Injection Of 3H-TdR Research Articles

Kinetics of neutrophil production following filgrastim sd/01

Filgrastim SD/01 is a new form of Filgrastim (r-metHuG-CSF) with sustained effective duration in vivo (Molineux et al. Exp Hemat 27,1999,1724). We have compared its ability with that of conventional Filgrastim to stimulate neutrophilia in normal and leukopenic mice. We have also measured the kinetics of neutrophil production in normal mice following stimulation with SD/01. SD/01 (1 g/kg) was injected iv to normal mice 3 d after injection of cyclophosphamide (200 mg/kg), and temozolamide (90 mg/kg). Subsequent WBCC and neutrophils were recorded daily. Conventional filgrastim was injected 2 × daily (125 mg/kg) for 3 d. For production kinetics, normal mice were injected with tritiated thymidine (3HTdR, 37KBq/g) 3 d after SD/01. Bone marrow cytospins (@ 1 h) and blood smears (@ 1 h to 7 d later) were autoradiographed. Differential cell and 3HTdR labelling indices were then counted. In normal mice, neutrophil counts increased 16 fold over 4 d. In leukopenic mice they increased from their 3 d nadir of 105/ml to 26 × 106/ml 4 d later. 3HTdR labelling of maturing granulocytic cells in the marrow was 50–100% higher than in non-stimulated mice. Labelled mature neutrophils peaked in the peripheral blood 24 h (compared to 3-4 d in normal animals) after injection of 3HTdR. Stimulation of neutrophil production by a single inoculum of SD/01 mirrors that of conventional G-CSF for which repeated injections over at least 3 d are necessary to maintain effective plasma levels. Analysis of the kinetics of granulocyte production shows accelerated proliferation in the marrow followed by rapid release into the peripheral blood of neutrophils with a normal peripheral half-life. We conclude that granulocytopoiesis is modulated by SD/01 in exactly the same way as the conventional formulation, and is considerably more manageable in its application.

Detection of a population of long-lived cells in mammary epithelium of the mouse.

The fate of dividing mouse mammary epithelial cells was followed by use of tritiated thymidine (3H-Tdr) autoradiography. Loss of label consistent with halving kinetics was observed at various times after injection; however, heavily labelled cells were frequently observed at two weeks and later, when none was expected. The grain count over these heavily labelled cells was often comparable with that 1 h after 3H-Tdr injection. Extensive serial sectioning revealed that the heavily labelled cells were often single cells surrounded by many unlabelled cells or that their label was in stark contrast (in excess of 20 reduced silver grains) to the surrounding group of cells whose label was just above background (a maximum of 3 grains). In addition, by injecting mice at different stages of oestrus, we demonstrated that these long-lived cells, although influenced by oestrus, replicated independently of the oestrogen peak. Our data support a model for mouse mammary epithelium that has a single 'stem' cell positioned within a group of its progeny to form a discrete proliferative unit. This model requires many such stem cells within the mammary epithelium and is consistent with similar models proposed for other tissues.

Age-related changes in replication of myogenic cells in mdx mice: Quantitative autoradiographic studies

Cell replication in muscle was measured by tritiated thymidine ( 3H-TdR) incorporation and autoradiography, in mdx mice from 2–44 weeks of age. Pre-mitotic labelling (within 1 h of 3H-TdR injection) was determined in 16 mice aged from 15 to 300 days. In 30 further mdx mice, one leg was irradiated 1 h after 3H-TdR injection to block DNA synthesis. Post-mitotic labelling was measured in both legs 10–15 days later. Between 20 and 60 days of age a very high proportion (up to 2%) of muscle (satellite cell) nuclei were replicating pre-mitotically; from 80–300 days cell replication was detectable but at much lower levels. Centrally placed nuclei within muscle fibres appeared at 24 days, increased rapidly to 50% by 50–100 days, declining thereafter to 25% at 300 days. In post-mitotic samples, labelled myotubes and labelled peripheral muscle nuclei (satellite cell nuclei and myonuclei) appeared at 28 days and were present in the mdx muscles through to 310 days, indicating continued cell replication and muscle regeneration. Myogenic cell replication was both retarded and inhibited by irradiation. These data demonstrate that muscle cell replication in mdx mice commences at about 3 weeks of age, is maximal at 4–8 weeks, but continues at lower levels until at least 44 weeks.

Haemopoietic cell kinetics in humans treated with rGM-CSF.

We have investigated the kinetics of myeloid cell proliferation in the marrow of patients with small-cell lung cancer and treated with 10 daily subcutaneous injections of granulocyte/macrophage colony-stimulating factor (GM-CSF). Bone marrow, obtained before and during treatment with the growth factor, was labelled in vitro with tritiated thymidine (3H-TdR). A 3rd bone-marrow sample was obtained 1 hr following an intravenous injection of 3H-TdR. Subsequent daily blood samples were also collected, and 3H-TdR labelling was assessed on these and the marrow preparations by autoradiography. GM-CSF treatment increased the peripheral granulocytic cells nearly 5-fold, but this included significant eosinophilia, so that the neutrophilic granulocytes increased only 3.3-fold. These cells were released from the marrow over a normal time scale, but their peripheral half-life was about 6 times longer than normal and they were probably functionally defective. Furthermore, significant numbers of immature cells were released from the marrow. Neutrophil production stimulated by GM-CSF was thus overestimated by measurement of the apparent peripheral granulocytosis. Increased labelling indices and grain counts in the proliferating granulocytic cells of the marrow indicate shortened cell-cycle times, and the excess granulocyte production appears to be the result of extra amplification divisions in the proliferative compartments.

Unscheduled DNA synthesis in various types of cells of the mouse brain in vivo.

Very low incorporation of 3H-thymidine (TdR) into neurons and non-proliferating glial and endothelial cells in various brain areas of the adult mouse after 3H-TdR injection and subsequent X-irradiation of the head with 45 Gy has been demonstrated autoradiographically after exposure times of 250 days. In accordance with biochemical studies this incorporation of 3H-TdR represents DNA repair synthesis or UDS (unscheduled DNA synthesis). However, 3H-TdR incorporation into nuclear DNA of non-proliferating cells in the brain was not only found in X-irradiated but also in sham-irradiated mice. This suggests that spontaneous UDS also occurs. Up to now spontaneous UDS has been shown only in HeLa cells in vitro. Nearly all the various types of brain cells tested exhibited UDS after X-irradiation as well as spontaneous UDS. After correcting the mean grain numbers per nucleus not only for background but also for beta-self-absorption, substantial differences became apparent in the extent of UDS between the individual types of cells. After X-irradiation, UDS was highest in Purkinje cells and hippocampal granular cells but comparable UDS was also found in endothelial cells, regardless of the different brain areas studied. The extent of spontaneous UDS is also quite different in the various cell types, being highest in neurons of different sites and considerably lower in endothelial and glial cells.

Three-dimensional presentation of cell migration in the periodontal ligament of the rat incisor.

The progenitor compartments and cell migration were examined in the tooth-related periodontal ligament (t-PDL) of rat incisors. A pulse injection of 3H-Tdr was administered to 15 rats (200 gm each) and the animals were killed in groups of five, at 1 hr and at 1 and 2 weeks after injection. Three-dimensional analysis of cell counts and labeling index demonstrated the existence of two progenitor compartments (PC). The apical PC (responsible for 70% of synthesizing cells) was concentrated in the apical 5 mm of the t-PDL. The paracemental PC (30% of synthesizing cells) was located along and around the cementum, occupying 24 micron of the t-PDL. The cells from the apical PC migrated incisally at a rate of 6 mm/week, which is a rate similar to that of tooth eruption. The cells from the paracemental PC moved in a transverse direction toward either bone or cementum at the much slower rate of 16 micron/week.

The fate of epidermal colcemid-arrested mitoses.

This study reports the fate of hairless mouse epidermal basal cells arrested in mitosis by a traditional stathmokinetic dose of 0.15 mg Colcemid. Epidermal basal cells in the S phase were labeled with 30 microCi (3H)TdR i.p. After 1 h, four animals from a cage of eight mice were given 0.15 mg Colcemid (Fluka) in 0.5 ml saline, and the other four mice were given saline only. Groups of eight mice (four experimental, four controls) were sacrificed 4, 9, 13, 21 and 25 h after (3H)TdR injection (i.e. 3, 8, 12, 16, 20 and 24 h after Colcemid). The following cell kinetic parameters were determined: the number of labeled basal and suprabasal cells, the mean grain count of the labeled cells, the specific activity, the mitotic count, the number of labeled mitoses, the fraction of labeled mitoses curve and the fraction of cells in S and in G2 as determined by flow cytometry. "Labeled paired twins", i.e. adjoining labeled cells with approximately the same grain count, were also scored. All the results taken together support the conclusion that cells labeled with (3H)TdR and arrested 1 h later with 0.15 mg Colcemid go through at least one subsequent cell division and thereafter some of them move out into the suprabasal layer at a normal rate. Hence, after this dose of Colcemid, cells arrested in mitosis for some hours do not die, and the Colcemid treatment does not seem to produce hyperploid cells. The study confirms the usefulness of this dose of Colcemid as a convenient tool for cell kinetic studies.

Compensatory proliferative response of the rat submandibular salivary gland to unilateral extirpation.

The present experiment was conducted in order to identify the progenitor compartment of the submandibular salivary gland (SSG) and to explore the proliferative activity of this gland in response to unilateral extirpation. Left submandibular and retrolingual glands were extirpated in 30 rats (B.W. 200 +/- 12 g). The rats were killed 0, 1, 2, 3, 5 and 7 days after surgery. Five intact rats served as controls. The animals were given intraperitoneal injections of 3HTdR (0.5 microCi/grB. W) 1 h before they were killed. The contralateral SSG's were subjected to routine histological procedures and embedded in glycol methacrylate. Selected sections (2 micron thickness) were processed for autoradiography. In each gland, labelled and unlabelled nuclei were counted in 50 random microscopic fields and sorted according to their parenchymal histomorphological features and "nuclear class" (number of nuclei/cross section/feature). In the control glands the total labelling index (LI) was 0.18%; during acute compensatory stimulation, however, the total LI reached a maximum of 0.86% on day 3 after surgery. suggesting that the SSG, which normally undergoes a slow turnover, is capable of elevated proliferation in response to a stimulus. In both normal and stimulated glands, the LI was higher in the intercalated ducts (1.1%-5.85%) than in the granular ducts (0.17%-0.93%) and acini (0.05%-0.36%). This consistency of LI ratio between the various histomorphological features in the normal and experimental glands indicates that the glandular progenitor compartment is located in the intercalated ducts, which supply cells to both the ductal system and acini.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effects of hydroxyurea on DNA synthesis in hairless mouse epidermis

The effect of 5 mg hydroxyurea (HU) i.p. on epidermal DNA synthesis in female hairless mice was assessed by measuring labelling indices and specific activity after 3HTdR injection, flow cytometry (FCM) and cell sorting of prelabelled basal cells. HU causes an almost immediate block in DNA synthesis lasting until 2-2.5 h. During this time the fraction of cells in S remains stationary, 1.20 of normal. From 2.5 to 12.5 h DNA synthesis is resumed, but in cells recruited from G1 or G0. The HU-blocked cells do not move out of S until after 12.5 h. Hence, from 2.5 to 12.5 h, the fraction of cells in S increases to 2.5 of normal, which means that entry into S is open, but exit is blocked. From 12.5 h flux through S is high. The blocked cells are now released and the fraction of cells in S falls to 0.7 of normal at 24.5 h. At 36.5 h a probable new wave of DNA synthesis is indicated. The results also show that 3HTdR is available for at least 20 min after i.p. injection. The consequences of these results for the interpretation of the effect of HU pretreatment on methylnitrosourea skin carcinogenesis are discussed.

Tissue concentrations and proliferative effects of massive doses of ascorbic acid in the mouse.

The effect of ascorbic acid supplementation on CF1 mice fed ascorbic acid for approximately six months at dose levels of 1%, 5%, and 10% of diet was investigated by analysis of tissue ascorbic acid concentration in the liver, kidney, stomach, small intestine, and large bowel. The effect on epithelial cell proliferation was also examined in the small and large bowel but only at the 5% level. In the control animals, ascorbic acid concentration was lowest in the liver (0.406 +/- 0.07 mg/g) and highest in the small bowel (0.754 +/- 0.16 mg/g). Dietary intake of 5% and 10% ascorbic acid significantly elevated levels in the liver (0.741 +/- 0.13; p less than 0.05), and all doses of ascorbic acid significantly raised tissue concentrations in the kidney and colon. No difference was observed in the percentage of DNA-synthesizing cells in the jejunum of controls or animals fed 5% ascorbic acid at 1 or 24 hours after 3HTdR injection. However, at 1 hour a significantly decreased level of proliferation was observed in the distal colon of ascorbic-acid-treated mice compared with controls (labeling index [L.I.] = 7.3 +/- 0.28 vs. 10.1 +/- 1.15; p less than 0.05), and an even greater suppression of DNA synthesis was achieved by 24 hours (L.I. = 11.4 +/- 1.06 vs. 18.6 +/- 1.61; p less than 0.01). None of the doses of ascorbic acid employed was toxic to the experimental mice.

Osteoclast cell‐surface specializations and nuclear kinetics during egg‐laying in Japanese quail

Medullary bone deposits serve as a reservoir of labile calcium for egg-shell calcification in birds. Quantitative transmission-electron-microscope methods and light-microscope autoradiographic cell-population-kinetic analyses were used to determine changes in cell-surface specializations and population dynamics of medullary bone osteoclasts during egg-laying in Japanese quail. Prior to egg-shell formation, from 0 to about 8 hours after the previous oviposition, very few osteoclast profiles had ruffled borders. The appearance of ruffled borders coincided with the beginning of egg-shell calcification, about 9-10 hours after the previous oviposition. During egg-shell calcification, about 10-21 hours after the previous oviposition, most osteoclast profiles had ruffled borders. Ruffled borders disappeared at the completion of egg-shell calcification and commencement of egg-shell pigmentation. Thus, functional activities of medullary bone osteoclasts appear to be closely synchronized with egg-shell calcification during egg-laying. From 1 to 48 hours after a single injection of 3H-thymidine (3H-TdR), very few labeled osteoclast nuclei were seen during egg-laying. Following multiple injections of 3H-TdR, the percentage of labeled nuclei reached a peak at about 170 hours after the first injection. At this peak-labeling time, relatively few of the osteoclast profiles that had labeled nuclei had two or more; although the average number of nuclei per osteoclast profile was about 3.6. These kinetic data suggest that the medullary bone osteoclast population has a prolonged rate of turnover compared to rapid changes in cell activities associated with each 24-hour egg-laying cycle; and collectively they would suggest that rapid changes in osteoclast functions occur independently of changes in cell-population dynamics.

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.

Autoradiographische Untersuchungen über die Zellkinetik der Kryptenepithelien im Jejunum von Ratten nach experimenteller Blockade des Ductus thoracicus in Höhe des linken Venenwinkels

These investigations were carried out on rats with a thoracic duct blockade at the base of the neck. In these animals we determined the labeling index, the mitotic index and the number of cells per villus and per crypt. Growth fraction (G. F.), the percentage of labelled mitoses (P.L.M.) curves, and the results of the 3H- 14C-TdR double labeling method were obtained from rats on the 7 th postoperative day. Availability was determined by counting silver grains above the nuclei of epithelial crypt cells 20, 40, 60, and 120 min. after a single injection of 3H-TdR. The availability of 3H-TdR is given by the time at which the curves reach a saturation level. In these experiments we found a depresssion in the 3H-TdR labeling index as well as in the mitotic index and in the number of epithelial cells per crypt and per villus. DNA synthesis time (Ts), generation (cycle) time (Tc), and G1 time (T G1) were increased whereas G2 plus mitotic time (T G2 + T M) and the availability remained unchanged. The G.F. decreased from 59% in the controls to 45% in the thoracic duct operated animals. The depression of the 3H-TdR index and the mitotic index was not due to an increase in Ts, T G1, and Tc, but to a decrease in the G.F. As a consequence of this decrease in the G.F. and the increase in cell cycle times, we found a reduction of the number of epithelial cells per crypt and per villus. After 2–3 weeks all parameters had returned to normal. These experiments therefore further strengthen our previous studies on liver and kidney of rats after thoracic duct blockade.

Double Labeling Autoradiography.Cell Kinetic Studies with 3H- and 14C-Thymidine 1.

Examples of the multiple applicability of the double labeling method with 3H- and 14C-TdR are demonstrated. Double labeling with 3H- and 14C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of 3H-TdR. This results in a considerable improvement of the resolution of the percent labeled mitoses curve. In addition, data is provided on the variances of the transit times of the cells through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the next S phase of the 3H-TdR-labeled S phase cells can be registered by injecting 14C-TdR at different time intervals following 3H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and 14C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. Up to now studies of the effect of cytotoxic drugs on cells in different cycle phases were confined to in vitro experiments, since such studies need synchronized cells. However, the double labeling method, which leads to well-defined subpopulations of differently labeled cells in different phases of the cycle, allows such studies to be carried out under in vivo conditions. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.

Differences in utilization of tritiated thymidine and tritiated deoxycytidine by rat lymph node cells

Large lymphoid cells and plasma cells in antigenically stimulated rat lymph nodes retain less label after injection of 3H-CdR than after injection of 3H-TdR. There is no difference amongst small lymphocytes. The data are consistent with a defect in the utilization of 3H-CdR in the late stages of B cell maturation in the rat.

Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. II. The irradiated testes.

In adult male mice exposed to 300 R X-irradiation, the spermatogonial population was selectively killed except for the radioresistant type As stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stage 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of 3HTdR were given between 2 and 3.5 days post-irradiation, 90-95% of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and at times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of 3HTdR prior to irradiation, there was appreciable retention of label by those type As spermatogonia that had originally incorporated 3HTdR in stages 2-4. This labeling pattern was identical to that of the long-cycling As stem cells in nonirradiated testes. Since the long-cycling As stem cells are thought to be characterized by a prolonged G1 or "A-phase" which is known to be a highly radioresistant portion of the cell cycle, it was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed that following germ cell depletion, as after irradiation injury, the long-cycling As survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population.

Growth fraction and cycle duration of hepatocytes in the three-week-old rat.

The proliferation of hepatocytes in the liver of 3-week-old rats has been investigated by autoradiographic methods. This investigation is a continuation of earlier work on the same topic (Schultze & Maurer, 1972; 1973). 21 days after birth, 102 rats received a single injection of 3H-TdR. The percentage of labelled mitoses was then determined 1 hr later and at various times throughout the interval up to 12 days after application of 3H-TdR. In agreement with earlier work, a first peak of labelled mitoses was found 7 hr after 3H-TdR injection. The area under the peak indicates an S phase duration of 8 hr. In addition a second very broad peak of labelled mitoses was found between 2 and 12 days after pulse labelling. The analysis of the results leads to the conclusion that the hepatocytes of the 3-week-old rat have a growth fraction close to 1 and a doubling time of 6-7 days. This is at variance with earlier results of Post, Huang & Hoffman (1963) and Grisham (1969) who had derived a value of 21.5 hr for the duration of the cell cycle and a value of only 0.1-0.2 for the growth fraction of the hepatocytes.

Effect of Cyclophosphamide on Interstitial Nephritis and Tubule Cell Proliferation in NZB/NZW Mice

Abstract The effect of cyclophosphamide on renal interstitial mononuclear cell infiltration and renal tubule cell (TC) proliferation has been examined in (NZB × NZW)F1 hybrid (B/W) and control mice. TC proliferation was measured by tritiated thymidine (3HTdr) injection, autoradiographic examination of kidney sections, and enumeration of labeled tubular cells. Cyclophosphamide administered 2 months before sacrifice virtually obliterated interstitial infiltration and significantly decreased tubule cell proliferation at 3, 5, 7, 9, and 12 months of age.

Age-related changes in numbers of newly-formed and long-lived small lymphocytes of normal and nude balb/c mice

To evaluate fluctuations in numbers of newly-formed and long-lived small lymphocytes by ageing, quantitations of cell numbers in the lymphomyeloid tissues of normal and congenitally athymic (nude) mice have been combined with autoradiographic results obtained 90 h after start of intensive 3H-TdR injections. It was shown that both the numbers of newly-formed and long-lived small lymphocytes of the lymph nodes and thymus declined by advancing age. However, high numbers of newly-formed small lymphocytes were present in bone marrow and spleen of old mice after a decline in younger adult mice. In the bone marrow a consistent age-related rise was seen in the numbers of long-lived small lymphocytes. High numbers of these cells were also found in the spleens of old mice. The production of small lymphocytes in the bone marrow of nude mice declined by ageing and was smaller than that of corresponding age groups of normal mice.