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Extracorporeal assist of anhepatic animals with liver slice perfusion
Extracorporeal Assist of Anhepatic Animals with Liver Slice Perfusion
T. Soyer, MD, Denver, Colorado M. Lempinen, MD, Denver, Colorado J. E. Walker, MD, Denver, Colorado P. Supanwanid, MD, Denver, Colorado B. Etseman, MD, FRCS, Denver, Colorado
Previously we have evaluated in vitro a number of perfusion devices using liver cell suspensions and thin liver slices with the ultimate aim of supporting patients in temporary hepatic failure [1,2]. The current studies involve testing the most promising of these devices on anhepatic animals.
Material and Methods Anhepatic Pig Model. Adult pigs weighing 25 to 35 kg are anesthetized with pentobarbital,25 mg/kg, and are given respiratoryassistwith room air.Hepatectomy is performed as described by us elsewhere [3].A 2.5 cm diameter expanded P.T.F.E. of Gore-TexO prosthetic graft is used to replace the excised vena ca~;afrom the renal veins to the diaphragm. A portacaval shunt is made between the leftside of the graft and the portal vein. No anticoagulantisused. BardicO catheters (numbers 16 and 18) are inserted into the right externaljugular vein and the carotid artery. The femoral artery is cannulated for blood pressure monitoring. A cystostomy is made and a Foley catheterinsertedintothe bladder. From the Departments of Sur¢mry and Neurology, Denver General Hospltal and the University of Colorado Medical School. Denver, Colorado. Thts work was supported by USPHS Grant =2R01 12126. NIH Fellowship =1 F05TW011776.01 (Dr Lempinen), and the John A. Hertford Foundation, Inc. Reprint requests should be addressed to Dr Eiseman, Department of Surgery, University of Colorado Medical Center, 4200 East 9th Avenue, Box 2333, Denver, Colorado 80220.
20
The animals are extubated immediately after the operation and no more respiratoryassistis given. A continuous 10 per cent glucose infusion(20 gtt/minute) is started during the operation and continued throughout the experimental period. Liver Slice Assist (LSA). Thin slicesof liverare prepared by excising the liver from the anesthetized (pentobarbital 25 mg/kg), heparinized (5 mg/kg) pigs (weighing 20 to 25 kg). The liver is prepared in situ through the portal vein with 1,000 to 1,500 ml of chilled (+4°C) Ringer's lactate solution with p H adjusted to 7.4 with sodium bicarbonate and 2 per cent procaine (6 ml/L) added. During the perfusionhepatectomy is completed. Thin slices(0.5 to 1.0 ram) are cut using a commercial Brown hand dermatome. The slices are further washed with cold buffered Ringer's lactate solution and placed within the perfusion chamber, which is filled with heparinized pig blood. Cold ischemia time of the liver slices varies from twenty to thirty minutes. The perfusion unit as previously described [I] is an acrylic cylinder 14 cm in diameter and 14 cm deep. (Figure 1.) A total of eighteen plates are housed in the chamber containing 200 to 250 gm of liver. Per[usion Circuit. The perfusion circuit is diagrammatically illustrated in Figure 2. Oxygenated blood from the right carotid artery flows through a collapsible plastic reservoir to the top of the chamber. From the bottom of the unit blood is led through a heat exchanger back to the pig's right external jugular vein using a roller pump.
The American Jouma! of Surgery
Liver Slice Assist In Anhepatic Animals
Regional heparinization is employed (10 mg of hepatin and 10 mg of protamine/hour) by using a HolterT M pump (model 905). Priming volume of the system is 2,500 ml. Initial perfusion is low (100 to 150 ml/minute) with gradual increase during the first hour to 200 ml/minute. Temperature in the chamber is kept between 37 and 39°C. Group I: Anhepatic Pig Control. Ten animals were hepatectomized as described and supported only with a 10 per cent glucose (20 gtt/minute) iP~fusion until the animals died.
Group II: LSA Two Hours post Hepatectomy. As soon as it was technically possible after hepatectomy (two hours), four anhepatic pigs were connected to the LSA apparatus and the assist was continued until the end of the experiment. This experiment was intended to determine if the LSA had benefit in anhepatic pigs before any sign of hepatic coma developed.
Group III: LSA Eighteen to Twenty.Two Hours post Hepatectomy. In this group, five anhepatic pigs were maintained overnight with 10 per cent glucose infusion. LSA was begun eighteen to twenty-two hours after hepatectomy, when clinical and electroencephalographic evidence of coma appeared. The LSA was continued until the pig's death. In each group recording was made at one to four hour intervals of vital signs, neurologic status, arterial pH, pO~, pCO2, and hematocrit. Electroencephalographic recordings (frontal, temporal, and occipital leads) were obtained in group I (six pigs) and group HI (five pigs) to monitor the reversal effect of LSA oll the electroencephalographic pattern of liver coma. Clotting time was measured using the microcapillary method [4] in group I (ten pigs). Serum and urine bilirubin was measured in group I (three pigs) and group H (three pigs). Serum urea concentration was measured in group I (six pigs) and group HI (four pigs) using Bertholot's method [5} modified to measure the component of nitrogen due to urea alone. Plasma ammonia was measured using ion exchange technics [6].
Figure 1. Perfuston chamber containing eighteen layers of thin pig fiver slices.
Although t h e r e was a positive correlation between the decreasing e l e c t r o e n c e p h a l o g r a p h i c act i v i t y a n d the clinical signs of coma, t h e r e were wide individual variations. S o m e a n i m a l s with alm o s t fla'c e.lectroencephalographic tracings s t a y e d relatively alert for several hours while i n others, despite essentially n o r m a l e l e c t r o e n c e p h a l o g r a m s , c o m a was progressive. Although t h e r e were individual differences, t h e r e - w e r e no statistically significant a l t e r a t i o n s in the length of survival between t h e controls of group I ( m e a n 24.5 hours) a n d those of groups II a n d III on e i t h e r early or late LSA ( m e a n survival 28 hours).
Electroencephalographic Studies. Group I (control group; six animals): In t h e i m m e d i a t e posta n e s t h e t i c period t h e r e was disorganized a c t i v i t y in t h e slow a l p h a range (7 to 10 cps). F r o m two to six hours postoperatively, increasing a m o u n t s of t h e t a a c t i v i t y were seen and the record b e c a m e progressively more disorganized. In t h r e e a n i m a l s CIRCUITRY OF EXTRACORPOREAL LIVER ASSIST
Results
T a b l e I s u m m a r i z e s the results of t h e experiments. Clinical and Neurologic Course. Characteristically the a n i m a l s awoke-from anesthesia a n d for a t i m e were a l e r t and reactive. Control a n i m a l s receiving only glucose b e c a m e decreasingly responsive a n d w e n t into c o m a in ten to f o u r t e e n hours. Blood pressure d r o p p e d gradually. R e s p i r a t i o n bec a m e superficial a n d the r a t e increased, p H rose regularly ( T a b l e II) a n d finally the a n i m a l s died of r e s p i r a t o r y arrest. M e a n survival was 24.5 hours after hepatectomy.
VOlume 12S, July 1973
L,~mewem,,miJ
Vein
Figure 2. Perluslon circuiL
21
Soyer et al
TABLE I
Summary of LSA Experiments
Parameter
Group I (10 experiments)
Group II (4 experiments)
Survival time =1: SD (hr) Arterial blood pH
21.5 =E 8 Gradually became alkalotic
19.5 :E 10 Remained within normal limit
Serum conjugated btlirubin concentration
Gradually increased to 1 to 2 mg per cent in 20 hr
Total urine conjugated bilirubin Serum urea concentration
Small amount was found in urine Decreased to 1 to ~.mg per cent after 12 to 20 hr Flat electroencephalogram between 12 and 24 hr after hepatectomy alternating with high voltage stow activity
Gradually increased to 1 to 2 mg per cent in 20 hr (more circulating volume) Greater amount as compared to that of group I (Figure 4) Decreased to 1 to 2 mg per cent after 12 to 20 hr
Electroencephalographic tracings
slow focal delta activity appeared in the left temporal region which later spread to" the frontal area on the same,side. Six to ten hours after hepatectomy the electroencephalographic tracing decreased in amplitude and interm~tently showed disorganized slow activity. In five to six animals a fiat electroencephalographic recording developed between twelve and twenty-four hours after hepatectomy. After that time, there were intermittent generalized runs of rhythmic, high amplitude biphasic slow waves at I ~ to 3 cps, usually lasting one to five seconds, but occasionally longer. No triphasic TABLE II
Arterial Blood pH in Groups I and II
Hours after Hepatectomy
Group I (Pig 18)
Group II (Pig 8)
pH
pH
0
i 2 4
6 8 10
pCO2
7.40 ___ 7.++ 7.58 __-
29 ... i.;; ;;" 34 7.47 27 24 . . . . . . 41 3'+ 7.44 4o . . . . . . .+'i.; 7.46 50 .
780
12 14 16
18 20 22 Survival time
22
.
.
pCO2
.
.
.
.
.
i.51 50 7.06 > I00 . . . . . . 21 hours
.
.
.
.
.
.
.
.
.
.
21; 122 •. 44 7.48 36 30 hours .
Group III (5 experiments) 30.5 =t: 6 Same as group I before connection to LSA, and was in the normal limit after the LSA assist
Increased to 3 to 4 mg per cent after LSA assist Same as group I before connection to LSA. After LSA, one of the animals, had return to alpha actfvtty (Figure 3) another three had some changes of electroencephalographic tracings toward normal
transients were observed. This type of alternating fiat and high voltage slow activity often persisted for many hours. One animal had persistence of disorganized theta activity at 5 to 8 cps for twenty-four hours. Group IH (LSA experiments): In group HI where L S A was started eighteen to twenty-two hours after hepatectomy, all pigs were comatose prior to LSA and had the electroencephalographic findings characteristic of coma. No change occurred in one animal's electroencephalographic record during LSA. Another animal showed a return of some 1 to 3 cps rhythms of varying voltage. In the other three animals, however, LSA resulted in significant electroencephalographic changes. In each, theta activity and runs of rhythmic slow activity reappeared after one htmr of LSA. After two to three hours on LSA, generally faster activity (6 to 8 cps) appeared with more normal voltage, b u t the tracings continued to be disorganized. Only one animal had return of alpha activity. (Figure 3.) Theta activity persisted in one animal for two hours and in the other for about twenty hours before the alternating fiat and rhythmic delta activity again became prominent. Completely fiat records similar to those prior to extracorporeal support returned after five hours of LSA in'one animal, but r~ot until after about thirty hours of LSA in another anhepatic pig. Biochemical Studies. No bleeding diathesis developed in any of the animals. Clotting times remained normal or only rose gradually to the upper limits of normal in control animals.
The American Joum4d of Su~llery
Liver Slice Assist In Anhepatlc Animals
t! Figure 3. Electroencephalogram of pig 14. Upper lractng was made twenty hours after hepatectomy while ihe pig was in coma. Lower tracing was made two hours later with animal on LSA,
Bilirubin: Bilirubin conjugation was studied in groups I and II. As seen in Figure 4, plasma concentration of conjugated bilirubin did not change during LSA due to the increase of circulating volume. However, total amounts of conjugated bilirubin were higher in treated animals. In the equivalent four hour period the conjugated bilirubin in urine was higher in animals treated by L S A than it was in untreated control animals. (Figure 4.) Urea: Serum urea concentration gradually decreased in hepatectomized control animals and stabilized at 1 to 2 rag per cent after twelve to twenty hours. This pattern also occurred in group H. In group HI, urea decreased during eighteen to twenty-two hours without L S A as it did in the control animals. During the first four hours of subsequent LSA, serum urea levels rose to two thirds the normal level and persisted for the remainder of liver slice perfusion. (Figure 5.) Urine output was constant (30to 70 ml/hour) in all animals. Ammonia: There was such wide variation in individual blood a m m o n i a concentration that no conclusions could be reached on the effect of L S A on a m m o n i a metabolism.
4 X~
~Treilod ~Cliniril
I
3exp 3illp
1
4
I!
12 16 HOURS
20
24
2
i j,
~
~rrelltelJ
L:.:"
3 llntlmill Q
ll
8 . 12 HOURS
16 -'~ll "
Figure 4. Effect of LSA (group II) on bilirubin con~uga(ion in anhepatic pigs. ;O| I ~81
UREA PRODUCTION IN ANHEPATIC PIGS CONTROL v l EXTRACORPOREAL LIVER SLICE ASSIST
Comments
These in vivo experiments confirm the metabolic effectiveness of t h i s simple LSA apparatus cn anhepatic pigs. But the limited length of the survival on LSA (a mean of thirty hours) and the
Volume 121~,July 1/173
4
8
12
i6 20 HOURS
24
28
32
36
Figure 5. Effect of LSA (group III) on blood urea concentratton.
23
Soyer et al
ultimate relapse into coma indicate t h a t the device as it currently exists is far from a complete substitute for a whole liver. The chamber itself holds up to 250 gm of liver. If all cells remained 100 per cent effective, this volume of liver (approximately one fourth the weight of the total organ) should totally support the animal because of the high metabolic reserve of the liver. Although these experiments confirm continued hepatocyte function in the extracorporeal device for as long as twelve hours, the hepatocytes are probably subject to the same "tiring" effect noted by Krebs [8] in both liver slices and with hepatocytes in culture. The best evidence of LSA effectiveness was its alteration of the electroencephalographic tracings toward normal in group lII. In four of five animals started on LSA when the animal was deeply comatose eighteen to twentytwo hours after hepatectomy, there was unquestionable although transient (two to five hours) alteration of the electroencephalographic tracings toward normal. LSA is unquestionably simpler to employ than is whole liver perfusion as a method of extracorporeal assistance. It can be used intermittently with ease employing a chronic arteriovenous s h u n t cannula. Of greatest importance is the effectiveness of LSA with limited flows when contrasted to t h a t of whole liver perfusion. Once an entire liver is on extracorporeal perfusion, a critical minimal flow of 200 to 500 ml/minu.te of oxygenated blood must constantly be maintained through its two perfusion lines (portal vein and hepatic artery) or irreversible ischem~c damage ensues and the entire complex apparatus must be disconnected. On the contrary, LSA requires but a single arterial line and can be maintained with lesser demands of flow. The chamber containing the liver slices can be switched with ease and new liver slices connected into the circuit. If the metabolic capacity of the device can be increased by improvements in chamber design, LSA should prove to be of clinical benefit in supporting patients in potentially reversible hepatic failure.
24
Conclusions 1. A total of nine anhepatic pigs were furnished support for periods up to twenty-one hours using a liver slice assist extracorporeal perfusion system. L S A was instituted two hours after hepatectomy in one group of four animals and eighteen to twenty-two hours after operation in an additional five animals. 2. Evidence of L S A function included a return toward normal electroencephalographic tracings, urea production, and bilirubin conjugation. 3. Although the current technic for L S A does not yet provide complete metabolic replacement of liver function in the anhepatic animal, its proved function provides a promising and relatively simple method for temporary liver support.
Summary Thin (0.5 to 1.0 mm) liver slices perfused with arterial blood have been used to provide temporary support of anhepatic pigs. The technic is far simpler t h a n is whole liver perfusion. It reverses the electroencephalographic abnormalities, and other metabolic functions are restored, but as yet it is not sufficient to provide total support.
References 1. Eiseman B, Soyer T: Prosthetics in hepatic assistance. Transplantation Proc 11: 1519. 1971. 2. Soyer T, Lempinen M, Bielanskl E, Eiseman B: Experimental extracorporeal hepatic assist using liver slices and ceil suspension. Surg Forum 23: 346, 1972. 3. Lempinen M, Soyer T, Eiseman B: A new technique for preparing total hepatectomized pigs, Submitted for publication. 4. Henry RJ: Clinical Chemistry; Principles and Techniques. New York, Harper & Row, 1964. 5. Chaney AL. Marbach EP: Modified reagents for determination of urea and ammonia. Clin Chem 8: 131, 1962. 6. Fenton JCB, Williams AH: Improved methods for the estimation of plasma ammonia by ion exchange. J Clin Path 21: 14, 1968. 7. Soyer T, Lempinen M, Eiseman B: In vitro extracorporeal liver slices and c~tl suspensions for temporary hepatic support'. Submitted for publication. 8. Krebs HA: Formation of ketone bodies in the perfused rat liver, p 219. Stoffnechsel der isoliert Perfundiertenlebet (Saib W and Scholz R, ed). Berlin, Springer-Verlag, 1968.
The American Journal of Surgery
T. Soyer, MD, Denver, Colorado M. Lempinen, MD, Denver, Colorado J. E. Walker, MD, Denver, Colorado P. Supanwanid, MD, Denver, Colorado B. Etseman, MD, FRCS, Denver, Colorado
Previously we have evaluated in vitro a number of perfusion devices using liver cell suspensions and thin liver slices with the ultimate aim of supporting patients in temporary hepatic failure [1,2]. The current studies involve testing the most promising of these devices on anhepatic animals.
Material and Methods Anhepatic Pig Model. Adult pigs weighing 25 to 35 kg are anesthetized with pentobarbital,25 mg/kg, and are given respiratoryassistwith room air.Hepatectomy is performed as described by us elsewhere [3].A 2.5 cm diameter expanded P.T.F.E. of Gore-TexO prosthetic graft is used to replace the excised vena ca~;afrom the renal veins to the diaphragm. A portacaval shunt is made between the leftside of the graft and the portal vein. No anticoagulantisused. BardicO catheters (numbers 16 and 18) are inserted into the right externaljugular vein and the carotid artery. The femoral artery is cannulated for blood pressure monitoring. A cystostomy is made and a Foley catheterinsertedintothe bladder. From the Departments of Sur¢mry and Neurology, Denver General Hospltal and the University of Colorado Medical School. Denver, Colorado. Thts work was supported by USPHS Grant =2R01 12126. NIH Fellowship =1 F05TW011776.01 (Dr Lempinen), and the John A. Hertford Foundation, Inc. Reprint requests should be addressed to Dr Eiseman, Department of Surgery, University of Colorado Medical Center, 4200 East 9th Avenue, Box 2333, Denver, Colorado 80220.
20
The animals are extubated immediately after the operation and no more respiratoryassistis given. A continuous 10 per cent glucose infusion(20 gtt/minute) is started during the operation and continued throughout the experimental period. Liver Slice Assist (LSA). Thin slicesof liverare prepared by excising the liver from the anesthetized (pentobarbital 25 mg/kg), heparinized (5 mg/kg) pigs (weighing 20 to 25 kg). The liver is prepared in situ through the portal vein with 1,000 to 1,500 ml of chilled (+4°C) Ringer's lactate solution with p H adjusted to 7.4 with sodium bicarbonate and 2 per cent procaine (6 ml/L) added. During the perfusionhepatectomy is completed. Thin slices(0.5 to 1.0 ram) are cut using a commercial Brown hand dermatome. The slices are further washed with cold buffered Ringer's lactate solution and placed within the perfusion chamber, which is filled with heparinized pig blood. Cold ischemia time of the liver slices varies from twenty to thirty minutes. The perfusion unit as previously described [I] is an acrylic cylinder 14 cm in diameter and 14 cm deep. (Figure 1.) A total of eighteen plates are housed in the chamber containing 200 to 250 gm of liver. Per[usion Circuit. The perfusion circuit is diagrammatically illustrated in Figure 2. Oxygenated blood from the right carotid artery flows through a collapsible plastic reservoir to the top of the chamber. From the bottom of the unit blood is led through a heat exchanger back to the pig's right external jugular vein using a roller pump.
The American Jouma! of Surgery
Liver Slice Assist In Anhepatic Animals
Regional heparinization is employed (10 mg of hepatin and 10 mg of protamine/hour) by using a HolterT M pump (model 905). Priming volume of the system is 2,500 ml. Initial perfusion is low (100 to 150 ml/minute) with gradual increase during the first hour to 200 ml/minute. Temperature in the chamber is kept between 37 and 39°C. Group I: Anhepatic Pig Control. Ten animals were hepatectomized as described and supported only with a 10 per cent glucose (20 gtt/minute) iP~fusion until the animals died.
Group II: LSA Two Hours post Hepatectomy. As soon as it was technically possible after hepatectomy (two hours), four anhepatic pigs were connected to the LSA apparatus and the assist was continued until the end of the experiment. This experiment was intended to determine if the LSA had benefit in anhepatic pigs before any sign of hepatic coma developed.
Group III: LSA Eighteen to Twenty.Two Hours post Hepatectomy. In this group, five anhepatic pigs were maintained overnight with 10 per cent glucose infusion. LSA was begun eighteen to twenty-two hours after hepatectomy, when clinical and electroencephalographic evidence of coma appeared. The LSA was continued until the pig's death. In each group recording was made at one to four hour intervals of vital signs, neurologic status, arterial pH, pO~, pCO2, and hematocrit. Electroencephalographic recordings (frontal, temporal, and occipital leads) were obtained in group I (six pigs) and group HI (five pigs) to monitor the reversal effect of LSA oll the electroencephalographic pattern of liver coma. Clotting time was measured using the microcapillary method [4] in group I (ten pigs). Serum and urine bilirubin was measured in group I (three pigs) and group H (three pigs). Serum urea concentration was measured in group I (six pigs) and group HI (four pigs) using Bertholot's method [5} modified to measure the component of nitrogen due to urea alone. Plasma ammonia was measured using ion exchange technics [6].
Figure 1. Perfuston chamber containing eighteen layers of thin pig fiver slices.
Although t h e r e was a positive correlation between the decreasing e l e c t r o e n c e p h a l o g r a p h i c act i v i t y a n d the clinical signs of coma, t h e r e were wide individual variations. S o m e a n i m a l s with alm o s t fla'c e.lectroencephalographic tracings s t a y e d relatively alert for several hours while i n others, despite essentially n o r m a l e l e c t r o e n c e p h a l o g r a m s , c o m a was progressive. Although t h e r e were individual differences, t h e r e - w e r e no statistically significant a l t e r a t i o n s in the length of survival between t h e controls of group I ( m e a n 24.5 hours) a n d those of groups II a n d III on e i t h e r early or late LSA ( m e a n survival 28 hours).
Electroencephalographic Studies. Group I (control group; six animals): In t h e i m m e d i a t e posta n e s t h e t i c period t h e r e was disorganized a c t i v i t y in t h e slow a l p h a range (7 to 10 cps). F r o m two to six hours postoperatively, increasing a m o u n t s of t h e t a a c t i v i t y were seen and the record b e c a m e progressively more disorganized. In t h r e e a n i m a l s CIRCUITRY OF EXTRACORPOREAL LIVER ASSIST
Results
T a b l e I s u m m a r i z e s the results of t h e experiments. Clinical and Neurologic Course. Characteristically the a n i m a l s awoke-from anesthesia a n d for a t i m e were a l e r t and reactive. Control a n i m a l s receiving only glucose b e c a m e decreasingly responsive a n d w e n t into c o m a in ten to f o u r t e e n hours. Blood pressure d r o p p e d gradually. R e s p i r a t i o n bec a m e superficial a n d the r a t e increased, p H rose regularly ( T a b l e II) a n d finally the a n i m a l s died of r e s p i r a t o r y arrest. M e a n survival was 24.5 hours after hepatectomy.
VOlume 12S, July 1973
L,~mewem,,miJ
Vein
Figure 2. Perluslon circuiL
21
Soyer et al
TABLE I
Summary of LSA Experiments
Parameter
Group I (10 experiments)
Group II (4 experiments)
Survival time =1: SD (hr) Arterial blood pH
21.5 =E 8 Gradually became alkalotic
19.5 :E 10 Remained within normal limit
Serum conjugated btlirubin concentration
Gradually increased to 1 to 2 mg per cent in 20 hr
Total urine conjugated bilirubin Serum urea concentration
Small amount was found in urine Decreased to 1 to ~.mg per cent after 12 to 20 hr Flat electroencephalogram between 12 and 24 hr after hepatectomy alternating with high voltage stow activity
Gradually increased to 1 to 2 mg per cent in 20 hr (more circulating volume) Greater amount as compared to that of group I (Figure 4) Decreased to 1 to 2 mg per cent after 12 to 20 hr
Electroencephalographic tracings
slow focal delta activity appeared in the left temporal region which later spread to" the frontal area on the same,side. Six to ten hours after hepatectomy the electroencephalographic tracing decreased in amplitude and interm~tently showed disorganized slow activity. In five to six animals a fiat electroencephalographic recording developed between twelve and twenty-four hours after hepatectomy. After that time, there were intermittent generalized runs of rhythmic, high amplitude biphasic slow waves at I ~ to 3 cps, usually lasting one to five seconds, but occasionally longer. No triphasic TABLE II
Arterial Blood pH in Groups I and II
Hours after Hepatectomy
Group I (Pig 18)
Group II (Pig 8)
pH
pH
0
i 2 4
6 8 10
pCO2
7.40 ___ 7.++ 7.58 __-
29 ... i.;; ;;" 34 7.47 27 24 . . . . . . 41 3'+ 7.44 4o . . . . . . .+'i.; 7.46 50 .
780
12 14 16
18 20 22 Survival time
22
.
.
pCO2
.
.
.
.
.
i.51 50 7.06 > I00 . . . . . . 21 hours
.
.
.
.
.
.
.
.
.
.
21; 122 •. 44 7.48 36 30 hours .
Group III (5 experiments) 30.5 =t: 6 Same as group I before connection to LSA, and was in the normal limit after the LSA assist
Increased to 3 to 4 mg per cent after LSA assist Same as group I before connection to LSA. After LSA, one of the animals, had return to alpha actfvtty (Figure 3) another three had some changes of electroencephalographic tracings toward normal
transients were observed. This type of alternating fiat and high voltage slow activity often persisted for many hours. One animal had persistence of disorganized theta activity at 5 to 8 cps for twenty-four hours. Group IH (LSA experiments): In group HI where L S A was started eighteen to twenty-two hours after hepatectomy, all pigs were comatose prior to LSA and had the electroencephalographic findings characteristic of coma. No change occurred in one animal's electroencephalographic record during LSA. Another animal showed a return of some 1 to 3 cps rhythms of varying voltage. In the other three animals, however, LSA resulted in significant electroencephalographic changes. In each, theta activity and runs of rhythmic slow activity reappeared after one htmr of LSA. After two to three hours on LSA, generally faster activity (6 to 8 cps) appeared with more normal voltage, b u t the tracings continued to be disorganized. Only one animal had return of alpha activity. (Figure 3.) Theta activity persisted in one animal for two hours and in the other for about twenty hours before the alternating fiat and rhythmic delta activity again became prominent. Completely fiat records similar to those prior to extracorporeal support returned after five hours of LSA in'one animal, but r~ot until after about thirty hours of LSA in another anhepatic pig. Biochemical Studies. No bleeding diathesis developed in any of the animals. Clotting times remained normal or only rose gradually to the upper limits of normal in control animals.
The American Joum4d of Su~llery
Liver Slice Assist In Anhepatlc Animals
t! Figure 3. Electroencephalogram of pig 14. Upper lractng was made twenty hours after hepatectomy while ihe pig was in coma. Lower tracing was made two hours later with animal on LSA,
Bilirubin: Bilirubin conjugation was studied in groups I and II. As seen in Figure 4, plasma concentration of conjugated bilirubin did not change during LSA due to the increase of circulating volume. However, total amounts of conjugated bilirubin were higher in treated animals. In the equivalent four hour period the conjugated bilirubin in urine was higher in animals treated by L S A than it was in untreated control animals. (Figure 4.) Urea: Serum urea concentration gradually decreased in hepatectomized control animals and stabilized at 1 to 2 rag per cent after twelve to twenty hours. This pattern also occurred in group H. In group HI, urea decreased during eighteen to twenty-two hours without L S A as it did in the control animals. During the first four hours of subsequent LSA, serum urea levels rose to two thirds the normal level and persisted for the remainder of liver slice perfusion. (Figure 5.) Urine output was constant (30to 70 ml/hour) in all animals. Ammonia: There was such wide variation in individual blood a m m o n i a concentration that no conclusions could be reached on the effect of L S A on a m m o n i a metabolism.
4 X~
~Treilod ~Cliniril
I
3exp 3illp
1
4
I!
12 16 HOURS
20
24
2
i j,
~
~rrelltelJ
L:.:"
3 llntlmill Q
ll
8 . 12 HOURS
16 -'~ll "
Figure 4. Effect of LSA (group II) on bilirubin con~uga(ion in anhepatic pigs. ;O| I ~81
UREA PRODUCTION IN ANHEPATIC PIGS CONTROL v l EXTRACORPOREAL LIVER SLICE ASSIST
Comments
These in vivo experiments confirm the metabolic effectiveness of t h i s simple LSA apparatus cn anhepatic pigs. But the limited length of the survival on LSA (a mean of thirty hours) and the
Volume 121~,July 1/173
4
8
12
i6 20 HOURS
24
28
32
36
Figure 5. Effect of LSA (group III) on blood urea concentratton.
23
Soyer et al
ultimate relapse into coma indicate t h a t the device as it currently exists is far from a complete substitute for a whole liver. The chamber itself holds up to 250 gm of liver. If all cells remained 100 per cent effective, this volume of liver (approximately one fourth the weight of the total organ) should totally support the animal because of the high metabolic reserve of the liver. Although these experiments confirm continued hepatocyte function in the extracorporeal device for as long as twelve hours, the hepatocytes are probably subject to the same "tiring" effect noted by Krebs [8] in both liver slices and with hepatocytes in culture. The best evidence of LSA effectiveness was its alteration of the electroencephalographic tracings toward normal in group lII. In four of five animals started on LSA when the animal was deeply comatose eighteen to twentytwo hours after hepatectomy, there was unquestionable although transient (two to five hours) alteration of the electroencephalographic tracings toward normal. LSA is unquestionably simpler to employ than is whole liver perfusion as a method of extracorporeal assistance. It can be used intermittently with ease employing a chronic arteriovenous s h u n t cannula. Of greatest importance is the effectiveness of LSA with limited flows when contrasted to t h a t of whole liver perfusion. Once an entire liver is on extracorporeal perfusion, a critical minimal flow of 200 to 500 ml/minu.te of oxygenated blood must constantly be maintained through its two perfusion lines (portal vein and hepatic artery) or irreversible ischem~c damage ensues and the entire complex apparatus must be disconnected. On the contrary, LSA requires but a single arterial line and can be maintained with lesser demands of flow. The chamber containing the liver slices can be switched with ease and new liver slices connected into the circuit. If the metabolic capacity of the device can be increased by improvements in chamber design, LSA should prove to be of clinical benefit in supporting patients in potentially reversible hepatic failure.
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Conclusions 1. A total of nine anhepatic pigs were furnished support for periods up to twenty-one hours using a liver slice assist extracorporeal perfusion system. L S A was instituted two hours after hepatectomy in one group of four animals and eighteen to twenty-two hours after operation in an additional five animals. 2. Evidence of L S A function included a return toward normal electroencephalographic tracings, urea production, and bilirubin conjugation. 3. Although the current technic for L S A does not yet provide complete metabolic replacement of liver function in the anhepatic animal, its proved function provides a promising and relatively simple method for temporary liver support.
Summary Thin (0.5 to 1.0 mm) liver slices perfused with arterial blood have been used to provide temporary support of anhepatic pigs. The technic is far simpler t h a n is whole liver perfusion. It reverses the electroencephalographic abnormalities, and other metabolic functions are restored, but as yet it is not sufficient to provide total support.
References 1. Eiseman B, Soyer T: Prosthetics in hepatic assistance. Transplantation Proc 11: 1519. 1971. 2. Soyer T, Lempinen M, Bielanskl E, Eiseman B: Experimental extracorporeal hepatic assist using liver slices and ceil suspension. Surg Forum 23: 346, 1972. 3. Lempinen M, Soyer T, Eiseman B: A new technique for preparing total hepatectomized pigs, Submitted for publication. 4. Henry RJ: Clinical Chemistry; Principles and Techniques. New York, Harper & Row, 1964. 5. Chaney AL. Marbach EP: Modified reagents for determination of urea and ammonia. Clin Chem 8: 131, 1962. 6. Fenton JCB, Williams AH: Improved methods for the estimation of plasma ammonia by ion exchange. J Clin Path 21: 14, 1968. 7. Soyer T, Lempinen M, Eiseman B: In vitro extracorporeal liver slices and c~tl suspensions for temporary hepatic support'. Submitted for publication. 8. Krebs HA: Formation of ketone bodies in the perfused rat liver, p 219. Stoffnechsel der isoliert Perfundiertenlebet (Saib W and Scholz R, ed). Berlin, Springer-Verlag, 1968.
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