Jun 17, 1986 - transplants when portal anastomosis became throm-. ' bosed.9 Thus, in .... Our previous studies with PBL in rats with different portacaval shunts.
Portal Branch Ligation in Reevaluation
of
a
the
Rat
Model
From the Department of Surgery, Lund University, Lund, Sweden
JACEK ROZGA, BENGT JEPPSSON, and STIG BENGMARK, MD
The effects of portal occlusion on the liver have been differently reported in different studies. The authors therefore reevaluated a model ofportal branch ligation (PBL) in the rat. Histologic appearance, DNA synthetic activity, labeling count, and mitotic index were serially evaluated in both ligated and nonligated parts of the liver after interruption of the portal flow to one fourth, one third, and two thirds of the liver mass. The authors confirmed the presence of compensatory hyperplasia induced in the nonligated liver lobe(s) by PBL, and its intensity was roughly proportional to the amount of liver tissue devoid of portal perfusion. Portal-deprived liver tissue un-
derwent a rapid and progressive atrophy, and, by the end of the first week, the weight of this part had decreased 10-fold. By a balance between atrophy and compensatory growth, the total liver weight was maintained at the level of sham-operated animals throughout the experiment. PBL invariably resulted in early centrilobular necrosis, which occupied 15-24% of the ligated lobe(s). However, already after 4 days it was almost totally resorbed and did not appear de novo. PBL was not followed by local collateralization. (Am J Pathol 1986, 125:300-308)
IT HAS BEEN SHOWN in different animal species that ligation of a branch of the portal vein induces atrophy of the affected part of the liver, while the remaining segments receiving all the portal flow undergo compensatory growth until the original mass of hepatic tissue is restored. Many studies have shown that, in the ligated part, the parenchymal cells diminish in size and appear to be crowded, but they remain intact and the lobular architecture is preserved.1-4 On the other hand, several authors have drawn attention to the appearance of multifocal necroses. They have been found in rats, cats, and rabbits.5-8 Some recent communications suggest that obstruction of intrahepatic portal branches also may lead to necrosis in man9 and that late pathomorphologic changes closely resemble observations made on experimental animals.6'10 In auxiliary liver transplants, thrombosis of the portal vein inevitably results in multifocal necroses of the graft."1 Because many studies report varying effects of portal branch ligation (PBL), we have found it desirable to reevaluate this model under rigidly controlled experimental conditions. The necessity of this study also arose from our increasing interest in extensive hepaticobiliary surgery for cancer in man, when ligation of the portal trunk or its branches is commonplace. The experiments include serial observations with
comparative pathomorphologic data after obstruction of the portal venous supply to one fourth, one third, and two thirds of the rat liver. Observations of other authors have been extended with the measurement of hypertrophy and hyperplasia in the nonligated liver lobes as well as the synthesis of DNA in the lobe(s) devoid of portal blood perfusion.
Materials and Methods Animals A total of 245 male Sprague-Dawley rats (250-280 g) were kept in a colony room at 21 C under 12 hours dark-light cycle (6 AM to 6 PM). They were housed four per cage and maintained on a standard food-pellet diet and tap water ad libitum.
Experimental Design Operations were carried out between 9 AM and noon
Accepted for publication June 17, 1986. Address reprint requests to Stig Bengmark, MD, Department of Surgery, Lund University, S-221 85 Lund, Sweden.
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Figures IA-C-The ligated and nonligated liver lobes 2 weeks after intervention.
while rats were under light ether anesthesia and with the use of clean but not sterile technique. So that hepatic denervation could be avoided and the artery and bile duct could be left intact, PBL was achieved with the use of 7-0 suture (Perma-Hand, Ethicon) under an operating microscope (Zeiss, magnification 10-25 x). Sham PBL involved laparotomy and dissection of the relevant ligaments without ligature. There were four groups of rats: Group 1- PBL to the right lobes (c:a 24% of liver mass); Group 2- PBL to the left anterior lobe (c:a 34% of liver mass); Group 3- PBL to the two anterior lobes (c:a 70Vo of liver mass); Group 4- sham-PBL controls. The rats were weighed daily and were sacrificed by aortic puncture in groups of 8-10 rats after 24 hours, 48 hours, 72 hours 96 hours, 1 week, and 2 weeks. Two subgroups of six rats were subjected to 70% ligation (Group 3) and sacrificed after 6 and 8 weeks, respectively, which confirmed that the histologic changes induced by PBL were completed within 2 weeks. One hour before rats were sacrificed, Me-3HThymidine (New England Nuclear, specific activity, 113.0 Ci per mmol) was injected intraperitoneally in a dose of 0.4 gCi per 1 g of body weight. Macroscopic appearance of the liver was noted, and the ligated part was cut so that bleeding could be observed. The site of ligature was inspected under an operating microscope. The exsanguinated liver was removed,
and both ligated and nonligated parts were weighed separately for measurement of their absolute and relative weights (weight/body weight ratios). In sham-PBL controls, all of the liver lobes were weighed separately, and the corresponding control ratios were calculated in correspondence to each group of test rats. Blocks of liver tissue were rapidly frozen and stored at - 80 C until measurement of DNA synthetic activity (DNASA). Representative biopsy specimens from the same lobes were washed three times in buffered formalin, fixed in the same agent, and processed with paraffin. DNA Synthetic Activity DNA synthetic activity was measured by the method of Scott et al.12 Standard curves were prepared with known quantities of DNA from calf thymus (Sigma Chemical Co., St. Louis, Mo). Radioactivity was measured in a liquid scintillation counter (TRI-CARB 460 CD, Packard). Quench correction was made with the use of external standard. Incorporation of tritiated thymidine into freshly synthesized DNA was expressed as dpm per 1 jig of purified DNA.
Autoradiographs Sections at 3 i were coated with Ilford K2 emulsion (Ciba-Geigy, England) exposed for 21 days and developed in Kodak D-19. Afterward, the slides were fixed in Kodak-Unifix, dehydrated, and stained with Mayer's
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hematoxylin. Hepatocytes with five or more silver grains over their nuclei were considered labeled. They were counted in 100 consecutive high-power fields (x400). Results are given as number of hepatocytes labeled per 1000 hepatic nuclei. Mitotic Activity Mitotic activity was determined in separately stained sections that were 5 thick (hematoxylin and eosin). Mitosing hepatocytes were sought in 100 consecutive high-power fields (x 400), and mitotic index was expressed per 1000 hepatic nuclei. Prophases before dissolution of nuclear membrane and late telophases were excluded. Necroses in the Portal-Deprived Lobes Necroses in the portal-deprived lobes were measured in all rats sacrificed 48 hours after PBL. Ten consecutive microscopic fields (x 100) were projected onto fine tracing paper. Necroses were outlined, cut out, and weighed. The extent of necrosis was expressed as a percentage of the total projected fields.
Statistical Methods Analysis of variance incorporating Newman-Keul's procedure was employed for detection of differences between any pair of means. For observation of changes in time sequences, the supplemental computations for simple effects were added to the analysis of variance. Significance was assumed to be if alpha 0.01 or less. Values are given as mean ± SEM.
Results There was no perioperative mortality, and all the rats regained their preoperative weight after 2-3 days. In rats that underwent PBL from groups sacrificed after 24-96 hours, the lobes deprived of portal flow were darkened. After 1 and 2 weeks, they were reduced to minute remnants with red appearance and a network of vessels visible through the residual parenchyma (Figure 1). In five of these rats, randomly chosen, the patency of intrahepatic arterial branches was confirmed
Table 1-Results of Liver Weight Measurement Where Comparisons Between Observed and Predicted Values Are Given with the Percentage of Liver Mass of the Ligated Lobe(s) at Different Time Intervals After PBL Percentage of Level of Ligated lobe(s) weight calculated significance Found Calculated Duration of lobe(s) weight (%) (alpha) (g) (g) experiment Group 24 hours
2.12 + 0.06
83.0 + 2.2
1.76 ± 0.04
0.001 48 hours
2.30 + 0.03
47.8 ± 1.7
1.10 ± 0.02 0.001
72 hours
2.23 :± 0.02
37.2 ± 1.9
0.83 ± 0.03
0.01 96 hours
2.41 + 0.07
29.5 ± 1.7
0.71 + 0.04
0.001 1 week
1
2.55 + 0.09
18.0 ± 0.9
0.46 ± 0.01 0.01
11
2 weeks 24 hours
2.94 + 0.10 3.45 + 0.11
0.33 + 0.02 2.50 ± 0.08
11
48 hours
3.77 + 0.13
2.24 ± 0.09
11
72 hours
11
96 hours
0.01
lI lI
59.4 + 2.5
0.001 3.72 + 0.09
37.6 ± 1.8
1.40 ± 0.05 0.001
3.84 + 0.10
23.2 ± 1.4
0.89 ± 0.04 0.001
11 11
11.2 + 0.4 72.5 + 2.1
1 week
3.85 ± 0.11
15.6
0.60 ± 0.03
+
0.8
0.01 2 weeks 24 hours
4.06 ± 0.13 7.33 ± 0.10
0.43 ± 0.02 5.27 + 0.06
48 hours
7.64 ± 0.12
4.40 ± 0.08
10.6 + 0.5 71.9 ± 1.8 0.001
57.6 ± 2.1 0.001
III
72 hours
7.83 ± 0.11
39.6
3.10 ± 0.04
+
1.7
0.001
III
96 hours
8.15 ± 0.14
28.6 ± 2.0
2.33 ± 0.05 0.001
III
1 week
8.55 ± 0.13
19.2 + 1.4
1.64 + 0.03 0.001
lil ~~~~~~~2 weeks
8.73
0. 14
1. 18 ± 0.03
13.5 +1.0
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The left anterior lobe ligated
The right liver lobes ligated
RL Sham
RL Sham
DL Sham
RL Sham
RL Sham
R}Sham
LRL Sham
LAL Sham
LAL Sham
LAnSham
24h
48h
72h
96h
1w
2w
24h
48h
72h
96h
LA!S.LnAB. han
1w
2w
The two anterior lobes ligated
U
OX
Relative weight of the non-ligated lobe(s) in PBL rats
3,
2' 1 nA V
Relative weight of the ligated lobe(s) in PBL rats
Um 111 .LI
as0 * 5, "1'
Sham'
AL Sham
AL Sham
AL
24h
48h
72h
AL Sham
96h
AL,Sham
1w
AL
Total LW.BW ratio in PBL rats
Relative weight of the sham-ligated lobe(s) in sham-PBL rats
Relative weight of the residual lobe(s) in sham-PBL rats Total LW:BW ratio in sham-PBL rats
Sham
2w
Figure 2-Graphic illustration of changes seen in the weights of ligated and nonligated liver lobes. Note that the total liver weights are maintained at the level of sham-operated controls at each time interval after PBL. (PBL, portal branch litagation; RL, right anterior and right posterior liver lobes; LAL, left anterior lobe; AL, the two anterior liver lobes; LW, liver weight; BW, body weight; W, weeks; h, hours)
on histologic
examination after injection of 20/ Evans Blue solution. All ligated lobes bled after incision. There were no venous collaterals overbridging the portal branch ligature when examination was performed under an operating microscope (25 x). A rapid and progressive atrophy of the ligated lobe(s) occurred. A 50%7 reduction in weight was found after 48 hours, and, by the 14th day, the difference was almost 10-fold. This was found in all groups of PBL rats and therefore it seemed independent of the amount of hepatic tissue devoid of portal blood supply (Table 1). The nonligated lobes were progressively enlarged, so that, by a balance of atrophy in the ligated part and compensatory growth, the total liver weight was maintained at the level of sham-PBL controls at each time interval (Figure 2). During the first 2 days after operation, DNA-SA in ligated lobes was significantly increased in all rats with PBL. The highest values were found at 24 hours. At 48 and 72 hours the DNA-SA was significantly greater than in sham-PBL controls only in rats with 70'70 ligation. The same group showed significantly larger DNA-
SA than did rats from Groups 1 and 2 (24 and 34% ligation), but only at 24 hours (Figure 3). At 1 week, the DNA-SA in the ligated lobes of all PBL rats was at the level of sham-operated controls. On autoradiography, no hepatic nuclei were found to be labeled, and, in the overall microscopic fields, only a few hepatocytes seemed to show karyomitosis. Thus, the labeling and mitotic count have not been made. Signs of compensatory growth, expressed by increased DNA-SA, labeled nuclei, and increased mitotic count, were found in all rats that underwent PBL in nonligated lobes (Figures 3, 4, and 5). The highest values of DNA-SA were found in rats with 70Ve ligation (Group 3), with the peak response at 24 hours. In these rats, an increased DNA-SA persisted at least until the termination of the experiment (2 weeks). Groups 1 and 2 rats showed significantly lower values of DNA-SA when compared with Group 3 rats during 4 days after portal occlusion. The mean values in Group 2 were larger than those of Group 1 rats; however, the differences did not reach the level of statistical significance. The peak responses were also observed at
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dpm/min/pg DNA
PBL to 1/4 of liver mass * PBL to 1/3 of liver mass I PBL to 2/3 of liver mass I Sham-PBL /
500
300 200 100 0 NS
NS
'CO.001 0.001
50
MIS
(-0.
001
NS
(0o.007
"o.oo
NS
not^s; 4g croses may be partly explained by the fact that$he concentration of oxygen in cells in this acinar zone is un,. a. . A .,. , der critically low physiologic conditions, which results , ~ * >;,@ .~ .- -,% in an increased susceptibility to hypoxia2l22 and toxic zSs ' :f:m1< V,* N 's *' *; ,t*,.t,,,*{;,s,agents.23 It is of interest that occlusion of portal inflow 2 o tg2 ''¢ttA to 70% of liver parenchyma is not followed by devel~t ..Q .,~ ~opment of collaterals overbridging the portal venous 4*,w ,, - *g X2S8,4,,pos,';§.;i _ ligature, but that they do appear after partial constriction of the main portal trunk.24 This suggests that the stimulus to collateralization is not only a function of '2. : ' , 4 ^ -. , , , , ; 2 - ~ pressure gradient, as it is usually regarded, and that nonFigure 8-Portal occlusion of the anterior lobes, 72 hours. Rest of cenl l ligated lobes may contribute to decompression of vetrilobular necrosis with macrophages. Portal zone to the left. (H&E, original magnification x 150) nous stasis by opening centrally situated pathways. The latter suggestion is in agreement with early suggestions by Daniel and Prichard.25 early reports of Weinbren, necroses were not described,2 but in 1964 the same laboratory reported their presence in rats sacrificed 48 hours after PBL'6 Recently, similar lesions were described in rats with one-stage size. P-. .. able constriction of the portal trunk,17 and large cen-'a. trizonal infarcts were regularly seen in auxiliary liver transplants when portal anastomosis became throm' bosed.9 Thus, in animals the pathomorphologic con4. * sequences of local portal occlusion are actually more .. A f dramatic than is generally assumed. In human beings, the early histologic changes after , ' portal branch occlusion are not known, although the z; % .,;; late macroscopic and microscopic features closely re,i' -' i semble those seen in animals.11 Segmental or lobar at- ' rophy of the liver that results from vascular or biliary '/ obstruction are important because they may create clinical and operative diagnostic problems.'8 The present study fully confirms the description given by Steiner and Martinez.8 In particular, the presence of early necrosis and its localization, distribution, and subsequent resolution are almost identical. The only remarkable difference includes the presence of contracted arterial branches in many of our sections obt 4 .iI . 4'. B tained after 72 hours. t * This finding is in agreement with observations of Ackerman et al, who found the arterial inflow to the hepatic tumors to be reduced after PBL,'9 and with those l of Daniel and Prichard, who described reflex vasocon7 striction after ligation of the portal branches.20 ; ; , * ", Thus, the absence of venous collaterals and the evi', ,, _ dence of reduced arterial inflow to the ligated lobe (s) 4 _ . (contraction of arterial branches) indicate that local Figures 9A and B-Portal occlusion, 2 weeks. Atrophy with portal tracts and central veins close to each other. In B, the fibrosis around the portal portal occlusion is not compensated by the hepatic arvein and the thickened hepatic artery and the low bile duct epithelium are tery, a phenomenon well documented after total porx 60; B, H&E; original magdemonstrated. (A, H&E original magnification, tal-systemic shunting. The possible role of inlet and nification, x 150) * s. i ,
3
I
A
V
-t
Vol. 125 * No. 2
Figure 10-Portal occlusion, 48 hours, nonligated lobes. Numerous mitoses. (H&E; original magnification, x 150)
In this study, the extent of necrosis was found to be somewhat smaller in rats with one lobe ligated compared with those with ligation of a branch supplying the two anterior lobes. We therefore do not support the statement by Weinbren et al,26 that posterior lobes are more resistent to ischemia because they are more solid and attached to the inferior vena cava. The final estimated loss of parenchyma was found to be similar in all PBL rats. The most rapid atrophy occurred during the first few days after operation. The atrophy was counterbalanced by restorative growth of the nonligated segments at each time interval, so that the total liver weight was maintained at the level of sham-PBL controls throughout the experiment. We did not observe a hyperplastic response (mitoses) in the portal-deprived tissue, which is in disagreement with data provided by Weinbren et al.27 However, we found an increased DNA synthesis during the first 3 postoperative days, which might be related at least in part to the activation of reticuloendothelial cells engaged in resorption of necroses. It seems that an increased DNA synthesis not followed by mitotic division may reflect a defensive response, and its presence in the lobes undergoing atrophy suggests, that these two phenomena are controlled by two different mechanisms, presumably humoral in nature. The DNA synthesis and nuclear labeling have not previously been measured in animals with portal branch ligation. In this study, the maximal values were observed in rats with 700o ligation after 24 hours, with a rapid reduction during the 3 subsequent days, but after 1 and 2 weeks there was still an elevated response. In rats with 24 and 34%o of the liver ligated, the synthesis of DNA, labeling index, and mitotic activity are lower, although the pattern of changes resembles that seen in rats with 70%0 ligation.
PORTAL BRANCH LIGATION
307
When Rous and Larimorel discussed their experiments with portai ligation in 1920, they were intrigued by the possibility that the hyperplastic response in the unmanipulated lobes was stimulated by humoral modulators present in the portal blood. After 65 years, it seems that PBL still constitutes the most simple animal preparation in favor of the hepatotrophic concept. Ligation of a branch of the portal vein in all likelihood results in similar hemodynamic alterations as excision of the same liver mass. A rapid rise in portal venous pressure and hyperperfusion of the remaining lobes may take place and constitute a primary determinant of growth response. It has been shown, however, by Rabinovici and Wiener,28 that from 12 hours after two-thirds hepatectomy the portal pressure is elevated only to a small extent, whereas the vascular space and hepatic blood flow per 1 g of liver tissue and per 100 g of body weight are below normal values. The absence of retrograde stasis together with decreased hepatic blood flow suggested that a significant amount of portal blood was shunted away from the liver via the preexisting portasystemic collaterals. Our previous studies with PBL in rats with different portacaval shunts also support the concept that the quality and not the quantity of portal blood is of importance.29 The PBL model does not support the hypothesis that the initiating factor in liver regeneration is the loss of an inhibitor,30 although several authors have described such substances.31'32 This might be true if excision of 70'70 of liver mass takes place, but not after portal occlusion when the total liver mass remains almost unchanged during the prereplicative period of reparative growth. Local portal occlusion induces a hyperplastic response as vigorous as excision of the corresponding liver mass.16 In parallel experiments, we have demonstrated that, if PBL rats are subjected to selective portasystemic shunting, the atrophy and necrosis in the ligated lobes are significantly inhibited.33 Thus, factors, in the portal blood exert hepatoprotective effect during hepatic arterial recirculation. In light of these observations, it seems that in rats with PBL alone the early increase in DNA synthesis in the ligated lobes may also reflect the influence of portal-borne hepatotrophic substances available via the hepatic artery. The atrophy of the portal-deprived tissue may be caused not only by ischemia, but also by a deficit of liver-supporting factors. This is supported by observations of Rabinovici and Vardi,34 who found that part of the arterial blood is shunted into the portal venous system before the sinusoid level, through hepatic artery-portal vein communications. This indicates that the contribution of both arterial and portal venous blood to the perfusion of the liver sinusoids is impor-
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ROZGA ET AL
tant. Consequently, it is difficult to explain the lack of atrophy and the normal regenerative capacity of the liver after hepatic artey ligation when compared with
AJP
20.
portal ischemia.35
References 1. Rous P, Larimore LD: Relation of the portal blood to liver maintenance: A demonstration of liver atrophy conditional on compensation. J Exp Med 1920, 31:609-632 2. Weinbren K: The portal blood supply and regeneration of the rat liver. Br J Exp Pathol 1955, 36:585-591 3. De Weese MS, Lewis C, Arbor A: Partial hepatectomy in the dog: An experimental study. Surgery 1951, 30: 642-651 4. Schalm L, Bax HR, Mansens BJ: Atrophy of the liver after occlusion of the bile ducts or portal vein and compensatory hypertrophy of the unoccluded portion and its clinical importance. Gastroenterology 1956, 31:131-155 5. Ehrhardt 0: Ueber die Folgen der Unterbindung grosser Gefasstamme in der Leber. Arch Klin Chir 1902, 68: 460-467 6. Milne LS: The histology of liver tissue regeneration. J
Pathol Bacteriol 1980-1909, 13:127-160 7. McMichael J: The oxygen supply of the liver. Q J Exp Physiol 1937-1938, 27:73-87 8. Steiner PE, Martinez JB: Effects on the rat liver of bile duct, portal vein and hepatic artery ligations. Am J Pathol 1961, 39:257-289 9. Walsh T, Eggelston JC, Cameron JL: Portal hypertension, hepatic infarction, and liver failure complicating pancreatic islet autotransplantation. Surgery 1982, 91:485-487 10. Benz EJ, Baggenstoss AH, Wollaeger EE: Atrophy of the left lobe of the liver. Arch Pathol 1952, 53:315-330 11. Price JB, Voorhees AB, Britton RC: The role of portal blood in regeneration and function of completely revascularized partial hepatic autografts. Surgery 1967, 62:195-203 12. Scott JF, Fraccastoro A, Taft EB: Studies in histochemistry: I. Determination of nucleic acids in microgram amounts of tissue. J Histochem Cytochem 1956, 4:1-10 13. Loeffler L: Factors determining necrosis or survival of liver tissue after ligation of hepatic artery. Arch Pathol 1936, 21:496-503 14. Lawrence W, Joly D, Brasfield R: A comparative study of various mechanisms of hepatic restoration in the rat. Surgery 1959, 45:543-551 15. Krauss GE, Beltran A: Effect of induced infarction on rat liver implanted with Walker carcinoma 256. Arch Surg 1959, 79:769-774 16. Weinbren K, Tarsh E: The mitotic response in the rat liver after different regenerative stimuli. Br J Exp Pathol 1964, 45:475-480 17. Myking AO, Halvorsen JF: Two-stage occlusion of the portaf vein in the rat: Survival related to weight variation and the interval between partial and total occlusion. Eur Surg Res 1975, 7:336-374 18. Ham JM: Segmental and lobar atrophy of the liver. Surg Gynecol Obstet 1974, 139:840-844 19. Ackerman NB, Lien WM, Silverman NA: The blood sup-
21. 22.
23. 24.
25.
26. 27. 28.
29.
*
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ply of experimental liver metastases: III. The effects of acute ligation of the hepatic artery or portal vein. Surgery 1972, 71:636-643 Daniel PM, Prichard NML: Effects of stimulation of the hepatic nerves and of adrenaline upon the circulation of portal venous blood within the liver. J Physiol 1951, 114:138-145 Rappaport AM, Hiraki GY: The anatomical pattern of lesions in liver. Acta Anat 1958, 32:126:140 Sies H: Oxygen gradients during hypoxic steady states in liver: Urate oxidase and cytochrome oxidase as intracellular 02 indicators. Hoppe-Seylers Z Physiol Chem 1977, 358:1021-1032 Gumucio JJ, Miller DL: Functional implications of liver cell heterogeneity. Gastroenterology 1981, 80:393-403 Rozga J, Jeppsson B, Hagerstrand I, Bengmark S: Acute portal vein stenosis: An experimental study on portal circulation and hepatosplenic function. Acta Chir Scand 1985, 151:125-131 Daniel PM, Prichard NML: Variations in the circulation of the portal venous blood within the liver. J Physiol 1951, 114:521-537 Weinbren K, Washington SLA, Smith CY: The response of the rat liver to alterations in total portal blood flow. Br J Exp Pathol 1975, 56:148-156 Weinbren K, Stirling GA, Washington SLA: The development of a proliferative response in liver parenchyma deprived of portal blood flow. Br J Exp Pathol 1972, 53:54-58 Rabinovici N, Wiener E: Hemodynamic changes in the hepatectomized liver of the rat and their relationship to regeneration. J Surg Res 1963, 3:3-8 Rozga J, Jeppsson B, Bengmark S: The effect of pancreatic and intestinal venous blood on hepatic atrophy and compensatory hyperplasia in the rat. (Manuscript
submitted)
30. Glinos AD: The mechanism of liver growth and regeneration, The Chemical Basis of Development. Edited by WD Mc Elroy, B Glass. Baltimore, Johns Hopkin's Press, 1958, p 813 31. Verly WG, Deschamps Y, Pushpathadan J, Desrosiers M: The hepatic chalone: I. Assay method for the hormone and purification of the rabbit liver chalone. Can J Biochem 1971, 49:1376-1383 32. Sekas G, Cook RT: The isolation of a low molecular weight inhibitor of (3H)TdR incorporation into hepatic DNA. Exp Cell Res 1976, 102:422-425 33. Rozga J, Jeppsson B, Bengmark S: Hepatotrophic factors in liver growth and atrophy. Br J Exp Pathol (In press) 34. Rabinovici N, Vardi J: The intrahepatic portal veinhepatic artery relationship. Surg Gynecol Obstet 1965, 120:38-44 35. Mizumoto R, Wexler M, Slapak M, Kojima Y, McDermott WV Jr: The effect of hepatic artery inflow on regeneration, hypertrophy and a portal pressure of the liver following 50%7o hepatectomy in the dog. Br J Surg 1970, 57:513-517
Acknowledgment Histologic examination and photography were performed by Inga Hagerstrand, MD, Department of Pathology, Lund University, Lund, Sweden.
