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An improvement on the Bollman method of restraining and collecting thoracic duct lymph from the rat
Journal of Immunological Methods, 110 (1988) 179-181
179
Elsevier JIM04770
An improvement on the Bollman method of restraining and collecting thoracic duct lymph from the rat D a v i d J. R e i c h * a n d Steven E. K e l l e r Department of Psychiatry, Mount Sinai School of Medicine, City University of New York, New York, N Y 10029, U.S.A.
(Received4 January 1988, accepted 8 January 1988)
A method of obtaining rat thoracic duct lymphocytes is described, including a review of the surgical technique, with several modifications, and including an outline of a newly designed rat restrainer that helps to triple thoracic duct output compared to the Bollman restrainer. The new restrainer allows the rat increased mobility but protects the thoracic duct cannula. Unanesthetized 200 g rats yielded 80 m l / d a y thoracic duct lymph containing 4.2 x 1 0 4 lymphocytes/ml. Key words: Thoracic duct; Lymph; Lymphocyte
Materials and methods Introduction
The design of a new type of rat restraining cage that helps to triple lymph flow through a thoracic duct cannula as compared to the flow when using the Bollman restrainer (1948) will be described. The rat thoracic duct may be cannulated by the methods of Bollman et al. (1948), Gowens (1959), and Ford (1978), as summarized below with several modifications. Cannulation of the thoracic duct allows for a relatively pure, voluminous and concentrated suspension of lymphocytes to be obtained and remains an important procedure used by cellular immunologists.
Correspondence to: S.E. Keller, Associate Professor, UMDNJ-New Jersey Medical School, Department of Psychiatry, 185 South Orange Avenue, Newark, NJ 07103-2757, U.S.A. * Present address: McGill University School of Medicine, Montreal, QC, Canada.
The rat is anesthetized with ether, A 25 gauge infusion set that is heparin primed should be placed in the tail vein immediately after induction of anesthesia. Afterwards, the vein contracts and is not significantly dilated even by warming. The rat is placed on its back with the lower limbs spread and taped to the operating board, and the left upper limb folded across the chest and taped with the fight upper limb next to the right ear. After shaving the skin an incision is made along the inferior border of the left costal margin. Retractors hold the muscle and skin apart, swabs tear fascia to mobilize the left kidney, and moist gauze retains the left kidney, liver, spleen, stomach and intestines on the right side of the abdomen. The aorta is separated from the psoas muscle with swabs and the 1.5 mm duct, which is almost transparent, is visualized between the renal vessels and the diaphragm. An operating microscope having 10 × magnification and good lighting are necessary equipment. A specially designed glass probe
0022-1759/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
180 //f~
with a narrow and curved tip is inserted between the duct and psoas, passed under the duct between the renal vessels and diaphragm, and then used to bore a hole from behind, between the duct and aorta, which are attached, without puncturing either vessel. The probe then fully mobilizes about 5 m m of the duct, a silk thread is passed under the duct, and a single knot is tied, but not fastened. A 50 cm length of polythene tubing PP50 with a U-shaped tip is prepared earlier by winding the tubing around a glass tube and heating in water. The tubing is passed through a trocar that is pushed through the back of the rat directly along the medial border of the psoas muscle to exit inferiorly. Later, movement of the rat will facilitate lymph flow via the pumping action of the psoas on the tubing. The trocar is removed and the cannula tip is cut with a 30 o bevel. Just prior to incising the duct the tail vein is infused with 2 ml of saline with 1 U / m l heparin, which will dilate the thoracic duct within 2 rain. Care must be taken not to infuse too much fluid, which will bloat the duct and cause it to rupture upon manipulation. Castroviejo microdissecting scissors are used to make a transverse cut through 1 / 3 of the circumference of the thoracic duct, 3 m m above the ligature. The tip of the cannula, primed with heparinized saline, is inserted with forceps into the vessel inferiorly and the ligature is tightened. Only one ligature around the thoracic duct, 3 m m below the sight of cannula insertion, is used to firmly secure the tubing. Additional sutures that tie the cannula to nearby musculature should not be used. These cause the cannula inside of the duct to exert tension on the duct wall when the animal moves because they inhibit the cannula outside of the duct from sliding in its track through the musculature. The organs are returned to their anatomical positions, the wound is sutured, the ether is removed and the animal is placed in a restrainer. The classical Bollman restraining cage almost completely immobilizes the rat. Instead, the rat should be kept in a larger cage with the dimensions shown in Fig. 1. The cage is made of clear lucite, 6 m m thick. It has three vertical slits in each of the two side walls into which a collar around the neck of the rat can slide. The three
holesforcannu]a. I ~'
~
+
""
slots for collar
bh,o0oforo en'ngcago aperalure for water bottle 5 and food
Fig. 1. Rat restraining cage made of lucite 6 mm thick, height 10 cm, width 10 cm, and length 20 cm.
pairs of slits are to accomodate different size rats. The collar is illustrated in Fig. 2. It is a flat square piece of plastic with a hole in the middle, which is cut in half. The two halves are placed around the neck of the rat and fastened so that the collar fits snugly. The collar should be shorter than the cage so that the rat can slide its neck up and down. It is easiest to apply the collar to the rat before putting it into the cage. Take note of the hinged roof in Fig. ] and of the adjustable collar fasteners in Fig. 2. A water bottle and food pellets are accessible through a window in the front wall. The cage has holes in the back wall through which the rat tail and the cannula can pass. The rat should be continuously infused with normal saline containing 2 U / m l heparin, at a rate of 6 m l / 2 4 h. L y m p h is collected from a level 15 cm below the rat, on ice, in medium containing 10 U / m l heparin.
Fig. 2. Rat collar made of plastic 2 mm thick, with height 6-8 cm, width 10 cm, and hole diameter 1-3 cm.
181
Results M e a s u r e m e n t s o n 15 u n a n e s t h e t i z e d , 200 + 50 g, m a l e W / F u rats ( M i c r o b i o l o g i c a l Assoc.) held in the new r e s t r a i n e r showed a m e a n t h o r a c i c d u c t l y m p h flow of 80 +_ 15 m l / d a y , as c o m p a r e d to a p p r o x i m a t e l y 25 m l / d a y ( B o l l m a n et al., 1948) o b t a i n e d f r o m unanesthetized, similar weight rats in a B o l l m a n restrainer. T h e l y m p h c o n t a i n s 4.2 _+ 0.8 )< 104 l y m p h o c y t e s / m l in k e e p i n g with the d a t a of M a n n et al. (1950). W i t h i n these values cellularity does n o t v a r y directly with flow volume. It is often c o n v e n i e n t to c a n n u l a t e the rat in the m o r n i n g , d i s c a r d l y m p h collected in the first 6 h as suggested b y S p r y (1972) a n d then harvest a p p r o x i m a t e l y 55 ml thoracic d u c t l y m p h , containing 2 x 106 cells, overnight. T h o r a c i c d u c t l y m p h can c o n t i n u e to b e collected with a similar yield for 3 days, after which t i m e t h o r a c i c d u c t l y m p h flow a n d l y m p h o c y t e c o n c e n t r a t i o n decline. T h e rat is a l l o w e d greater f r e e d o m in the m o d ified cage t h a n in the B o l l m a n cage. It c a n m o v e its h e a d a n d b o d y up, down, a n d sideways. O n l y f o r w a r d a n d b a c k w a r d m o v e m e n t is i m p e d e d . I n spite of the i n c r e a s e d m o b i l i t y the rat is r e s t r a i n e d a d e q u a t e l y to p r o t e c t the cannula.
Discussion T h e m o s t likely r e a s o n that t h o r a c i c d u c t l y m p h flow is m o r e t h a n t r i p l e d w h e n the n e w r e s t r a i n e r is used is that the rat can b e t t e r exercise its muscles which has b e e n shown to increase b o t h the f o r m a t i o n a n d p r o p u l s i o n o f l y m p h ( H u d a c k et al., 1933; W h i t e et al., 1933; M c M a s t e r , 1937). By i m p r o v i n g rat m o b i l i t y the new, m o r e a n i m a l f r i e n d l y r e s t r a i n e r also enables the r a t to d r i n k m o r e easily which increases t h o r a c i c d u c t l y m p h flow ( G l e n n et al., 1949; S h r e w s b u r y et al., 1952). F u r t h e r m o r e , greater f r e e d o m lessens the stress on
the rat, a n d stress decreases t h o r a c i c duct o u t p u t (Spry, 1972; H e d m a n , 1980). I n c r e a s e d thoracic d u c t l y m p h flow aids in the m a i n t e n a n c e of a p a t e n t c a n n u l a b y i n h i b i t i n g clotting. O b t a i n i n g t h o r a c i c d u c t l y m p h f r o m the rat is a difficult t a s k t h a t c a n b e facilitated b y using b o t h the c a n n u l a t i o n m e t h o d d e s c r i b e d above, a n d the new r e s t r a i n e r that i m p r o v e s that m o b i l i t y a n d triples t h o r a c i c d u c t l y m p h flow.
References Bollman, J.L. (1948) A cage which limits the activity of rats. J. Lab. Clin. Med. 33, 1348. Bollman, J.L, Cain, J.C., and Grindley, J.H. (1948) Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat. J. Lab. Clin. Med. 33, 1349. Ford, W.L. (1978) In: D.M. Weir (Ed.), Handbook of Experimental Immunology, Vol. 2, Cellular Immunology. Blackwell, Oxford, p. 23.1. Glenn, W.W.L., Cresson, S.L., Bauer, T.X., Goldstein, F., Hoffman, O. and Healey, J.E. (1949) Experimental Thoracic Duct Fistula - Observations on the technique, the absorption of fat and fluid from the intestine, and protein depletion. Surg. Gynecol. Obstet. 89, 200. Gowans, J.L. (1959) The recirculation of lymphocytes from blood to lymph in the rat. J. Physiol. 146, 54. Hedman, L.A. (1980) The effect of steroids on the circulating lymphocyte population, IV. The effect of stress on the thoracic duct lymphocyte population in normal and neonatally thymectomized rats. Lymphology 13, 34. Hudack, S. and McMaster, P.D. (1933) The lymphatic participation in human cutaneous phenomena. A study of the minute lymphatics of the living skin. J. Exp. Med. 57, 751. McMaster, P.D. (1937) Changes in the cutaneous lymphatics of human beings and in the lymph flow under normal and pathological conditions. J. Exp. Med. 65, 347. Shrewsbury, M.M. and Reinhardt, W.O. (1952) Comparative metabolic effects of ingestion of water or 1% sodium chloride solution in the rat with a thoracic duct fistula. Am. J. Physiol. 168, 366. Spry, C.J.F. (1972) Inhibition of lymphocyte circulation by stress and corticotropin. Cell. Immunol. 4, 86. White, J.C., Field M.E. and Drinker, C.K. (1933) On the protein content and normal flow of lymph from the foot of the dog. Am. J. Physiol. 103, 34.
179
Elsevier JIM04770
An improvement on the Bollman method of restraining and collecting thoracic duct lymph from the rat D a v i d J. R e i c h * a n d Steven E. K e l l e r Department of Psychiatry, Mount Sinai School of Medicine, City University of New York, New York, N Y 10029, U.S.A.
(Received4 January 1988, accepted 8 January 1988)
A method of obtaining rat thoracic duct lymphocytes is described, including a review of the surgical technique, with several modifications, and including an outline of a newly designed rat restrainer that helps to triple thoracic duct output compared to the Bollman restrainer. The new restrainer allows the rat increased mobility but protects the thoracic duct cannula. Unanesthetized 200 g rats yielded 80 m l / d a y thoracic duct lymph containing 4.2 x 1 0 4 lymphocytes/ml. Key words: Thoracic duct; Lymph; Lymphocyte
Materials and methods Introduction
The design of a new type of rat restraining cage that helps to triple lymph flow through a thoracic duct cannula as compared to the flow when using the Bollman restrainer (1948) will be described. The rat thoracic duct may be cannulated by the methods of Bollman et al. (1948), Gowens (1959), and Ford (1978), as summarized below with several modifications. Cannulation of the thoracic duct allows for a relatively pure, voluminous and concentrated suspension of lymphocytes to be obtained and remains an important procedure used by cellular immunologists.
Correspondence to: S.E. Keller, Associate Professor, UMDNJ-New Jersey Medical School, Department of Psychiatry, 185 South Orange Avenue, Newark, NJ 07103-2757, U.S.A. * Present address: McGill University School of Medicine, Montreal, QC, Canada.
The rat is anesthetized with ether, A 25 gauge infusion set that is heparin primed should be placed in the tail vein immediately after induction of anesthesia. Afterwards, the vein contracts and is not significantly dilated even by warming. The rat is placed on its back with the lower limbs spread and taped to the operating board, and the left upper limb folded across the chest and taped with the fight upper limb next to the right ear. After shaving the skin an incision is made along the inferior border of the left costal margin. Retractors hold the muscle and skin apart, swabs tear fascia to mobilize the left kidney, and moist gauze retains the left kidney, liver, spleen, stomach and intestines on the right side of the abdomen. The aorta is separated from the psoas muscle with swabs and the 1.5 mm duct, which is almost transparent, is visualized between the renal vessels and the diaphragm. An operating microscope having 10 × magnification and good lighting are necessary equipment. A specially designed glass probe
0022-1759/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
180 //f~
with a narrow and curved tip is inserted between the duct and psoas, passed under the duct between the renal vessels and diaphragm, and then used to bore a hole from behind, between the duct and aorta, which are attached, without puncturing either vessel. The probe then fully mobilizes about 5 m m of the duct, a silk thread is passed under the duct, and a single knot is tied, but not fastened. A 50 cm length of polythene tubing PP50 with a U-shaped tip is prepared earlier by winding the tubing around a glass tube and heating in water. The tubing is passed through a trocar that is pushed through the back of the rat directly along the medial border of the psoas muscle to exit inferiorly. Later, movement of the rat will facilitate lymph flow via the pumping action of the psoas on the tubing. The trocar is removed and the cannula tip is cut with a 30 o bevel. Just prior to incising the duct the tail vein is infused with 2 ml of saline with 1 U / m l heparin, which will dilate the thoracic duct within 2 rain. Care must be taken not to infuse too much fluid, which will bloat the duct and cause it to rupture upon manipulation. Castroviejo microdissecting scissors are used to make a transverse cut through 1 / 3 of the circumference of the thoracic duct, 3 m m above the ligature. The tip of the cannula, primed with heparinized saline, is inserted with forceps into the vessel inferiorly and the ligature is tightened. Only one ligature around the thoracic duct, 3 m m below the sight of cannula insertion, is used to firmly secure the tubing. Additional sutures that tie the cannula to nearby musculature should not be used. These cause the cannula inside of the duct to exert tension on the duct wall when the animal moves because they inhibit the cannula outside of the duct from sliding in its track through the musculature. The organs are returned to their anatomical positions, the wound is sutured, the ether is removed and the animal is placed in a restrainer. The classical Bollman restraining cage almost completely immobilizes the rat. Instead, the rat should be kept in a larger cage with the dimensions shown in Fig. 1. The cage is made of clear lucite, 6 m m thick. It has three vertical slits in each of the two side walls into which a collar around the neck of the rat can slide. The three
holesforcannu]a. I ~'
~
+
""
slots for collar
bh,o0oforo en'ngcago aperalure for water bottle 5 and food
Fig. 1. Rat restraining cage made of lucite 6 mm thick, height 10 cm, width 10 cm, and length 20 cm.
pairs of slits are to accomodate different size rats. The collar is illustrated in Fig. 2. It is a flat square piece of plastic with a hole in the middle, which is cut in half. The two halves are placed around the neck of the rat and fastened so that the collar fits snugly. The collar should be shorter than the cage so that the rat can slide its neck up and down. It is easiest to apply the collar to the rat before putting it into the cage. Take note of the hinged roof in Fig. ] and of the adjustable collar fasteners in Fig. 2. A water bottle and food pellets are accessible through a window in the front wall. The cage has holes in the back wall through which the rat tail and the cannula can pass. The rat should be continuously infused with normal saline containing 2 U / m l heparin, at a rate of 6 m l / 2 4 h. L y m p h is collected from a level 15 cm below the rat, on ice, in medium containing 10 U / m l heparin.
Fig. 2. Rat collar made of plastic 2 mm thick, with height 6-8 cm, width 10 cm, and hole diameter 1-3 cm.
181
Results M e a s u r e m e n t s o n 15 u n a n e s t h e t i z e d , 200 + 50 g, m a l e W / F u rats ( M i c r o b i o l o g i c a l Assoc.) held in the new r e s t r a i n e r showed a m e a n t h o r a c i c d u c t l y m p h flow of 80 +_ 15 m l / d a y , as c o m p a r e d to a p p r o x i m a t e l y 25 m l / d a y ( B o l l m a n et al., 1948) o b t a i n e d f r o m unanesthetized, similar weight rats in a B o l l m a n restrainer. T h e l y m p h c o n t a i n s 4.2 _+ 0.8 )< 104 l y m p h o c y t e s / m l in k e e p i n g with the d a t a of M a n n et al. (1950). W i t h i n these values cellularity does n o t v a r y directly with flow volume. It is often c o n v e n i e n t to c a n n u l a t e the rat in the m o r n i n g , d i s c a r d l y m p h collected in the first 6 h as suggested b y S p r y (1972) a n d then harvest a p p r o x i m a t e l y 55 ml thoracic d u c t l y m p h , containing 2 x 106 cells, overnight. T h o r a c i c d u c t l y m p h can c o n t i n u e to b e collected with a similar yield for 3 days, after which t i m e t h o r a c i c d u c t l y m p h flow a n d l y m p h o c y t e c o n c e n t r a t i o n decline. T h e rat is a l l o w e d greater f r e e d o m in the m o d ified cage t h a n in the B o l l m a n cage. It c a n m o v e its h e a d a n d b o d y up, down, a n d sideways. O n l y f o r w a r d a n d b a c k w a r d m o v e m e n t is i m p e d e d . I n spite of the i n c r e a s e d m o b i l i t y the rat is r e s t r a i n e d a d e q u a t e l y to p r o t e c t the cannula.
Discussion T h e m o s t likely r e a s o n that t h o r a c i c d u c t l y m p h flow is m o r e t h a n t r i p l e d w h e n the n e w r e s t r a i n e r is used is that the rat can b e t t e r exercise its muscles which has b e e n shown to increase b o t h the f o r m a t i o n a n d p r o p u l s i o n o f l y m p h ( H u d a c k et al., 1933; W h i t e et al., 1933; M c M a s t e r , 1937). By i m p r o v i n g rat m o b i l i t y the new, m o r e a n i m a l f r i e n d l y r e s t r a i n e r also enables the r a t to d r i n k m o r e easily which increases t h o r a c i c d u c t l y m p h flow ( G l e n n et al., 1949; S h r e w s b u r y et al., 1952). F u r t h e r m o r e , greater f r e e d o m lessens the stress on
the rat, a n d stress decreases t h o r a c i c duct o u t p u t (Spry, 1972; H e d m a n , 1980). I n c r e a s e d thoracic d u c t l y m p h flow aids in the m a i n t e n a n c e of a p a t e n t c a n n u l a b y i n h i b i t i n g clotting. O b t a i n i n g t h o r a c i c d u c t l y m p h f r o m the rat is a difficult t a s k t h a t c a n b e facilitated b y using b o t h the c a n n u l a t i o n m e t h o d d e s c r i b e d above, a n d the new r e s t r a i n e r that i m p r o v e s that m o b i l i t y a n d triples t h o r a c i c d u c t l y m p h flow.
References Bollman, J.L. (1948) A cage which limits the activity of rats. J. Lab. Clin. Med. 33, 1348. Bollman, J.L, Cain, J.C., and Grindley, J.H. (1948) Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat. J. Lab. Clin. Med. 33, 1349. Ford, W.L. (1978) In: D.M. Weir (Ed.), Handbook of Experimental Immunology, Vol. 2, Cellular Immunology. Blackwell, Oxford, p. 23.1. Glenn, W.W.L., Cresson, S.L., Bauer, T.X., Goldstein, F., Hoffman, O. and Healey, J.E. (1949) Experimental Thoracic Duct Fistula - Observations on the technique, the absorption of fat and fluid from the intestine, and protein depletion. Surg. Gynecol. Obstet. 89, 200. Gowans, J.L. (1959) The recirculation of lymphocytes from blood to lymph in the rat. J. Physiol. 146, 54. Hedman, L.A. (1980) The effect of steroids on the circulating lymphocyte population, IV. The effect of stress on the thoracic duct lymphocyte population in normal and neonatally thymectomized rats. Lymphology 13, 34. Hudack, S. and McMaster, P.D. (1933) The lymphatic participation in human cutaneous phenomena. A study of the minute lymphatics of the living skin. J. Exp. Med. 57, 751. McMaster, P.D. (1937) Changes in the cutaneous lymphatics of human beings and in the lymph flow under normal and pathological conditions. J. Exp. Med. 65, 347. Shrewsbury, M.M. and Reinhardt, W.O. (1952) Comparative metabolic effects of ingestion of water or 1% sodium chloride solution in the rat with a thoracic duct fistula. Am. J. Physiol. 168, 366. Spry, C.J.F. (1972) Inhibition of lymphocyte circulation by stress and corticotropin. Cell. Immunol. 4, 86. White, J.C., Field M.E. and Drinker, C.K. (1933) On the protein content and normal flow of lymph from the foot of the dog. Am. J. Physiol. 103, 34.