Journal of Immunological Methods, 12 (1976) 377--385 Q North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
377
Short communication RAPID, SAFE AND SIMPLE METHOD FOR GRAFTING WHOLE THYMUS IN THE MOUSE
D.A. COHN Department of Anatomy, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, N.Y. 11203, U.S.A.
(Received 8 February 1976, accepted 5 March 1976)
A rapid, safe and simple method is presented for obtaining and grafting whole neonatal thymuses in mice. No anesthesia, sutures or incisions are required for implantation. The grafting technique involves no special skills except the ability to inject mice subcutaneously. Thymuses grafted by the method described show normal architecture, survive lethal irradiation and bone marrow reconstitution, repopulate thymic dependent areas of peripheral lymphoid organs and restore normal inflammatory cell responses to thymic deprived mice. INTRODUCTION T h e c o n t i n u e d i n t e r e s t in isolating B a n d T cell f u n c t i o n a n d t h e wides p r e a d use o f t h y m i c - d e p r i v e d m i c e , m a n y o f w h i c h h a v e b e e n l e t h a l l y irrad i a t e d , m a k e it p r a c t i c a l t o h a v e a fast, safe m e t h o d o f i m p l a n t i n g w h o l e t h y m u s . T h e c u r r e n t r e p o r t d e s c r i b e s a p r o c e d u r e f o r grafting w h o l e t h y m u s w h i c h p r e s e n t s n o risks t o t h e r e c i p i e n t since i m p l a n t a t i o n o f t h e g r a f t requires n o a n e s t h e s i a , incisions, s u r g e r y or sutures. MATERIALS AND METHODS O b t a i n i n g t h y m u s grafts
C57B1/6J f e m a l e s , p r e g n a n t b y D B A / 2 m a l e s w e r e o b t a i n e d f r o m t h e J a c k s o n L a b o r a t o r y , Bar H a r b o r , Me. T h e i r o f f s p r i n g ( B D F I m a l e s a n d females) w e r e u s e d at o n e or t w o d a y s o f age as t h y m u s d o n o r s . N e o n a t a l tissue was c h o s e n b e c a u s e o f its great g r o w t h p o t e n t i a l (Metcalf, 1 9 6 6 ) . Clean b u t n o t sterile p r o c e d u r e s w e r e f o l l o w e d . D o n o r s w e r e chilled d i r e c t l y o n ice f o r at least 2 m i n t o p r o d u c e cardiac arrest a n d m i n i m i z e bleeding. F o r e x c i s i o n o f t h e t h y m u s each d o n o r was c e l l o p h a n e - t a p e d , v e n t r a l side up, o n t o t h e t o p c o v e r o f a Petri dish c o n t a i n i n g ice. A dissecting m i c r o s c o p e at l o w p o w e r was used t o facilitate t h y m e c t o m y . T h e fastest m e t h o d o f excising t h e t h y m u s r e q u i r e d t h a t d o n o r s be sacrificed. A t r a n s v e r s e incision t h r o u g h skin, m u s c l e and p e r i t o n e u m was m a d e
378
Fig. 1. Incisions used for the most rapid method of obtaining donor thymus; one transverse incision through skin and peritoneum, and two oblique incisions through skin and thorax. Fig. 2. The thymus is exposed as xiphoid process is grasped with forceps, and the anterior thorax is lifted.
at t h e level o f t h e liver. This was f o l l o w e d b y t w o ascending diagonal incisions t h r o u g h t h e skin and ribs to t h e m i d p o i n t s o f t h e clavicles (fig. 1). T h e x i p h o i d process was grasped with forceps and the wedge-shaped section o f t h o r a c i c cage f o r m e d b y these t h r e e incisions was t h e n freed f r o m t h e diap h r a g m and m e d i a s t i n u m . As the t h o r a c i c cage was elevated, the t h y m u s was clearly visible adhering t o t h e u n d e r surface o f the r e f l e c t e d s t e r n u m (fig. 2). Branches o f t h e internal t h o r a c i c b l o o d vessels and o t h e r vessels s u p p l y i n g t h e gland were separated by b l u n t dissection, and the t h y m u s was s c o o p e d o u t with a forceps. A m o d i f i e d p r o c e d u r e was p e r f o r m e d if t h e survival o f the d o n o r was required. D o n o r s were r e m o v e d f r o m the ice within 2 min. One descending longitudinal incision was m a d e t h r o u g h the skin and s t e r n u m r u n n i n g f r o m t h e jugular n o t c h d o w n t h r e e - f o u r t h s o f t h e length o f the s t e r n u m . T h e split s t e r n u m and u n d e r l y i n g fascia were separated with forceps, t h e submaxillary gland r e t r a c t e d and the t h y m u s e x p o s e d . T h e t h y m u s was r e m o v e d o n e side at a t i m e b y m e a n s o f a suction a p p a r a t u s a t t a c h e d t o a suitable trap f o r c a t c h i n g the organ. T h e t h o r a x was t h e n closed with i n t e r r u p t e d 6--0 silk sutures and c o l l o d i o n applied t o the incision site. N e o n a t a l d o n o r t h y m u s e s weighed a p p r o x i m a t e l y 5.0 mg and m e a s u r e d
379
1
2
Fig. 3 Above, A 14-gauge needle containing an entire neonatal thymus. Below, The stylet withdrawn from the needle, but positioned at the correct angle for expelling the thymus, B) Gross appearance of thymus graft (arrow) at autopsy 43 days after implantation. The skin of the recipient has been reflected. The thymus graft lies in the superficial fascia. Blood vessels can be seen within and entering the capsule of the grafted thymus.
1 - - 2 m m in l e n g t h {fig. 3 A ) . F o r m o s t p r o c e d u r e s , 6 o r 8 t h y m u s e s w e r e e x c i s e d a t a t i m e a n d h e l d in a c o v e r e d P e t r i d i s h o n f i l t e r p a p e r s o a k e d in i c e d saline until needed for implantation. The interval between excision and implantation did not exceed 20 min.
380 I m p la n ta tio n
Recipients were 44 neonatally t h y m e c t o m i z e d BDFI males who received grafts at either two or four weeks of age. Complete removal of their thymuses was later confirmed at autopsy by gross and histologic examination of the mediastinum. A clean # 1 4 or #16 2-inch thin wall needle (BectonDickinson #1307 or #1310) was assembled with its bevel up and its stylet retracted about 1/4 of an inch. The bevel of the stylet was positioned parallel to the bevel of the needle. Each half of the t h y m u s was loaded into the front end of the needle (fig. 3A), and the stylet was r e t r a c t e d a n o t h e r inch to draw the tissue further into the needle. The recipient was grasped with one hand in the usual position for giving a subcutaneous flank injection, and the skin was held taut. The needle with retracted stylet and bevel turned up punctured the skin easily. It was then pushed forward for about an inch under the skin. The stylet was advanced without rotating it, expelling the t h y m u s from the needle and depositing it under the skin at least an inch away from the entry site. Needle and stylet were then carefully withdrawn and the puncture wound in the skin closed in i m m e d i a t e l y . The needle was rinsed with distilled water, then with chilled saline and was ready for the next graft. No sutures or dressings were required. The animal appeared unaffected by this procedure and was retured immediately to its cage. Procedures for evaluating the grafts
The criteria used to evaluate the success of the grafts were compared in intact mice, thymic-deprived mice and thymic-deprived mice bearing t h y m u s grafts. The parameters were: 1) gross and histologic appearance of the grafts following lethal irradiation and bone marrow reconstitution; 2) histology of the thymic dependent areas; 3) the restoration of the T cell dependent inf l a m m a t o r y cell response (Walls et al., 1971; Cohn et al., 1974; Ponzio and Speirs, 1975). The experimental schedule is shown in table 1. Group 1 cofisisted of 18 neonatally t h y m e c t o m i z e d recipients grafted at 14 days of age, and exposed two weeks later, to 1000 R whole body irradiation from a 250 KV Maximar III X-ray machine containing a 0.25 mm Cu and 1.0 mm A1 filter operating at 15 MA. These lethally irradiated mice were reconstituted with 106 syngeneic bone marrow cells. After an interval of 18 days for hemopoietic recovery, 12 of these grafted mice were primed subcutaneously (s.c.) with tetanus toxoid, challenged intraperitoneally (i.p.) 21 days later and assayed for their inflammatory cell response 4 days after challenge. The remaining 6 irradiated mice were not immunized. Control animals consisting of 10 intact mice and 10 neonatally t h y m e c t o m i z e d non-grafted mice were each irradiated, reconstituted, immunized and assayed as described. The procedures for immunization and for quantifying the inflammatory cell response have previously been described (Cohn et al., 1974).
381 TABLE 1 Experimental design. Number of mice
Procedure 1 day *
14 days
28 days
46 days
67 days
71 days
Group 1 12 6 10 (controls) 10 (controls)
tx ** tx -tx
graft graft ---
irrad. irrad. irrad. irrad.
prime -prime prime
challenge -challenge challenge
autopsy autopsy autopsy autopsy
Group 2 12 14 10 (controls) 10 (controls)
tx tx -tx
-----
graft graft ---
prime -prime prime
challenge -challenge challenge
autopsy autopsy autopsy autopsy
BMC BMC BMC BMC
* age o f m i c e .
** neontally thymectomized. Graft: whole thymus; irrad. BMC: lethally irradiated and reconstituted with 106 bone marrow cells;prime and challenge: tetanus toxoid.
G r o u p 2 consisted o f 26 recipients, w h o were grafted at 28 d a y s o f age, and n o t irradiated. O f these mice, 12 were p r i m e d (s.c.) with t e t a n u s t o x o i d at 46 d a y s o f age, challenged (i.p.) 21 days later a n d assayed f o r their inf l a m m a t o r y cell response 4 d a y s after challenge. T h e r e m a i n i n g 14 nonirradiated n e o n a t a l l y t h y m e c t o m i z e d , grafted recipients were n o t imm u n i z e d . C o n t r o l animals consisting o f 10 i n t a c t mice and 10 n e o n a t a l l y t h y m e c t o m i z e d mice were also i m m u n i z e d and assayed as described. Spleens, l y m p h n o d e s and t h y m u s e s to be e x a m i n e d m i c r o s c o p i c a l l y were fixed in g l u t e r a l d e h y d e , s e c t i o n e d at 5 m i c r o n s , and stained with Geimsa. RESULTS A m o n g t h e 44 n e o n a t a l l y t h y m e c t o m i z e d mice receiving t h y m u s grafts b y t h e m e t h o d described, t h e r e were n o signs o f wasting, n o fatalities and 40 o f the 44 t h y m u s grafts were retrieved at a u t o p s y . Since t h e i m p l a n t s were inserted into ~he same site (the right flank), t h e r e were n o p r o b l e m s in l o c a t i n g t h e grafts at a u t o p s y . T h e t h y m u s grafts l o o k e d like f l a t t e n e d disks o u t l i n e d against the u n d e r l y i n g external o b l i q u e muscle (fig. 3B}. Vascular c o n n e c tions were e v i d e n t o n gross e x a m i n a t i o n . Histologic sections o f all t h y m u s grafts s h o w e d n o r m a l a r c h i t e c t u r e regardless o f w h e t h e r the grafted recipients were u n t r e a t e d , i m m u n i z e d o r lethally irradiated and r e c o n s t i t u t e d . T h e cortices were d e e p and densely p o p u l a t e d with small l y m p h o c y t e s (figs. 4 and 5). L y m p h a t i c n o d u l e s and foci o f e x t r a m e d u l l a r y granulopoiesis w e r e - n o t observed. N o r m a l cellular
Fig. 4. Histologic appearance of the thymus graft showing normal capsule. Blood vessels are seen entering the cortex. (Geimsa, x 30.)
Fig. 5. High power view of the blood vessels seen in fig. 4. Note vessels to supply the cortex and medulla (Geimsa, X 100.)
cortex,
medu dla and
the branching
c If t1lese
383
Fig. 6. High power view of the cortex and connective tissue septum in thymus graft. Cells characteristically present in the septum (granulocytes and mast cells) can be seen. A large blood vessel is also present. The cortex is densely populated with small lymphocytes. (Geimsa, x 450.)
c o m p o n e n t s were present in the medulla, and the grafts were well vascularized (figs. 4, 5 and 6). Thymic dependent zones in the spleens and lymph nodes, which are depleted of cells in t h y m e c t o m i z e d and t h y m e c t o m i z e d irradiated animals, were well populated in all animals bearing t h y m u s grafts, regardless of whether the recipients were untreated, immunized or lethally irradiated and reconstituted. The presence of these t h y m u s grafts in our experiments restored the normal inflammatory cell responses to antigen that are known to be depressed in thymic-deprived mice (Walls et al., 1971; Cohn et al., 1974) and are particularly reduced 4 days after a secondary injection of antigen (Cohn et al., 1974). As shown in table 2, compared to thymic-deprived mice which had a depressed inflammatory cell response, non-irradiated and irradiated thymectomized mice bearing thymus grafts had normal numbers of each type of inflammatory cell responding after a secondary injection of tetanus toxoid.
384 TABLE 2 Number of peritoneal exudate cells (× 106 ± S.E.) responding to a challenging injection of tetanus toxoid.
Neutrophils
Irradiated bone marrow
Not irradiated
Intact 0.79 _+0.10 Mononuclear 23.86 cells _+1.95 Eosinophils 10.98 +-1.08 Total cells 35.63 +-2.33
Tx *
Grafted** F ***
Intact
1.62 ±0.40 10.27 a -+3.02 2.34 a -+0.91 14.23 a -+4.02
1.08 +~0.23 22.46 b -+1.82 12.22 b +-0.69 35.76 b -+2.10
0.81 -+0.22 14.34 _+1.35 7.72 ±0.22 22.88 ±1.47
NS 8.95 23.31 19.01
Tx 0.66 _+0.18 5.09 a -+0.60 0.31 a ±0.12 6.06 a ±0.65
Grafted ** F *** 0.99 +0.44 12.99 b +_2.16 10.31 b ±1.51 24.30 b _+1.47
NS 10.93 22.91 64.36
* Neonatally thymectomized mice. ** Neonatally thymectomized mice bearing thymus grafts. *** One-way ANOVA: F significant at P < 0.05. Multiple comparisons tests: a Significantly different from intact mice P < 0.05. b Significantly different from thymectomized mice P < 0.05. DISCUSSION T h e c u r r e n t r e p o r t describes a m e t h o d f o r t h y m u s grafting w h i c h s e e m s t o solve s o m e of t h e p r o b l e m s e n c o u n t e r e d b y o t h e r s . T h e s e p r o b l e m s include: fatalities a m o n g t h e r e c i p i e n t s (Pantelouris, 1 9 7 1 ) ; m i n i m a l regenerat i o n o f t h e d e p l e t e d t h y m i c d e p e n d e n t areas (Hays, 1 9 6 9 ; T a k a d a et al., 1 9 7 1 ) ; d i f f i c u l t y in retrieving t h e t h y m u s grafts ( H a l l e n b e c k et al., 1 9 6 8 ) ; a n d q u e s t i o n a b l e r e c o v e r y o f i m m u n o l o g i c c a p a c i t y ( H a l l e n b e c k et al., 1 9 6 8 ; T a k a d a et al., 1968). S u b c u t a n e o u s grafts in p a r t i c u l a r were f o u n d t o be r e t a r d e d in g r o w t h (Metcalf, 1 9 6 6 ) , p o o r at p r e v e n t i n g wasting, a n d o n l y p a r t l y successful at r e s t o r i n g n o r m a l a r c h i t e c t u r e to p e r i p h e r a l l y m p h a t i c tissue ( E a s t and P a r r o t t , 1 9 6 4 ) . I r r a d i a t i o n also i n t e r f e r e d w i t h g r o w t h o f t h e t h y m u s grafts and w i t h t h e r e s t o r a t i o n o f i m m u n o l o g i c a l c o m p e t e n c e ( D u k o r et al., 1 9 6 5 ; Miller et al., 1 9 6 6 ; Davies et al., 1971). A p r e d i c t a b l e s e q u e n c e o f events o c c u r s f o l l o w i n g t h y m u s grafting. O n t h e first and s e c o n d d a y s , necrosis o c c u r s at t h e c e n t e r o f t h e graft, leaving o n l y a t h i n p e r i p h e r a l r i m o f epithelial cells and d o n o r l y m p h o c y t e s surviving. F o l l o w i n g intense m i t o t i c activity, t h e c o r t e x is r e s t o r e d b y 5 o r 6 days, a n d t h e a r c h i t e c t u r e is n o r m a l in a p p e a r a n c e at 8 days. By 15 days, t h e t h y m u s is r e p o p u l a t e d b y h o s t cells ( b o n e m a r r o w s t e m cells). T h u s , w h e n assessing t h e f u n c t i o n a l c a p a c i t y o f t h e t h y m u s g r a f t t h e t i m i n g o f t h e assay is critical. This is p a r t i c u l a r l y t r u e o f i r r a d i a t e d animals f o r w h i c h s u f f i c i e n t t i m e m u s t be a l l o t t e d so t h a t b o n e m a r r o w s t e m cells can r e p o p u l a t e the d e p l e t e d t h y m u s tissue.
385 F o r s a t i s f a c t o r y g r o w t h and f u n c t i o n , t h e early plasmatic circulation, u p o n which the i m p l a n t d e p e n d s , and the later vascularization are b o t h critical. R e f e r e n c e s in the l i t e r a t u r e have suggested t h a t failure o f s o m e t h y m u s grafts m a y have resulted f r o m i n a d e q u a t e vascularization ( D u k o r et al., 1 9 6 5 ; Metcalf, 1 9 6 6 ; T a k a d a et al., 1 9 7 1 ) . In t h e c u r r e n t s t u d y , gross and histologic e x a m i n a t i o n s h o w e d t h a t all t h y m u s e s grafted b y t h e p r e s e n t m e t h o d were well vascularized. This i m p r o v e m e n t m a y have resulted f r o m (1) t h e p l a c e m e n t o f the graft at a substantial distance f r o m the skin o p e n i n g w h e r e the needle e n t e r e d ; and (2) absence o f t h e local i n f l a m m a t i o n t h a t w o u l d have a c c o m p a n i e d t h e use o f sutures o r w o u n d clips. In earlier r e p o r t s , regardless o f w h e t h e r the t h y m u s e s were grafted subc u t a n e o u s l y , i n t r a p e r i t o n e a l l y or u n d e r t h e k i d n e y capsule, t h e p r o c e d u r e s f o l l o w e d generally i n c l u d e d anesthesia, incisions, w o u n d clips o r sutures. All o f these increased the t i m e r e q u i r e d and possibly j e o p a r d i z e d the graft o r t h e recipient. T h e p r e s e n t m e t h o d is simpler, faster and less h a z a r d o u s to t h e r e c i p i e n t t h a n a n y t e c h n i q u e f o r t h y m u s grafting which requires anesthesia, o p e n i n g the b o d y wall or e x t e r i o r i z i n g an organ. This has distinct advantages w h e n w o r k i n g with surgically m a n i p u l a t e d , w e a k e n e d or valuable recipients such as r a d i a t i o n chimeras or n u d e mice. Moreover, t h e m e t h o d requires n o t e c h n i c a l skill e x c e p t the ability t o give injections s u b c u t a n e o u s l y . REFERENCES Cohn, D.A., T.J. Athanassiades and R.S. Speirs, 1974, J. Reticuioendothelial Soc. 15, 199. Davies, A.J.S., E. Leuchars, V. Wallis and R.L. Carter, 1971, in: Cell interactions and receptor antibodies in immune responses, eds. O. Makela, A. Cross and T.U. Kosunen (Academic Press, London and New York) p. 311. Dukor, P., J.F.A.P. Miller, W. House and V. Allman 1965, Transplantation 3,639. East, J. and D.M.V. Parrott, 1964, J. Nat. Cancer Inst. 30, 673. Hallenbeck, G.A., TP. Kubista and R.G. Shorter, 1969, Proc. Soc. Exp. Biol. Med. 130, 1142. Hays, E.F., 1969, J. Exp. Med. 129, 1235. Metcalf, D., 1966, The thymus (Springer Verlag, New York). Miller, J.F.A.P., P.M. DeBurgh, P. Dukor, G. Grant, V. Allman and W. House, 1966, Clin. Exp. Immunol. 1, 61. Ponzio, N.M. and R.S. Speirs, 1975, Immunology 28,243. Takada, A, Y. Takada and J.L. Ambrus, 1971, J. Immunol. 107, 1185. Walls, R.S., A. Basten, E. Leuchars and A.J.S. Davies, 1971, Brit. Med. J. 3,157.
377
Short communication RAPID, SAFE AND SIMPLE METHOD FOR GRAFTING WHOLE THYMUS IN THE MOUSE
D.A. COHN Department of Anatomy, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, N.Y. 11203, U.S.A.
(Received 8 February 1976, accepted 5 March 1976)
A rapid, safe and simple method is presented for obtaining and grafting whole neonatal thymuses in mice. No anesthesia, sutures or incisions are required for implantation. The grafting technique involves no special skills except the ability to inject mice subcutaneously. Thymuses grafted by the method described show normal architecture, survive lethal irradiation and bone marrow reconstitution, repopulate thymic dependent areas of peripheral lymphoid organs and restore normal inflammatory cell responses to thymic deprived mice. INTRODUCTION T h e c o n t i n u e d i n t e r e s t in isolating B a n d T cell f u n c t i o n a n d t h e wides p r e a d use o f t h y m i c - d e p r i v e d m i c e , m a n y o f w h i c h h a v e b e e n l e t h a l l y irrad i a t e d , m a k e it p r a c t i c a l t o h a v e a fast, safe m e t h o d o f i m p l a n t i n g w h o l e t h y m u s . T h e c u r r e n t r e p o r t d e s c r i b e s a p r o c e d u r e f o r grafting w h o l e t h y m u s w h i c h p r e s e n t s n o risks t o t h e r e c i p i e n t since i m p l a n t a t i o n o f t h e g r a f t requires n o a n e s t h e s i a , incisions, s u r g e r y or sutures. MATERIALS AND METHODS O b t a i n i n g t h y m u s grafts
C57B1/6J f e m a l e s , p r e g n a n t b y D B A / 2 m a l e s w e r e o b t a i n e d f r o m t h e J a c k s o n L a b o r a t o r y , Bar H a r b o r , Me. T h e i r o f f s p r i n g ( B D F I m a l e s a n d females) w e r e u s e d at o n e or t w o d a y s o f age as t h y m u s d o n o r s . N e o n a t a l tissue was c h o s e n b e c a u s e o f its great g r o w t h p o t e n t i a l (Metcalf, 1 9 6 6 ) . Clean b u t n o t sterile p r o c e d u r e s w e r e f o l l o w e d . D o n o r s w e r e chilled d i r e c t l y o n ice f o r at least 2 m i n t o p r o d u c e cardiac arrest a n d m i n i m i z e bleeding. F o r e x c i s i o n o f t h e t h y m u s each d o n o r was c e l l o p h a n e - t a p e d , v e n t r a l side up, o n t o t h e t o p c o v e r o f a Petri dish c o n t a i n i n g ice. A dissecting m i c r o s c o p e at l o w p o w e r was used t o facilitate t h y m e c t o m y . T h e fastest m e t h o d o f excising t h e t h y m u s r e q u i r e d t h a t d o n o r s be sacrificed. A t r a n s v e r s e incision t h r o u g h skin, m u s c l e and p e r i t o n e u m was m a d e
378
Fig. 1. Incisions used for the most rapid method of obtaining donor thymus; one transverse incision through skin and peritoneum, and two oblique incisions through skin and thorax. Fig. 2. The thymus is exposed as xiphoid process is grasped with forceps, and the anterior thorax is lifted.
at t h e level o f t h e liver. This was f o l l o w e d b y t w o ascending diagonal incisions t h r o u g h t h e skin and ribs to t h e m i d p o i n t s o f t h e clavicles (fig. 1). T h e x i p h o i d process was grasped with forceps and the wedge-shaped section o f t h o r a c i c cage f o r m e d b y these t h r e e incisions was t h e n freed f r o m t h e diap h r a g m and m e d i a s t i n u m . As the t h o r a c i c cage was elevated, the t h y m u s was clearly visible adhering t o t h e u n d e r surface o f the r e f l e c t e d s t e r n u m (fig. 2). Branches o f t h e internal t h o r a c i c b l o o d vessels and o t h e r vessels s u p p l y i n g t h e gland were separated by b l u n t dissection, and the t h y m u s was s c o o p e d o u t with a forceps. A m o d i f i e d p r o c e d u r e was p e r f o r m e d if t h e survival o f the d o n o r was required. D o n o r s were r e m o v e d f r o m the ice within 2 min. One descending longitudinal incision was m a d e t h r o u g h the skin and s t e r n u m r u n n i n g f r o m t h e jugular n o t c h d o w n t h r e e - f o u r t h s o f t h e length o f the s t e r n u m . T h e split s t e r n u m and u n d e r l y i n g fascia were separated with forceps, t h e submaxillary gland r e t r a c t e d and the t h y m u s e x p o s e d . T h e t h y m u s was r e m o v e d o n e side at a t i m e b y m e a n s o f a suction a p p a r a t u s a t t a c h e d t o a suitable trap f o r c a t c h i n g the organ. T h e t h o r a x was t h e n closed with i n t e r r u p t e d 6--0 silk sutures and c o l l o d i o n applied t o the incision site. N e o n a t a l d o n o r t h y m u s e s weighed a p p r o x i m a t e l y 5.0 mg and m e a s u r e d
379
1
2
Fig. 3 Above, A 14-gauge needle containing an entire neonatal thymus. Below, The stylet withdrawn from the needle, but positioned at the correct angle for expelling the thymus, B) Gross appearance of thymus graft (arrow) at autopsy 43 days after implantation. The skin of the recipient has been reflected. The thymus graft lies in the superficial fascia. Blood vessels can be seen within and entering the capsule of the grafted thymus.
1 - - 2 m m in l e n g t h {fig. 3 A ) . F o r m o s t p r o c e d u r e s , 6 o r 8 t h y m u s e s w e r e e x c i s e d a t a t i m e a n d h e l d in a c o v e r e d P e t r i d i s h o n f i l t e r p a p e r s o a k e d in i c e d saline until needed for implantation. The interval between excision and implantation did not exceed 20 min.
380 I m p la n ta tio n
Recipients were 44 neonatally t h y m e c t o m i z e d BDFI males who received grafts at either two or four weeks of age. Complete removal of their thymuses was later confirmed at autopsy by gross and histologic examination of the mediastinum. A clean # 1 4 or #16 2-inch thin wall needle (BectonDickinson #1307 or #1310) was assembled with its bevel up and its stylet retracted about 1/4 of an inch. The bevel of the stylet was positioned parallel to the bevel of the needle. Each half of the t h y m u s was loaded into the front end of the needle (fig. 3A), and the stylet was r e t r a c t e d a n o t h e r inch to draw the tissue further into the needle. The recipient was grasped with one hand in the usual position for giving a subcutaneous flank injection, and the skin was held taut. The needle with retracted stylet and bevel turned up punctured the skin easily. It was then pushed forward for about an inch under the skin. The stylet was advanced without rotating it, expelling the t h y m u s from the needle and depositing it under the skin at least an inch away from the entry site. Needle and stylet were then carefully withdrawn and the puncture wound in the skin closed in i m m e d i a t e l y . The needle was rinsed with distilled water, then with chilled saline and was ready for the next graft. No sutures or dressings were required. The animal appeared unaffected by this procedure and was retured immediately to its cage. Procedures for evaluating the grafts
The criteria used to evaluate the success of the grafts were compared in intact mice, thymic-deprived mice and thymic-deprived mice bearing t h y m u s grafts. The parameters were: 1) gross and histologic appearance of the grafts following lethal irradiation and bone marrow reconstitution; 2) histology of the thymic dependent areas; 3) the restoration of the T cell dependent inf l a m m a t o r y cell response (Walls et al., 1971; Cohn et al., 1974; Ponzio and Speirs, 1975). The experimental schedule is shown in table 1. Group 1 cofisisted of 18 neonatally t h y m e c t o m i z e d recipients grafted at 14 days of age, and exposed two weeks later, to 1000 R whole body irradiation from a 250 KV Maximar III X-ray machine containing a 0.25 mm Cu and 1.0 mm A1 filter operating at 15 MA. These lethally irradiated mice were reconstituted with 106 syngeneic bone marrow cells. After an interval of 18 days for hemopoietic recovery, 12 of these grafted mice were primed subcutaneously (s.c.) with tetanus toxoid, challenged intraperitoneally (i.p.) 21 days later and assayed for their inflammatory cell response 4 days after challenge. The remaining 6 irradiated mice were not immunized. Control animals consisting of 10 intact mice and 10 neonatally t h y m e c t o m i z e d non-grafted mice were each irradiated, reconstituted, immunized and assayed as described. The procedures for immunization and for quantifying the inflammatory cell response have previously been described (Cohn et al., 1974).
381 TABLE 1 Experimental design. Number of mice
Procedure 1 day *
14 days
28 days
46 days
67 days
71 days
Group 1 12 6 10 (controls) 10 (controls)
tx ** tx -tx
graft graft ---
irrad. irrad. irrad. irrad.
prime -prime prime
challenge -challenge challenge
autopsy autopsy autopsy autopsy
Group 2 12 14 10 (controls) 10 (controls)
tx tx -tx
-----
graft graft ---
prime -prime prime
challenge -challenge challenge
autopsy autopsy autopsy autopsy
BMC BMC BMC BMC
* age o f m i c e .
** neontally thymectomized. Graft: whole thymus; irrad. BMC: lethally irradiated and reconstituted with 106 bone marrow cells;prime and challenge: tetanus toxoid.
G r o u p 2 consisted o f 26 recipients, w h o were grafted at 28 d a y s o f age, and n o t irradiated. O f these mice, 12 were p r i m e d (s.c.) with t e t a n u s t o x o i d at 46 d a y s o f age, challenged (i.p.) 21 days later a n d assayed f o r their inf l a m m a t o r y cell response 4 d a y s after challenge. T h e r e m a i n i n g 14 nonirradiated n e o n a t a l l y t h y m e c t o m i z e d , grafted recipients were n o t imm u n i z e d . C o n t r o l animals consisting o f 10 i n t a c t mice and 10 n e o n a t a l l y t h y m e c t o m i z e d mice were also i m m u n i z e d and assayed as described. Spleens, l y m p h n o d e s and t h y m u s e s to be e x a m i n e d m i c r o s c o p i c a l l y were fixed in g l u t e r a l d e h y d e , s e c t i o n e d at 5 m i c r o n s , and stained with Geimsa. RESULTS A m o n g t h e 44 n e o n a t a l l y t h y m e c t o m i z e d mice receiving t h y m u s grafts b y t h e m e t h o d described, t h e r e were n o signs o f wasting, n o fatalities and 40 o f the 44 t h y m u s grafts were retrieved at a u t o p s y . Since t h e i m p l a n t s were inserted into ~he same site (the right flank), t h e r e were n o p r o b l e m s in l o c a t i n g t h e grafts at a u t o p s y . T h e t h y m u s grafts l o o k e d like f l a t t e n e d disks o u t l i n e d against the u n d e r l y i n g external o b l i q u e muscle (fig. 3B}. Vascular c o n n e c tions were e v i d e n t o n gross e x a m i n a t i o n . Histologic sections o f all t h y m u s grafts s h o w e d n o r m a l a r c h i t e c t u r e regardless o f w h e t h e r the grafted recipients were u n t r e a t e d , i m m u n i z e d o r lethally irradiated and r e c o n s t i t u t e d . T h e cortices were d e e p and densely p o p u l a t e d with small l y m p h o c y t e s (figs. 4 and 5). L y m p h a t i c n o d u l e s and foci o f e x t r a m e d u l l a r y granulopoiesis w e r e - n o t observed. N o r m a l cellular
Fig. 4. Histologic appearance of the thymus graft showing normal capsule. Blood vessels are seen entering the cortex. (Geimsa, x 30.)
Fig. 5. High power view of the blood vessels seen in fig. 4. Note vessels to supply the cortex and medulla (Geimsa, X 100.)
cortex,
medu dla and
the branching
c If t1lese
383
Fig. 6. High power view of the cortex and connective tissue septum in thymus graft. Cells characteristically present in the septum (granulocytes and mast cells) can be seen. A large blood vessel is also present. The cortex is densely populated with small lymphocytes. (Geimsa, x 450.)
c o m p o n e n t s were present in the medulla, and the grafts were well vascularized (figs. 4, 5 and 6). Thymic dependent zones in the spleens and lymph nodes, which are depleted of cells in t h y m e c t o m i z e d and t h y m e c t o m i z e d irradiated animals, were well populated in all animals bearing t h y m u s grafts, regardless of whether the recipients were untreated, immunized or lethally irradiated and reconstituted. The presence of these t h y m u s grafts in our experiments restored the normal inflammatory cell responses to antigen that are known to be depressed in thymic-deprived mice (Walls et al., 1971; Cohn et al., 1974) and are particularly reduced 4 days after a secondary injection of antigen (Cohn et al., 1974). As shown in table 2, compared to thymic-deprived mice which had a depressed inflammatory cell response, non-irradiated and irradiated thymectomized mice bearing thymus grafts had normal numbers of each type of inflammatory cell responding after a secondary injection of tetanus toxoid.
384 TABLE 2 Number of peritoneal exudate cells (× 106 ± S.E.) responding to a challenging injection of tetanus toxoid.
Neutrophils
Irradiated bone marrow
Not irradiated
Intact 0.79 _+0.10 Mononuclear 23.86 cells _+1.95 Eosinophils 10.98 +-1.08 Total cells 35.63 +-2.33
Tx *
Grafted** F ***
Intact
1.62 ±0.40 10.27 a -+3.02 2.34 a -+0.91 14.23 a -+4.02
1.08 +~0.23 22.46 b -+1.82 12.22 b +-0.69 35.76 b -+2.10
0.81 -+0.22 14.34 _+1.35 7.72 ±0.22 22.88 ±1.47
NS 8.95 23.31 19.01
Tx 0.66 _+0.18 5.09 a -+0.60 0.31 a ±0.12 6.06 a ±0.65
Grafted ** F *** 0.99 +0.44 12.99 b +_2.16 10.31 b ±1.51 24.30 b _+1.47
NS 10.93 22.91 64.36
* Neonatally thymectomized mice. ** Neonatally thymectomized mice bearing thymus grafts. *** One-way ANOVA: F significant at P < 0.05. Multiple comparisons tests: a Significantly different from intact mice P < 0.05. b Significantly different from thymectomized mice P < 0.05. DISCUSSION T h e c u r r e n t r e p o r t describes a m e t h o d f o r t h y m u s grafting w h i c h s e e m s t o solve s o m e of t h e p r o b l e m s e n c o u n t e r e d b y o t h e r s . T h e s e p r o b l e m s include: fatalities a m o n g t h e r e c i p i e n t s (Pantelouris, 1 9 7 1 ) ; m i n i m a l regenerat i o n o f t h e d e p l e t e d t h y m i c d e p e n d e n t areas (Hays, 1 9 6 9 ; T a k a d a et al., 1 9 7 1 ) ; d i f f i c u l t y in retrieving t h e t h y m u s grafts ( H a l l e n b e c k et al., 1 9 6 8 ) ; a n d q u e s t i o n a b l e r e c o v e r y o f i m m u n o l o g i c c a p a c i t y ( H a l l e n b e c k et al., 1 9 6 8 ; T a k a d a et al., 1968). S u b c u t a n e o u s grafts in p a r t i c u l a r were f o u n d t o be r e t a r d e d in g r o w t h (Metcalf, 1 9 6 6 ) , p o o r at p r e v e n t i n g wasting, a n d o n l y p a r t l y successful at r e s t o r i n g n o r m a l a r c h i t e c t u r e to p e r i p h e r a l l y m p h a t i c tissue ( E a s t and P a r r o t t , 1 9 6 4 ) . I r r a d i a t i o n also i n t e r f e r e d w i t h g r o w t h o f t h e t h y m u s grafts and w i t h t h e r e s t o r a t i o n o f i m m u n o l o g i c a l c o m p e t e n c e ( D u k o r et al., 1 9 6 5 ; Miller et al., 1 9 6 6 ; Davies et al., 1971). A p r e d i c t a b l e s e q u e n c e o f events o c c u r s f o l l o w i n g t h y m u s grafting. O n t h e first and s e c o n d d a y s , necrosis o c c u r s at t h e c e n t e r o f t h e graft, leaving o n l y a t h i n p e r i p h e r a l r i m o f epithelial cells and d o n o r l y m p h o c y t e s surviving. F o l l o w i n g intense m i t o t i c activity, t h e c o r t e x is r e s t o r e d b y 5 o r 6 days, a n d t h e a r c h i t e c t u r e is n o r m a l in a p p e a r a n c e at 8 days. By 15 days, t h e t h y m u s is r e p o p u l a t e d b y h o s t cells ( b o n e m a r r o w s t e m cells). T h u s , w h e n assessing t h e f u n c t i o n a l c a p a c i t y o f t h e t h y m u s g r a f t t h e t i m i n g o f t h e assay is critical. This is p a r t i c u l a r l y t r u e o f i r r a d i a t e d animals f o r w h i c h s u f f i c i e n t t i m e m u s t be a l l o t t e d so t h a t b o n e m a r r o w s t e m cells can r e p o p u l a t e the d e p l e t e d t h y m u s tissue.
385 F o r s a t i s f a c t o r y g r o w t h and f u n c t i o n , t h e early plasmatic circulation, u p o n which the i m p l a n t d e p e n d s , and the later vascularization are b o t h critical. R e f e r e n c e s in the l i t e r a t u r e have suggested t h a t failure o f s o m e t h y m u s grafts m a y have resulted f r o m i n a d e q u a t e vascularization ( D u k o r et al., 1 9 6 5 ; Metcalf, 1 9 6 6 ; T a k a d a et al., 1 9 7 1 ) . In t h e c u r r e n t s t u d y , gross and histologic e x a m i n a t i o n s h o w e d t h a t all t h y m u s e s grafted b y t h e p r e s e n t m e t h o d were well vascularized. This i m p r o v e m e n t m a y have resulted f r o m (1) t h e p l a c e m e n t o f the graft at a substantial distance f r o m the skin o p e n i n g w h e r e the needle e n t e r e d ; and (2) absence o f t h e local i n f l a m m a t i o n t h a t w o u l d have a c c o m p a n i e d t h e use o f sutures o r w o u n d clips. In earlier r e p o r t s , regardless o f w h e t h e r the t h y m u s e s were grafted subc u t a n e o u s l y , i n t r a p e r i t o n e a l l y or u n d e r t h e k i d n e y capsule, t h e p r o c e d u r e s f o l l o w e d generally i n c l u d e d anesthesia, incisions, w o u n d clips o r sutures. All o f these increased the t i m e r e q u i r e d and possibly j e o p a r d i z e d the graft o r t h e recipient. T h e p r e s e n t m e t h o d is simpler, faster and less h a z a r d o u s to t h e r e c i p i e n t t h a n a n y t e c h n i q u e f o r t h y m u s grafting which requires anesthesia, o p e n i n g the b o d y wall or e x t e r i o r i z i n g an organ. This has distinct advantages w h e n w o r k i n g with surgically m a n i p u l a t e d , w e a k e n e d or valuable recipients such as r a d i a t i o n chimeras or n u d e mice. Moreover, t h e m e t h o d requires n o t e c h n i c a l skill e x c e p t the ability t o give injections s u b c u t a n e o u s l y . REFERENCES Cohn, D.A., T.J. Athanassiades and R.S. Speirs, 1974, J. Reticuioendothelial Soc. 15, 199. Davies, A.J.S., E. Leuchars, V. Wallis and R.L. Carter, 1971, in: Cell interactions and receptor antibodies in immune responses, eds. O. Makela, A. Cross and T.U. Kosunen (Academic Press, London and New York) p. 311. Dukor, P., J.F.A.P. Miller, W. House and V. Allman 1965, Transplantation 3,639. East, J. and D.M.V. Parrott, 1964, J. Nat. Cancer Inst. 30, 673. Hallenbeck, G.A., TP. Kubista and R.G. Shorter, 1969, Proc. Soc. Exp. Biol. Med. 130, 1142. Hays, E.F., 1969, J. Exp. Med. 129, 1235. Metcalf, D., 1966, The thymus (Springer Verlag, New York). Miller, J.F.A.P., P.M. DeBurgh, P. Dukor, G. Grant, V. Allman and W. House, 1966, Clin. Exp. Immunol. 1, 61. Ponzio, N.M. and R.S. Speirs, 1975, Immunology 28,243. Takada, A, Y. Takada and J.L. Ambrus, 1971, J. Immunol. 107, 1185. Walls, R.S., A. Basten, E. Leuchars and A.J.S. Davies, 1971, Brit. Med. J. 3,157.
