Chronobiology of the proliferative events related to angiogenesis in mice liver regeneration after partial hepatectomy.

In liver regeneration the formation of new capillary blood vessels is a fundamental requirement for cellular proliferation. Vascular endothelial growth factor (VEGF) is involved in the events of angiogenesis, the mRNA of which is expressed in both hepatocytes and non-parenchymal cells. In this experimental design we try to establish if during liver regeneration in mouse, the expression of VEGF is produced before or after the hepatocytes proliferation. C3H/S adult male mice were divided in three groups in order to study: VEGF expression; S-phase index (SI); and mitotic activity (MA) of hepatocytes. The results that were analyzed by ANOVA, show that VEGF expression starts to increase 26 h after PH with a peak at 28 h. Furthermore, the DNA synthesis (DNAs) reaches maximal level 42 h after pH, meanwhile the MA of the hepatocytes shows an increase 8h after the DNAs peak. In conclusion, it could be argued that the chronobiology of the events related to liver regeneration in mice started with a release of VEGF by the hepatocytes, followed by its DNAs and mitosis.

Development of compensatory hepatic hyperplasia in mice carrying the hepatocellular carcinoma ES12a.

In a previous paper we reported that the presence of the hepatocellular carcinoma SS1K in host mice resulted in an earlier appearance of the hepatocyte mitotic peak during liver regeneration after a partial hepatectomy as well as in an increase in the amplitude of that mitotic wave. In the present work we analyse the effect of another hepatocellular carcinoma, the ES12a (HCES12a). Adult male mice of the C3H/S strain standardised for circadian-periodicity analysis, were used. One group received a subcutaneous graft of the HCES12a tumor, while another group served as control. Fifteen days later, all animals were submitted to a partial (70%) hepatectomy at 10:00 h and beginning at 16:00 h lots of between 5 and 9 host and control animals each were sacrificed at 4 h intervals until 16:00 h on the third day thereafter. All mice were injected with 2 microg/g colchicine 4 hrs before killing, and samples of livers were processed for hematoxylin-eosin staining. We determined the hepatocyte mitotic index for each animal and the mean value +/- the standard error of the mean for each lot. The peak of mitotic activity in the tumor-bearing animals took place four hours earlier than in control mice but the average values of hepatocytic mitotic activity were similar in both groups

Effect of partial and sham hepatectomy on the growth of a hepatocellular carcinoma.

We examined the effect of partial hepatectomy on the proliferation of hepatoma ES12a grafted into C3H/S mice compared to tumor growth in sham-hepatectomized controls. The animals were sacrificed every 4 hrs during three days from the 6th to the 78th h following each type of surgery. Unoperated tumor-bearing mice were likewise killed as controls, but only during one complete circadian period. All animals received 2 microg of colchicine per g of body weight intraperitoneally 4 hrs before decapitation. Measurement of mitotic indices in hematoxylineosin-stained tumor samples revealed a decrease in proliferation and a modification of the diurnal mitotic-activity profile in the hepatectomized and sham-operated animals from the first day after surgery. These differences persisted by the third postoperative day only in the hepatectomized animals. Thus, although surgical stress may initially affect tumor growth, the latter results must be the effect of the influence of the hepatic regeneration.

Cytokeratin immunoreactivity in mouse tissues: study of different antibodies with a new detection system.

The cross-reactivity of a group of monoclonal antibodies (MABs) generated against human cytokeratins (CKs) was investigated in mouse tissues. Formalin-fixed and paraffin-embedded sections of lung, stomach, small and large intestine, liver, and kidney were immunostained with MABs after epitope retrieval with enzyme digestion. AE1/AE3, a "cocktail" of two MABs that recognizes basic and acidic CKs, 5D3 MAB to low molecular weight CKs (8, 18, and 19), and monospecific MABs to CK 7 and 20 were tested. Additionally, CK 17 and 34betaE12 MABs to high molecular weight CKs were evaluated in the same organs and in sections from skin and preputial glands. We employed the new universal animal system (ARK) as the detection system. The results showed intense reactivity for the first group of antibodies used, with topographic distribution similar to that in human tissues, with the exception of CK 7 in lung parenchyma, which displayed reactivity only in type II pneumocytes, with negativity of adjacent bronchial epithelium. Also of note was the lack of reaction of liver hepatocytes and renal tubular cells to AE1/AE3 and 5D3 MABs. Regarding the second group of antibodies, no reaction was obtained for CK 17 in the tissues tested. On the contrary, 34betaE12 MAB yielded intense reactivity in cells of epidermis and hair follicles. Compared to other detection systems used previously in this animal, ARK produced a well-defined reactivity at the cellular level without any background. We conclude that a useful panel of anti-CK antibodies commonly used in human pathology can be applied successfully to mouse tissues after enzyme digestion, leading to a more accurate definition of cellular populations in this laboratory animal.

Circadian rhythm of DNA synthesis and mitotic activity in tongue keratinocytes.

Tongue keratinocytes have a high mitotic index (MI) with an evident circadian variation. Our study set out to compare and contrast two phases of the cell cycle: DNA synthesis (S-phase), with inmunocytochemical detection by bromodeoxyuridine (BrdU), and mitosis (M-phase), by the colchicine-arrest of metaphase method, exploring both the dorsal and ventral surfaces of the mouse tongue throughout a circadian period. Adult male mice standardized for light periodicity used for MI experiment were injected intraperitoneally with colchicine. Other animals were injected intraperitoneally with 5-BrdU for S-phase determination. Animals given both treatments were divided into six groups and killed at 4 h intervals until 20:00 h. Tongue samples were processed for histology and immuno-histochemistry. S and M indices were expressed as labelled nuclei or colchicine metaphases, respectively, per 1000 nuclei. Peak MI occurred at 12:00, with the minimum value at 20:00 on dorsal and ventral tongue surfaces. Peak S-phase was at 04:00, whereas the minimum value was at 16:00 for both surfaces. These results show that the proliferative activity of the tongue epithelium is of similar intensity and temporal distribution on both surfaces.

Mitotic activity of the pars intermedia in the female mouse: age-associated variations in proliferation rate and circadian periodicity.

We previously reported daily variations in the mitotic activity of the endocrine cells in the pars intermedia of 21- and 28-day-old male mice. Since cellular proliferation might be affected by factors such as sex and age, we undertook the present experiments to study the mitotic activity of the pars intermedia from 14-, 28-, and 150-day-old female mice. Inbred C3H/S mice, grouped according to age, were housed under standard conditions (12h each of light and dark [LD 12:12]) for periodicity analysis and were killed in lots of 5-11 animals every 4h over a single 24h cycle, with each mouse receiving 2 microg/g of colchicine 4h before decapitation. Pituitaries were excised, extracted, fixed in buffered formaldehyde, embedded in celloidin-paraffin, sectioned at 5 microm, and stained with hematoxylin and eosin. We counted the total number of nuclei to estimate the total number of cells monitored and then calculated the mitotic index (metaphases/1000 nuclei). Differences were analyzed for statistical significance by the Student t test. While the 14-day-old animals manifested no significant changes in mitotic activity during the 24h cycle, the 28- and 150-day-old mice showed higher mitotic indices during the period of darkness. The average mitotic activity over the entire cycle, however, was higher in the two groups of younger animals than in the 150-day-old mice. Moreover, the averages for the 28-day-old females were higher than the corresponding values previously reported by us for male mice of the same age.

DNA synthesis and nucleolar organizer regions circadian rhythm in mouse regenerating liver hepatocytes.

DNA synthesis and Nucleolar Organizer Regions (NORs) were studied in C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) incorporated into DNA with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections of regenerating liver. Tissue samples were obtained at different times after hepatectomy along a circadian span. The results showed a strong correlation of values between DNA synthesis (BrdU labelling index) and AgNOR numbers, with higher counts during the activity period of animals at 00:00/38 and 04:00/42 hours Time of Day/Hours Post-Hepatectomy (TD/HPH), being the differences with other time points highly significant. Our observations demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU incorporation and AgNOR numbers with a defined circadian rhythm in mouse regenerating hepatocytes.

DNA synthesis and nucleolar organizer regions circadian rhythm in mouse regenerating liver hepatocytes.

DNA synthesis and Nucleolar Organizer Regions (NORs) were studied in C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) incorporated into DNA with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections of regenerating liver. Tissue samples were obtained at different times after hepatectomy along a circadian span. The results showed a strong correlation of values between DNA synthesis (BrdU labelling index) and AgNOR numbers, with higher counts during the activity period of animals at 00:00/38 and 04:00/42 hours Time of Day/Hours Post-Hepatectomy (TD/HPH), being the differences with other time points highly significant. Our observations demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU incorporation and AgNOR numbers with a defined circadian rhythm in mouse regenerating hepatocytes.

Effect of normal and tumor factors on different phases of cell populations cycle.

In the present experiments we studied the effect of extracts from intact liver (LE), ES2 tumor extract (TE), plasmas from intact mice (PI), and from tumor bearing animals (PT) on different phases of hepatocytes and renocytes cell cycles. C3HS 28-day-old male mice, standardized for periodicity analysis, were injected at 16:00 hours and killed every 4 hours during a circadian cycle at 20:00/04; 00:00/08; 04:00/12; 08:00/16; 12:00/20 and 16:00/24 (time of day/hours post treatment). Colchicine (2 microg/g) was injected 4 hours before killing them. Samples of livers and kidneys were processed for histology and mitotic index determinations. The results were expressed as colchicine arrested metaphases per 1000 nuclei. The TE, LE and PI had a promoting effect on the mitotic activity of hepatocytes during the first 12 hours post treatment. During the subsequent 12 hours, not only these treatments but also the PI had an inhibiting effect on the mitotic activity of the same cell population. Also the TE and the PT had a promoting effect on the mitotic activity of the renocytes during the first 12 hours while the effect of all treatments showed a clear inhibition of the mitotic activity studied during the last 12 hours. 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.

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.

Effect of tissue and plasma factors on kidney proliferation.

Liver extract, plasma from intact mice, ES2 tumour extract and plasma from tumour bearing mice has an inhibiting effect on the mitotic activity of hepatocytes and duodenal enterocytes. In the present experiments, the effect of these treatments on the mitotic activity of renal tubular cells was studied. C3HS 28 day-old male mice, standardized for periodicity analysis were used. The determination of normal mitotic circadian curve of the renocytes was done. A second batch of mice were injected with 0.01 ml/gr of either liver extract, plasma from intact mice, ES2 tumour extract or plasma from tumour bearing mice, at 16:00 hours and controlled at 08:00, 12:00 and 16:00 hs during 2 consecutive days post treatment. Colchicine (2 micrograms/gr) was injected 4 hours before killing. Kidneys were processed for histology and mitotic index determinations. Results were expressed as colchicine metaphases per 1000 nuclei, and showed that mitotic activity values of treated animals were significantly lower than those of controls. In conclusion, mitotic activity inhibition of renocytes may be due to some non specific plasmatic and/or tissue factors.

[Action of liver extract from partially hepatectomized mice on the mitotic activity of young mouse enterocytes]. / Accion del extracto de hígado de ratones parcialmente hepatectomizados sobre la actividad mitótica de los enterocitos del ratón joven.

We have previously demonstrated that adult mouse plasma obtained 36 hours post partial hepatectomy has an inhibitory effect on the mitotic activity of enterocytes from young mouse duodenal cripts. In this paper we investigate whether this effect is derived from any regenerating liver factor. Accordingly, we studied the action of adult mouse (90 days old) liver extract obtained 36 hours after partial hepatectomy (70%), on the mitotic activity of young mouse enterocytes, considering 3 cellular levels of the duodenum cripts. Thirty six C3H/S inbred female mice (27 days old) were employed. Half of them received at 16:00 hour an intraperitoneal injection of saline and the other half received liver extract (0.01 ml/g). Animals from each group were sacrificed at 08:00/ 16, 12:00/20 and 16:00/24 (time of day/hours post treatment). All the animals received an intraperitoneal dose of colchicine (2 micrograms/g) 4 hours before sacrifice. The results are expressed as colchicine metaphases/1000 nuclei and show that the mitotic activity is significantly lower in the animals treated with the extract than in the controls. This inhibiting effect is observed at the levels from 1 to 4 and from 5 to 12 cells of the analyzed cripts. At the superficial level from 13 to 20 cells there is no modification of the proliferative activity. This inhibiting effect on the mitotic activity of duodenum enterocytes from the basal and intermedial zone of the cripts is probably due to a liver diffusing factor.

[Mitotic activity of duodenal-crypt enterocytes in mice with hepatocarcinoma]. / Actividad mitótica de los enterocitos de las criptas duodenales en ratones portadores de un hepatocarcinoma.

Tumors grafted into mice may modify the proliferation of normal cell populations. In this paper, we have studied the evolution of mitotic activity (MA) in duodenal-crypt enterocytes of ES12a hepatocarcinoma-bearing mice; a total of 87 28-day-old female animals of the C3H/S strain were used after standardization for circadian-periodicity analysis. The mice were distributed into two groups: those remaining intact and those receiving tumor grafts. Each group was then divided into six batches (n = 6-10), one of which was sacrificed every 4 h over a period of one day. A dose of colchicine (2 micrograms/g) was administered to each animal 4 h before killing. Samples of duodenum were fixed in 10% (v/v) buffered formalin and processed for assessment of mitotic activity. The number and topographic localization of the colchicine-arrested metaphases were recorded among the entero-cytes within 20 longitudinal sections of the duodenal crypts in each animal. From these data the mitotic indices over the total crypt-cell population as well as within each previously-established zone were determined along with mean +/- SEM for each experimental group. The statistical significance of the differences among the data were analyzed by Student t test. The results show that the presence of ES12a tumor inhibits the mitotic activity of the duodenal-crypt enterocytes and produces an apparent temporal shift in the peak and trough within the circadian curve for this growth parameter.

An initial characterization of a mitostatic activity for hepatocytes and renocytes in extracts from adult mouse liver.

We have previously demonstrated that liver extract, when administered at 16:00 hr, inhibits the mitotic activity of hepatocytes and renocytes at 08:00, 12:00 and 16:00 hr. during the following 2 days. The experiments reported here were designed to achieve an initial characterization of this liver mitostatic activity. For these studies we used 28-days-old male C3HS mice injected with either saline, liver extract (LE), mitochondrial-pellet extract (MPE), or postmitochondrial supernatant (PMS) at a dose of 1.9 mg of protein/g body weight. Colchicine (2 microg/g) was injected 4 hrs. before sacrifice and the number of arrested metaphases within the hepatocyte and renocyte population was estimated. The mice treated with LE were killed at (time of day/hr. post injection) 16/08:00, 20/12:00 and 24/16:00; while those injected with MPE or PMS were sacrificed at 20/12:00. We found that both the LE and the PMS inhibited the mitotic activity of hepatocytes and renocytes, while MPE reduced the mitotic activity of the liver cells without affecting that of the renal population.

Circadian rhythm in the mitotic activity of the pars intermedia endocrine cell population of the mouse.

A mitotic circadian rhythm is reported for the endocrine cells of the pars intermedia of mouse pituitary. The peak is observed at 0000 and the trough at 1600. This curve is about 12h out of phase with the curves of mitotic activity of most of the cell populations of the organism of the mouse which show peaks about noon (1200). A similar inverted mitotic rhythm has been reported for corticoadrenal since a long time ago. The results are discussed in relation with the contradiction existing between proliferation and function in cell populations. The conclusion is drawn that the endocrine cells of the pars intermedia could represent another link in the circadian system controlling periodicity.

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver.

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian 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.