The re-establishment of hypersensitive cells in the crypts of irradiated mouse intestine

Within 3-6 h of small doses of radiation (gamma-rays) the number of dead cells (apoptotic cells) in the crypts of the small intestine reaches peak values. These return to normal levels only after times later than 1 day. After higher doses elevated levels of cell death persist for longer times. The dead cells first occur most frequently at the lower positions of the crypt (median value for the distribution of apoptotic fragments is about cell position 6). At later times more dead cells are observed at higher positions. Two doses of radiation separated by various time intervals have been used to investigate when after irradiation the cell population susceptible to acute cell death is re-established. Dead cells were scored 3 or 6 h after the second dose. The yield of dead cells after two doses represents the sum of the dead cells produced by, and persisting from, the first dose and new apoptotic cells induced by the second dose. Since the temporal and dose-dependence aspects of the dead-cell yield after the first dose alone is known, the additional dead cells attributable to the second dose alone can be determined by subtraction. Within 1-2 days of small doses (0.5 Gy) the sensitive cells, recognized histologically as apoptotic cells, are re-established at the base of the crypt (around cell position 6). After higher doses (9.0 Gy) they are not re-established until about the fourth day after irradiation. Even in the enlarged regenerating crypts the sensitive cells are found at the same position at the crypt base. It has been estimated that the crypt contains five or six cells that are susceptible to low doses (0.5 Gy) (hypersensitive cells) and up to a total of only seven or eight susceptible cells that can be induced by any dose to enter the sequence of changes implicit in apoptosis. Between 4 and 10 days after an initial irradiation of 9.0 Gy the total number of susceptible cells increased from seven to eight to about 10 to 13 per crypt.     

Effect of the time of administration of 5-hydroxytryptophan on the restoration of circadian stimulation of ACTH secretion in rats treated with p-chlorophenylalanine

In female rats kept under a photoperiod of 12L-12D (50 lux from 07.00-19.00 h) the pharmacological blockade of serotonin synthesis by pCPA (2 X 300 mg/kg i.p.) obliterated the diel ACTH stimulation, which could, however be restored by an additionnal 5-HTP injection (60 mg/kg i.p.), provided that the serotonin precursor was administered at 11.00 h. If injected at 23.00 h the same dosage of 5-HTP failed to elicit any increase in plasma ACTH. The circadian ACTH rhythm appears, therefore to depend upon a daily activation of the serotoninergic system occurring 4 h after the onset of the light phase.     

Determination of parameters of the survival curve of the stem cells of the intestinal crypts by LD50 and the regenerated crypt count. Determination of the RBE of p(65) + Be neutrons

The early effects of an irradiation on the intestinal epithelium have been evaluated, at the tissular level, by LD50 after single and multifraction irradiation, and, at the cellular level, by numeration of the regenerated intestinal crypts (Withers technique) after a single fraction irradiation. From the set of informations provided by both criteria, one derived the values of the parameters defining the survival curve of the intestinal clonogenic crypt cells after irradiation by gamma-rays (two component model): D0 = 1.5 Gy, 1D0 = 4.5 Gy, nD0 = 2.25 Gy and n = 20. In other respects, the p(65) + Be neutrons RBE (ref. 60-Cobalt) after a single fraction irradiation is equal to 1.75 +/- 0.2 and 1.64 +/- 0.25 for the LD50 at the 5th day and for the regeneration of 50 crypts after 3.5 days respectively.     

Circadian variation of the cell proliferation in the jejunal epithelium of rats at weaning phase

Circadian variation in cell proliferation of the jejunal epithelium of 18-day-old rats was studied using the 2-h arrested metaphase score and crypt isolation method. A continuous decrease in the arrested metaphases occurred from 07.00 h to 13.00 h. From 17.00 h arrested metaphase values increased and were maintained at the higher level during the dark period as showed by Cosinor analyses (P < 0.05). These results indicate that in the young rat there is already a circadian variation in jejunal epithelial cell proliferation as early as 18 days. We can even suggest that the presence of a circadian rhythm at weaning contributes to the steady state of cell proliferation in the intestinal epithelium observed in adult life.     

Estimates of the number of clonogenic cells in crypts of murine small intestine

There is a proliferative cell hierarchy in the mouse intestinal crypt with ancestral stem cells which can regenerate all components of the lineage after injury (clonogenic cells). The number of these clonogenic or regenerative cells per crypt can be estimated from radiobiological experiments where doses of radiation are used to kill cells and ablate crypts. Various approaches can be adopted which provide different estimates of this number of cells. One of the conventional approaches used in the past provided estimates of about 70-80 clonogenic cells per crypt (i.e. about 50% of the proliferative or 30% of all crypt cells). A technically simpler approach has recently been suggested. This has been used here to provide many independent estimates of the number of crypt clonogenic cells. These suggest about 32 clonogenic cells exist per crypt i.e. about half the previous estimate and about twice the number of putative "functional" stem cells (those which operate as stem cells in the normal steady-state crypt). The reasons for the differences are discussed. The new estimates are compatible with the hypothesis that the crypt contains a ring of about 16 functional stem cells which are expected to be clonogenic, besides which there is a second ring of 16 clonogenic cells which represent early transit cells (the immediate daughters of the stem cells) which can act as clonogenic cells if required after radiation injury.     

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.     

CIRCADIAN RHYTHMS OF PRESUMPTIVE STEM CELLS IN THREE DIFFERENT EPITHELIA OF THE MOUSE

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with ³H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.     

Radiobiology and stathmokinetics of intestinal crypts associated with patches of Peyer

The stathmokinetics and radiobiology of intestinal crypts directly adjoining the lymphoid patches of Peyer, have been compared with those of non-patch-associated crypts. Patch crypts contain an additional one to two rings of cells, the Mitotic Index for the whole crypt is higher than in non-patch crypts, and the apparent cell cycle time is insignificantly lower. Using single and split doses of gamma-rays, dose-survival curves were obtained for whole intestinal crypts, from which single-cell survival curves were derived for the clonogenic cells of the crypt. For a single-hit, multitarget, model, the extrapolation numbers of the cell survival curves for patch and non-patch crypts were the same (approximately 35) but the final D0 for cells of the patch crypts was significantly higher (2.1 versus 1.7 Gy). A linear-quadratic fit gave a similar ratio of alpha/beta (approximately 10) for the two curves. For a given level of crypt depletion, the number of clonogenic cells per crypt derived by the use of equal split doses of radiation, was the same for patch and non-patch crypts. This number is a function of the dose regime employed: the higher the level of crypt depletion, the higher the derived number of cells (range 10 to 45, for non-patch crypts).     

The clonogen content of murine intestinal crypts: dependence on radiation dose used in its determination.

The number of colony-forming (clonogenic) cells in each of the crypts in mouse small intestine was deduced using a two-dose irradiation technique. The number was 7.5 +/- 0.8 cells using two equal doses each less than 9 Gy and 38 +/- 7 cells using 9 Gy or more per dose. The significant dose dependence could not be accounted for by considerations of intra- or intercrypt variability, or by the factor introduced to correct the sampling frequency for the influence of crypt size. The results suggest that more colony-forming cells may be recruited when the injury is more severe.     

Protection of mouse jejunum against lethal irradiation by Podophyllum hexandrum

Radiation induced gastrointestinal damage occurs due to the destruction of the clonogenic crypt cells and eventual depopulation and denudation of the villi. P. hexandrum, a plant, known for its antitumour activity, has been shown to protect the mice against whole body lethal (10 Gy) irradiation. Present study was undertaken to investigate the radioprotective effect of P. hexandrum on jejunal villi cells, crypt cells, their proliferative capacity and mitigation of apoptosis.

In an in vivo micro colony survival assay, pre-irradiation administration of P. hexandrum (–2 h) increased the number of surviving crypts in the jejunum by a factor of 3.0 (P < 0.05) and villi cellularity by 2.7 (P < 0.05) fold in comparison to irradiated control. Pre-irradiation administration of P. hexandrum reduced the incidence of apoptotic bodies in the crypts (P < 0.05) in a time dependent manner and depicted a mitotic arrest till the 24 h. However, after 84 h the percentage of mitosis was observed to be nearly similar to that of unirradiated control.

This study suggests that arrest of cell division may help in protecting the clonogenic cells against radiation. It would be interesting to investigate further the role of P. hexandrum in influencing various cell cycle regulators like bcl-2, TGF-β, Cyclin-E etc.     

p53 and cell-cycle-regulated protein expression in small intestinal cells after fast-neutron irradiation in mice

The involvement of the p53 gene in apoptosis of many cell types towards -radiation is well established. However, little information is available on the relationship between p53 status and cells ability to undergo apoptosis following exposure to fast neutrons. The aim of this study was to characterize the apoptotic pathway traveled by neutrons in mouse intestinal crypt cells. Each mouse received whole body doses of 0.25–8 Gy fast neutrons and were sacrificed 0, 4, 6, 12, 24, 48, and 72 h, respectively, after irradiation. Apoptosis of crypt cells and expression of p53, cyclin A, cyclin B, cyclin D, and cyclin E were measured. The apoptosis in crypt cells was maximal at 4 and 6 h after irradiation, showing a gradual decline at 24 h. The highest frequency of apoptosis was seen at a 1 Gy dose and then declined gradually beyond a 2 Gy dose with high levels of damage. In immunoblot analysis, apoptosis was confirmed to be dependent on p53 function after fast-neutron irradiation. In addition, cyclin B1, cyclin D, and cyclin E were overexpressed in intestinal cells after fast-neutron irradiation and their immunoreactivities were increased strongly in round and oval cells of laminar propria in villi core and crypts. The results of the current study suggest that apoptosis in crypt cells shows a time- and dose-dependent increase after fast-neutron irradiation. In addition, fast-neutron-induced apoptosis in mouse intestinal crypt cells appears to be related to the increase in functional p53 proteins to a level sufficient to initiate apoptosis and up-regulation of cell-cycle-regulated proteins, which may lead to resistance to DNA damage through cell cycle arrest, is involved deeply in protection of gastrointestinal cells after low doses of fast-neutron irradiation. (Mol Cell Biochem 270: 21–28, 2005)     

The proliferative status of intestinal epithelial clonogenic cells: sensitivity to S phase specific cytotoxic agents.

High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3 1/2 days after a range of radiation doses indicates that a majority of the clonogenic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.     

Daily fluctuations of nucleoli in neurosecretory cells of the rat supraoptic nucleus

Summary Daily fluctuations of nucleoli and nucleolar fibrillar centres in neurosecretory cells from the supraoptic nucleus (SON) were investigated in rats artificially synchronized for 3 weeks to a set 12 h light/12 h dark cycle with free access to food and water. Groups of 3 animals were sacrificed by intracardiac perfusion every 4 h for a 24-h period and every 2 h between 22.00 h and 07.00 h. The SON of each animal was removed, and the mean nucleolar volume and the mean volume of the nucleolar fibrillar centres were estimated by a stereological analysis. The quantitative data showed that the fluctuations in the nucleolar volume of SON neurons depend on the time of sacrifice. A peak value was found in animals sacrificed at 03.00 h which was 1.5 times the value found in animals sacrificed at 19.00 h. The volume of fibrillar centres underwent small, but not significant changes over the 24-h period. None of the large fibrillary centres that can be observed in the superior cervical ganglion were found in the SON. Our results demonstrate that in these neurons the size of the nucleolus undergoes daily fluctuations. These results are discussed in the light of previous studies conducted at our laboratory on the circadian rhythm of nucleolar volume and of nucleolar components in neurons of the superior cervical ganglion.     

The gastrointestinal syndrome and mucosal clonogenic cells: relationships between target cell sensitivities, LD50 and cell survival, and their modification by antibiotics

The sensitivity of the target cells responsible for the gastrointestinal syndrome in mice was deduced from the steepness of the dose-survival curve for mice assessed on Day 7 after irradiation. The D0 value was 1.25 +/- 0.22 Gy, virtually identical to the value of 1.23 +/- 0.08 measured for microcolony-forming cells (clonogens) over about the same range of dose in concurrent experiments. The survival of clonogens was similar when assayed in mice surviving to Days 3, 4, or 5, but clonogenic sensitivity was lower when assessed on Day 7. This was shown at one dose to be due largely to a selection of mice with high colony counts with only a small contribution from crypt budding. The LD50 for mice corresponded to a surviving fraction of crypts of about 0.35. An injection of 5 mg streptomycin sulphate ip daily for 5 days after irradiation increased the latent period by about 1 day, increased the LD50 by about 1.4 Gy, but did not significantly change the survival of clonogens. These studies are the first to test and satisfy the interpretation of a dose-response curve for animal survival in terms of "target cell" survival, where measurements of both are made over a similar range of dose in concurrent experiments.     

Gender Dependency of Circadian Blood Pressure and Heart Rate Profiles in Spontaneously Hypertensive Rats: Effects of Beta‐Blockers

This study investigated (i) blood pressure (BP), heart rate (HR), and their relation to urinary NOx and eNOS protein expression in male and female spontaneously hypertensive rats (SHR), as well as (ii) gender‐dependent cardiovascular effects of nebivolol (NEB) in comparison to metoprolol (MET) in SHR. BP and HR were measured telemetrically after a single intraperitoneal application of NEB or MET at 07.00 and 19.00 h in male rats and at 19.00 h in proestrus female rats. The two β‐blockers varied in time of decreasing BP and HR and also in duration. In males, MET decreased BP and HR for few hours exclusively when applied at the onset of the activity phase (i.e., at 19.00 h), while after its application at 07.00 h, BP and HR were unchanged. In females, MET also caused a short‐lasting BP and HR reduction, with the effect being more pronounced than in males. In males, NEB at either dosing time decreased HR and BP to a greater extent than did MET. This effect was evident both during the activity and rest periods and persisted for at least five days. In females, NEB provoked a similar, but more pronounced, effect on BP and HR in comparison to males. These findings demonstrate that significant gender‐dependent differences in the circadian profile of BP and HR exist. BP and urinary NOx as well as eNOS expression are inversely correlated, and the cardiovascular effects of NEB and MET vary, depending on the time of application as well as gender.     

Postnatal development of TRH and TSH rhythms in the rat

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.     

Gender dependency of circadian blood pressure and heart rate profiles in spontaneously hypertensive rats: effects of beta-blockers

This study investigated (i) blood pressure (BP), heart rate (HR), and their relation to urinary NOx and eNOS protein expression in male and female spontaneously hypertensive rats (SHR), as well as (ii) gender-dependent cardiovascular effects of nebivolol (NEB) in comparison to metoprolol (MET) in SHR. BP and HR were measured telemetrically after a single intraperitoneal application of NEB or MET at 07.00 and 19.00 h in male rats and at 19.00 h in proestrus female rats. The two β-blockers varied in time of decreasing BP and HR and also in duration. In males, MET decreased BP and HR for few hours exclusively when applied at the onset of the activity phase (i.e., at 19.00 h), while after its application at 07.00 h, BP and HR were unchanged. In females, MET also caused a short-lasting BP and HR reduction, with the effect being more pronounced than in males. In males, NEB at either dosing time decreased HR and BP to a greater extent than did MET. This effect was evident both during the activity and rest periods and persisted for at least five days. In females, NEB provoked a similar, but more pronounced, effect on BP and HR in comparison to males. These findings demonstrate that significant gender-dependent differences in the circadian profile of BP and HR exist. BP and urinary NOx as well as eNOS expression are inversely correlated, and the cardiovascular effects of NEB and MET vary, depending on the time of application as well as gender.     

Detailed profile of prolactin secretion in the immature female rat: evidence for the existence of an ultradian rhythm

The detailed profile of prolactin (PRL) secretion in 22-24 and 29-31 days old female rats was investigated by serial blood sampling through an intracardiac cannula at 15-min intervals for each of the 9 or 10-h periods beginning at 09.00 or 10.00 and 22.00 h. By analysis of the power spectrum and the least squares method the time series of PRL concentrations which were measured by RIA were found to have approximately a 3-h period ultradian rhythm in either sampling period of both the 22-24 and 29-31 days old rats. The peak times calculated based on the acrophase estimated through the calculation of periodicity were concentrated around 12.00, 15.00 and 18.00 h for the sampling period 10.00-19.00, and 24.00, 03.00 and 06.00 h for the sampling period 22.00-07.00 h. However, in more than half of the animals at 22-24 days of age, one secretory episode around 12.00 h, and two secretory episodes around 24.00 and 03.00 h had markedly small amplitudes, making the remaining secretory episodes distinct diurnal and nocturnal surges, respectively. In the animals at 29-31 days of age, the amplitudes of the PRL episodes occurring around 12.00 h were markedly small, making the remaining two episodes as diurnal surges, whereas the amplitudes of PRL secretory episodes during the period 22.00-07.00 h were analogous to each other. These findings indicate that the semicircadian rhythm of PRL secretion is established on the basis of PRL secretion with the 3.0-h period ultradian rhythm.     

Modification of radiation induced damage in mouse intestine by WR-2721

Intestinal protection in mice against radiation injury by WR-2721 (300 mg/kg body wt, i.p., 30 min before irradiation) was studied after whole body gamma irradiation (0.5, 1.5, 3.0, 4.5, or 6.0 Gy). Crypt survival and induction of apoptosis, and abnormal mitoses in crypt cells in the jejunum were studied on day 1, 3 and 7 after irradiation. Irradiation produced a significant decrease in crypt survival, whereas apoptosis and abnormal mitoses showed a significant increase from sham-treated control animals. Maximum changes in all the parameters were observed on day 1 after irradiation and the effect increased linearly with radiation dose. There was recovery at later intervals, which was inversely related to radiation dose. WR-2721 pre-treatment resulted in a significant increase in the number of surviving crypts, whereas the number of apoptotic cells in the crypts showed a significant decrease from respective irradiated controls on day 1 after exposure. The recovery was also faster in WR-2721 pre- treated animals. It is concluded that WR-2721 protects against gastrointestinal death by reducing radiation induced cell death, thereby maintaining a higher number of stem cells in the proliferating compartment.     

The thyroid hormone receptor-alpha (TRalpha) gene encoding TRalpha1 controls deoxyribonucleic acid damage-induced tissue repair

The thyroid hormone (TH) controls, via its nuclear receptor, TH receptor-alpha1 (TRalpha1), intestinal crypt cell proliferation in the mouse. In order to understand whether this receptor also plays a role in intestinal regeneration after DNA damage, we applied a protocol of gamma-ray irradiation and monitored cell proliferation and apoptosis at several time points. In wild-type mice, the dose of 8 Gy induced cell cycle arrest and apoptosis in intestinal crypts a few hours after irradiation. This phenomenon reverted 48 h after irradiation. TRalpha(0/0) mutant mice displayed a constant low level of proliferating cells and a high apoptosis rate during the period of study. At the molecular level, in TRalpha(0/0) animals we observed a delay in the p53 phosphorylation induced by DNA damage. In our search for the expression of the protein kinases responsible for p53 phosphorylation upon irradiation, we have focused on DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The number of cells expressing DNA-PKcs in crypts remained high 48 h after irradiation, specifically in TRalpha mutants. Altogether, in TRalpha(0/0) animals the rate of apoptosis in crypt cells remained high, apparently due to an elevated number of cells still presenting DNA damage. In conclusion, the TRalpha gene plays a role in crypt cell homeostasis by regulating the rate of cell renewal and apoptosis induced by DNA damage.