Injection Of Thymidine Research Articles

Labeling of differentiated rat purkinje cells after [3H]thymidine injection

Labeling of differentiated rat purkinje cells after [3H]thymidine injection

DNA synthesis in atrial myocytes of rats with aortic stenosis.

Indices of labeled myonuclei have been determined in hypertrophying hearts of adult Wistar rats by autoradiography after single-pulse or repeated [3H]thymidine administration. After single [3H]thymidine injections, only 1.36 +/- 0.66 and 1.32 +/- 0.87% labeled myonuclei were observed in the left and right atria, respectively. In the experiments with multiple [3H]thymidine administration, the first injection of this precursor was given on the seventh day after aortic constriction; thereafter, 30 or 42 injections of [3H]thymidine were given at 12-hr intervals up to the fourth postoperation week. Following 30 repeated [3H]thymidine injections, 29.75 +/- 4.65 and 16.78 +/- 3.33% labeled myonuclei were visible in left and right atrial muscle cells, respectively. The cumulative labeling index for left atrium myocytes clearly correlates (r = 0.65-0.73) with an increase in the weight of the heart. Increase in heart weight to more than 160% of controls corresponds to [3H]thymidine labeling of 38.06 +/- +/- and 21.67 +/- 4.16% in left and right atrial myocytes, respectively, whereas in hearts weighing less than 140% of controls, [3H]thymidine labels only 8.20 +/- 1.93% in the left atrium and 3.94 +/- 1.57% in the right one. In the ventricles, cumulative indices of myonuclear labeling do not exceed 0.217 +/- 0.11% even in hearts weighing nearly 180% of controls. Cumulative frequencies of labeling for AV system myocytes are almost ten times higher (1.97 +/- 0.38). These results, together with our data concerning mycocardial infarction (27-29,31), make it necessary to reconsider the role of cardiomyocyte hyperplasia in different experimental and pathological conditions, paying special attention to the proliferative behavior of the atrial muscle cells. DNA synthesis in atrial myocytes seems to be stimulated by heart hyperfunction.

Autoradiographic study of neurogenesis in the duck olfactory bulb.

Neurogenesis was studied in the duck olfactory bulb by injection of tritiated thymidine into the eggs at 53 h and at the 3rd, 5th, 8th, 10th, 12th, 14th and 18th days of incubation. The large neurons appear before the small ones: the mitral cells arise between embryonic day 3 (E3) and E5, the tufted cells between E5 and E8 and the granular cells between E12 and E14. The periglomerular cells could be formed after E18. The order of appearance of this 4 main neurons of the duck olfactory bulb is the same as that in the mouse. All the neurons, except maybe for the periglomerular cells, are principally formed before the hatching of the duckling and the olfactory sense seems to have acquired most of its principal functional aptitudes at this moment.

Recovery kinetics of epidermal cell proliferation after two intraperitoneal injections of epidermal extracts (chalone).

The cell kinetic effect of two intraperitoneal (IP) injections of 5 mg of crude lyophilized skin extracts given 12 h apart was assessed during the recovery period (5 to 52 h after last injection) by measuring epidermal labelling indices and the specific activity after tritiated thymidine (3HTdR) injection, and by determining the cell cycle phase distribution by flow cytometry. The skin extracts produced an epidermal chalone effect and inhibited both DNA synthesis and mitosis. A slow recovery took place from 5 to between 22 and 36 h after the last chalone injection. During this period the cell flux into DNA-synthesis and mitosis slowly recovered, but the exits were blocked and cells accumulated in the respective phases. The fluxes out opened up at the S phase about 22 h, and at the M phase about 30 h after the second chalone injection. A secondary inhibitory effect was observed at about 40 h, followed by a subsequent recovery to normal at 52 h. The similarity between the recovery kinetics after chalone and that observed after hydroxyurea (HU) is emphasised.

A Simple Method for Determining Epithelial Cell Turnover in Small Intestine

Epithelial cell migration in animal gut classically is measured by autoradiography after systemic administration of tritiated thymidine. A migration rate is calculated from observations of the leading edge of villus epithelial cells containing labeled nuclei at varying time periods after tritiated thymidine injection. This method is very accurate, but laborious, time consuming, and costly. A new, simplified method for measuring epithelial cell measurement of [3H] deoxyribonucleic acid specific activity in frozen sections of intestine cryostat-cut from villus tip to crypt base. The leading edge of [3H]deoxyribonucleic acid can be detected reproducibly (coefficient of variation 11.25%). Autoradiographic and chemical methods for determining the leading edge of labeled cells compared favorable (r=0.848). Epithelial cell migration rates in barrier-reared Fisher 344 aging (27 mo) and young control (4-5 mo) rats were compared and no differences were detected in duodenum, jejunum, or ileum. Labeled epithelial cells reached the villus tip sooner in the ileum than in duodenum or jejunum. However, crypt-villus column heights were greater in duodenum and jejunum and the calculated epithelial cell migration rate was faster in upper intestine than in the ileum. Although ileal epithelial cells are shed from the villus sooner than cells in the upper intestine, the rate of cell migration in ileum is slower.

Sensitivity of follicular melanoblasts in newborn mouse skin to tritiated thymidine: Evidence for a long term retention of label.

Injection of tritiated thymidine into newborn mice results in a progressive greying of hair that does not begin until after the first hair coat is grown. After a year the depigmentation is appreciable (about 60% of the hair are white). The effect cannot be simulated by external irradiation of newborn mice or by the administration of radioactive uridine or methionine. The effect can best be explained by a long-term retention of radioactivity in the DNA of melanocyte stem cells (melanoblasts) in spite of several rounds of cell division. This could be achieved by labelling the strands of DNA destined to act as templates throughout life by being selectively retained in the stem line as described in Cairns' hypothesis.

Psoriasiform Dermatosis in a Rhesus Monkey

We have previously described the clinical and dermatopathologic features of a psoriasiform dermatosis in a rhesus monkey (Macaca mulatta). This animal has been studied further in attempts to characterize its skin disease. Epidermal cell DNA synthesis was measured following the intradermal injection of tritiated thymidine. Epidermal cell labeling indexes in the involved and uninvolved skin of the affected monkey and in the skin of a normal monkey were compared. The labeling indexes in the involved skin were significantly increased as compared with uninvolved skin; they were also significantly higher than in normal skin. Epidermal polyamine assays showed increased epidermal putrescine in involved skin together with increased spermidine and an increased spermidine-spermine ratio compared with uninvolved and normal skin sites.

Localization and mechanism of thymidine transport in the central nervous system.

The localization and mechanism of thymidine and deoxyuridine transport in the central nervous system were studied in vivo and in vitro. Previous studies have shown that thymidine enters brain from blood in part via the CSF. In vitro, isolated adult bovine cerebral microvessels, which readily concentrated and phosphorylated deoxyglucose, were unable to concentrate thymidine and deoxyuridine. In vivo, [3H]thymidine (0.2 microM) and [3H]deoxyuridine (0.4 microM) were not extracted more readily than [14C]sucrose in a single pass through the cerebral circulation of rats. In vivo, [3H]thymidine retention in CSF and brain after entry from blood was increased when the efflux of [3H]thymidine from CSF and the phosphorylation of [3H]thymidine in brain were depressed by the intraventricular injection of unlabeled thymidine. These studies and previous work suggest that the transfer of thymidine (and deoxyuridine) through the blood-brain barrier in either direction must be extremely low. The present studies are consistent with the postulate that thymidine is transported by an active transport system in the choroid plexus that transfers thymidine from blood into the CSF; from the CSF, the thymidine enters brain cells and is phosphorylated.

Self-replication of somatostatin cells in the antral mucosa of rodents.

The possibility that antral somatostatin cells have a self-replicating activity has been studied in three species of rodents: mice, rats and guinea-pigs, after a flash tritiated thymidine injection. The immunocytochemical staining of somatostatin cells, using specific antiserum, was combined with radioautographic procedures. The labelling index for somatostatin cells--and for gastrin cells identified on serial sections--was established after counting a large number of cells at the optical microscope level, on parallel tissue strips removed throughout the entire antrum. A significant percentage of the somatostatin cell population synthesized DNA. Values were similar for the three species of rodents ranging from 0.8 to 1.1%, that is slightly higher than the percentage of labelled gastrin cells, which was 0.67-0.7%. After a 36-hr continuous infusion of radioactive precursor in one rat, the labelling index observed remained low; 2.33% for somatostatin cells and 1.68% for gastrin cells. Colchicine injection in mice allowed the observation of mitotic figures in well differentiated somatostatin cells. Four hours after that injection, the mitotic index was estimated roughly at 0.3%. Thus, evidence has been presented that in rodents a fraction of the antral somatostatin cell population is capable of dividing, similar to the situation in gastrin cells.

An autoradiographic study of methyl-azoxy-methanol acetate-induced cortical malformation.

A completely disorganized pattern of neuronal migration was observed in intracortical neuronal nodules induced by the injection of methyl-azoxy-methanol acetate (MAM) in the rat on the day 14 of gestation. Labeled neurons were found at different cortical levels after a single injection of tritiated thymidine on days 14, 16, or 18 of gestation. Preservation of the ascending migratory progress of neuroblasts toward the cerebral cortex is, however, seen in the largest areas in which no intracortical nodules are found in spite of the abnormal structure of these areas. Neurons labeled on day 16 of gestation occupy deep regions of the cortex, while neurons labeled on day 18 are found at superficial levels. These results indicate variable degrees of disturbance in the migratory pattern in spite of the disorganization of the cortical structure in brains injured at an early stage before the cortical plate is formed.

Hypertrophic and hyperplastic effects of thyroxine on the submandibular gland of the mouse.

The nature of the trophic response of the mouse submandibular gland to thyroxine (T4) was examined. Adult female Swiss-Webster mice were given daily subcutaneous injections of T4 (1 microgram/gm body weight) for two or four days; two injections of tritiated thymidine (3H-TdR) were given 24 and 29 hours after the last injection of hormone, and the mice were killed one hour after the last injection of 3H-TdR. One gland was analyzed chemically for DNA content and for incorporation of 3H-TdR, while the other was used to prepare autoradiograms. The cellular composition of each gland was analyzed by counting 1000 nuclei, and the frequency and labelling index (LI) of six cell types were established. A rise in specific activity of DNA and a fall in its concentration were seen in response to T4. The LI for the entire gland more than doubled. The LIs and frequencies of granular convoluted tubule and granular intercalated duct cells were increased more than those of acinar and nongranular intercalated duct cells; striated and excretory duct cells were not affected. It is concluded that the enlargement of the submandibular gland of the mouse caused by T4 is due to both hyperplastic and hypertrophic effects.

Effects of Neutron and X Irradiation on Cell Proliferation in Mouse Lung

Studies of animal survival following split-dose lung irradiation have shown the existence of an extended process which followed X irradiation but did not occur after neutrons. Since the type II pneumonocyte is strongly implicated in the response of lung to irradiation, the possibility that a proliferative response is involved either in the changing effectiveness of split doses of radiation with dose separation or in the differences between the effects of neutrons and X rays has been investigated. Repeated injections of tritiated thymidine have been given to show proliferative changes after doses of either 7.7 Gy neutrons or 10 Gy X rays. The study clearly demonstrates that proliferative responses after equivalent doses of neurons and X rays are indistinguishable and are incapable of explaining differences in the response of lung to the two radiations, i.e., slow repair after X rays but not after neutrons. Both kinds of irradiation affected proliferation, with more marked changes seen in the ...

The effect of a liquid elemental diet on cell proliferation in the colon of rats.

A liquid elemental diet (Vivonex) was given to rats for 6 days while control animals received a normal diet. At the end of the experiment each animal received one intraperitoneal injection of tritiated thymidine at 8 a.m. Animals from each group were killed hourly during the first 24 h after the injection and the proliferative activity was studied by autoradiography of the mucosa of the colon using the labeled mitoses-wave method. The epithelial cell proliferation was significantly decreased in the colon of the Vivonex-fed animals.

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The effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on gastroduodenal epithelial proliferation p]rior to the development of frank neoplasia was studied in inbred LEW rats with or without gastric ulcers. The rats received either MNNG (100 gm/liter) in the drinking water or plain water. After 4 weeks, some rats in the MNNG-treated and control groups were given injections of tritiated thymidine and killed 1 hour later. In other rats, either an ulcer of the fundic mucosa was formed by a suction biopsy tube at laparotomy or a sham operation was performed. At 2 and 4 weeks after the operation, these rats were given injections of tritiated thymidine and killed 1 hour later. Sections of fundus, antrum, and duodenum were prepared for light autoradiography. MNNG treatment stimulated gastroduodenal epithelial proliferation, expanded the proliferative zone (PZ), and in the duodenum caused marked villus blunting and elongation of the crypts. No additional effect of the fundic ulcer or sham operation on gastroduodenal proliferation could be determined. The MNNG-induced expansion of the PZ occurred in a downward direction. Thus theories of carcinogenesis should include not only the expansion of the PZ toward the mucosal surface but also the possibility of expansion of the PZ toward the base of the mucosa.

Effect of a lithogenic diet on cell proliferation in the murine gallbladder epithelium.

A cholesterol-cholic acid lithogenic diet was administered to mice for periods from 2 days to 12 weeks. Intraperitoneal injections of tritiated thymidine and autoradiographs were used for the estimation of the proliferative parameters in the gallbladder epithelium. The total DNA content of the gallbladder was determined. Significantly higher labeling (p less than 0.01) and mitotic (p less than 0.01) indices were observed from the 2nd day and the total DNA content of the gallbladder was increased (p less than 0.01) from the 2nd week of the diet. Increased proliferative activity was followed by hyperplasia of the gallbladder epithelium. A significant dilatation of the gallbladders was also noted from the 2nd day of the diet (p less than 0.01). Gallstones appeared after at least 4 weeks. Our results indicate that gallstone formation is preceded by a higher proliferative activity in the gallbladder mucosa. Distension of the gallbladder could be one of the factors explaining this phenomenon in mice, but the influence of intraluminal modifications in the bile salt composition cannot be excluded.

Malignant bone tumours induced by a local injection of colloidal radioactive 144Cerium in rats as a model for human osteosarcomas.

Lung metastases were observed in 80% to 85% of rats bearing advanced malignant bone tumours (osteogenic osteosarcomas and angiosarcomas). These tumours were induced in 2-month-old Sprague-Dawley rats by inoculation of a colloidal suspension of radioactive cerium (144Ce) into the hind leg, in close contact to the bones of the knee joint. Twenty-eight rats were killed or died spontaneously shortly after detection of palpable tumours at the site of injection: the incidence of lung metastases was 73.3% and 53.8%, respectively, for osteogenic sarcomas and angiosarcomas, showing that most lung metastases are present at the time of diagnosis of the primary tumour. Tumour-cell kinetic parameters were studied in 49 rats bearing tumours following intraperitoneal injection of [3H]thymidine. The labelling index (LI) of the primary tumours was significantly lower in advanced tumours (7.2% for osteosarcomas and 10.1% for angiosarcomas) than than in tumors examined at the time of detection (12.2% and 13.5%, respectively). Mitotic indices (MI) of all tumours were less than 1%. From the curve of the percentage of labelled mitoses (PLM) at different times after [3H]thymidine injection, Ts (6.5 h) and TG2 (1.75 h) were determined. TC and TG1 were also evaluated (18 h and 9.25 h, respectively). These results show that malignant bone tumours induced in rats with 144Ce may be a good model for human osteosarcomas and may be useful in studying the numerous problems in the therapy of malignant bone tumours in man.

The cell cycle kinetics of human breast cancer.

Cell kinetic parameters were determined in 32 patients with breast cancer following intravenous [3H]thymidine injection. The mean labelling index (LI) was 8.6, range 1.2 to 24.1. The LI increased significantly with increased stage of disease. The highest values were noted in patients with chest wall metastases with a mean LI of 15.9. In one patient with multiple discrete tumor foci, the larger foci had significantly decreased LI consistent with classic cytokinetic theory. The intralesional variation in LI was measured in 31 lesions. The average variation of LI/lesion was 1.2. Therefore, three samples should provide a representative LI. The interlesional LI was compared by taking multiple samples of two or three lesions per patient in seven patients. For each patient, there was little variation in mean LI from lesion to lesion. The data suggest that higher LI are associated with a poorer prognosis. Among patients with advanced T4 or metastatic disease, the median survivals for patients with LI less than 8 and greater than or equal to 8 were 79 weeks and 21 weeks, respectively. For patients with metastatic disease, the survivals were 56 and 9 weeks, respectively. The tumor growth fraction analyzed in one patient was 40%. In summary, the growth rate of this cancer in an individual patient is relatively homogeneous and LI may provide important prognostic information. Further studies are required to establish the value of LI in the staging and treatment of breast cancer.

Toxicologic effects of a high dose of thymidine in mice

The pharmacologic effects and plasma concentrations produced by the ip administration of high doses of thymidine were determined in CDF 1 mice. Within 15 min after the injection of thymidine at a dose of 4.075 g/kg (between LD0 and LD50), mean arterial blood pressure and heart rate fell precipitously and remained depressed for over 6 hr. During the experimental period, sedation and anuresis were also consistently observed in thymidine-treated mice. After a dose of 4.7 g/kg, millimolar plasma thymidine concentrations were maintained for 16 to 20 hr; by contrast, at a lower but nonlethal dose (3.8 g/kg), millimolar plasma thymidine concentrations were maintained for only 9 hr. It is suggested that sustained elevation of circulating thymidine in the range 1–10 m m for periods greater than 1 2 day may be associated with toxicity in mice.

Bone cell kinetics during longitudinal bone growth in the rat.

The purpose of this work was to provide futher knowledge about bone cell kinetics in the metaphysis of the growing long bone. Seventy rats were sacrificed from 1 to 120 h after injection of tritiated thymidine. Autoradiographs of 3 micrometers thick sections of the proximal tibial metaphysis were studied in a manner which allowed evaluation of labeled cell nuclei as a function of increasing age of metaphyseal tissue. A cell cycle duration study for osteoprogenitor cells was done. Labeled osteoprogenitor cells and osteoblasts first appeared at 1 h post-injection. The great majority of all labeled osteoprogenitor cells and osteoblasts was found within 1 mm of the growth cartilage-metaphyseal junction (GCMJ) at all times, apparently migrating with the moving GCMJ. In contrast, labeled osteoclast nuclei first appeared at 24 h post-injection within 0.3 mm of the GCMJ and remained always with the area of bone surface with which they were first associated, even as the GCMJ migrated away. By 5 days post-injection, the source of new labeled osteoclast nuclei in the metaphysis near the GCMJ was depleted, whereas that for the osteoblasts remained. The existence of two kinetically different, as well as ultrastructurally different, members of the metaphyseal osteoprogenitor cells population is postulated. A cell cycle time of 39 +/- 18 h was found for the osteoprogenitor cell population, but has limited meaning. A schema for metaphyseal bone cell movements during longitudinal bone growth is presented.

Thymidine transport in the central nervous system.

The mechanisms by which thymidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [3H]thymidine intravenously and intraventricularly. [3H]thymidine, with and without unlabeled thymidine, was infused at a constant rate into conscious adult rabbits. At 150 min, [3H]thymidine readily entered CSF, choroid plexus, and brain. In brain, approximately 45% of the nonvolatile radioactivity was [3H]thymidine phosphates. The addition of 0.21 mmol/kg unlabeled thymidine to the infusion syringe decreased the phosphorylation of [3H]thymidine in brain by approximately 85%; the addition of 2.1 mmol/kg of unlabeled thymidine to the infusion syringe decreased the relative entry of [3H]thymidine into CSF and brain by 40 and 78%, respectively. Two h after intraventricular injection of [3H]thymidine, [3H]thymidine was rapidly cleared from CSF, in part, to brain, where approximately 40% of the [3H]thymidine was converted to [3H]thymidine phosphates. The intraventricular injection of unlabeled thymidine (21 mumol) with the [3H]thymidine abolished the phosphorylation of [3H]thymidine in brain and significantly decreased the clearance of [3H]thymidine from the CSF. Rabbit brain slices accumulated [3H]thymidine by an energy-dependent, saturable high-affinity system that depended, in part, on intracellular phosphorylation of the [3H]thymidine. These results were interpreted as showing that the entry of thymidine from blood into CSF and presumably the extracellular space of brain and then into brain cells involves one or more saturable transport and/or metabolic steps.