1 Laboratory of Experimental Surgery and 2 Diabetes and Nutrition Unit, University of Louvain Medical School, B-1200 Brussels, Belgium
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ABSTRACT |
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To examine the role of the early changes occurring in the liver within the first hours after a partial hepatectomy and in an attempt to demonstrate the involvement of subsequent regulatory mechanisms, the size of the remnant liver was modified at various times and by different surgical techniques. Male Wistar rats were submitted to a two-thirds "temporary partial hepatectomy" produced by a 3-h occlusion of the pedicle of the anterior lobes protected by local hypothermia. Various indexes of cell proliferation ([3H]thymidine uptake and 5-bromo-2'-deoxyuridine and proliferating cell nuclear antigen labeling) were not increased despite a c-myc expression as high as that observed after a two-thirds partial hepatectomy. The temporary partial hepatectomy and a sham operation induced modifications of the hepatocytes, allowing rapid DNA synthesis after a subsequent two-thirds partial hepatectomy. After this initial nonspecific response, the extent of the regenerative response is determined according to the size of the liver mass present approximately from the 10th to the 18th hour after the initial stimulus. For instance, when a one-third partial hepatectomy was converted into a two-thirds partial hepatectomy at the 10th hour, the DNA synthesis at the 24th hour reached the value observed after a straightforward two-thirds partial hepatectomy. Inversely, the regenerative response was significantly reduced when additional liver lobes were connected to neck vessels between the 14th and the 18th hour after a two-thirds partial hepatectomy. In conclusion, the actual liver mass present during the period corresponding to mid- to late G1 appears to control the magnitude of the proliferative response, which is not the simple consequence of the early changes following a partial hepatectomy.
liver regeneration; c-myc; DNA synthesis
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INTRODUCTION |
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THE HEPATIC REGENERATION following a two-thirds partial
hepatectomy (PH) in the rat is a well-defined phenomenon that starts within a few minutes and leads, after a "latent phase" of ~16 h, to an increased synthesis of DNA followed by a first wave of mitosis. The mechanisms initiating and controlling this regenerative process, which restores quite precisely the original liver mass, remain
poorly understood despite numerous studies performed in vivo and in
vitro (see Refs. 12, 28, and 29 for reviews). In recent years, a great
deal of emphasis has been placed on events occurring very rapidly after
PH. Within a short time period, the hepatocytes should be modified
(primed) by a reduction of the liver mass to become more responsive to
various growth factors produced at a higher rate or, at least, present
at a greater concentration (12, 25, 28). Various modifications have
indeed been observed quite early in the remaining lobes. For instance,
a rise of the hepatocyte membrane potential has been detected as soon
as 20 min after PH (11). The increased expression of the protooncogenes c-jun,
c-fos, and
c-myc during the first hours (36), as
well as the rapid DNA binding of nuclear factor B (10, 14), lends weight to the hypothesis that the proliferative response not only starts but is determined during this early phase. The role of positive
feedback systems, such as the secondary production within the liver of
growth factors amplifying initial stimuli, is well established (27,
43), whereas until now, negative controls have been mainly implicated
in the cessation of proliferation after a few days.
In this study, we have attempted to demonstrate in vivo the existence and time of occurrence of various control mechanisms and their possible dependence on the reduction of the liver mass. To address these questions, the size of the remnant liver mass was modified by different surgical techniques during the first 24 h after a one-third or a two-thirds PH. We initially developed a model of "temporary partial hepatectomy" (TPH) in which the liver lobes normally resected in a two-thirds PH [left lateral and median lobes, from now on designated as anterior lobes (AL)] were simply excluded from the circulation by occluding their vascular pedicle and kept in hypothermia for 3 h. Removing the vascular clamp reverses the PH, allowing investigation of the effect of the initial stimuli in the presence of a normal liver mass at a later time (35). A regenerative response evaluated by [3H]thymidine uptake (TdrU) or 5-bromo-2'-deoxyuridine (BrdU) or proliferating cell nuclear antigen (PCNA) labeling did not appear despite an increased expression of the c-myc protooncogene. To examine a possible persistence of modifications induced by TPH, the AL were resected after being reperfused for various periods of time. The kinetics of DNA synthesis following this resection were compared with the response to a simple two-thirds PH or to a two-thirds PH preceded by a sham operation, a procedure that is known to accelerate this response (30, 34). The progressive nature of the change induced by a previous stimulus was also examined by varying the interval between sham operation and PH. The results obtained suggested that the initial modifications of the hepatocytes after PH are quite independent of the reduction of the liver mass and thus that the magnitude of the regenerative response should be determined at a later time. To verify this hypothesis, we modified the liver mass present between the 10th and the 24th hour after an initial PH. A secondary reduction of the liver mass was obtained by converting a one-third PH into a two-thirds PH, 10 or 14 h after the original operation. To increase the liver mass, posterior lobes taken from an intact rat were placed as an auxiliary graft on the neck vessels 14 or 18 h after a twothirds PH.
At the end of this work, the following model is proposed: PH initially induces nonspecific changes that prepare the hepatocytes to proliferate; however, these changes, which are not related to the reduction of the liver mass, are not sufficient to secure a regenerative response. In a second stage, the extent of the proliferative response is determined by factors sensitive to the amount of liver tissue present and acting apparently at restriction points in mid- and late G1.
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MATERIALS AND METHODS |
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Male Wistar rats (weight 220-240 g), obtained from the University breeding facilities, were maintained under constant room temperature, with a lighting schedule from 0700 to 1900 and with free access to standard food and water. PH, sham operation, or TPH were all performed in the morning. All animals received humane care in compliance with the University of Louvain regulations.
The following experiments summarized in Table 1 were performed.
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TPH
Animals were divided into seven groups.Group 1: TPH. Male Wistar rats were anesthetized with Thalamonal (1.6 ml/kg; Janssens Pharmaceutica, Beerse, Belgium). After a median laparotomy, the hepatic pedicle was occluded "en bloc" with a vascular clamp above the posterior lobes, as previously described (2). The median and left lateral lobes (AL), resected in the classic Higgins-Anderson two-thirds hepatectomy (17), were thus devascularized, while the posterior lobes remained perfused. The wound was partially closed with the devascularized lobes exteriorized and immersed in a circulating bath filled with 0.9% NaCl solution at 8°C (Fig. 1). The body temperature of the rat was maintained at 37°C with heating lamps. After 3 h, the clamp was removed and the AL were replaced in the abdomen (n = 11).
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Group 2. The same protocol as in group 1 was used, but after the 3-h cold ischemia the devascularized lobes were resected, converting the TPH into a standard two-thirds PH (n = 6).
Group 3: Sham operation. The same anesthesia, median laparotomy, and lobe handling as in groups 1 and 2 were used, but without vascular exclusion (n = 13).
Group 4. A classic two-thirds PH was performed to act as the control group (n = 11).
Three animals from groups 1, 3, and 4 were killed 2 h after the start of the procedure to measure c-myc expression. A further three were killed at 24 h, 1 h after intraperitoneal administration of BrdU (Sigma Chemical, St. Louis, MO).
All other animals were killed under ether anesthesia 24 h after the start of the procedure, 1 h after administration of 50 µCi of [6-3H]thymidine (Amersham, Buckinghamshire, UK) into the femoral vein.
In addition, the following experiments were also performed.
Group 5. The pedicle of the AL was ligated, and the lobes were left in situ (n = 4).
Group 6. The procedure was identical to that for group 1, but the [3H]thymidine was injected at 26 h and rats were killed 1 h later (n = 6).
Group 7. Again, the procedure was identical to that for group 1, but at the end of TPH, the posterior lobes were removed. Survival of the animals was therefore dependent on the AL recovering from the cold ischemic challenge (n = 5).
Effect of a Preceding Operation on the Peak of DNA Synthesis
In a first series of experiments, a TPH or sham operation was performed as described above. Six, ten, or fourteen hours after the start of either TPH or sham operation, the rats were reanesthetized with ether and a classic two-thirds PH was performed. The posterior lobes were removed 24 h after the first operation, 1 h after [3H]thymidine administration (six groups of at least five rats).In a second series of experiments, to study the influence of the interval between sham operation and PH, sham operations were followed 6, 10, 14, or 24 h later by a classic PH. The animals were killed 14, 18, 24, or 30 h after this second operation, 1 h after [3H]thymidine administration.
Conversion of a One-Third PH into a Two-Thirds PH
A one-third PH was initially performed by excising the left lateral lobe while the rats were under ether anesthesia. Ten or fourteen hours later, again while the rats were under ether anesthesia, the laparotomy was reopened and the median lobe excised, converting the one-third PH into a two-thirds PH. In control animals, the median lobe was simply manipulated as a sham operation. Finally, the posterior lobes were removed 14, 18, or 24 h after the second operation (conversion), 1 h after [3H]thymidine administration (n = at least 5 for each time point).Auxiliary Liver Graft
Male Wistar rats, weighing ~200-300 g for donors and 350-450 g for recipients, were used. The donor and recipient animals were prepared simultaneously to reduce the ischemic time of the organ. The technique of grafting, as well as the evaluation of the function of the grafted liver, has been described in detail (4).Donor preparation. The donor was anesthetized with ether, and the liver was removed, including portal vein and hepatic artery, with an abdominal aortic patch. The infrahepatic vena cava was ligated, and the supradiaphragmatic inferior vena cava was prepared. A two-thirds PH was performed ex vivo, removing the left lateral and median lobes.
Recipient
preparation. Anesthesia was initiated
with ether and maintained with intravenous propofol (0.05-0.5
mg · kg1 · h
1)
(Diprivan; Zeneca, Destelbergen, Belgium) administered by a pump
(Perfusor IV; Braun, Melsungen, Germany) through a catheter inserted
into the femoral vein. The right external jugular vein and the right
common carotid artery were dissected. The reduced liver graft was
placed upside down on the cervical region of the recipient. The
suprahepatic inferior vena cava was anastomosed to the proximal
right external jugular vein, the portal vein to the distal right
external jugular vein, and the donor aorta to the common carotid
artery, with the use of the cuff technique. Liver reperfusion was
rapid, with recovery of normal color taking 10-20 s and bile flow
3-5 min. The recipient rat remained sedated with propofol, and the
skin was left open. After a predetermined time lapse, the auxiliary
liver was removed, the vessels were ligated, the skin was closed, and
the anesthesia was discontinued.
The heart transplants were performed in a similar manner, according to the method of Heron (16), with the distal part of the jugular vein being ligated.
The experiments were divided into five groups of six rats each. A two-thirds PH was performed in all animals, and the posterior liver lobes were resected 24 h later, 1 h after a [3H]thymidine injection.
Group 1: Control. A two-thirds PH was performed in rats of the same age and weight (350-450 g) as in groups 2-5.
Group 2. Posterior liver lobes were transplanted onto the neck vessels 14 h after a two-thirds PH and remained connected for 4 h.
Group 3. A heart was transplanted with the same protocol as in group 2.
Group 4. The same protocol as in group 2 was used, but the liver graft was transplanted after 18 h and left in situ for 5 h.
Group 5. A heart was transplanted with the same protocol as in group 4.
Analytical Methods
Cell proliferation. In the majority of the experiments, DNA synthesis was evaluated by the incorporation of [3H]thymidine into the DNA. The posterior liver lobes were snap frozen in liquid nitrogen and kept atCell proliferation was also evaluated by immunocytochemical determination of BrdU or PCNA/cyclin labeling. Twelve serial sections of the liver were cut for each animal studied. Four sections were stained with hemotoxylin, and the total hepatocyte number was evaluated from five fields per section. Four other sections were used for the determination of BrdU nuclei labeling (23), with a monoclonal anti-BrdU antibody (Amersham). This was counterstained with an immunoperoxidase system, using the streptavidin-biotin complex (Boehringer Mannheim, Mannheim, Germany). Four sections were used for the determination of the PCNA labeling index (15). The monoclonal antibody 19A2 anti-PCNA/cyclin (Coulter Electronics, Luton, UK) was detected by the same immunoperoxidase system. The results are expressed as a BrdU or PCNA/cyclin labeling index, which corresponds to the ratio between the positive nuclei and the total hepatocyte population.
Thymidine kinase (TK) activity was evaluated by measuring the amount of [3H]thymidine phosphate produced by a liver homogenate according to the method described by Kahn et al. (19). The results are expressed as dpm per microgram protein per minute.
RNA extraction and Northern blot analysis. RNA was extracted from liver tissue using the guanidine thiocyanate and cesium chloride method (8). Twenty micrograms of RNA were size-fractionated on 1% agarose gels containing 1% formaldehyde and transferred to Hybond-N filters (Amersham). After ultraviolet fixation, filters were hybridized with the c-myc 32P-labeled cDNA (39). The c-myc cDNA probe is a 1.8-kB EcoR I-Cla I human c-myc exon 3 fragment (Oncor, Gaithersburg, MD). The results are expressed in arbitrary densitometric units.
Statistical analysis. Student's t-test or, for multiple group comparisons, one-way analysis of variance was used. A P value of <0.05 was considered significant.
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RESULTS |
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TPH
After TPH, DNA synthesis, as estimated by TdrU at 24 h, was the same as after a sham operation (11.6 ± 1.1 vs. 9.7 ± 2.0 dpm/µg DNA; means ± SE, n = 5 and 7, respectively). This was markedly lower than after a classic two-thirds PH (317.8 ± 42.9 dpm/µg DNA; n = 5; P < 0.001) or than after a TPH immediately followed by a PH (205 ± 17.2 dpm/µg DNA; n = 6; P < 0.001) (Table 2).
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Similar results were obtained when cell proliferation was estimated by BrdU or PCNA labeling index. Again, no proliferative response was found after TPH or sham operation, compared with a significant response after PH (Table 2). On the other hand, the expression of c-myc at the second hour increased from a control value of 117 ± 35 arbitrary densitometric units to a value of 189 ± 42 arbitrary densitometric units after sham operation [not significant (NS)] and to values of 440 ± 70 and 509 ± 96 arbitrary densitometric units following a TPH and a PH, respectively (P < 0.05 vs. control and sham operation groups) (Fig. 2).
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The TdrU, 27 h after TPH, was not higher than at 24 h, indicating that TdrU was not simply delayed. In group 5, in which the AL remained permanently excluded, TdrU in the posterior lobes did not differ from that obtained after excision of the AL. The resection of the posterior lobes at the end of a TPH caused no mortality, but, in the absence of local cooling of the AL and when their occlusion exceeded 120 min, the mortality rate reached 93% (2).
Influence of a Preceding TPH or Sham Operation on DNA Synthesis
The reconnection of the AL for 3 h after a TPH (resection at the 6th h) did not decrease the TdrU measured at 24 h. If the AL were reperfused during 7 h and resected at the 10th hour, this TdrU was decreased, and the TrdU was nearly suppressed when the lobes remained present until the 14th hour (Fig. 3, lower time scale).
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If one considers the response following the definitive PH (upper time scale on Fig. 3), it can be noticed that the peak of TdrU already occurred at the 18th hour instead of the 24th hour after a simple PH. At the 14th hour, the DNA synthesis was significantly greater than after the simple PH, but a sham operation produced the same acceleration as a TPH.
Varying the time interval between a sham operation and a subsequent PH
was found to alter the timing of TdrU following this latter operation
(Fig. 4). As already mentioned, if the
interval was 6 h, the peak uptake, equivalent to the one obtained at 24 h without a preceding stimulus, was already obtained at the 18th hour.
As another indication of the accelerated response, the TK activity
measured at 18 h was twice the value obtained at that time after a
simple two-thirds PH: 8.82 ± 1.02 and 4.19 ± 1.26 dpm · µg
protein1 · min
1,
respectively (P < 0.05).
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The acceleration seemed to reach a maximal value when the interval
between the sham operation and the PH was 10 h. The TdrU measured at
the 14th hour was greater than after the reference two-thirds PH but
also significantly greater than after a PH following a sham operation
by 6 or 14 h. When a sham operation preceded the PH by 24 h, the
initial response was on the contrary markedly delayed or depressed; for
instance, at the 24th hour, the TdrU reached only one-half the value
observed after a simple PH, whereas the values at 18 and 30 h were not
increased. This reduced response was also shown by a lower rise in TK
activity at 24 h (8.71 ± 1.21 and 14.88 ± 2.02 dpm · µg
protein1 · min
1,
respectively; P < 0.05) and at 30 h
(6.6 ± 1.7 and 21.02 ± 2.8 dpm · µg
protein
1 · min
1,
respectively; P < 0.01).
Conversion of a One-Third PH into a Two-Thirds PH
When a one-third PH (removal of the left lateral lobe) was converted into a two-thirds PH at 10 h (by the resection of the median lobe), TdrU measured 20 h after the initial operation was similar to the values observed at the same time after an unmodified one-third PH. However, at the 24th hour, TdrU had increased to reach a peak value equivalent to the one obtained at that time with a straightforward two-thirds PH (Fig. 5). In contrast, a sham operation performed at 10 h had no effect on the TdrU curve (data not shown). When conversion was delayed until the 14th hour, the response was much slower and the TdrU peak was not reached before the 32nd hour.
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Late Restoration of Liver Mass by an Auxiliary Graft
The restoration of liver mass using an auxiliary graft connected to rats from the 14th to the 18th hour after a two-thirds PH decreased TdrU to 16 ± 8% of the value obtained in controls (P < 0.001), whereas a heart graft did not modify the response (123 ± 47%, NS) (Fig. 6). This reduction of TdrU was not seen if the auxiliary liver was present only later, between the 18th and the 23rd hours. The TdrU values were 109 ± 30% and 70 ± 13% of the control group, for the auxiliary liver and heart groups, respectively.
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DISCUSSION |
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The experiments reported here demonstrate that it is possible to alter the regenerative response of rat liver to PH by modifying at various times the amount of liver tissue present or by superimposing the effect of a preceding surgical stress. The results obtained suggest that the importance and specificity of the early changes following PH should be reevaluated. They also indicate that control mechanisms influence the regenerative response at different moments within the first 24 h after liver resection.
The regenerative response was mainly estimated by the incorporation of 3H-labeled thymidine into DNA, which allows easy quantification and comparison. Because the experiments were limited to the first 24 h after a PH, this method essentially evaluates the hepatocyte response, with other cells starting the DNA synthesis at a later time. In agreement with other studies (7, 34), the first peak of TdrU was observed ~24 h after a one-third or a two-thirds PH. In some experiments, results obtained with [3H]thymidine have been confirmed by BrdU labeling as well as by an independent method, the measurement of PCNA (15, 41). The TK activity was also measured in an attempt to detect changes that probably precede and prepare the DNA synthesis, although it appeared that the peak of TK activity is reached after the peak of TdrU (13).
TPH: the Early Response to a PH
The first part of this work was aimed at evaluating the importance and specificity of the changes occurring within the first 3 h after PH. To determine whether the early changes by themselves can induce the whole regenerative response, we developed a new model, the TPH. This model involves clamping of the vascular pedicle of the anterior (left lateral and median) lobes for a period of 3 h, followed by reperfusion. With this procedure, the remaining liver lobes, as well as the entire animal, are placed in the same situation as during the first 3 h after a classic PH. However, the regenerative response evaluated by the TdrU or the BrdU and PCNA labeling indexes at 24 h was not greater than after a sham operation.The inhibition of the DNA synthesis, in comparison to a classic PH, cannot be explained by a toxic effect caused by the presence of the recovering ischemic lobes, because we and Lawrence et al. (24) have found that the regenerative response following the excision or devascularization of the AL is identical. On the other hand, the marked proliferative response obtained when the temporarily excluded lobes were resected at the end or 3 h after the end of the cold ischemic period clearly demonstrates that the absence of response after TPH is not due to the stress imposed by the procedure. Measurement of TdrU later than 24 h after TPH also indicates that the response is not simply delayed.
The absence of proliferation after TPH occurs despite an initial response that can be assumed to be identical to the one elicited after a two-thirds PH. Indeed, we observed the same increase in membrane potential after PH and reversible vascular occlusion (data not shown). We also found that the rise in c-myc expression in the posterior lobes after 2 h was similar in permanently or temporarily hepatectomized rats, and greater in these two groups than after a sham operation. The rise in c-myc expression, considered a good marker of the priming state, has been obtained after a two-thirds PH but not after a sham operation (26). A similar increase in protooncogene expression, without ensuing DNA synthesis, has been reported after dietary manipulation or surgical operation performed on another organ, namely the kidney (3, 18, 26). It could be argued that the changes produced by such manipulations do not include a specific response needed to initiate liver regeneration. However, in the TPH model, the stimulus, a reduction in liver mass, is identical to the stimulus produced by PH during the first 3 h. Therefore, the absence of cell proliferation has to be related to some control mechanisms operative at a later time. It is clearly demonstrated that the early changes are not sufficient to sustain a full regenerative response.
The TPH nevertheless leaves some persisting modifications of the hepatocytes. They are quite similar to those produced by a sham operation, namely a laparotomy of the same size with dissection of the hepatic pedicle. Both TPH and sham operation do not markedly stimulate DNA synthesis but accelerate it when produced by a subsequent PH or, for the TPH, by its equivalent, the secondary removal of the excluded lobes after a few hours of reperfusion (30, 34). An earlier rise in TK activity, as well as TdrU, was indeed obtained. This effect of a preceding sham operation was described years ago by Moolten et al. (30) as the induction of a "state of preparation" of the hepatocytes. By varying the interval between the initial stimulus and the PH, we demonstrated that this state of preparation progressively reaches a maximal value within 10 h. For instance, the TdrU measured 14 h after a two-thirds PH, barely detectable in absence of a preceding stimulus, increases slightly when a sham operation is performed 6 h before the PH and markedly when the interval is extended to 10 h but decreases if the interval becomes larger. This state of preparation appears to be followed by a refractory state: when the sham operation precedes the PH by 24 h, both the TdrU at 24 h and the TK activity at 24 h and 30 h are significantly reduced. This inhibition, the nature of which remains unknown, has not been reported previously, to our knowledge, and should be kept in mind when interpreting the effect of a manipulation performed on an animal during the day preceding a PH (22).
There are some differences between the effect of a sham operation and
that of a PH. For instance, only PH increases the expression of
c-myc or the sensitivity of
hepatocytes to hepatocyte growth factor (HGF) administration (21, 40).
It can nevertheless be considered that even after PH, the initial
changes that prepare the hepatocytes to proliferate are not related to
the reduction of the liver mass and thus can be considered as part of a
nonspecific response. This interpretation is supported by the recent
demonstration of the importance of the two proinflammatory cytokines
interleukin-6 and tumor necrosis factor (TNF)-, also responsible for
the acute-phase response, in the initiation of liver regeneration (1,
9, 42). Some of their effects, namely the activation of
STAT3, can be observed within the first 2 h after PH; they
should thus be present after TPH and perhaps also, in a limited manner,
after sham surgery (9). Although these initial changes are needed to
induce a proliferative competence, they seem unable to produce a full
regenerative response.
The Regulation of the Magnitude of the Response in Mid- and Late G1
As a consequence of these findings, it can be proposed that the role of the liver mass reduction only becomes critical after the 6th to 10th hour. This period corresponds to mid-G1 for the cell taking part in the first round of division. To demonstrate the existence of control mechanisms depending on the liver mass, one-third PH have been transformed into two-thirds PH, 10 h after the initial operation. The peak value of TdrU is obtained at the 24th hour and is equivalent to the one observed after a classic two-thirds PH. To explain how this level of DNA synthesis was obtained in such a short time, 14 h, instead of the usual 20-24 h, it was postulated that an amount of cells in excess of that needed to achieve an adequate response to a one-third PH has already reached a state of preparation allowing them to synthesize DNA rapidly. We therefore propose that the number of cells allowed to progress to S phase and thereafter to cell division is determined around the 10th hour, according to the liver mass present at that time. The response can be minimal or absent after a sham operation or a TPH, moderate after a one-third PH, and maximal after a two-thirds PH. The responsible control mechanism could be either internal (a self-extinguishing reaction in the absence of further stimuli) or external, due to inhibitory factors such as transforming growth factor (TGF)-A second phase in the process leading to cell proliferation thus seems to start around the 10th hour. At that time, various mediators are still able to influence the regenerative response; for instance, the administration of prostaglandin I2 at the 8th hour suppresses the inhibition produced by indomethacin (5). The present experiments suggest that the second phase of liver regeneration is not simply the continuation of the initial process but a response to other stimuli more directly related to the reduction of the liver mass; the progressive development of "metabolic overload" could be one of them (31).
A late control mechanism is suggested by the experiments in which the
functional liver mass present after a two-thirds PH was increased by
grafting to the cervical vessels the posterior hepatic lobes removed
from another rat. A reduction of the regenerative response was clearly
seen when this graft was connected during the period corresponding to
late G1 (from the 14th to the 18th hour) but did not appear
when the graft was present at the start of the S phase (from the 18th
to the 23rd hour). This inhibition was not obtained when a heart was
similarly transplanted to provide an equivalent surgical stress without
changing the amount of hepatic tissue. A reduction of the liver mass
has to be performed around the 10th hour to increase the regenerative
response, probably by allowing more cells to enter the second part of
G1. By contrast, the reverse
transformation, increasing the liver mass, performed at a later time is
still able to reduce this response. Two distinct control mechanisms
thus seem to exist: the first one, in
mid-G1, can be compared with point
V described for fibroblasts (32). This analogy was already evoked by
Mead et al. (26). The second could be compared with a final restriction
point acting on cells that have progressed to late
G1 but before they have entered
the S phase. Examples of similar late inhibition have been described: for instance, the administration of TGF- blocks the replication process of hepatocytes in vivo and in vitro in late
G1 but becomes ineffective during
the S phase (33, 37). The nature of the mechanisms relating the
proliferative response to the amount of remaining liver tissue remains
speculative. It could be internal processes dependent on the imposed
workload, but the release of autocrine, paracrine, or endocrine
factors, including both stimulators and inhibitors, such as TGF-
,
HGF, TNF-
, TGF-
, activin, and the hypothetical chalone, has to be
considered but is beyond the scope of this paper.
In conclusion, it is proposed that after PH, but also after a sham operation, hepatocytes enter G1 and prepare themselves to rapidly synthesize DNA, but the initial stimuli are insufficient to lead to a proliferative response. In a second stage, the size of the reduction of the liver mass around the 10th hour determines the number of cells allowed to progress in the second part of G1. The amount of functioning liver mass is also critical at a later time, immediately before the start of the DNA synthesis. After these two "restriction points," the process becomes independent of the liver mass.
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ACKNOWLEDGEMENTS |
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This work was supported by Grants 3.4624.92 and 3.4559.93 from the Fund for Scientific Medical Research (Belgium). E. M. Tagliaferri was supported by a grant from the Commissariat Général aux Relations Internationales de la Communauté Française de Belgique (91-92) and a grant from the University of Louvain, Louvain-la-Neuve, Belgium (92-93). A. P. Barker was supported by the P. F. Sobotka Postgraduate Fellowship from the University of Western Australia, The Travelling Fellowship from the Royal Australian College of Surgeons, and the Nestle Travelling Fellowship from the Australian College of Paediatrics. A. G. Baranski was supported by the Euroliver Foundation.
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FOOTNOTES |
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Part of this work was presented at the American Gastroenterological Association/American Association for the Study of Liver Diseases meeting in Boston, MA, in May 1993, and published in abstract form in Gastroenterology 104: A934, 1993, and at the American Association for the Study of Liver Diseases in Chicago, IL, in November 1993, and published in Hepatology 18: 163A, 1993.
Present address of E. M. Tagliaferri: Hospital Italiano Garibaldi, Virasoro 1249, 2000 Rosario, Argentina.
Present address of A. P. Barker: Princess Margaret Hospital for Children, Subiaco, Western Australia 6008.
Present address of A. G. Baranski: Warsaw Univ. Medical School, Dept. of Vascular Surgery and Transplantology, 1A Banacha St., 02-097 Warsaw, Poland.
Address for reprint requests: L. Lambotte, Laboratory of Experimental Surgery, Univ. of Louvain Medical School, Ave. Hippocrate 55, UCL/5570, B-1200 Brussels, Belgium.
Received 31 December 1996; accepted in final form 28 June 1997.
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