Injection Of Thymidine Research Articles

Induction of antral gastrin cell proliferation by refeeding of rats after fasting

The proliferation of antral gastrin cells after fasting and refeeding of rats was studied by using a quantitative histologic method for determining the gastrin cell number and a radioautographic technique after injections of tritiated thymidine for recognizing and quantitating the newly formed gastrin cells. The total number of gastrin cells decreased 68% (P < 0.01) after a 4-day fasting period, whereas refeeding of rats during 6 days after a 4-day fasting period resulted in a 79% (P < 0.01) increase of the gastrin cell mass. The labeling index of gastrin cells after six daily injections of tritiated thymidine given during this period of refeeding was significantly (P < 0.01) increased when compared with this value in control animals. The observed intensity of the proliferative response during refeeding was compatible with the increase in gastrin cell number observed during refeeding. These observations indicate that a new population of gastrin cells is formed in the antral glands when rats are refed after fasting.

Kinetics of deoxyribonucleic acid synthesis and replication of thyrotrophs and somatotrophs during development of hypothyroidism and L-triiodothyronine treatment of hypothyroid rats.

The rates of DNA synthesis and cell replication were measured in somatotrophs (St) and thyrotrophs (T3) in vivo during the development of hypothyroidism and during T3 treatment of hypothyroid rats. Groups of rats were killed at different times after [3H]thymidine injection for 30 days after thyroidectomy and during 20 days of T3 treatment (10 μg/100 g BW day) of rats thyroidectomized for 30 days. The number of St and Tt per pituitary was determined by a method combining cell counting and DNA measurement in dispersed cells as well as the percentage distribution of cells at the electron microscopic level. [3H]Thymidine incorporation into pituitary DNA was measured, and the distribution of incorporated counts among anterior pituitary cells was assessed by electron microscopic autoradiography. [3H]Thymidine incorporation into St and Tt DNA and the relative specific activity of these cell populations were then calculated. After thyroidectomy, the number of St decreased at a constant fractional rate (1.3% day1)...

Cell Renewal in Noncornified and Cornified Buccal Epithelium in the Rabbit

This study was undertaken to compare some aspects of cell renewal in the noncornified and cornified epithelia that are juxtaposed in the buccal mucosa of the rabbit. Specimens were analyzed by autoradiography and scintillation counting at various times after the injection of tritiated thymidine or triated proline. In the noncornified epithelium, the labeling index in the proliferative compartment at 1 hr was 15.5%; in the entire cell population, it was 5.8% at 1 hr, 10.4% at 24 hr, and 20.2% at 72 hr. The leading edge of labeled cells reached the surface by 96 hr. In the cornified epithelium, the labeling index in the proliferative compartment was 8.7% at 1 hr; in the entire nucleated cell population, it was 4.2% at 1 hr, 9.2% at 24 hr, and 12.1% at 96 hr. The leading edge of labeled cells reached the stratum corneum by 96 hr and the surface by 144 hr. It was concluded that renewal occurs at a more rapid rate in the noncornified than in the cornified epithelium under study.

The leucocyte alkaline phosphatase activity in mature neutrophils of different ages.

The marrow myeloid precursor cells of a haematologically normal patient were labelled by intravenous injection of tritiated thymidine. Young labelled segmented neutrophils were then released from the marrow into the blood by an injection of cortisol. These PMN had a significantly lower LAP activity than the older blood neutrophils. It is shown that within the morphologic boundaries of the segmented PMN, the cells are still in different stages of cytoplasmic maturation. In addition, labelled and unlabelled neutrophils showed a linear and parallel increase of LAP activity during the 24 h following cortisol injection. This observation suggests that the neutrophil prematurely released in the blood can mature into normal LAP=PMN and more generally that LAP activity of a blood neutrophil increases with time.

Repeated injection (continuous labelling) experiments in mouse epidermis

Theoretical labelling index curves for epidermis have been generated under conditions of repeated tritiated thymidine injection. These curves take into account different injection intervals, circadian fluctuations in labelling and two different models for epidermal proliferation; one based on a homogeneous basal layer with “random” loss initially (later, loss was restricted to late G 1), and the other based on a programmed sequential aging of proliferative cells in a compartment derived from a minority class of stem cells. These curves have been compared with previously published experimental results and with results from some new experiments. Both models fit the data to some extent provided a mean value of T c of about 140 h is assumed. However, the sequential aging model provides a slightly better overall fit. A further conclusion is that it is impossible to make any accurate statements on the epidermal growth fraction from repeated labelling data.

On the influence of topically applied drugs on cell kinetics in the sebaceous gland. Investigations with the example of selenium disulfide in the Syrian hamster ear.

60 male Syrian hamsters were treated on one ear with 2.5% selenium disulfide in a surfactant base and on the other ear with the same surfactant base. After treatment 15 animals were investigated by 3H-thymidine autoradiography, 15 animals by the colcemid method and 15 animals by the in vivo double labelling technique with 3H- and 14C-thymidine. In 15 animals the porportion of labeled cells was determined which still had contact with the basal lamina in the sebaceous gland 6 days after 3H-thymidine injection. Besides an increase in sebaceous gland size and in cell proliferation, a shortening of the S phase and a reduction of the labeled sebaceous gland cells which still have contact with the basal lamina of the sebaceous gland 6 days after 3H - thymidine injection were determined.

Cell Proliferation Kinetics in the Human Hair Root

The kinetics of cell proliferation in the matrix cells of human scalp hair were determined in vivo. Autoradiographic analysis was employed on biopsy specimens taken at specific time intervals after intradermal injection of tritiated thymidine (3H-TdR) into the scalps of volunteer subjects. The durations of the S and G2 phases (Ts and Tg2) were 11.1 and 3.6 hr respectively, obtained from the composite percent labeled mitosis curve. The labeling index of the hari matrix cells was 28.6% +/- 2.2%. The durations of mitosis and of the total cell cycle (Tm and Tgc) were calculated to be 0.8 hr and 38.8 hr respectively. The growth fraction of hair root germinative cells was estimated as 75-100% on the basis of "continuous" 3H-TdR exposure over the duration of the cell cycle. It is noted that the kinetics of human hair root cells are quite similar to the kinetics of psoriatic epidermal cells, suggesting that both tissues are approaching the maximum proliferative rate of keratinogenic epithelial cells.

Selective labeling of mesenteric lymph nodes: cell production and emigration of newly formed lymphocytes to other organs.

Lymphocyte production by mesenteric lymph nodes of normal young pigs was studied by intranodal injections of either tritiated thymidine or tritiated deoxycytidine as DNA precursors. One or two days after selective labeling of the mesenteric lymph nodes the relative and absolute number of lymphocytes derived from mesenteric lymph nodes were determined autoradiographically in the following organs: mesenteric, cervical and inguinal lymph nodes, spleen, thymus, bone marrow, Peyer's patches, tonsil, different regions of the gut, lung and liver. The overall cell production of mesenteric lymph nodes, as derived from the sum of all labeled cells one day after labeling, was estimated to be about 7 X 10(9) lymphocytes. Up to 40% of all newly formed lymphocytes had already left the lymph nodes within one day and were found in all organs studied. There was a preferential homing to the mucosa of the small intestine, but a considerable number migrated to the spleen and even to the thymus and bone marrow. In lymphoid organs all labeled cells were small and medium-sized lymphocytes one and two days after labeling. In cervical lymph nodes, spleen, tonsil and Peyer's patches the relative distribution to T and B cell areas was determined. There was an obvious preference of newly formed lymph node cells to home to T cell areas. The differences of labeling between thymidine or deoxycytidine were surprisingly low.

Specific triggering of macrophage accumulation at the site of secondary tumor challenge in mice with concomitant tumor immunity

Intraperitoneal injection of tumor cells triggers a greater influx of macrophages into the peritoneal cavity of mice with concomitant immunity than into control mice. The specificity of the reaction parallels the specificity of concomitant immunity. The newly arrived cells were classified as young monocyte-derived macrophages on the basis of morphology, position, and change in sedimentation velocity profiles, chemotaxis, and labeling in vivo after tritiated thymidine injection.

Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet.

The feeding of a high-fat diet to adult rats was shown to increase the incorporation of [3H]thymidine into DNA of the adipocyte and stromal fractions. After only 2 days on a high-fat diet there was a marked increase in the incorporation of label. When a 2-week period was interposed between [3H]thymidine administration and determination of DNA specific activity, the greatest increase in incorporation of label was found after 1 week on the diet, when incorporation increased 6-fold or more in both adipocytes and stroma and subsequently decreased to stabilize at a level two or three times that of chow-fed rats in the adipocyte fraction. Rats labeled when young and later placed on a high-fat diet showed a decrease in DNA specific activity in both adipocytes and stroma, confirming that cellular proliferation had occurred in both fractions. The specific activities of both stromal and adipocyte DNA were very similar at all time points studied. An attempt to increase the difference in specific activities by waiting many weeks after [3H]thymidine injection before isolating DNA was not successful. This may be because the total amount of DNA in the stromal and adipocyte fractions increases in parallel on the diet. The significance of these findings in terms of the normal turnover of adipose tissue DNA and the responsiveness to diet is discussed.

DURATION OF EGG-CHAMBER GROWTH IN YOUNG IMAGINES OF DROSOPHILA MELANOGASTER MEIG.

After [3 H] thymidine injection to new-hatching Drosophila melanogaster females the migration of labelled egg-chambers through the ovarioles was studied by autoradiographies of the ovaries at daily intervals. The growth duration of egg-chambers which progressed during the first 48 hr was 24 hr longer than the growth duration of chambers progressing thereafter. This temporary delay of ovarian activity was related to endocrine modifications occuring in female during this s period and particularly to a JH-ecdysone interaction.

Genesis of the primate neostriatum: [ 3H]thymidine autoradiographic analysis of the time of neuron origin in the rhesus monkey

The time of neuron origin and the spatio-temporal pattern of neuronal distribution in the caudate nucleus and putamen were examined in autoradiograms from 2- to 5- month-old monkeys that were exposed to a single pulse of [ 3H]thymidine on various embryonic or early postnatal days. Heavily labeled neurons, representing cells that entered their last cell division at the time of [ 3H]thymidine injection, were found in both the caudate and putamen in the animals injected between embryonic days 36 and 80, but not in monkeys injected at earlier or later stages. A peak in neuronal proliferation occurs between embryonic days 43 and 50. Thus, all of the neurons that ultimately compose the neostriatum are generated during a 45-day period in the first half of gestation, which lasts 165 days in the Rhesus monkey. Two classes of neurons, distinguished by their size and morphology, were discernible in autoradiograms stained with toluidine blue: small neurons with maximum cell body diameters ranging from 13 to 22 μm and larger cells with maximum diameters of 25–40 μm. The larger cells are generated over the first 7–8 days of striatal neurogenesis, while small cells are produced throughout the entire period. In agreement with previous [ 3H]thymidine autoradiographic studies in rodents, no spatio-temporal gradient related to the time of neuron origin was found in the primate neostriatum. However, small neurons generated on a given day were usually arranged in scattered clusters which, in a single coronal section, consisted of 5 to over 40 labeled neurons. These isochronously generated cell aggregates may represent a basic structural unit of the neostriatum.

Structural defects in rat liver deoxyribonucleic acid. Endogenous single-strained regions in comparison with damage induced in vivo by a carcinogen.

Rat liver DNA may be separated into two fractions by stepwise elution from benzoylated-DEAE-cellulose with NaCl and caffeine solutions respectively. Other studies using bacterical and yeast DNA suggested that the first fraction contains native DNA, whereas the second may exhibit some degree of single-stranded character. In the present experiments, chromatography of DNA was monitored by labelling in vivo with [methyl-3H]thymidine in rats previously subjected to partial hepatectomy. In animals killed up to 1 h after thymidine injection, radioactivity eluted in the second fraction was inversely related to the incorporation time, being greatest when animals were killed 10 min after radioisotope injection. However, for most experiments, animals were allowed to survive 2-4 weeks after surgery before use, analysis being made on non-dividing DNA. Under these conditions, the proportion of caffeine-eluted DNA was decreased by subjecting the preparation to shear, before chromatography. A procedure that resulted in 12% of the recovered radioactivity being eluted with caffeine was adopted for experiments involving comparisons of the two DNA fractions. Under these conditions, cross-contamination could be detected by rechromatography, but this did not preclude distinction being made between the two fractions in terms of DNA structure. NaCl-eluted DNA did not bind to nitrocellulose filters. Caffeine-eluted DNA was retained by the filters and released by washing with 3mM-Tris/HCl,pH9.4. The fractions did not differ in terms of isopycnic centrifugation in CsCl. The NaCl-eluted fraction migrated as a single band in polyacrylamide gels, and this pattern was not modified by prior digestion with Neurospora crassa endonuclease. In contrast, caffeine-eluted DNA contained a minor component having a wide molecular-weight distribution and was subject to limited digestion by the endonuclease. The kinetics of denaturation of NaCi-eluted DNA in the presence of formaldehyde, in common with unfractionated DNA, were consistent with double-stranded structure. The same analysis of caffeine-eluted DNA revealed structural abnormality equivalent to two defects per 10000 base-pairs. The data are consistent with the minor fraction of rat liver DNA, separated by using benzoylated-DEAE-cellulose, containing regions of local denaturation. We previously showed that administration of the hepatocarcinogen dimethylnitrosamine is associated with an increase in the proportion of caffeine-eluted DNA. In terms of most analysis, differences between DNA fraction from nitrosamine-treated rats were similar to differences exhibited by preparations from control animals. However, structural analysis using denaturation kinetics indicated defects in both the NaCl- and caffeine-eluted DNA isolated from nitrosamine-treated rats. The two fractions differed from each other in that caffeine-eluted DNA exhibited a degree of structural damage far greater than that detected in any preparation from control animals...

Histogenesis of the superior colliculus of the albino rat: A tritiated thymidine study

The pattern of generation of neurons in the albino rat superior colliculus has been studied in adult and fetal material. Neurons are generated between embryonic days 12 to 17, with rostrolateral colliculus in advance of caudomedial parts. More of the cells contributing to the deeper layers are generated early, while more of the later generated cells are located superficially. The cells of individual laminae are not formed on specific days as in the cortex, nor are the complicated gradients described previously for the chick optic tectum 18 evident. While the largest cells (found deep in the colliculus) are among the first formed and the small marginal cells among the last, there is in general a broad range of cell size being generated at any one time. The observed patterns are consistent with the concept of simultaneous production of several cell types from the ventricular epithelium on any given day. Studies of material at short times after [ 3H]thymidine injection allow correlation of the time of arrival of cells in their appropriate layer with time of arrival of afferents. In addition they suggest that factors controlling the final placement of cells in the mature nervous system is a very complex process and may involve some form of intercellular recognition.

Proliferative and migratory activity of glial cells in the partially deafferented hippocampus.

The proliferative response of the glial cell population of the adult rat hippocampus deafferented by unilateral lesion of the entorhinal cortex was studied using 3H-thymidine autoradiography. Two experimental paradigms were used, involving: (1) intraventricular 3H-thymidine injection at a number of post-lesion intervals with sacrifice six hours later and (2) intraventricular injection at 30 hours post-lesion with sacrifice at 6, 96, or 192 hours later. The first increase in the number of labeled glial cells was obtained at 20 hours post-lesion and was confined to areas of degenerating axons. By 30 hours a large and uniformly dense proliferative response was observed throughout the ipsilateral, and medial aspects of the contralateral, hippocampus encompassing both deafferented and intact regions. Cell division continued through 50 and 65 hours post-lesion particularly in directly deafferented regions, but diminished to control levels by 80 hours. Although oligodendroglia and astrocyte-like cells were sometimes found to have incorporated the label the most common proliferative element within the hippocampus corresponded to previous light microscopic descriptions of "microglial" cells. The experiments using thymidine injection given at the peak proliferative period followed by survival periods of varying lengths indicated that a progressive redistribution of labeled nuclei occurred resulting in an accumulation of labeled cells in the zones of deafferentation. Multiple division of cells within these areas as well as the migration of nuclei from non-deafferented regions was found to contribute to this effect. The possible involvement of glial proliferation with other morphological effects of deafferentation, including the sprouting response of intact afferents, is discussed.

Autoradiographic study of the development of the neostriatum in the rabbit.

Autoradiographic labelling has been employed to analyze the morphogenesis of the neostriatum. Pregnant rabbits received a single intraperitoneal injection of tritiated thymidine at different stages of gestation. Careful microscopical observation of the autoradiographs shows that cellular components of the neostriatum originate between days 15 and 18 of the intrauterine life from a layer of proliferating matrix cells that lies on the floor of the anterior part of the lateral ventrical (ganglionic eminence). From this proliferating layer, precursor cells migrate outwards to reach the developing neostriatum in a sequential fashion according to two gradients of histogenesis. Thus, it was found that neurons formed at early stages occupy a ventromedial position in the neostriatum, while those formed at later stages occupy a dorsolateral position (ventromedial to dorsolateral gradient). Furthermore, the present study indicates that the rostral regions of the neostriatum arise somewhat later than the caudal ones, demonstrating the existence of a caudocephalic gradient of cytogenesis.

Growth and cell replacement in the new-born pig intestine.

Both macroscopical and microscopical aspects of growth in the pig small intestine have been measured and related to the kinetics of cell turnover and changing physiological function in the period immediately following birth. The first 10 days of post-natal life was associated with an 80 % increase in length and a 30 % increase in diameter of the small intestine. The absorptive area doubled during this period of post-natal growth. Villi formed at birth were 29, 50 and 75 % longer by day 10 (proximal, mid and distal regions respectively). Maximal elongation occurred on day 6 of post-natal life. Thymidine labelling of cells showed mitosis to be confined to the crypts. Newly formed cells migrated onto the villi. The mean rate of cell appearance in a section was 0.4 cell/h. This rate remained unchanged and independent of the area of the small intestine chosen for study during the first 4 days of post-natal life. ^Examination of villus sections at different times following injection of radioactive thymidine showed considerable cell mixing to occur. Labelled cells, which never formed protein-filled vacuoles, became mixed with vacuolated and non-vacuolated, unlabelled cells. There was also a redistribution of labelled cells upon the villus. The level of radioactivity within cells did not always correspond to that predicted from their respective positions on the villus. Two alternative theories are put forward to account for these findings. The time taken for complete cell replacement is approximately 19 days. The mucosal uptake of macromolecules finally ceases and the resistance of piglets to enteric viral infections increases at about this time. It is suggested that both events are dependent on the final disappearance of cells originally present in the pig intestinal mucosa at birth.

The relationship of cell division to the acquisition of adrenergic characteristics by developing sympathetic ganglion cell precursors

The relationship of the acquisition of defining characteristics by precursor cells of sympathetic ganglia to the withdrawal of these cells from the cell cycle was investigated in the developing chick. The characteristics studied included the ability to synthesize catecholamines (CA), the development of characteristic subcellular storage granules, and the specific uptake of norepinephrine (NE). All were present in presumptive sympathicoblasts and adrenal medullary precursors, which also became labeled after the injection of tritiated thymidine and so retained the ability to divide. These dividing CA-containing cells were found in both primary ganglia and secondary preand paravertebral ganglia. The developing sympathetic neuronal population was found to be a heterogeneous one. Some sympathetic precursor cells appeared to become postmitotic (or to enter a pause in division) early in ontogeny, while others continued to divide throughout the time of hatching. As embryogenesis proceeded, the proportion of CA-containing cells or their precursors which were dividing decreased. However, those cells which did divide probably divided repeatedly. It is concluded that some of the definitive characteristics of mature neurons are expressed by dividing precursor cells. The specific characteristic that marks the transition from immature dividing cells to mature postmitotic neurons has not yet been determined.

The development of the cerebral cortex in the embryonic mouse: an electron microscopic serial section analysis.

AbstractThe techniques of reconstructions of cells from serial thin sections and autoradiography after tritiated thymidine injections have been employed to study the early histogenesis of the cerebral cortex in the embryonic day‐15 (E15) mouse. The autoradiographic studies show that cells below the E15 cortical plate in the intermediate layer are destined to migrate through the preexisting cortical plate cells to take up a more superficial position. Having this information, it has been possible, through reconstructions of large numbers of cells (more than 150) throughout the thickness of the cerebral vesicle, to identify some of the important morphogenetic events of cortical histogenesis. The following scheme is proposed. The first step in neuronal differentiation involves the detachment of the ventricularly directed process of the ventricular cell from the junctional region next to the ventricle. In thin sections, these junctions have the appearance of zonulae adherentes, but freeze cleavage experiments performed in this study show that, in addition, some of them resemble small gap junctions while others appear to be remnants of tight junctions or possibly linear gap junctions. Detachment of the ventricular process accompanies the migration of the nucleus and perikaryon through the ventricular layer. Within the intermediate layer the migrating cells become rounded and sprout numerous processes. Some cells may undergo a mitotic division at this stage. Eventually the differentiating cells sprout a longer lateral process which is oriented tangentially to the pial surface. This process originates from the anterior surface of the soma and at its tip has the characteristics of an axonal growth cone. The cells migrate externally and radially with simultaneous elongation of the primitive axon. In the subcortical plate region of the intermediate layer all cells contain an anteriorly directed axon. Subsequently the cells sprout an apical process which extends into the cortical plate, and the nucleus and perikaryon apparently migrate radially within this process. The result is that the primitive axon first descends into the intermediate layer proper before turning to run tangentially. Dendritic growth and further differentiation begins once the cells reach their definitive position in the cortical plate.One interesting finding is the presence of eight cells in the cortical plate without long anteriorly directed axons. Yet, autoradiographic data show that subcortical plate cells are the immediate precursors of cortical plate cells, and all 28/s28 reconstructed subcortical plate cells have long anteriorly directed axons. Thus, it is possible that the long axon of some cells may be lost as the cells continue to differentiate in the cortical plate. In fact, one cell has been found which appears to be in the process of losing its anteriorly directed axon.A number of molecular layer cells have also been reconstructed. These cells have several processes oriented tangentially to the pial surface. The identity of these processes could not always be determined. Occasional asymmetric synapses have been found between unidentified axons and the horizontal cell soma or its processes. Autoradiographic studies show that horizontal cells have the earliest time of origin of any cortical cell type.

Localization of tagged cells in the islets, acini, and peri-insular zone of the mouse pancreas after resection

[3H]Thymidine was injected five times in the course of the 24-h period into CBA x C57BL/6 hybrid mice in which about 40% of the tissue of the pancreas has been resected. The number of tagged cells in the islets and acini did not change with an increase in the time elapsing after the last injection of thymidine (5–18 days). The localization of the tagged cells in the pancreatic islets in the later stages did not differ significantly from that observed 2 h after injection of the isotope into the animals. On examination of medium-sized and large islets as far removed as possible from the site of injury, a very similar number of tagged cells per unit area was found in the central, middle, and peripheral zones of the islets 2 h and 18 days after the last injection of the isotope.