Injection Of Tritiated Thymidine Research Articles

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.

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.

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.

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.

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.

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.

An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats.

Satellite cells were traced autoradiographically during the regeneration of skeletal muscle in young Sprague-Dawley rats. Approximately 31% of the satellite cells in uninjured muscles appeared labelled after three injections of tritiated thymidine; none of the myonuclei were labelled in the same muscles. Four to six days after transplanting the radioactive muscles to non-radioactive littermates, regenerating myotube nuclei in the host appeared labelled. Thus, this study confirms that satellite cells in young rats can differentiate into multinucleated myotubes following muscle injury.

Cell proliferation and morphogenesis of the apical ectodermal ridge in the pectoral fin bud of the trout embryo (Salmo trutta fario L.)

Cell proliferation has been studied in the pectoral fin bud of the brown trout (Salmo trutta fario L.). A single injection of tritiated thymidine into the coelom was performed at stages 160, 180 or 200. Fixations were made at increasing intervals between 30 min and 12 days after the injection. The incubation temperature was 7° C, stage 244 being attained 12 days after injection at stage 160.Graphs of the variations of the labelling index, the mitotic index and the percentage of labelled mitoses as a function of the time after injection of tritiated thymidine at stage 160 have been drawn for the periderm, the basal layer of the ectoderm and the mesoderm. The duration of the cell cycles and of their phases were deduced from the graphs. Results are summarized in the table.Knowledge of the parameters of cell proliferation in the mesoderm and the ectoderm of the fin bud is of great interest for the analysis of morphogenetic mechanisms leading to the formation of the apical ectodermal ridge. The dorsal ectoderm of the bud proliferates intensely between stages 158 and 167, and this might lead to the initiation of the formation of the apical ectodermal ridge. Following that first period, the ventral ectoderm, in turn, proliferates more actively than the dorsal one between stages 170 and 209. In the subjacent mesoderm two proliferative crises occur at stages 161.75 and 170, thus probably causing or permitting the extension of the dorsal and ventral parts of the ectoderm. This alternative proliferative activity of dorsal and ventral ectoderm finally results in the formation of an apical ectodermal ridge that becomes elongated into an apical fold, which itself will give rise to the swimming paddle of the fin including its skeletal elements (lepidotrichia andactinotrichia).

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.

Histochemical and autoradiographic study of injured and uninjured dystrophic ( [formula omitted]) and normal muscle

A combination of morphological, histochemical, and autoradiographic techniques was used to study muscle regeneration in the soleus and gastrocnemius muscles of normal ( C57BL 6J +/+ ) and dystrophic ( C57BL 6J dy 2 J dy 2 J ) mice. The present study is the first demonstration that dy 2 J dy 2 J muscle can successfully regenerate in vivo. The presence of labeled nuclei in dy 2 J dy 2 J muscle fibers as long as 42 days after injection of tritiated thymidine confirmed the ability of dy 2 J dy 2 J muscle to support both spontaneous as well as injury-induced muscle regeneration. Many of these regenerating fibers were capable of becoming histochemically “normal” (i.e., red, intermediate, white) in terms of their succinic dehydrogenase, alkali-stable ATPase, and acid-stable ATPase staining. The comparative pattern and rate of histochemical and morphological changes in the labeled regenerating normal and dy 2 J dy 2 J muscle fibers was similar. After a crush injury, the dystrophic muscle was able to regenerate approximately the same percentage of histochemically “normal” and “abnormal” type fibers as in its contralateral uninjured control muscle. The injured dy 2 J dy 2 J muscle did not appear to become more severely “dystrophic” as a result of the injury. The results reported here for the dy 2 J dy 2 J mice were compared with similar studies dealing with the dy dy form of muscular dystrophy.

An autoradiographic analysis of postnatal cell proliferation in the normal and degenerative mouse retina.

AbstractLight microscopic autoradiography, following injection of tritiated thymidine, was used to study the postnatal development of cells in the inner nuclear layer (INL) of the normal and rd (C57BL/6J‐rd le) mouse retina. Specific attempts were made to determine whether some bipolar cell degeneration occurs in conjunction with previously noted photoreceptor cell degeneration in the retina of the mutant (rd) mouse. A differential count of cell types in the INL of both the central and peripheral retina indicates that the same ratio of bipolar cells to Müller cells (3:1) exists in the normal and the rd retina during postnatal development. The density of cells in the INL was similar in both groups during development and similar decreases in width of the INL occurred in the normal and rd mouse during maturation. In addition, autoradiographic analysis of the proliferation of postnatally forming bipolar cells suggested that the rd gene is not lethal to the bipolar cell population which forms after birth. The labeling index (percent labeled cells) in the INL, determined as a function of the date of 3H‐thymidine injection and assessed in the mature retina (day 41), was similar in both mice. Analysis of the number of silver grains/nucleus presented a more complete picture of how the progenitor pool of cells contributes to the cell types formed postnatally in the retinas of both mice.The conclusion that bipolar cell death does not occur to any significant degree in the retina of the rd also suggests that transneuronal degeneration does not occur in the retina of the mutant mouse during the period (up to day 41) when most of the photoreceptor cells die. The observation in both groups of mice that cell density remains constant between postnatal day 14 and day 41, while the width of the INL changes tremendously, suggests that there is a significant redistribution of cells during growth of the eyeball and/or cell death which occurs in all cell types found in the INL in both the normal and mutant mouse.

Actinic keratoses and the epidermis on which they arise.

The appearance of the epidermis and epidermal autoradiographic labelling indices were compared in actinic keratoses and paralesional skin in seventeen patients after injection of tritiated thymidine. The skin on the buttock area was also studied as a "non-sun exposed" control. Labelling indices obtained from skin removed after 1 hwere as follows: actinic keratoses 17-4%; paralesional skin 11-2%; buttock skin 5-4%. Many of the epidermal cells in the keratoses that incorporated tritiated thymidine were bizarre and not restricted to the basal or suprabasal regions but were scattered through the thickness of the epidermis. Paralesional skin also showed epidermal thickening and cytological abnormalities including the formation of multinucleate epidermal cells. These findings suggest that the development of actinic keratoses takes place in epidermis that is itself abnormal. The changes suggest that there is a gradual stepwise progression of sun damage epidermis via the clinically obvious keratosis to squamous cell epithelioma.

The epidermis in thyroid disease.

Epidermal dimensions, replication and anabolic activity have been studied in thirteen patients with thyrotoxicosis and seven patients with hypothyroidism. Epidermal thickness and rete pattern were significantly reduced in hypothyroidism. The rates of epidermal cell division and anabolic activity in the epidermis were estimated by measuring the rates of incorporation of tritiated precursor compounds and were found to be increased in thyrotoxicosis. The mean autoradiographic labelling indices after the intracutaneous injection of tritiated thymidine were 9-1% in thyrotoxic patients and 4-3% in the hypothyroid group. None of the changes observed could be correlated accurately with the degree of thyroid gland activity, but increased thyroid activity was reflected in the epidermal changes better than decreased thyroid activity.

Activation of T lymphocytes to M locus determinants in vivo. I. Quantitation of T cell proliferation and migration into thoracic duct lymph.

Proliferative responses to M locus (Mls) determinants were investigated in vivo by injecting T cell-containing populations intravenously into irradiated H2-identical, Mls-incompatible mice. Responses were studied 1-6 days later by measuring levels of radioactivity in the spleen and lymph nodes of the recipient 45 min after intravenous injection of tritiated thymidine. High responses were observed against Mlsa and Mlsd determinants, i.e. determinants which give strong proliferative responses in vitro. With injection of thoracic duct lymphocytes (TDL), lymph node or thymus cells, the responses, both in the spleen and the lymph nodes, reached a peak at day 3-4 and then fell sharply. This was in marked contrast to the stimulation observed with H-2-incompatible mice where, with TDL or lymph node cells (though not with thymus cells), responses were low at day 3-4 but reached high levels at day 6. Significant responses were also observed against Mlsb and Mlsc determinants, i.e. determinants which stimulate poorly in vitro. The kinetics of this response differed from that against Mlsa or Mlsd determinants in in that proliferation was very low at day 4 but high at day 6. No evidence was found that the responses observed reflected "back-stimulation" by the host. Since congenic resistant strains were not used, the possibility that the stimulation observed was in fact due to minor transplantation antigens rather than Mls determinants could not be excluded. Large numbers of blast cells appeared in thoracic duct lymph of mice sustaining responses to Mlsa or Mlsd determinants. Nearly all of these cells had recently synthesized DNA and entered the lymph after day 3.

Periodontal ligament cell kinetics following orthodontic tooth movement.

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (3H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with 3H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. 3H-TdR-labeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bbone forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G2 block by the orthodontic stimulation.