Diurnal biological rhythms in the cells of granulation tissue in a liver injury]

Under study were the diurnal biological rhythms in 37 young mature male Wistar rats and in 44 young mature female Wistar rats in the cells of the granulation tissue (fibroblasts) 4 and 9 days respectively after injuring the liver. The animals were killed in groups every 4 h during 1 day, and in the second series--during 2 days. The diurnal rhythm was found in the mitotic activity. The first peak of mitoses was observed at midday and the second one--at midnight. The second peak may be absent. It has been shown autoradiographically that the maximum intensive synthesis of DNA took place at 8'clock a. m., i. e. 4th. before the appearance of the first maximum of mitoses. The most intensive synthesis of proteins and sulphated mucopolysaccharides in fibroblasts took place at 4 p. m.     

Circadian rhythms of the parameters of the mouse esophagus epithelial cell kinetics

Circadian rhythms of the mitotic activity, DNA synthesis and the parameters of the mitotic cells of the mouse esophagus epithelium were studied during the periods of maximum and minimum proliferation. The number of mitoses and DNA-synthetizing cells increases rhythmically at 1--7 a. m. from 22 p. m. to 4 a. m., respectively. When 3H-thymidine was injected to the mice at 2 a. m., tG2min was 1h; tG2+1/2 M was 2h; tS was 7.1; tG1+1/2 M was 2h; tS was 7.1; tG1+1/2 M was 15.9h. When 3H-thymidine was injected at 2 p. m., tS rose up to 8.2 and tG1+1/2 M up to 14.8h. The mitotic cycle in both series of experiments totalled 25 h. Thus, the duration of various phases of the mitotic cycle depends on the time of the day and correlates with circadian rhythms of the mitotic activity and the number of DNA-synthetizing cells. Duration of the mitotic cycle of the cells passing through it at varying time of the day is the same and approximates the period of the circadian rhythm of mitoses and DNA synthesis in esophagus epithelium.     

Circadian Rhythm of the Liver of Male Rats Dosed with Phenobarbital—I. Organ Weight,Cellular Structures,Glycogen Contents and Mitotic Activity

The circadian rhythm of the liver, namely organ weight, cellular structures (by light-microscopy), glycogen content (by periodic acid-Schiff (PAS) reaction) and mitotic activity, was studied in 166 male Sprague-Dawley rats orally treated daily at 0800-0900 with 70 (study 1) or 50 (study 2) mg/kg phenobarbital (PB) for 7days. Thereafter, eight (study 1) or five (study 2) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through till the following day. The lighting schedule in the colony was 12:12, light:dark (light from 0600 to 1800). The liver weight was raised in PB-treated rats at all times of the day compared to controls and showed a distinct circadian rhythm with a peak at 1000 and a minimum at 2200 in PB-treated rats and the controls. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which was in good agreement with the controls, but at 1400 and 1800 the glycogen particles were more distinctly diminished in the enlarged centrilobular hepatocytes of PB-treated rats. The mitotic activity of hepatocytes was markedly increased in rats treated with PB but showed the same circadian rhythm as controls with a peak at 1000.     

Circadian Rhythm of the Liver of Male Rats Dosed with Phenobarbital—I. Organ Weight, Cellular Structures, Glycogen Contents and Mitotic Activity

The circadian rhythm of the liver, namely organ weight, cellular structures (by light-microscopy), glycogen content (by periodic acid-Schiff (PAS) reaction) and mitotic activity, was studied in 166 male Sprague-Dawley rats orally treated daily at 0800-0900 with 70 (study 1) or 50 (study 2) mg/kg phenobarbital (PB) for 7days. Thereafter, eight (study 1) or five (study 2) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through till the following day. The lighting schedule in the colony was 12:12, light:dark (light from 0600 to 1800). The liver weight was raised in PB-treated rats at all times of the day compared to controls and showed a distinct circadian rhythm with a peak at 1000 and a minimum at 2200 in PB-treated rats and the controls. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which was in good agreement with the controls, but at 1400 and 1800 the glycogen particles were more distinctly diminished in the enlarged centrilobular hepatocytes of PB-treated rats. The mitotic activity of hepatocytes was markedly increased in rats treated with PB but showed the same circadian rhythm as controls with a peak at 1000.     

Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration

Partial hepatectomy (PH) triggers hepatocyte proliferation-mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively. CONCLUSION: PH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.     

Circadian rhythm of the mitotic activity of pulmonary interalveolar septum cells in rats of different ages]

The daily rhythm of mitotic activity in the lungs of the 20-day-dd embryo coincides with the rhythm of the adult organism. The mitotic activity of the 1-, 3- and 10-day-old animals was the maximum in the evening and the minimum-in the morning hours. A definitive rhythm of cells division (with the maximal mitotic activity in the morning and the minimal-in the evening) is established beginning from the 17th day of the postnatal development. The average mitotic activity is very high in the embryos, but it falls immediately after birth. It rises on the 3rd day, and begins to decrease again from the 7th day after birth.     

Combined effect of epinephrine and chalone extracted from Ehrlich ascites carcinoma administered during different time of day on cell division in the tumor

The biphasic circadian rhythm of mitotic activity has been demonstrated in a 5-day Ehrlich's ascites carcinoma (EAC) in mice. Adrenaline injected intraperitoneally in a dose of 1.5 micrograms/g bw produced an inhibitory effect on cell division that lasted over 4 hours and reached maximum at injection to mice during light time of the day. EAC extract in a dose of 1 ml also inhibited the mitosis during 4 hours, but the greatest fall in the mitotic activity was observed during the minimum mitotic activity in the control animals. Combined administration of adrenaline and the extract resulted in the phenomenon of prolonged inhibition of cell division, that persisted for maximum 6-8 hours, if the preparations were injected in the middle of the day light time. Of definite importance was the rhythm of changes in the sensitivity of proliferating tumor cells.     

Circadian Rhythm of Liver Parameters (Cellular Structures, Mitotic Activity, Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Circadian Rhythm of Liver Parameters (Cellular Structures,Mitotic Activity,Glycogen and Lipids in Liver and Serum) During Three Consecutive Cycles in Phenobarbital-Treated Rats

The circadian rhythm of gastric content, serum alkaline phosphatase (alk.P.), serum lipids, body weight (wt), relative (rel.) liver wt, cellular structures (by light- and electron-microscopy), mitotic activity of hepatocytes, glycogen content, protein and lipids in liver was studied in 180 male Sprague-Dawley rats orally treated at 0830-1030 with 50 mg/kg phenobarbital (PB) for 7 days. Thereafter, five PB-treated males and five controls each were studied at 4-hr intervals at 0600, 1000, 1400, 1800, 2200 and 0200 on 3 consecutive days. The lighting schedule in the colony was 12:12 = light/dark (light from 0600 to 1800). Following the rhythm of gastric emptying, the rel. liver wt showed a clear circadian rhythm with a peak at 0800. The rel. liver wt was raised in PB-treated rats at all times of the day. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which exhibited a clear rhythm with the peak also at 0800, but lowered values were found in PB-treated rats. The mitotic activity of hepatocytes was significantly increased in PB-treated rats but displayed the same circadian rhythm as controls with peaks at noon and troughs at midnight. The well-known hypertrophy of the smooth endoplasmic reticulum in PB-treated rats was not found at 0600, but was fully developed at 1400 and 2200. PB-treatment increased significantly the liver content of cholesterol, triglycerides and phospholipids. Liver cholesterol showed a clear circadian rhythm with peaks at 1800. No rhythm of liver protein, triglycerides and phospholipids was observed. In serum, levels of cholesterol were significantly elevated, those of triglycerides and alk.P. significantly lowered, while those of phospholipids were not affected by the treatment. The three serum lipids, alk.P. and beta-lipoprotein exhibited a clear circadian rhythm, while serum glucose and non-esterified fatty acids did not.     

Influence of the seasons on the circadian rhythm of blood glucose and tissue glycogen in male Wistar rats

Adult, ad libitum fed male Wistar rats were adapted for three weeks to standard laboratory conditions and a 12:12 h light:dark regimen (7 a.m. to 7 p.m., 7 p.m. to 7 a.m.). In January, in April, in July and in October they were decapitated at 3-hour intervals and the glucose level was determined in their blood and the glycogen concentration in their liver, skeletal muscle, heart and white and brown adipose tissue. The influence of the seasons on circadian variation of the given parameters was not the same in all the tissues. Circadian variation of the liver and heart glycogen concentration was the least affected. The liver glycogen curves attained the maximum at the beginning of the light part of the day in all the seasons except the autumn; the rhythm was present everywhere. The rhythm was not demonstrated in the heart in the winter only and except for the summer the curves attained the maximum at the end of the dark part of the day. Pronounced seasonal changes were observed in the blood glucose and skeletal muscle glycogen concentration, whose curves reached the maximum in various parts of the day and where the rhythm was absent in the autumn - and in the case of skeletal muscle in the summer as well. The glycogen concentration in the two adipose tissues displayed the greatest seasonal changes. The shape of the circadian oscillation curves and their maxima in the various seasons were very different, with absence of the rhythm in the autumn. Determination of the influence of the photoperiodic reactions and of changes in hormones and the key enzymes regulating the main metabolic routes might help to explain the basis of circaannual variations in the metabolism of the laboratory rat.     

Relationship between the proliferation waves of the hepatocytes after hepatectomy and the waves of diurnal rhythm of mitotic activity. 1. Dependence of the form of the mitotic wave and the time of attaining its maximal degree on the time of operation]

The diurnal rhythm of mitotic activity (MA) of intact animal hepatocytes and the proliferative wave of hepatocytes after partial hepatactomy at time t0 are thought to appear as a result of formation of an initial proliferative wave, Pk-wave, within the G0-phase at constant moments of the day--time tk+1=tk+TMA(TMA=24hrs/K, k=1, or 2, . . ., or K) under the influence of the regulating system of the organism. Cells of the Pk-wave pass during a short time deltat (deltat less than TMA) from the G0-phase into the transformation phase, and then into the G1-phase. The 1st stimulated proliferative wave is formed at time tk, if tk--TMA less than t0 less than or equal to tk; its intensity depends most likely on the intensity of the corresponding Pk-wave of the intact liver. It was noted that time t0 of partial hepatectomy was necessary to coordinate with tk, but not with the time of the maximal mitotic activity, and that it was necessary to hepatectomize all animals within the interval from time tk--TMA to time tk. The model was shown to compare well with data by Post et al. (1963), Barbason (1970), and Van Cantfort and Barbason (1972) for hepatocytes of Wistar rats with TMA=8hrs, and tk (k=1,2,3) within intervals (2 a.m.; 4 a.m), (10 p.m.; noon), and (6 p.m. 8 p.m.). The maximal rate of liver generation was observed for all the hepactomized animals with the time of operation being between 8 p. m. and 2 a.m.     

Twenty-four-hour rhythm in the mitotic activity and in the water and dry matter content of regenerating liver

Summary A twenty-four hour rhythm is described in the variations of mitotic activity, dry weight, and water content of the regenerating liver of mice hepatectomized about noon. These variables have their maximal value at the onset of the rest periods in the morning and their minimal values at the initiation of the body activity periods in the evening. The rhythm is well defined on the second day and is repeated on the third day on a higher level.We acknowledge the assistance of Mrs. R. Cavoura in performing the hepatectomies and that of Mrs. M. A. V. Albornoz in making the histologic slides.The present work was carried out with the help of grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and Liga Mendocina de Lucha contra el Cáncer.     

CORRELATION BETWEEN TISSULAR AND DIVISION FUNCTIONS IN THE LIVER OF YOUNG RATS

The division and tissue specific functions are studied in the liver of young rats during the first post-natal weeks. The division function is tested by the mitotic and the ³H-thymidine labelling index, the specific tissular function by the cholesterol-7 alpha-hydroxylase activity. The nycthemeral rhythm is triggered simultaneously for the two functions at the 20th day of life. From this time, spontaneous or induced mitoses occur during the day (rest period) and the cholesterol-7-alpha-hydroxylase during the evening (period of activity). The results are explained by the influence of the hypothalamoadrenal axis, which presents a circadian activity with a maximum in the evening.     

Developmental stages of Hepatozoon hemprichii sp. nov. infecting the skink Scincus hemprichii and the tick Hyalomma impeltatum from Saudi Arabia

The life cycle of Hepatozoon hemprichi n. sp. is described; the vertebrate host is Scincus hemprichii and it is vectored by Hyalomma impeltatum. Erythrocytic stages of 18 ± 1.8 × 4 ± 0.8 μm developed in the hemocoel of ticks to sporozoites within 16-18 days. Schizogony occurred in the liver parenchyma and the endothelial cells of blood capillaries in lung and spleen. Mature schizonts measuring 27 ± 3.11 × 20.13 ± 3.0 μm produced 28 merozoites (on average). The merozoites were 13 ± 1.21 × 1.21 ± 0.72 μm with nuclei 5 ± 0.65 × 2.1 ± 0.51 μm. Syzygy and differentiation of gamonts took place in tick's hemocoel up to the third day post-infection (PI). The microgamont (16 ± 0.31 × 18 ± 0.42 μm) produced 4, uniflagellated microgametes at 4-5 days PI. The microgamete measured 15.2 ± 0.31 μm while the flagellum was always at least 26 μm. The macrogamete was very large in size (31 ± 3.11 μm) with a central nucleus. After fertilization, (5-6 days PI) zygotes developed into oocysts (55 ± 3.41 × 52 ± 4.11 μm) in which repeated mitotic divisions with centripetal invaginations occurred; each contained 18 banana-shaped sporozoites, 13.61 ± 0.8 × 1.2 ± 0.31 μm in size. Experimental transmission was successfully carried out by oral administration or by intra-peritoneal inoculation of the infective stages (sporozoites) to uninfected skinks and led to the appearance of blood stages after 5 wk and 4 wk, respectively.     

Mitotic activity and its 24-hour rhythm in the ovarian follicles during the estrous cycle of a wild rat, Bandicota bengalensis

Mitotic activity in ovarian follicles was studied in relation to the size of the follicles during a 24-hour period (10.00, 16.00, 22.00 and 04.00 h) throughout the estrous cycle of the wild bandicoot rat (Bandicota bengalensis) to ascertain the cell proliferation rate and its 24-hour rhythm in the follicular tissue. In the bandicoot ovary, mitotic activity in the granulosa and thecal cells was highest in the follicles ranging from 201 to 400 micron in diameter. During the estrous cycle, mitotic activity of the granulosa cells was highest at estrus in follicles less than 601 micron, and at diestrus in follicles greater than 600 micron; while the mitotic trough was at proestrus in all the follicles. Thecal mitosis was significantly lower than mitosis of the granulosa cells. In most of the follicles, mitotic activity in the thecal cells was highest at diestrus and lowest at metestrus. In both the granulosa and thecal cells, the timing of mitotic peaks and troughs varied according to the size of the follicles and the stages of the estrous cycle. In the granulosa cells mitotic peaks were maximal in the daytime (10.00 h, 16.00 h) and in some cases at night (04.00 h); and mitotic troughs were primarily during the night (22.00 h, 04.00 h) and in some cases in the day (10.00 h). In the thecal cells, however, mitotic activity in most of the follicles was distinctly higher in the daytime (16.00 h) than at night (22.00 h, i.e., evening). Thus, a prominent 24-hour mitotic rhythm was noticed in the ovarian follicles of the bandicoot rat.     

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).     

GROWTH AND MITOTIC ACTIVITY OF LUNG COLONIES DERIVED FROM MOUSE TERATOCARCINOMAS

This study dealt with the mitotic activity of lung colonies in the process of differentiation of mouse teratocarcinoma cells injected in vivo . The results showed that three stages were identifiable in respect to the mitotic activity: 1) a lag stage for a few days after injection, 2) a stage during which maximum mitotic activity is observed for several days following the first stage and 3) a third stage displaying a decrease in mitotic activity. There is a correspondence between these three steps in mitotic activity and differentiation in lung colonies previously identified by histological observations.     

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).     

Changes in intestinal and hepatic thyroid hormone deiodination during spontaneous metamorphosis of the sea lamprey, Petromyzon marinus

We measured microsomal low-K(m) outer-ring deiodination (ORD) and inner-ring deiodination (IRD) activities for thyroxine (T(4)) and 3, 5,3'-triiodothyronine (T(3)) in intestine and liver in nonmetamorphosing (undersized) larvae, immediately premetamorphic larvae, animals in stages 1-7 of metamorphosis, and immediately postmetamorphic sea lampreys (Petromyzon marinus). For intestine: T(4)ORD activity was relatively low in nonmetamorphosing larvae, increased in premetamorphic individuals, was highest in stages 1 and 2 and was very low during stages 3-7; T(4)IRD activity was negligible until stage 3 but increased 4.7-fold through stages 3 to 7 such that T(4)IRD activity was 14 times T(4)ORD activity at stage 6; T(3)ORD activity was undetectable; T(3)IRD activity was not measured through stages 3-7 but correlated with T(4)IRD activity at other stages. For liver: deiodination was only measured up to stage 2 and in postmetamorphic animals; in contrast to intestine, T(4)ORD activity fell to low levels at stage 2 and was low during postmetamorphosis; T(4)IRD and T(3)IRD activities were very low and uninfluenced by developmental stage; T(3)ORD activity was undetectable. We conclude that (1) deiodination activity is usually much higher in intestine than in liver, (2) intestinal ORD and IRD activities change reciprocally so that ORD predominates in early metamorphosis but IRD predominates in mid and late metamorphosis, and (3) changes in intestinal deiodination may contribute to the characteristic depression of plasma T(4) and T(3) levels during spontaneous metamorphosis. J. Exp. Zool. 286:305-312, 2000.     

Effect of thyroxine on the diurnal rhythm of the mitotic activity and parameters of the life cycle of cells of the liver and esophagus]

The duration of the cellular cycle and the diurnal rhythm of the amount of mitosis were studied in young rats in normality and under the influence of thyroxin. The parenchymal and connective-tissue cells of the liver and cells of the liver and the cells of the oesophagus epithelium basal layer were studied. It was found that under the influence of thyroxin there occured a shortening of the periods of the cellular cycle and a 3--6 h shift to the left of the diurnal rhythm curve of the amount of mitoses. In thyroxinized animals the 21--95% increase of the amount of mitoses in the period of maximum values of the mitotic index during a day was observed as compared with control animals. A conclusion is made about the diurnal rhythm of sensitivity of G0-phase cells to the synchronizing factor, suggesting the decisive significance of the state of the cell population in the interaction of the tissue and hormone cells. The data obatained in the work show that the thyroid hormones regulate the cellular reproduction in the organism by stimulating the cells in the division cycle, synchronization of greater amount of cells by the moment of beginning of the mitotic cycle at a definite time of day and by shortening the period of the cell mitotic cycle.