The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

G2 restriction point within populations of hepatocytes and tongue keratinocytes in young, growing mice

The authors studied the effect of either extracts from liver (LE) or the malignant tumour ES2 (TE) or plasma from intact mice (PI) or tumour-bearing animals (PT) on the mitotic activity of the hepatocytes and tongue keratinocytes in young, growing C3H/s male mice (28+/-1 days old). Animals standardized for periodicity analysis were injected intraperitoneally with either TE, LE, PI, PT, or saline (S) at 16:00 h with 0.01 ml of sample/g of body weight and were then killed at (time of day/h post-injection) 20:00/04, 00:00/08, and 04:00/12. Colchicine (2 microg/g) was injected 4 h before death. Samples of the liver and tongue from each animal were processed for histology and assessment of mitotic index. The results were expressed as colchicine-arrested metaphases/1000 nuclei. The TE and LE stimulated the mitotic activity of hepatocytes and tongue keratinocytes. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.     

The possible cyclic AMP-dependence of an early prereplicative event that determines mitosis in regenerating rat liver

The beta-adrenergic blocker dl-propranolol prevented a large proportion of regenerating rat liver cells from entering the mitotic phase of their first cell division cycle without affecting their ability to initiate or complete DNA replication. The drug, at a dose of 20 or 50 mg/kg of body weight, was most effective in reducing mitosis when injected between 1 and 2 hours after the proliferatively activating partial hepatectomy, which was 22 to 23 hours before the peak of DNA-synthetic activity. Propranolol also inhibited the early prereplicative surge of total liver cyclic AMP, which occurs shortly after partial hepatectomy, but this effect was not correlated to the mitosis-inhibiting activity. However, cyclic AMP or dibutyryl cyclic AMP completely reversed propranolol's mitosis-inhibiting action when injected between 1.5 and 2 hours (but not sooner or later) after partial hepatectomy, which was just before the total liver cyclic AMP content began to rise. Thus, there appears to be a transient, propranolol-inhibitable, probably cyclic AMP-initiated event in the early prereplicative development of rat hepatocytes that determines entry into mitosis rather than the initiation of DNA replication.     

The control of liver regeneration by parathyroid hormone and Calcium

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA-synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium-mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium-dependent stage near the end of the pre-replicative phase of development.     

The control of liver regeneration by parathyroid hormone and calcium.

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA-synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium-mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium-dependent stage near the end of the pre-replicative phase of development.     

Inhibiting effect of a hepatoma extract on the mitotic rate of regenerating liver

Aqueous tumor extracts were prepared by the homogenization of a fast-growing, undifferentiated, transplantable malignant murine hepatoma in distilled water. After centrifugation, an aliquot of 0.01 ml of the supernatant g body weight was injected intraperitoneally into partially hepatectomized mice. Control animals were injected with saline. Groups of mice were killed at various times in relation to the hepatectomy. Four h before killing the animals were given Colcemid (1 microgram/g body weight). The number of Colcemid-arrested mitoses in the hepatocytes and in the littoral cells, respectively, were counted in 140 microscopic fields. The extract significantly inhibited the mitotic rate in hepatocytes when the injection was given between 22 h before, and up to 26 h after hepatectomy. In the littoral cells, a slight initial stimulation was followed by a slight but significant inhibition which occurred when the injection was given at hepatectomy or until 18 h after hepatectomy. The effect was not modified by exposing the extracts to temperatures of 47 degrees C for 30 min or 22 degrees C for 24 h, but 10 min of boiling destroyed their inhibitory effect. Lyophilization and storing at -18 degrees C for up to 4 weeks did not modify the effect. The mitosis-inhibiting effect was also measurable when the extract was injected subcutaneously. There was an almost linear dose-response curve. The results are discussed in relation to circadian rhythms, the pattern of liver cell proliferation after hepatectomy, and recent similar reports from the literature. The conclusion is drawn that extracts of a hepatoma contain one or more growth-inhibitory factors significantly active on regenerating liver cells, and less significantly on littoral cells.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Variations in DNA Synthesis and Mitotic Indices in Hepatocytes and Sinusoid Litoral Cells of Adult Intact Male Mouse Along a Orcadian Time Span

Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.     

A correlative study between cortisol and ACTH in Cushing's disease following bilateral adrenalectomy and in Nelson's syndrome.

Correlation analysis was used to investigate the interrelation between plasma ACTH and serum cortisol concentrations determined at 8:00, 12:00, 16:00 and 22:00 h in 48 patients bilaterally adrenalectomized for Cushing's disease, including 23 patients with a pituitary adenoma (Nelson's syndrome). In the patients without evidence of a pituitary adenoma a significant inverse correlation was found at 8:00, 16:00, 22:00 h and additionally when all the pairs of estimations were analyzed. In a full-blown Nelson's syndrome an inverse correlation was not proved (p = 0.05). During remission in Nelson's syndrome an inverse correlation between cortisol and ACTH concentrations was stated at 8:00 h and after the evaluation of all the pairs of estimations. The results of our studies have shown that exogenous cortisol exerts a partial inhibitory action on ACTH secretion in patients bilaterally adrenalectomized for Cushing's disease. In active Nelson's syndrome this influence is questionable, it comes however into prominence during remission.     

Complete change of the cell composition of the liver parenchyma following exposure to dipin and partial resection

The phenomenon of total replacement of preexisting and damaged hepatocytes in mice were demonstrated by the method of autoradiography. Adult mice were injected an alkylating drug Dipin 2 h prior to partial hepatectomy and then proliferating cells were labelled by means of multiple injections of 14C-thymidine. Dipin in combination with mitotic stimulation induced multiple mitotic aberrations in proliferating hepatocytes resulting in degeneration, death and then elimination of prelabelled liver cells. New parenchymal tissue originated from non-labelled preneoplastic nodules. These hepatocyte nodules grew in size, propagated and 8-10 months later completely replaced the preexisting hepatocytes.     

DNA synthesis in tongue keratinocytes of hepatectomized and tumor-bearing mice

Tongue keratinocytes have high S-phase and mitotic indices with evident circadian variation. Transplanted tumors modify the intensity and temporal structure of the S-phase index in cell populations in tumor-bearing animals; also, partial hepatectomy changes the concentrations of substances involved in cellular proliferation, leading to compensatory liver hyperplasia. The aim of our study was to analyze the interaction between tumor growth and the liver regeneration that follows partial hepatectomy, and the effects of both these processes on lingual keratinocytes. We used 380 adult male mice divided into six groups: tumor-free and tumor-bearing mice without surgery, with sham hepatectomy, and with partial hepatectomy. Each group was divided into six subgroups, which were killed at 4-h intervals until a circadian cycle was completed (from 26 until 50h post-surgery in the operated animals). Each animal was injected with 5-bromodeoxyuridine (50mg/kg) 1h before it was killed, and tongue samples were obtained and processed for histology. The sections were placed on silanized slides and incubated with the primary antibody Bu 20a (1/100 dilution). The reaction was developed using diaminobenzidine and staining was detected visually. SIs were measured as the number of labeled nuclei per thousand cells. The mean+/-S.E. of each group was calculated. Differences among experimental groups were analyzed by ANOVA and the Student-Newman-Keuls Multiple Comparisons Test. The results show that the presence of a tumor alters the normal circadian curve of SI in lingual keratinocytes, irrespective of whether the mice underwent surgery. This finding has to be considered in drug treatments for neoplasms and in experiments related to growth.     

Variations in DNA Synthesis and Mitotic Indices in Hepatocytes and Sinusoid Litoral Cells of Adult Intact Male Mouse Along a Orcadian Time Span

Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.     

Lithium: Short-term and chronic effects on plasma testosterone and luteinizing hormone concentrations in mice

The effects of short-term and chronic lithium administration on the concentrations of plasma testosterone (T) and luteinizing hormone (LH) were evaluated in C57BL/6 mice, maintained on a fixed photo-period of LD 14:10 (white lights on at 06:00 h, CST). Lithium chloride was injected intraperitoneally twice daily (at 09:00 and 16:00 h) in groups of adult male mice at a dosage of 2.5 meq/kg for 7 days, and 1.25 meq/kg for 21 days. Circulating levels of T and LH were measured by standard radioimmunoassay (RIA) methods. Plasma T levels showed a significant increase in mice treated with lithium for 7 days as compared to those in saline-injected control animals. However, there was no significant difference in the concentrations of plasma T between chronic (21 days) lithium-treated mice and the matched control. Plasma LH levels remained unchanged following both short-term and chronic lithium treatment.     

Circadian rhythm of VEGF expression in the liver of hepatectomized-tumor-bearing adult and young mice

We analyzed VEGF expression in regenerating liver (after partial hepatectomy) of tumor-bearing adult and young mice, throughout one complete circadian cycle. The animals were sacrificed every 4 h, from 26 to 46 h post-hepatectomy. Liver samples were processed for immunohistochemistry. The results showed circadian variation of VEGF expression in hepatectomized mice (control group) and hepatectomized-tumor-bearing mice. The maximum value of VEGF expression was found at 16:00/30 h of day/hour post-hepatectomy (HD/HPH) in tumor-bearing young mice, and at 20:00/34 HD/HPH in the controls. In adult mice the maximum values of VEGF expression were at 16:00/30 HD/HPH in tumor-bearing mice, and at 08:00/46 HD/HPH in the controls. Young tumor-bearing mice showed significantly higher mean values than the controls. In conclusion, the presence of the tumor in the animals induces modifications in the intensity and the temporal distribution of the circadian curves of VEGF expression.     

Evidence for a noradrenergic transmission in the control of parturition in the rat

6-Hydroxydopamine, when injected at 14:00 h on Days 21 and 22 of pregnancy in the rat (2 X 50 mg/kg), markedly decreased plasma and uterine noradrenaline concentrations (-60% and -82% respectively; P less than 0.001). As a consequence of this treatment, there was severe disturbance in the distribution pattern of parturitions: 61% of rats had suppressed parturition and 31% of rats displayed a lengthened or interrupted labour. A bolus dose of prazosin (3 mg/kg) administered at 12:00 h on Day 22 completely blocked the normal process of parturition throughout the next 6 h, a result which is compatible with the half-life of the drug (2.9 +/- 0.8 h). Administration of phentolamine (3 mg/kg) at term induced a significant decrease of uterine activity (frequency X duration of bursts of spike potentials) as revealed by electromyographic recordings in vivo. These results suggest that noradrenaline released from sympathetic nerve terminals interacts with alpha-adrenoceptors located post-synaptically to improve the overall excitability of the myometrium at the onset of labour.     

Circadian variations of serotonin in plasma and different brain regions of rats

Most of the physiological processes that take place in the organism follow a circadian rhythm. Serotonin is one of the most important neurotransmitters in our nervous system, and has been strongly implicated in the regulation on the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN). The present study analysed the levels of serotonin over a period of 24 h in the plasma and in different brain regions. The model used was of male Wistar rats, 14 +/- 2 weeks of age (n = 120), maintained under conditions of 12 h light and 12 h dark, and food and water ad libitum. The serotonin levels were measured by ELISA every hour at night (20:00-08:00 h) and every 4 h during the daytime (08:00-20:00 h). Ours results show that the maximum levels of serotonin in plasma were obtained at 09:00 and 22:00 and a minor peak at 01:00 h. In hypothalamus there was a significant peak at 22:00 and two minor peaks at 17:00 and 02:00 h; the same occurred in hippocampus with a significant peak at 21:00, and two secondary peaks at 24:00 and 05:00 h; in cerebellum there were two peaks at 21:00 and 02:00 h, while in striatum and pineal there were peaks at 21:00 h and 23:00, respectively. In conclusion, the higher levels of serotonin were during the phase of darkness, which varies depending on the region in which it is measured.     

Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy.

We have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co-cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen-NPCs), the response of hepatocytes from resting liver to EGF, TGF-alpha, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell-to-cell contact, and conditioned-medium from regen-NPCs, but not normal NPCs, inhibited EGF-induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS-PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin-sensitive but relatively heat-stable. The effects of blocking antibodies established that it was not TGF-beta 1, IL1-beta, or IL6. Investigations of regen-NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy.     

Ketone-body metabolism in tumour-bearing rats.

During starvation for 72 h, tumour-bearing rats showed accelerated ketonaemia and marked ketonuria. Total blood [ketone bodies] were 8.53 mM and 3.34 mM in tumour-bearing and control (non-tumour-bearing) rats respectively (P less than 0.001). The [3-hydroxybutyrate]/[acetoacetate] ratio was 1.3 in the tumour-bearing rats, compared with 3.2 in the controls at 72 h (P less than 0.001). Blood [glucose] and hepatic [glycogen] were lower at the start of starvation in tumour-bearing rats, whereas plasma [non-esterified fatty acids] were not increased above those in the control rats during starvation. After functional hepatectomy, blood [acetoacetate], but not [3-hydroxybutyrate], decreased rapidly in tumour-bearing rats, whereas both ketone bodies decreased, and at a slower rate, in the control rats. Blood [glucose] decreased more rapidly in the hepatectomized control rats. Hepatocytes prepared from 72 h-starved tumour-bearing and control rats showed similar rates of ketogenesis from palmitate, and the distribution of [1-14C] palmitate between oxidation (ketone bodies and CO2) and esterification was also unaffected by tumour-bearing, as was the rate of gluconeogenesis from lactate. The carcinoma itself showed rapid rates of glycolysis and a poor ability to metabolize ketone bodies in vitro. The results are consistent with the peripheral, normal, tissues in tumour-bearing rats having increased ketone-body and decreased glucose metabolic turnover rates.