[4]
THE P E R F U S E D RAT LIVER
25
[4] T h e P e r f u s e d R a t L i v e r
By JOHN H. EXTON The perfused liver preparation offers many advantages in the study of intermediary metabolism. It preserves the control systems associated with intact cells and provides a milieu similar to that in the intact animal while retaining the advantages of an in vitro system, i.e., strict control of substrate and hormone concentrations and elimination of complicating interactions with other tissues. The technique of liver perfusion was first utilized extensively by Embden and his co-workers in their studies of lipid and carbohydrate metabolism at the beginning of this century. It was later used by Lundsgaard and his students in investigations of hormone effects on liver metabolism. In more recent years, the technique has come back into vogue due to the efforts of Miller l and Mortimore2 The method of liver perfusion described in this chapter is based on that of Mortimore. Although developed originally for rats a it is also being used for mice with minor modifications: Apparatus. The perfusion apparatus described in this section was originally designed by Dr. Howard E. Morgan, then of the Physiology Department, Vanderbilt University School of Medicine, and Mr. Bailey F. Moore, head of the Apparatus Shop at the Vanderbilt University School of Medicine. The prototype has been modified several times, but the basic system of perfusion remains unchanged. The latest model will be described. As shown in Figs. 1-4, the apparatus consists of a Plexiglas box (120 cm long, 40 cm high, 40 cm deep) placed on a Formica-coated wooden base containing two light bulbs for heating (150 W, severe service), the blowers for circulation of air (Dayton 2C782, W. W. Grainger, Inc., Chicago, Illinois), the motor for rotating the oxygenation chambers (Bodine 2246 E-30, Bodinc Electric Co., Chicago, Illinois), the temperature control system (Thermistemp model 63Ra, Yellow Springs Instrument Co., Yellow Springs, Ohio), and switches. The top of the Formica base can be removed for servicing the motors and heating system, and the front panel of the Plexiglas box can be readily removed to allow the 1L. L. Miller, C. G. Bly, M. L. Watson, and W. F. Bale, J. Exp. Med. 94, 431 (1951). " G. E. Mortimore, Amer. J. Physiol. 200, 1315 (1961). 3j. H. Exton and C. R. Park, J. Biol. Chem. 242, 2622 (1967). 4 F. Assimacopoulos-Jeannet, J. H. Exton, and B. Jeanrenaud, Amer. J. Physiol. 225, 25 (1973).
26
MVEa
[4]
various components of the perfusion system proper to be dismantled (Fig. 1). The air in the Plexiglas box is circulated continuously during perfusion and is kept at 38.5 ° by a thermostat, the probe (model 621, Yellow Springs Instrument Co.) of which is placed near the livers. Each of the two oxygenation chambers (Fig. 2) consists of a Plexiglas cylindrical drum (9 cm internal diameter), the external face of which is removable and allows penetration of a Teflon-sealed metal shaft carrying four stainless steel tubes for influent and effluent perfusion media and gases (Figs. 1 and 2). The drum rotates around the metal shaft at 60 rpm being driven by a gear system (model 133, Crown Gear Co., Worcester, Massachusetts) coupled to the Bodine motor described above2 The animals whose livers are to be perfused in situ are placed on stainless steel trays (13 cm X 27 cm) which can be slid out of the Plexiglas box through removable windows to facilitate cannulation and other procedures involved in setting up the perfusion (Figs. 1 and 3). The trays have two blocks to which the cannulas and tubing can be anchored by adhesive tape during perfusion (Fig. 3). Perfusion medium is pumped by means of a peristaltic pump (model 1201, Harvard Apparatus Co., Millis, Massachusetts) from the oxygenation chamber through a filter assembly (Fig. 4), containing nylon gauze (100 mesh), and a bubble trap (Fig. 5) to the liver. Tygon tubing ( ~ o.d. X ~ 6 i.d., Formula $50 HL, Norton Plastics and Synthetics Division, Akron, Ohio) is used throughout the perfusion circuit except in the pumps, where Silastic tubing (ST 430, Extracorporeal Medical Specialties, Inc., King of Prussia, Pennsylvania) is used. The size of the pump tubing can be varied together with the pump rate to give the desired flow rate (generally 1-2 ml per minute per gram of liver). For purposes of sampling or infusing substrates or hormones, 2.5-cm lengths of latex tubing ( ~ 6 o.d. X ~ i.d.) are inserted into the perfusion circuit proximal to the pump (sampling) and proximal to the portal cannula (infusion). The medium is equilibrated with humidified 0..,-C02 (95:5) which enters the oxygenation chamber at a rate of about 200 ml per minute. In experiments with 14C-labeled substrates in which 14CO2 is to be measured, the gas mixture may be withdrawn through a series of 3 tubes, each containing 25 ml of 2 M NH4OH. Aliquots from each tube are mixed with scintillation fluid and counted2 An alternative method of measuring 14CO2 formation is described later. The cannulas have short bevels and may be of stainless steel tubing 5Other fabrication and specification details of the perfusion apparatus are available upon request from Mr. Bailey F. Moore, The Apparatus Shop, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232.
[41
THE PERFUSED RAT LIVER
27
(Eisele Co., Nashville, Tennessee) or Teflon tubing (available from Sargent-Welch Scientific Co., Skokie, Illinois). The portal cannulas may be gauge 18 for rats or gauge 20 for mice. The vena cava cannulas are notched for tying in place and may be gauge 12 for rats or gauge 17 for mice. PerJusion Media. The media used in the author's laboratory generally contain red blood cells because the increased 02 carrying capacity permits low flow rates, which are more desirable in experiments using nonrecirculating medium. In some experiments (e.g., studies of ion fluxes, lactate transport or glucose transport), it is preferable to omit red cells and use high flow rates (4-6 ml per minute per gram of liver). Bovine erythrocytes are routinely used in the author's studies, although rat, aged human, or sheep cells may be employed.6 Beef blood (approximately 5 liters) obtained from the jugular vein of stunned animals at a commercial slaughterhouse is immediately mixed with 0.2 volume of anticoagulant solution consisting of 13.2 g of glucose, 13.2 g of sodium citrate, 4.4 g of citric acid, and 150 mg of chloramphenicol per liter. The anticoagulant solution is prepared on the day of collection and tile anticoagulated blood is kept at 2°C before being used within 5 days. The blood is centrifuged, and the plasma is discarded. The cells are washed three times in 2 volumes of saline (9 g of NaC1 per liter) and twice in 2 volumes of bicarbonate buffeV containing 1 g of bovine serum albumin per liter. Tile white cell layer is removed by aspiration at each wash step. The packed red cells are stored at 2 ° and used within 2 days. Perfusion medium is made up by resuspending the red cells in oxygenated bicarbonate buffer; containing 30 g of bovine serum albumin (Cohn fraction V) ~ per liter. Sufficient cells are added to give a hematocrit of 18-22% (for experiments employing recirculating medium) or 36-407c (for experiments with nonrecirculating medium). The pH of the medium is then adjusted to 7.4. The required volume of medium (50-70 ml if recycled and 130 ml or more if not recycled) is pmnped into the oxygenation chamber and is recirculated through the system for 15-20 minutes to bring the pH, 0=, tension and temperature to the required values (7.4, 250 mM Hg, and 37.5°, respectively~. In the case of perfusion at slow flow rates, it may be necessary to pass the medium which is returning from the pumps through a coiled length of tubing immersed in water at 37.5 °. GBovine ceils are preferred, especially in studies of hepatic carbohydrate metal',olism, because of their low rate of glycolysis. H. A. Krebs and K. Henseleit, Hoppe-Seyler's Z. Physiol. Chem. 210, 33 (1932). Albumin purchased from any of the major supply houses is usually satisfactory. H u m a n serum albumin may also be used if available?
28
Liv~a
[4]
Fro. 1. Upper panel: General view of liver perfusion apparatus. The circuit tubing, filter assemblies and bubble traps have been omitted for clarity. The left-
[4l
THE P E R F U S E D RAT LIVER
29
D
C
\~
r
I-.~.
10.6 cm--
FIG. 2. Cross section of oxygenation chamber. A, tube for withdrawing medium; B, gas inlet tube; C, tube for returning medium. D, gas outlet tube; E, Teflon seal; F, medium; G, stainless steel arm to keep tube cluster fixed while chamber is rotating.
Technique of PerIusion. The procedure of Mortimore 2 is followed. The rat (90-150 g) is injected intraperitoneally with 6 mg of sodium pentobarbital per 100 g. The anesthetized animal is placed on the mobile tray and fastened in place using adhesive tape (Fig. 3). The abdomen is opened widely and the gut is reclined to the left to expose the portal vein and inferior vena cava. Using surgical silk (4/0) mounted on a curved needle, three loose ligatures are set: (a) around the portal vein just before its entry into the liver (inclusion of the bile duct must be a~:oided); (b) around the superior mesenteric and celiac arteries; (c~ around the inferior vena cava above the right renal vein (Fig. 3). Circulation of medium through the perfusion system is continued durhand animal lray (A) has been slid out of Plexiglas box through the removable window to its position during the eannulation procedures; the right-hand tray (B) is in its normal position during perfusion. The Plexiglas oxygenation chambers (C) and the gear system (D) which drives them are shown. The four front inlet ports (E) to the blowers for circulation of air are visible, the four rear outlet ports are obscured. The temperature control system is housed behind panel F. Lower panel: View of exposed base of perfusion apparatus. Visible are the electric motor (A) which rotates the oxygenation chambers, the blowers (B) for circulation of air, the temperature control system (C), the switch and cooling grille (D) for the electric motor, and the switch (E) for the temperature control system.
30
LIVER
[4]
FIG. 3. Diagram depicting cannulation and perfusion procedures. A, ligature tying portal vein eannula and placed above entry of splenic vein; B, inferior vena cava and abdominal aorta sectioned after insertion of portal cannula; C, infusion tubing and cannula inserted into rubber tubing insert in inflow line; D, inflow line taped to aluminum block to hold portal cannula in place; E, ligature around celiac axis'and superior mesenteric arteries, tied off after insertion of portal cannula; F, ligature around inferior vene, cava above R renal vein, tied off after insertion of vena cava cannula; G, vena cava caanula inserted through right atrium into inferior vena cava, not tied; H, outflow line taped to aluminum block to hold vena cava cannula in place. ing the above procedures b y t e m p o r a r i l y connecting portal and vena c a v a carmulas. I t is then stopped by t u r n i n g off the p u m p and clamping the tubing proximal to the portal cannula and distal to the vena c a v a cannula. T h e cannulas are then separated and the portal cannula is inserted
[4]
THE PERFUSED RAT LIVER
31
E
//"
4cm
8
FIG. 4. Cross section of filter assembly. A, Plexiglas outer casing; B, nylon gauze (100 mesh); C, brass screw carrying stainless steel inflow tube; D, rubber O-ring seals; E, anodized aluminum screw for holding outflow assembly in place F, Teflon outflow assembly carrying stainless steel outflow tube. into the portal vein, Immediately following this, the proximal clamp is removed, and the pump is restarted. Speed is essential at this stage in order to minimize interruption of blood flow to the liver. The vena cava is then sectioned below the set ligature so that perfusion medium flowing through the liver may escape via this route. The portal cannula is fixed in place with adhesive tape and the ligatures around the portal vein and the arteries are tied. The chest is then opened widely and the heart is exposed. The vena cava cannula is inserted through the right atrium into the inferior vena cava. The ligature around the lower part of the inferior vena cava is tightened, thus closing the circuit (Fig. 3). In experiments with recirculating medium, the perfusate leaving the liver flows by gravity back to the oxygenation chamber, which is 15 cm below the liver2 In experiments with nonrecirculating medium, commencement of the flow of effluent 9A negative hydrostatic pressure must be maintained in the portal cannula to prevent swelling and deterioration of the liver.
32
Liven
[4]
3cm
/
\
Fin. 5. Cross section of bubble trap assembly. A, clamp; B, rubber tubing, C, glass outer ground joint, standard taper 1%o; D, stainless steel inflow and outflow tubes; E, Teflon inner ground joint carrying inflow and outflow tubes; F, rubber O-ring seal; G, medium.
medium from the liver is facilitated by mild suction. The effluent medium is collected in sequential samples and is not returned to the oxygenation chamber. If 14C02 production is to be measured in this type of experiment, samples (1 ml) of effluent perfusate are drawn directly from the tubing and injected into Erlenmeyer flasks (25 ml) sealed with rubber stoppers (Kontes Glass Co., Vineland, New Jersey) and equipped with plastic center wells (Kontes) containing filter paper wicks impregnated with 0.3 ml of 1 M NaOH. To measure 14C02, 0.5 ml of 30% HCIO~ is injected into the flasks which are then incubated with shaking for 2 hours at 37 ° . The center wells are transferred to scintillation fluid and counted.3 To prevent the liver from drying during perfusion, it is routinely coy-
[4]
THE PERFUSED RAT LIVER
33
ered with a gauze pad soaked in warm saline. This is removed at intervals for inspection of the liver. In experiments with recirculating medium, samples are withdrawn from the oxygenation chambers before including the liver in the perfusion circuit and usually at 20-minute intervals thereafter. Since loss of perfusate during the cannulation procedures is variable, the volume of recirculating medium is measured by pumping the medium out of the system into a measuring cylinder at the end of perfusion and adding to this volume the residual volume (usually 1 ml) and the volume of the samples withdrawn during perfusion. In experiments with nonrecirculating medium, it is necessary to know the flow rate of the medium accurately. This is measured at the beginning of each perfusion experiment. Cleaning. Bacterial contamination is a complication of all perfusion studies and must be vigorously searched for and prevented if possible. Some bacterial infection of blood is almost inevitable during collection at the slaughterhouse. This is minimized by routine sterilization of the container, inclusion of chloramphenicol in the anticoagulant solution, and instruction of the personnel at the slaughterhouse. Spot checks of bacterial contamination and of the antibiotic sensitivity of contaminating organisms are also undertaken. If sterility is important, aged human blood may be used. At the end of a single perfusion, the circuit is cleaned by pumping through it at least 500 ml of saline at the rate of 30 ml per minute. At the end of a day of perfusion, the perfusion circuit, including all tubing and all components, is completely dismantled and soaked in bactericidal detergent (Dreft, Proctor and Gamble, Cincinnati, Ohio). Tubing is also routinely discarded after 1 month of use. General Considerations. Livers perfused by this technique for up to 2 hours show no deterioration, as evidenced by their gross and electron microscopic appearanceA ~ There is negligible edema as shown by the low water content of livers perfused for 1 hour (0.765 ± 0.003 ml per gram, wet weight)i° and by the absence of an increase in liver weight during perfusion for 2 hours2 Bile production is maintained and release of K + into the medium is minimal." Release of enzymes (alkaline phosphatase, glutamate oxaloacetate transaminase, lactate dehydrogenase) into the perfusate is absent or negligible. Oxygen consumption is steady 3,''-' and levels of ATP are maintained. 4,'3 Steady rates of glycogeloL. E. Mallette, J. H. Exton, and C. R. Park, J. Biol. Chem. 244, 5713 (1969). ~ T. F. Williams,J. H. Exton, N. Friedmann, and C. R. Park, Amer. J. Physiol. 221, 1645 (1971). 12j. H. Exton, J. G. Corbin, and S. C. Harper, J. Biol. Chem. 247, 4996 (1972). '~J. H. Exton and C. R. Park, J. Biol. Chem. 244, 1424 (1969).
34
LIVER
[4]
nolysis, gluconeogenesis, ureogenesis, and steady levels of cyclic A M P are attained within 15 minutes of completion of the perfusion circuit and are maintained for at least 60 minutes2 ,4,1°,12,14 Steady rates of utilization and oxidation of substrates such as lactate and alanine are also observed, 3,4,1°,12 The rates of most metabolic processes appear to be similar to those occurring in the intact animal 3,4 with the exception of glycogen synthesis which is low 3,1~ and proteolysis which is increased after 30 minutes of perfusion without added amino acids or insulin. 1~ Livers perfused by this technique also respond rapidly to physiological levels of hormones 4,~°,1',~4,~6-~s or to changes in the substrate concentration within the physiological range. 3,4,1°,13,19 The perfusion technique 2 described here has been successfully employed in a wide variety of studies of hepatic metabolism. The following is a list of selected references to such studies. Carbohydrate metabolism, s,4,~°-~,~7-26 protein metabolism, 1°,~2,~6,~ lipid metabolism, 3,~,27-3° glucagon, catecholamine, or cyclic A M P action, 4,11-14,16,17,19,26,23-25'29's1'32 insulin action, 2,11,12,1s,19,21,25,31-33 steroid action, 34-s6 membrane transport, ~2,37 ion fluxes, ~,~,~s,~,~°,3',3s and hormone assay. ~,39,~° 19j. H. Exton, G. A. Robison, E. W. Sutherland, and C. R. Park, J. Biol. Chem. 246, 6166 (1971). ,5 The low rate of glycogen synthesis observed in earlier studies~ was probably due to the low level of glucose in the perfusion medium since this substrate markedly affects the rate of glycogen deposition (H. Buschiazzo, J. H. Exton, and C. R. Park, Proc. Nat. Acad. Sci. U.S. 65, 383 (1970); W. Glinsmann, G. Pauk, and E. Hern, Biochem. Biophys. Res. Commun. 39, 774 (1970). ,e C. E. Mondon and G. E. Mortimore, Amer. J. Physiol. 212, 173 (1968). "J. H. Exton and C. R. Park, J. Biol. Chem. 243, 4189 (1968). ,8 G. E. Mortimore, Amer. J. Physiol. 204, 699 (1963). ,gj. H. Extort, J. G. Corbin, and C. R. Park, J. Biol. Chem. 244, 4095 (1969). 20j. H. Exton, S. B. Lewis, R. J. Ho, G. A. Robison, and C. R. Park, Ann. N.Y. Acad. Sci. 185, 85 (1971). 21G. E. Mortimore, E. King, Jr., C. E. Mondon, and W. Glinsmann, Amer. J. Physiol. 212, 179 (1967). 2~T. F. Williams, J. H. Exton, C. R. Park, and D. M. Regen, Amer. J. Physiol. 215, 1200 (1968). 2sT. H. Claus, D. M. Regen, J. H. Exton, and C. R. Park, J. Biol. Chem. Submitted for publication. M. Ui, T. H. Claus, J. H. Exton, and C. R. Park, J. Biol. Chem. 248, 5344 (1973). W. Glinsmann and G. E. Mortimore, Amer. J. Physiol. 215, 553 (1968). 2'K. It. Woodside and G. E. Mortimore, J. Biol. Chem. 247, 6474 (1972). ~'J. D. McGarry and D. W. Foster, J. Biol. Chem. 246, 1149 (1971). 's J. D. McGarry and D. W. Foster, J. Biol. Chem. 246, 6247 (1971). 29D. M. Regen and E. B. Terrell, Biochim. Biophys. Acta 170, 95 (1968). ,o R. L. Hamilton, D. M. Regen, M. E. Gray, and V. S. LeQuire, Lab. Invest. 16, 305 (1967).
[4]
~ H E PERFUSED
RAT LIVER
35
I n most instances, perfusion with nonrecirculating medium is preferred. With this method, substrates and hormones can be maintained at constant physiological concentrations, and rapid changes in substrate utilization or product formation can be readily measured. Complications due to the transformation of substrates or products in the medium itself are also avoided with this method because substrates can be infused into the influent medium just prior to the portal cannulas and effluent medium can be immediately deproteinized. The disadvantages are t h a t more medium is needed and t h a t some metabolic changes are too small to produce measurable alterations in substrates or products during a single passage through the liver. In this description, attention has been focused on the use of the perfused liver for the study of short-term aspects of hormonal control of metabolism. I t has not as yet proved feasible to perfuse livers satisfactorily for more than 3 hours with the unsupplemented medium described in this chapter. The reasons for the deterioration of the liver during longer perfusions are not known, but it is suspected t h a t the lack of certain plasma constituents and excessive bacterial growth m a y be factors. I t has been found in the author's laboratory t h a t livers can be perfused satisfactorily for up to 6 hours with medimn consisting of equal parts of rat blood collected under sterile conditions and bicarbonate buffer. Inclusion of antibiotics in this medium might result in satisfactory perfusions of even longer duration. Such perfusions would be desirable in the study of the mechanisms of action of slowly acting hormones, of the induction of enzymes and of the regulation of the synthesis and release of proteins such as albumin and lipoproteins. ~ L. S. Jefferson, J. H. Exton, R. W. Butcher, E. W. Sutherland, and C. R. Park. J. Biol. Chem. 243, 1031 (1968). ~-"J. H. Exton, J. G. Hardman, T. F. Williams, E. W. Sutherland, and C. R Park. J. Biol. Chem. 246, 2658 (1971). ~ J. H. Exton, S. C. Harper, A. L. Tucker and R. J. Ho, Biochim. Biophys. Act~ 329, 23 (1973). '~'J. H. Exton, N. Friedmann, E. H. A. Wong, J. P. Brineaux, J. D. Corbin, and C. R. Park, J. Biol. Chem. 247, 3579 (1972). ~ J. H. Exton, S. C. Harper, T. B. Miller, Jr., and C. R. Park, Biochim. Biophys. Acta. Submitted for publication. ~ J. H. Exton, S. C. Harper, A. L. Tucker, J. L. Flagg, and C. ]~. Park, Biochim. Biophys. Acta 329, 41 (1973). ~TT. H. Claus, D. M. ttegen, and A. tloos, Amer. J. Physiol. Submitted for publication. ~ N. Friedmann and C. R. Park, Proc. Nat. Acad. Sci. U.S. 6I, 504 (1968). ~"R. H. Unger, A. Ohneda, I. Valverde, A. M. Eisentraut, and J. H. Exton, J. Clin. Invest. 47, 48 (1968). 4oj. Marco, G. R. Faloona, and R. I-I. Unger, J. Clin. Invest. 50, 1650 (1971).
36
LIVER
[5]
Addendum. A recent study 41 indicates that fluorocarbons are satisfactory substitutes for erythrocytes in perfusion experiments. Livers perfused with the fluorocarbon FC-47 (3M Company, St. Paul, Minnesota) emulsified with bicarbonate buffer~ and Pluronic polyol F-68 (BASF Wyandotte Corp., Wyandotte, Michigan) were virtually identical in their metabolic behavior to livers perfused with bicarbonate buffer containing 15% erythrocytes.
41M. N. Goodman, R. Parrilla, and C. J. Toews, Amer. J. Physiol. 225, 1384 (1973).
[5] T h e F e t a l R a t L i v e r E x p l a n t as a M o d e l S y s t e m in t h e S t u d y o f H o r m o n e A c t i o n I B y EDWARD BRESNICK and KURT BURKI2
Any study of the mechanism of action of a particular hormone in the intact animal may be complicated by different effects upon different tissues, environmental influences, varying rates of absorption from the site of entry, etc. Consequently, it is sometime necessary to isolate a particular tissue for a detailed study of its biochemistry as influenced by hormonal administration. In this regard, organ or tissue culture has been of value as a biological model system. Although many techniques are available for maintenance of tissue explants, they depend upon growth on either solid or fluid media. The latter have been modified such that the tissue is grown at an interphase between the gas and liquid phases; the method of Trowell 3 has been useful in the investigation on liver tissue. Liver is a particularly difficult tissue to study by the explant technique partly because of its heterogeneity with regard to types of cells. Adult liver is composed of connective tissue elements, a variety of reticuloendothelial cells including Kupffer cells, as well as the hepatic parenchymal cells, i.e., hepatocytes. Under most circumstances, the parenchymal cells of adult liver tissue will not proliferate under culture conditions and, in fact, die. However, fetal liver adapts quite well to primary explanation, i.e., cultivation of pieces of tissues fresh from the organism. The fetal liver explant system has several other desirable properties which make it well suited for studying problems of development, cell 1The work presented in this article was supported by grants from the National Institutes of Health (GM 18623 and CA 12609), the National Science Foundation, Pharmaceutical Manufacturers Association, and Brown-Hazen Fund. Present address: Institute of Pathology, University of Bern, Bern, Switzerland. s O. A. Trowell, Exp. Cell Res. 6, 246 (1954).
THE P E R F U S E D RAT LIVER
25
[4] T h e P e r f u s e d R a t L i v e r
By JOHN H. EXTON The perfused liver preparation offers many advantages in the study of intermediary metabolism. It preserves the control systems associated with intact cells and provides a milieu similar to that in the intact animal while retaining the advantages of an in vitro system, i.e., strict control of substrate and hormone concentrations and elimination of complicating interactions with other tissues. The technique of liver perfusion was first utilized extensively by Embden and his co-workers in their studies of lipid and carbohydrate metabolism at the beginning of this century. It was later used by Lundsgaard and his students in investigations of hormone effects on liver metabolism. In more recent years, the technique has come back into vogue due to the efforts of Miller l and Mortimore2 The method of liver perfusion described in this chapter is based on that of Mortimore. Although developed originally for rats a it is also being used for mice with minor modifications: Apparatus. The perfusion apparatus described in this section was originally designed by Dr. Howard E. Morgan, then of the Physiology Department, Vanderbilt University School of Medicine, and Mr. Bailey F. Moore, head of the Apparatus Shop at the Vanderbilt University School of Medicine. The prototype has been modified several times, but the basic system of perfusion remains unchanged. The latest model will be described. As shown in Figs. 1-4, the apparatus consists of a Plexiglas box (120 cm long, 40 cm high, 40 cm deep) placed on a Formica-coated wooden base containing two light bulbs for heating (150 W, severe service), the blowers for circulation of air (Dayton 2C782, W. W. Grainger, Inc., Chicago, Illinois), the motor for rotating the oxygenation chambers (Bodine 2246 E-30, Bodinc Electric Co., Chicago, Illinois), the temperature control system (Thermistemp model 63Ra, Yellow Springs Instrument Co., Yellow Springs, Ohio), and switches. The top of the Formica base can be removed for servicing the motors and heating system, and the front panel of the Plexiglas box can be readily removed to allow the 1L. L. Miller, C. G. Bly, M. L. Watson, and W. F. Bale, J. Exp. Med. 94, 431 (1951). " G. E. Mortimore, Amer. J. Physiol. 200, 1315 (1961). 3j. H. Exton and C. R. Park, J. Biol. Chem. 242, 2622 (1967). 4 F. Assimacopoulos-Jeannet, J. H. Exton, and B. Jeanrenaud, Amer. J. Physiol. 225, 25 (1973).
26
MVEa
[4]
various components of the perfusion system proper to be dismantled (Fig. 1). The air in the Plexiglas box is circulated continuously during perfusion and is kept at 38.5 ° by a thermostat, the probe (model 621, Yellow Springs Instrument Co.) of which is placed near the livers. Each of the two oxygenation chambers (Fig. 2) consists of a Plexiglas cylindrical drum (9 cm internal diameter), the external face of which is removable and allows penetration of a Teflon-sealed metal shaft carrying four stainless steel tubes for influent and effluent perfusion media and gases (Figs. 1 and 2). The drum rotates around the metal shaft at 60 rpm being driven by a gear system (model 133, Crown Gear Co., Worcester, Massachusetts) coupled to the Bodine motor described above2 The animals whose livers are to be perfused in situ are placed on stainless steel trays (13 cm X 27 cm) which can be slid out of the Plexiglas box through removable windows to facilitate cannulation and other procedures involved in setting up the perfusion (Figs. 1 and 3). The trays have two blocks to which the cannulas and tubing can be anchored by adhesive tape during perfusion (Fig. 3). Perfusion medium is pumped by means of a peristaltic pump (model 1201, Harvard Apparatus Co., Millis, Massachusetts) from the oxygenation chamber through a filter assembly (Fig. 4), containing nylon gauze (100 mesh), and a bubble trap (Fig. 5) to the liver. Tygon tubing ( ~ o.d. X ~ 6 i.d., Formula $50 HL, Norton Plastics and Synthetics Division, Akron, Ohio) is used throughout the perfusion circuit except in the pumps, where Silastic tubing (ST 430, Extracorporeal Medical Specialties, Inc., King of Prussia, Pennsylvania) is used. The size of the pump tubing can be varied together with the pump rate to give the desired flow rate (generally 1-2 ml per minute per gram of liver). For purposes of sampling or infusing substrates or hormones, 2.5-cm lengths of latex tubing ( ~ 6 o.d. X ~ i.d.) are inserted into the perfusion circuit proximal to the pump (sampling) and proximal to the portal cannula (infusion). The medium is equilibrated with humidified 0..,-C02 (95:5) which enters the oxygenation chamber at a rate of about 200 ml per minute. In experiments with 14C-labeled substrates in which 14CO2 is to be measured, the gas mixture may be withdrawn through a series of 3 tubes, each containing 25 ml of 2 M NH4OH. Aliquots from each tube are mixed with scintillation fluid and counted2 An alternative method of measuring 14CO2 formation is described later. The cannulas have short bevels and may be of stainless steel tubing 5Other fabrication and specification details of the perfusion apparatus are available upon request from Mr. Bailey F. Moore, The Apparatus Shop, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232.
[41
THE PERFUSED RAT LIVER
27
(Eisele Co., Nashville, Tennessee) or Teflon tubing (available from Sargent-Welch Scientific Co., Skokie, Illinois). The portal cannulas may be gauge 18 for rats or gauge 20 for mice. The vena cava cannulas are notched for tying in place and may be gauge 12 for rats or gauge 17 for mice. PerJusion Media. The media used in the author's laboratory generally contain red blood cells because the increased 02 carrying capacity permits low flow rates, which are more desirable in experiments using nonrecirculating medium. In some experiments (e.g., studies of ion fluxes, lactate transport or glucose transport), it is preferable to omit red cells and use high flow rates (4-6 ml per minute per gram of liver). Bovine erythrocytes are routinely used in the author's studies, although rat, aged human, or sheep cells may be employed.6 Beef blood (approximately 5 liters) obtained from the jugular vein of stunned animals at a commercial slaughterhouse is immediately mixed with 0.2 volume of anticoagulant solution consisting of 13.2 g of glucose, 13.2 g of sodium citrate, 4.4 g of citric acid, and 150 mg of chloramphenicol per liter. The anticoagulant solution is prepared on the day of collection and tile anticoagulated blood is kept at 2°C before being used within 5 days. The blood is centrifuged, and the plasma is discarded. The cells are washed three times in 2 volumes of saline (9 g of NaC1 per liter) and twice in 2 volumes of bicarbonate buffeV containing 1 g of bovine serum albumin per liter. Tile white cell layer is removed by aspiration at each wash step. The packed red cells are stored at 2 ° and used within 2 days. Perfusion medium is made up by resuspending the red cells in oxygenated bicarbonate buffer; containing 30 g of bovine serum albumin (Cohn fraction V) ~ per liter. Sufficient cells are added to give a hematocrit of 18-22% (for experiments employing recirculating medium) or 36-407c (for experiments with nonrecirculating medium). The pH of the medium is then adjusted to 7.4. The required volume of medium (50-70 ml if recycled and 130 ml or more if not recycled) is pmnped into the oxygenation chamber and is recirculated through the system for 15-20 minutes to bring the pH, 0=, tension and temperature to the required values (7.4, 250 mM Hg, and 37.5°, respectively~. In the case of perfusion at slow flow rates, it may be necessary to pass the medium which is returning from the pumps through a coiled length of tubing immersed in water at 37.5 °. GBovine ceils are preferred, especially in studies of hepatic carbohydrate metal',olism, because of their low rate of glycolysis. H. A. Krebs and K. Henseleit, Hoppe-Seyler's Z. Physiol. Chem. 210, 33 (1932). Albumin purchased from any of the major supply houses is usually satisfactory. H u m a n serum albumin may also be used if available?
28
Liv~a
[4]
Fro. 1. Upper panel: General view of liver perfusion apparatus. The circuit tubing, filter assemblies and bubble traps have been omitted for clarity. The left-
[4l
THE P E R F U S E D RAT LIVER
29
D
C
\~
r
I-.~.
10.6 cm--
FIG. 2. Cross section of oxygenation chamber. A, tube for withdrawing medium; B, gas inlet tube; C, tube for returning medium. D, gas outlet tube; E, Teflon seal; F, medium; G, stainless steel arm to keep tube cluster fixed while chamber is rotating.
Technique of PerIusion. The procedure of Mortimore 2 is followed. The rat (90-150 g) is injected intraperitoneally with 6 mg of sodium pentobarbital per 100 g. The anesthetized animal is placed on the mobile tray and fastened in place using adhesive tape (Fig. 3). The abdomen is opened widely and the gut is reclined to the left to expose the portal vein and inferior vena cava. Using surgical silk (4/0) mounted on a curved needle, three loose ligatures are set: (a) around the portal vein just before its entry into the liver (inclusion of the bile duct must be a~:oided); (b) around the superior mesenteric and celiac arteries; (c~ around the inferior vena cava above the right renal vein (Fig. 3). Circulation of medium through the perfusion system is continued durhand animal lray (A) has been slid out of Plexiglas box through the removable window to its position during the eannulation procedures; the right-hand tray (B) is in its normal position during perfusion. The Plexiglas oxygenation chambers (C) and the gear system (D) which drives them are shown. The four front inlet ports (E) to the blowers for circulation of air are visible, the four rear outlet ports are obscured. The temperature control system is housed behind panel F. Lower panel: View of exposed base of perfusion apparatus. Visible are the electric motor (A) which rotates the oxygenation chambers, the blowers (B) for circulation of air, the temperature control system (C), the switch and cooling grille (D) for the electric motor, and the switch (E) for the temperature control system.
30
LIVER
[4]
FIG. 3. Diagram depicting cannulation and perfusion procedures. A, ligature tying portal vein eannula and placed above entry of splenic vein; B, inferior vena cava and abdominal aorta sectioned after insertion of portal cannula; C, infusion tubing and cannula inserted into rubber tubing insert in inflow line; D, inflow line taped to aluminum block to hold portal cannula in place; E, ligature around celiac axis'and superior mesenteric arteries, tied off after insertion of portal cannula; F, ligature around inferior vene, cava above R renal vein, tied off after insertion of vena cava cannula; G, vena cava caanula inserted through right atrium into inferior vena cava, not tied; H, outflow line taped to aluminum block to hold vena cava cannula in place. ing the above procedures b y t e m p o r a r i l y connecting portal and vena c a v a carmulas. I t is then stopped by t u r n i n g off the p u m p and clamping the tubing proximal to the portal cannula and distal to the vena c a v a cannula. T h e cannulas are then separated and the portal cannula is inserted
[4]
THE PERFUSED RAT LIVER
31
E
//"
4cm
8
FIG. 4. Cross section of filter assembly. A, Plexiglas outer casing; B, nylon gauze (100 mesh); C, brass screw carrying stainless steel inflow tube; D, rubber O-ring seals; E, anodized aluminum screw for holding outflow assembly in place F, Teflon outflow assembly carrying stainless steel outflow tube. into the portal vein, Immediately following this, the proximal clamp is removed, and the pump is restarted. Speed is essential at this stage in order to minimize interruption of blood flow to the liver. The vena cava is then sectioned below the set ligature so that perfusion medium flowing through the liver may escape via this route. The portal cannula is fixed in place with adhesive tape and the ligatures around the portal vein and the arteries are tied. The chest is then opened widely and the heart is exposed. The vena cava cannula is inserted through the right atrium into the inferior vena cava. The ligature around the lower part of the inferior vena cava is tightened, thus closing the circuit (Fig. 3). In experiments with recirculating medium, the perfusate leaving the liver flows by gravity back to the oxygenation chamber, which is 15 cm below the liver2 In experiments with nonrecirculating medium, commencement of the flow of effluent 9A negative hydrostatic pressure must be maintained in the portal cannula to prevent swelling and deterioration of the liver.
32
Liven
[4]
3cm
/
\
Fin. 5. Cross section of bubble trap assembly. A, clamp; B, rubber tubing, C, glass outer ground joint, standard taper 1%o; D, stainless steel inflow and outflow tubes; E, Teflon inner ground joint carrying inflow and outflow tubes; F, rubber O-ring seal; G, medium.
medium from the liver is facilitated by mild suction. The effluent medium is collected in sequential samples and is not returned to the oxygenation chamber. If 14C02 production is to be measured in this type of experiment, samples (1 ml) of effluent perfusate are drawn directly from the tubing and injected into Erlenmeyer flasks (25 ml) sealed with rubber stoppers (Kontes Glass Co., Vineland, New Jersey) and equipped with plastic center wells (Kontes) containing filter paper wicks impregnated with 0.3 ml of 1 M NaOH. To measure 14C02, 0.5 ml of 30% HCIO~ is injected into the flasks which are then incubated with shaking for 2 hours at 37 ° . The center wells are transferred to scintillation fluid and counted.3 To prevent the liver from drying during perfusion, it is routinely coy-
[4]
THE PERFUSED RAT LIVER
33
ered with a gauze pad soaked in warm saline. This is removed at intervals for inspection of the liver. In experiments with recirculating medium, samples are withdrawn from the oxygenation chambers before including the liver in the perfusion circuit and usually at 20-minute intervals thereafter. Since loss of perfusate during the cannulation procedures is variable, the volume of recirculating medium is measured by pumping the medium out of the system into a measuring cylinder at the end of perfusion and adding to this volume the residual volume (usually 1 ml) and the volume of the samples withdrawn during perfusion. In experiments with nonrecirculating medium, it is necessary to know the flow rate of the medium accurately. This is measured at the beginning of each perfusion experiment. Cleaning. Bacterial contamination is a complication of all perfusion studies and must be vigorously searched for and prevented if possible. Some bacterial infection of blood is almost inevitable during collection at the slaughterhouse. This is minimized by routine sterilization of the container, inclusion of chloramphenicol in the anticoagulant solution, and instruction of the personnel at the slaughterhouse. Spot checks of bacterial contamination and of the antibiotic sensitivity of contaminating organisms are also undertaken. If sterility is important, aged human blood may be used. At the end of a single perfusion, the circuit is cleaned by pumping through it at least 500 ml of saline at the rate of 30 ml per minute. At the end of a day of perfusion, the perfusion circuit, including all tubing and all components, is completely dismantled and soaked in bactericidal detergent (Dreft, Proctor and Gamble, Cincinnati, Ohio). Tubing is also routinely discarded after 1 month of use. General Considerations. Livers perfused by this technique for up to 2 hours show no deterioration, as evidenced by their gross and electron microscopic appearanceA ~ There is negligible edema as shown by the low water content of livers perfused for 1 hour (0.765 ± 0.003 ml per gram, wet weight)i° and by the absence of an increase in liver weight during perfusion for 2 hours2 Bile production is maintained and release of K + into the medium is minimal." Release of enzymes (alkaline phosphatase, glutamate oxaloacetate transaminase, lactate dehydrogenase) into the perfusate is absent or negligible. Oxygen consumption is steady 3,''-' and levels of ATP are maintained. 4,'3 Steady rates of glycogeloL. E. Mallette, J. H. Exton, and C. R. Park, J. Biol. Chem. 244, 5713 (1969). ~ T. F. Williams,J. H. Exton, N. Friedmann, and C. R. Park, Amer. J. Physiol. 221, 1645 (1971). 12j. H. Exton, J. G. Corbin, and S. C. Harper, J. Biol. Chem. 247, 4996 (1972). '~J. H. Exton and C. R. Park, J. Biol. Chem. 244, 1424 (1969).
34
LIVER
[4]
nolysis, gluconeogenesis, ureogenesis, and steady levels of cyclic A M P are attained within 15 minutes of completion of the perfusion circuit and are maintained for at least 60 minutes2 ,4,1°,12,14 Steady rates of utilization and oxidation of substrates such as lactate and alanine are also observed, 3,4,1°,12 The rates of most metabolic processes appear to be similar to those occurring in the intact animal 3,4 with the exception of glycogen synthesis which is low 3,1~ and proteolysis which is increased after 30 minutes of perfusion without added amino acids or insulin. 1~ Livers perfused by this technique also respond rapidly to physiological levels of hormones 4,~°,1',~4,~6-~s or to changes in the substrate concentration within the physiological range. 3,4,1°,13,19 The perfusion technique 2 described here has been successfully employed in a wide variety of studies of hepatic metabolism. The following is a list of selected references to such studies. Carbohydrate metabolism, s,4,~°-~,~7-26 protein metabolism, 1°,~2,~6,~ lipid metabolism, 3,~,27-3° glucagon, catecholamine, or cyclic A M P action, 4,11-14,16,17,19,26,23-25'29's1'32 insulin action, 2,11,12,1s,19,21,25,31-33 steroid action, 34-s6 membrane transport, ~2,37 ion fluxes, ~,~,~s,~,~°,3',3s and hormone assay. ~,39,~° 19j. H. Exton, G. A. Robison, E. W. Sutherland, and C. R. Park, J. Biol. Chem. 246, 6166 (1971). ,5 The low rate of glycogen synthesis observed in earlier studies~ was probably due to the low level of glucose in the perfusion medium since this substrate markedly affects the rate of glycogen deposition (H. Buschiazzo, J. H. Exton, and C. R. Park, Proc. Nat. Acad. Sci. U.S. 65, 383 (1970); W. Glinsmann, G. Pauk, and E. Hern, Biochem. Biophys. Res. Commun. 39, 774 (1970). ,e C. E. Mondon and G. E. Mortimore, Amer. J. Physiol. 212, 173 (1968). "J. H. Exton and C. R. Park, J. Biol. Chem. 243, 4189 (1968). ,8 G. E. Mortimore, Amer. J. Physiol. 204, 699 (1963). ,gj. H. Extort, J. G. Corbin, and C. R. Park, J. Biol. Chem. 244, 4095 (1969). 20j. H. Exton, S. B. Lewis, R. J. Ho, G. A. Robison, and C. R. Park, Ann. N.Y. Acad. Sci. 185, 85 (1971). 21G. E. Mortimore, E. King, Jr., C. E. Mondon, and W. Glinsmann, Amer. J. Physiol. 212, 179 (1967). 2~T. F. Williams, J. H. Exton, C. R. Park, and D. M. Regen, Amer. J. Physiol. 215, 1200 (1968). 2sT. H. Claus, D. M. Regen, J. H. Exton, and C. R. Park, J. Biol. Chem. Submitted for publication. M. Ui, T. H. Claus, J. H. Exton, and C. R. Park, J. Biol. Chem. 248, 5344 (1973). W. Glinsmann and G. E. Mortimore, Amer. J. Physiol. 215, 553 (1968). 2'K. It. Woodside and G. E. Mortimore, J. Biol. Chem. 247, 6474 (1972). ~'J. D. McGarry and D. W. Foster, J. Biol. Chem. 246, 1149 (1971). 's J. D. McGarry and D. W. Foster, J. Biol. Chem. 246, 6247 (1971). 29D. M. Regen and E. B. Terrell, Biochim. Biophys. Acta 170, 95 (1968). ,o R. L. Hamilton, D. M. Regen, M. E. Gray, and V. S. LeQuire, Lab. Invest. 16, 305 (1967).
[4]
~ H E PERFUSED
RAT LIVER
35
I n most instances, perfusion with nonrecirculating medium is preferred. With this method, substrates and hormones can be maintained at constant physiological concentrations, and rapid changes in substrate utilization or product formation can be readily measured. Complications due to the transformation of substrates or products in the medium itself are also avoided with this method because substrates can be infused into the influent medium just prior to the portal cannulas and effluent medium can be immediately deproteinized. The disadvantages are t h a t more medium is needed and t h a t some metabolic changes are too small to produce measurable alterations in substrates or products during a single passage through the liver. In this description, attention has been focused on the use of the perfused liver for the study of short-term aspects of hormonal control of metabolism. I t has not as yet proved feasible to perfuse livers satisfactorily for more than 3 hours with the unsupplemented medium described in this chapter. The reasons for the deterioration of the liver during longer perfusions are not known, but it is suspected t h a t the lack of certain plasma constituents and excessive bacterial growth m a y be factors. I t has been found in the author's laboratory t h a t livers can be perfused satisfactorily for up to 6 hours with medimn consisting of equal parts of rat blood collected under sterile conditions and bicarbonate buffer. Inclusion of antibiotics in this medium might result in satisfactory perfusions of even longer duration. Such perfusions would be desirable in the study of the mechanisms of action of slowly acting hormones, of the induction of enzymes and of the regulation of the synthesis and release of proteins such as albumin and lipoproteins. ~ L. S. Jefferson, J. H. Exton, R. W. Butcher, E. W. Sutherland, and C. R. Park. J. Biol. Chem. 243, 1031 (1968). ~-"J. H. Exton, J. G. Hardman, T. F. Williams, E. W. Sutherland, and C. R Park. J. Biol. Chem. 246, 2658 (1971). ~ J. H. Exton, S. C. Harper, A. L. Tucker and R. J. Ho, Biochim. Biophys. Act~ 329, 23 (1973). '~'J. H. Exton, N. Friedmann, E. H. A. Wong, J. P. Brineaux, J. D. Corbin, and C. R. Park, J. Biol. Chem. 247, 3579 (1972). ~ J. H. Exton, S. C. Harper, T. B. Miller, Jr., and C. R. Park, Biochim. Biophys. Acta. Submitted for publication. ~ J. H. Exton, S. C. Harper, A. L. Tucker, J. L. Flagg, and C. ]~. Park, Biochim. Biophys. Acta 329, 41 (1973). ~TT. H. Claus, D. M. ttegen, and A. tloos, Amer. J. Physiol. Submitted for publication. ~ N. Friedmann and C. R. Park, Proc. Nat. Acad. Sci. U.S. 6I, 504 (1968). ~"R. H. Unger, A. Ohneda, I. Valverde, A. M. Eisentraut, and J. H. Exton, J. Clin. Invest. 47, 48 (1968). 4oj. Marco, G. R. Faloona, and R. I-I. Unger, J. Clin. Invest. 50, 1650 (1971).
36
LIVER
[5]
Addendum. A recent study 41 indicates that fluorocarbons are satisfactory substitutes for erythrocytes in perfusion experiments. Livers perfused with the fluorocarbon FC-47 (3M Company, St. Paul, Minnesota) emulsified with bicarbonate buffer~ and Pluronic polyol F-68 (BASF Wyandotte Corp., Wyandotte, Michigan) were virtually identical in their metabolic behavior to livers perfused with bicarbonate buffer containing 15% erythrocytes.
41M. N. Goodman, R. Parrilla, and C. J. Toews, Amer. J. Physiol. 225, 1384 (1973).
[5] T h e F e t a l R a t L i v e r E x p l a n t as a M o d e l S y s t e m in t h e S t u d y o f H o r m o n e A c t i o n I B y EDWARD BRESNICK and KURT BURKI2
Any study of the mechanism of action of a particular hormone in the intact animal may be complicated by different effects upon different tissues, environmental influences, varying rates of absorption from the site of entry, etc. Consequently, it is sometime necessary to isolate a particular tissue for a detailed study of its biochemistry as influenced by hormonal administration. In this regard, organ or tissue culture has been of value as a biological model system. Although many techniques are available for maintenance of tissue explants, they depend upon growth on either solid or fluid media. The latter have been modified such that the tissue is grown at an interphase between the gas and liquid phases; the method of Trowell 3 has been useful in the investigation on liver tissue. Liver is a particularly difficult tissue to study by the explant technique partly because of its heterogeneity with regard to types of cells. Adult liver is composed of connective tissue elements, a variety of reticuloendothelial cells including Kupffer cells, as well as the hepatic parenchymal cells, i.e., hepatocytes. Under most circumstances, the parenchymal cells of adult liver tissue will not proliferate under culture conditions and, in fact, die. However, fetal liver adapts quite well to primary explanation, i.e., cultivation of pieces of tissues fresh from the organism. The fetal liver explant system has several other desirable properties which make it well suited for studying problems of development, cell 1The work presented in this article was supported by grants from the National Institutes of Health (GM 18623 and CA 12609), the National Science Foundation, Pharmaceutical Manufacturers Association, and Brown-Hazen Fund. Present address: Institute of Pathology, University of Bern, Bern, Switzerland. s O. A. Trowell, Exp. Cell Res. 6, 246 (1954).
