Barbul, A., Sisto, D.A. and Efron, G. (1979) A safe method of thymectomy in adult rats. Transplantation 28, 427. Borst, G.H., Haverich, A., Walterbusch G. and Maatz, W. (1982) Fibrin adhesive: An important hemostatic adjunct in cardiovascular operations. J. Thorac. Cardiovasc. Surg. 84, 548. Finkel, T.H., Cambier, J.C., Kubo, R.T., Born, W.K., Marrack, P. and Kappler, J.W. (1989) The thymus has two functionally distinct populations of immature alpha-beta + T cells: One population is deleted by ligation of alpha-beta TcR. Cell 58, 1047. Ford, W.L. (1978) In: D.M. Weie (Ed.), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, p. 231. Haimov, M., Wong, D., Burrows, L. et al. (1971) Thymectomy in the adult mouse. Transplantation 11, 100. Ham, A.C., Kort, W.J., Weijima, I.M., Ingh, H.F.G.M. and Jeekel, H. (1992) Effect of antibiotics in fibrin sealant on healing colonic anastomoses in the rat. Br. J. Surg. 79, 525. Kappler, J.W., Roehm, N. and Marrack, P.(1987) T cell tolerance by clonal elimination in the thymus. Cell 49, 273. Kisielow, P., Bluthmann, H., Staerz, U.D. et al. (1988) Tolerance in T-ceil-receptor transgenic mice involves deletion of nonmature CD4+ CD8 + thymocytes. Nature 333, 742. Kobayashi, E., Igarashi, Y., Kawai, K., Watanabe, H., Matumoto, Y. and Fujiwara, M. (1991) Experience of use of fibrin glue (Beriplast R P). Shinryou to Shinyaku (Jpn.) 28, 869. Metcalf, D. (1965) Delayed effect of thymus in adult life on immunological competence. Nature 208, 1336. Miller, J.F.A.P. (1961) Immunological functions of the thymus. Lancet ii, 748. Miller, J.F.A.P. (1962) Immunological significance of the thymus of adult mouse. Nature 195, 1318.
JOURNALOF IMMUNOLO61CAL METHODS Journal of ImmunologicalMethods 171 (1994) 33-36
A technique for complete thymectomy in adult rats Eiji Kobayashi *'a'b, Naoshi Kamada b, Shin Enosawa c, Nobuyuki Toyama a,b, Luc Delriviere h, Shigeru Goto b, Roger Lord b, Steve Stamatiou b, Michio Miyata
a
a Department of Surgery, Omiya Medical Center, Jichi Medical School, Amanuma 1-847, Omiya 330, Japan, b Department of Surgery, Universityof Queensland and QIMI~ Brisbane QLD 4029, Australia, c Department of Experimental Surgery, National Children's Medical Research Center, Tokyo 154, Japan
(Received 23 August 1993; revised received 19 November 1993; accepted 15 December 1993)
Abstract
50 open thymectomies were performed in adult rodents using intubation combined with a fibrin glue able to prevent hemorrhage and pulmonary air leakage. This method had a 100% success rate and lower mortality than the ordinal suction procedure. Although the conventional suction thymectomy has been widely used, the open thymectomy method would permit more complete thymectomies for immunological studies. Key words: Thymectomy; Tracheal intubation; Suction method; Fibrin glue
1. Introduction
It is well known that the thymus is indispensable for T cell maturation and tolerance induction (Kappler et al., 1987; Kisielow et al., 1988; Finkel et al., 1989). In immunological studies of adult rodents suction thymectomy is a widely used procedure. However, there are problems associated with this method, since it has a high mortality rate and it is difficult to ensure that complete ablation has been achieved. In 1979, Barbul et al. introduced a method of open thymectomy using tracheal intubation combined with positive pressure. Unfortunately, although a wide sternotomy gives a full view of the thymus,
* Corresponding author. Tel.: (81-48) 647-2111; Fax: (81-48) 648-5188.
this technique has not proved to be successful in our hands. In particular, we experienced problems of both h e m o r r h a g e and p n e u m o t h o r a x possibly attributable to our lack of experience with this technique. Accordingly, we have developed a modification of this procedure using a fibrin glue which controls bleeding and pulmonary air leaks (Borst et al., 1982). Here, we describe in detail a satisfactory method of complete thymectomy which gives a high success rate, and we compare it to the ordinal suction method.
2. Materials and methods 2.1. Rats
Male and female P V G ( R T 1 c) and ( L E W x BN) F 1 hybrid (RT1 l/n) rats (6-12 weeks old, weighing 125-330 g) were used.
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00016-P
34
E. Kobayashi et al. /Journal of lmmunological Methods 171 (1994) 33-36
2.2. Surgical procedure
E t h e r anesthesia was used because the depth of anesthesia could easily controlled. Sodium pentobarbitorate may also be used in place of ether for anesthesia in laboratories where the use of ether is restricted or unavailable. However, rats should be anesthetized slowly, since rapid induction increases the production of secretions. After controlled anesthesia, a 4 - 5 cm middle neck incision was carried out. Two surgical procedures were used. (1) The ordinal suction method as described by Ford (1987) was performed in 20 rats. (2) The tracheal intubation procedure was performed in 50 rats using a modification of the method of Barbul et al. (1979). The pretracheal muscles were separated until the trachea was exposed. The separated muscles were ligated with 4-0 Dexson (American Cyanamid, Pearl River, NY, USA) to permit traction in operation time and final rapid approximation. After traction of the ligated muscles, the trachea was separated from its surroundings and a 2-0 silk suture was passed around it. The trachea was punctured with a fine steel wire between two intratracheal rings and the wire was passed retrograde through the mouth. Occasionally there was some difficulty in passing the wire, but the process was unusually assisted by the traction of the rat's tongue. A 4 F G polyethylene tube (Portex, UK) was then introduced into the trachea using the wire as a guide (Fig. 1). The intubated tube was tied to the lower jaw of the rat in order to fix it in place. A 1.5 cm (approximately) midline sternotomy was carried out to expose the thymus (Fig. 2). The u p p e r poles of the thymus were then gently teased until free using iris forceps. The thymus was then removed by separating it from the pleural linings. Care was taken not to injure the superior vena cava or internal carotid artery, when cauterizing the tissue (Fig. 3a). It was essential to remove the thymus completely and without trauma (Fig. 4). When the pleural cavity was open, respiration was controlled by positive pressure. Pressure was applied to evacuate air from the pleural cavity, and fibrin glue (Beriplast P, Hoechst Japan, Tokyo) was then applied to the separated muscle tissue (Fig. 3b). The pin hole at
(a)
(b)
Fig. 1. Illustration of the incubation method and use of the guide wire. a: exposure of trachea; b: passing a fine steel wire through the mouth; c: intubation using the wire as a guide.
the punctured trachea was also closed using this glue. A few minutes were sufficient to allow tight closure. Only one ligation was required between the muscle using the tensioned suture. The skin was then closed with a running 4-0 Dexson suture.
Fig. 2. Full view of the thymus after median sternotomy.
35
E. Kobayashi et al. /Journal of Immunological Methods 171 (1994) 33-36
hl,iI LII,,, ,' ' ii
,
!
', ,/1
(a)
(b) Fig. 3. Removal of the thymus and closure using fibrin glue. a: complete thymectomywithout bleeding; b: quick closure and sealing of air leakage using fibrin glue.
2.3. A s s e s s m e n t
The success of the thymectomy was confirmed 1-10 weeks later both macroscopically and histologically.
3. Results A n open thymectomy procedure was performed in 50 rats by the same operator (E.K.). The results obtained are shown in Table 1. In the case of three rats in the intubation method group, the trachea was damaged, nasal bleeding was observed and eventually the rats died of bleeding and air leakage from the punctured trachea. We believe, therefore, that in the event of injury or bleeding due to such complications the animals should be killed. However, there were no cases of damage to the trachea in another series of 50 rats
Fig. 4. Resected thymus. Complete thymuswithout injury.
undergoing thymectomy using this method. The operation time required was 14 + 2 min in the surviving 47 cases. 94% of rats survived and complete thymectomy was confirmed macroscopically. Histological examination confirmed the absence of thymic tissue and revealed that a number of mononuclear cells had infiltrated the tissue surrounding the fibrin glue 7 - 1 4 days after the operation. As previously reported with mice, no glue was observed 3 weeks postoperatively (Kobayashi et al., 1991). 20 thymectomies were carried out using the suction method and four of these animals died post-operatively. The causes of death were hemorrhage from the branch of the superior vena cava in two cases and pneumothorax in the other two rats. The total operating time for the suction procedure was 5 min. 16 rats survived and in five cases (31%) remnants of the thymic organ, including epithelial tissue, remained in situ.
Table 1 Comparison of suction and intubation methods of thymectomy Procedure n Mortality
Complete rate
Operation time
Suction method Intubation method
69% (11/16) 100% (47/47)
within 5 min 11-17 min
20 50
20% (4*/20) 6% (3**/50)
* Fatal hemmorrhage (2 cases) and pneumothorax (2 cases). ** Damage to the trachea caused by difficulties in passing the guide wire. *'** There was no statistically significant difference between the groups (Fisher's exact test, t = 0.0967).
36
E. Kobayashi et al. /Journal of Immunological Methods 171 (1994) 33-36
4. Discussion Tolerance induction by neonatal thymectomy has been well documented (Miller, 1961), and the effect of thymectomy in adults has also been reported (Miller, 1962; Metcalf, 1965). The surgical technique of suctionthymectomy is the conventional method in general use and has been well standardized (Ford, 1978). However, this procedure is performed 'blind' and carries a high mortality rate in adult rodents. In particular, there is a danger of animals developing a pneumothorax because the thymic capsule is intimately attached to the pleural lining (Haimov et al., 1971). To overcome these problems, open-thymectomy was introduced by Barbul et al. in 1979. This technique offers a full view of the thymus using tracheal intubation. However, there are still some problems with the technique and air leakage can occur from the opened pleura and from the hole punctured by the guide wire. The survival of the rat requires the maintenance of positive pressure and this can be achieved either manually or with a respirator. Fibrin sealant made from concentrated fibrinogen has been introduced as a multicomponent biological adhesive. It can be used to establish hemostasis and also as an adhesive in wound repair. The sealing effect of fibrin glue has also been advocated in cardiovascular (Borst et al., 1982) and digestive operations (Ham et al., 1992) to prevent leakage. We have shown that open thymectomy using fibrin glue protects the rat from bleeding and air leakage after each operation. Comparing the intubation method and the ordinal suction technique of thymectomy showed that the suction method took less time and was easier to perform, but the thymus was not always completely removed. Although the intubation method appears difficult to perform, complete thymectomy using this technique was made easier because it en-
sured a full view of the organ. Open thymectomy was associated with a lower mortality rate and, in our hands, was always confirmed histologically.
Acknowledgements The authors thank Mrs. M. Saul for her excellent illustration and Mrs. H. Matthews for her precise photography.
References Barbul, A., Sisto, D.A. and Efron, G. (1979) A safe method of thymectomy in adult rats. Transplantation 28, 427. Borst, G.H., Haverich, A., Walterbusch G. and Maatz, W. (1982) Fibrin adhesive: An important hemostatic adjunct in cardiovascular operations. J. Thorac. Cardiovasc. Surg. 84, 548. Finkel, T.H., Cambier, J.C., Kubo, R.T., Born, W.K., Marrack, P. and Kappler, J.W. (1989) The thymus has two functionally distinct populations of immature alpha-beta + T cells: One population is deleted by ligation of alpha-beta TcR. Cell 58, 1047. Ford, W.L. (1978) In: D.M. Weie (Ed.), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, p. 231. Haimov, M., Wong, D., Burrows, L. et al. (1971) Thymectomy in the adult mouse. Transplantation 11, 100. Ham, A.C., Kort, W.J., Weijima, I.M., Ingh, H.F.G.M. and Jeekel, H. (1992) Effect of antibiotics in fibrin sealant on healing colonic anastomoses in the rat. Br. J. Surg. 79, 525. Kappler, J.W., Roehm, N. and Marrack, P.(1987) T cell tolerance by clonal elimination in the thymus. Cell 49, 273. Kisielow, P., Bluthmann, H., Staerz, U.D. et al. (1988) Tolerance in T-ceil-receptor transgenic mice involves deletion of nonmature CD4+ CD8 + thymocytes. Nature 333, 742. Kobayashi, E., Igarashi, Y., Kawai, K., Watanabe, H., Matumoto, Y. and Fujiwara, M. (1991) Experience of use of fibrin glue (Beriplast R P). Shinryou to Shinyaku (Jpn.) 28, 869. Metcalf, D. (1965) Delayed effect of thymus in adult life on immunological competence. Nature 208, 1336. Miller, J.F.A.P. (1961) Immunological functions of the thymus. Lancet ii, 748. Miller, J.F.A.P. (1962) Immunological significance of the thymus of adult mouse. Nature 195, 1318.