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PubMed1983

Assay of anticancer drugs in tissue culture: cell cultures of biopsies from human astrocytoma.

A method has been developed for measuring the drug sensitivity of human gliomas in short-term culture, using scintillation counting or autofluorography. Cell cultures prepared from malignant astrocytomas were treated with anticancer drugs whilst in exponential growth in microtitration plates. After drug treatment and a recovery period, residual viability was measured by [3H] leucine incorporation followed by scintillation counting or by [35S] methionine incorporation and autofluorography in situ. In 5 glioma cell lines tested against 6 drugs, the microtitration method correlated well with monolayer cloning. Although replicate samples of the same tumour showed little variation in chemosensitivity, there was marked variation between the chemosensitivities of cultures derived from the tumours of different patients. However, as variability between replicates was apparent during drug exposure or shortly after, it is important to allow the assay to run as long as possible after drug removal. It is hoped that this assay may provide the basis of a method for the prediction of in vivo chemosensitivity or the screening of potential chemotherapeutic drugs.

Antineoplastic Agents↗

PubMed1983

The scintillometric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity.

The induction of DNA repair synthesis by UV radiation and methylmethane sulphonate (MMS) in mammalian cell lines of human (EUE, HeLa, FT, KB) and hamster (CHO, BHK) origin has been evaluated by means of autoradiography and the scintillometric procedure which implied the use of hydroxyurea (HU) to suppress DNA replication. While with UV radiation both methods produce concordant positive results, in the case of MMS the evidence of DNA repair synthesis obtained from the autoradiograms is occasionally accompanied by a lack of increase of DNA radioactivity in the treated cultures, as detected by scintillation counting. In such instances MMS is shown to reverse the enhancement of pool radioactivity in the cultures incubated with HU and even to reduce the radioactivity of thymidine pool below control values. By normalizing DNA radioactivities on the basis of pool variations, the discrepancy between autoradiography and scintillation counting is solved. The chromatographic analysis of thymidine pool components justifies the normalization procedure as it demonstrates that also in cultures treated with MMS or MMS + HU pool variations closely parallel the variations of thymidine triphosphate (dTTP) level. The normalization of DNA radioactivities based on the overall pool radioactivities gives an improved evaluation of the actual rate of DNA synthesis. It can be recommended for screening studies of DNA repair inducers because it allows one to correct false negative results without producing false positive data. Compared with the dTTP levels, overall pool radioactivities used as normalizing factors still produce an underestimate of DNA repair when high doses of MMS are applied to hamster cell cultures.

Animals↗

PubMed1994

A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-beta-D-maltoside.

Glycogenin is a self-glycosylating protein that catalyzes early glucosyl transfer steps in the biosynthesis of glycogen. In currently used assays of glycogenin activity, the enzyme is incubated with radioactive UDP-glucose, and the labeled reaction product is then isolated by precipitation with trichloroacetic acid. A new assay is reported here which is based on the observation that glycogenin is not only self-glycosylating but may also use exogenous alkyl maltosides as substrates. After incubation of the enzyme with n-dodecyl-beta-D-maltoside and UDP-[3H]glucose, the radioactivity in the resultant n-dodecyl-beta-D-[3H]maltotrioside is determined by any one of the following three procedures, which all rely on the hydrophobic properties conferred on the reaction product by the alkyl aglycone: (i) adsorption of the product to a Sep-Pak C18 cartridge and elution with 70% ethanol; (ii) biphasic liquid scintillation counting in ScintiLene/25% isoamyl alcohol, without isolation of the product, and (iii) precipitation with trichloroacetic acid in the presence of carrier protein. The Sep-Pak C18 procedure has the advantage that it allows essentially quantitative isolation of the reaction product, while, under the conditions chosen, only about 50% of the product is precipitated by trichloroacetic acid. For most applications, however, biphasic liquid scintillation counting is the method of choice, since close to 90% of the labeled product is extracted into the organic phase and can be counted directly without interference from the labeled nucleotide sugar which remains in the aqueous phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Effects of disodium ascorbyl phytostanol phosphates (FM-VP4) on cholesterol accumulation within rat intestinal cells.

The objective of this study was to determine whether FM-VP4, a novel compound derived from plant sterols, can effectively reduce cholesterol accumulation within rat intestinal epithelial crypt (IEC-6) cells. EC-6 cells were cultured in Dulbecco's minimal essential medium (DMEM) containing 5% fetal bovine serum, 100 U/mL penicillin, 100 micro g/mL streptomycin, and 0.1 units/mL insulin at 37 degrees C under a humidified 5% CO2 atmosphere and seeded at 6.4 x 10(4) cells/well in 48-well plates. Experiments were initiated 14 days postconfluence. IEC-6 cells were exposed to [3H]cholesterol micelles (containing oleic and taurcholic acids), co-incubated with FM-VP4 (0, 10, 50, and 100 micro M) in Hepes Buffered Sterile Saline (HBSS). Cells were also preincubated with FM-VP4 prior to [3H]cholesterol micelle incubation to determine whether its effects are elicited intracellularly. The cellular localization of cholesterol was determined using digitonin. To determine the effects of cholesterol on the extent of FM-VP4 accumulation within IEC-6 cells, [3H]FM-VP4 was incubated with IEC-6 cells in the presence of unlabeled cholesterol micelles (0, 10, and 50 micro M). The extent of [3H]cholesterol or [3H]FM-VP4 associated with cell monolayers was determined after cell lysis using liquid scintillation counting in a Beckman LS6500 Scintillation Counter. Dose-response and time course studies were performed in which control (no FM-VP4 treatment) and FM-VP4 (10-100 micro M) were co-incubated with 50- micro M [3H]cholesterol micelles from 1 minute to 24 hours. Incubation with only 50- micro M FM-VP4 for less than 24 hours resulted in a 50% to 60% reduction (n = 6, P <.05) in [3H]cholesterol associated with the monolayer compared with control (n = 6). Preincubation of FM-VP4 did not elicit a significant reduction in cholesterol accumulation compared with control (n = 6). Approximately 25% of the total [3H]cholesterol associated with the cells was determined to be cytosolic, while 75% was noncytosolic in the presence and/or absence of FM-VP4. [3H]FM-VP4 was also shown to associate with IEC-6 cells at similar concentrations to cholesterol with the most pronounced inhibition of FM-VP4 accumulation occurring at a cholesterol concentration of 50 micro M. However, cholesterol-induced inhibition was detectable only after 1 hour of incubation. FM-VP4 inhibits cholesterol accumulation within IEC-6 cells and is most effective at equimolar concentrations with cholesterol. Our findings further suggest that the action of FM-VP4 is likely at the cell surface and not elicited intracellularly.

Animals↗

PubMed1976

Attachment of Mycoplasma pneumoniae to respiratory epithelium.

The attachment of radioisotope-labeled Mycoplasma pneumoniae to hamster tracheal rings in organ culture was examined by radioautography and liquid scintillation counting. Radioautographs of individual rings exposed for 8 h to (3H) thymidine-labeled virulent M. pneumoniae revealed a dense extracellular collection of emulsion grains along the luminal surface of epithelial cells. Similar exposure of rings to isotope-labeled avirulent M. pneumoniae resulted in no accumulation of emulsion grains. The numbers of attached virulent mycoplasmas, as measured by liquid scintillation counting of infected rings, were found to increase in a nearly linear fashion over an 8-h incubation period. Viability of the mycoplasmas and metabolic integrity of the tracheal rings were important for optimal attachment. Pretreatment of rings with neuraminidase or sodium periodate significantly impaired orgainism adherence. These data suggest a specificity of interation between virulent M. pneumoniae and tracheal epithelial cells that can be further examined through the use of isotopically labeled mycoplasmas.

Animals↗

PubMed1980

Tissue distribution of 14C-lynestrenol in pregnant rats.

The tissue distribution of 4-14C-lynestrenol (17 alpha-ethynyl-oestr-4-en-17 beta-ol) following oral administration to pregnant rats was studied by whole body autoradiography and liquid scintillation counting. Pregnant females were sacrificed on days 10, 12, 14 and 19 of gestation, in each case 5 hours after oral administration of 43 microCi 4-14C-lynestrenol per animal. The isotopelabelled compound was distributed throughout most tissues, including the fetuses. The highest concentrations were found in the liver, while there was lower activity in the fatty tissues and the activity in the fetuses was comparable with that in the brain. The placental transfer was verified by the results of liquid scintillation counting. The concentration of labelled substance in the fetuses increased with the duration of pregnancy.

Administration, Oral↗

PubMed2004

A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate-polyacrylamide gels.

We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate-polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70 degrees C overnight followed by liquid scintillation counting. H(2)O(2) had no effect on the count rates of [(14)C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70 degrees C resulted in incomplete extraction of radioactivity from gels containing [(14)C]BSA, but there was also a significant reduction in count rates in samples incubated at 80 degrees C. At 70 degrees C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89-94%, and the coefficient of variation for five replicate samples was 5-10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein.

Animals↗

PubMed1996

A method of quantitative autoradiography for the spatial localization of proteoglycan synthesis rates in cartilage.

Incorporation of [35S]-sulfate into cartilage tissue indicates the synthesis of aggrecan, the large aggregating proteoglycan (PG) that endows cartilage with resistance to compression. Scintillation counting of tissue digests provides a quantitative measure of incorporated sulfate but does not provide information on the spatial location of synthesis within the tissue. Such spatially specific information is necessary to determine which cell populations respond to diffusible factors and to correlate local mechanical events (e.g., deformation, interstitial fluid stress) to cellular biosynthetic responses. The aim of this study was to develop and characterize a liquid emulsion autoradiography technique, including an automated grain counting procedure, to derive spatial profiles of aggrecan synthesis rates in cartilage. We chose mature 10-14-month-old bovine humeral head articular cartilage as a model system and applied a liquid emulsion autoradiography technique to [35S]-sulfate-labeled, resin-embedded, and semithin-sectioned tissue explants. High-magnification light microscopy color images were captured on a computer. Automated image analysis for grain number determination included a color thresholding procedure to discriminate grains from the lightly stained structural image and computation of the average area of a single grain from each image. Determination of grain number, whether originating from single grains or grain clusters, was performed by dividing the total grain area in the image by the average area of a single grain in the same image. This procedure largely eliminated the effects of variations of microscope light intensity, camera performance, image focus, section stain intensity, and thresholding on the resulting grain numbers. By altering the specific activity of the medium radiolabel and the emulsion exposure times, we demonstrated a linear dose dependence, without saturation, of grain number on radioactive content in the underlying section. By cutting specimens in half and performing liquid scintillation counting on one half and autoradiography on the other half, we found that each disintegration occurring in the section during exposure resulted in 0.67 +/- 0.21 grains (mean +/- SD; n = 58). Therefore, counted grain numbers can be directly converted to incorporated sulfate, largely reflecting the synthesis of the PG aggrecan. As an example of calculated intratissue profiles of aggrecan synthesis rates, we found for the mature bovine tissue in serum-free medium that aggrecan synthesis increases monotonically from the articular surface to the radial zone by as much as tenfold.

Animals↗

PubMed1986

Effect of peroxisome proliferator carcinogens on unscheduled DNA synthesis in rat hepatocytes determined by autoradiography.

The potent peroxisome proliferator hepatocarcinogens WY-14,643, BR-931, and nafenopin, as well as mono(ethylhexyl)phthalate (MEHP), the principle metabolite of the weaker hepatocarcinogen di(2-ethylhexyl)phthalate) (DEHP), were evaluated in the in vitro rat hepatocyte DNA repair assay by quantitative autoradiography. None of these carcinogens induced unscheduled DNA synthesis (UDS). These results failed to confirm the previously reported induction of UDS by WY-14,643 and BR-931 as determined by nuclear liquid scintillation counting. Hydroxyurea (HU, 10 mM) is commonly employed to inhibit replicative DNA synthesis (RDS) when using scintillation counting techniques for UDS. Autoradiographs revealed incomplete inhibition of RDS by HU.

Animals↗

PubMed1981

Cellular localization of 2-[3H]deoxy-D-glucose from paraffin-embedded brains.

Results of experiments in which regional neuronal activity is revealed by a 2-[3H]deoxy-D-glucose (3H-2-DG)-paraffin section-emulsion autoradiography method are described. The trigeminal pathway of freely behaving mice was activated differentially by selective patterns of whisker removal. One hour after injection of concentrated 3H-2-DG, the animals were perfused systemically with a periodate/lysine/paraformaldehyde mixture (McLean, I. W., and P. K. Nakane (1974) J. Histochem. Cytochem. 22: 1077-1083), the brains were embedded in paraffin, and serial sections were taken and coated with emulsion for autoradiography. Diffusion of the isotope out of the tissue was assessed visually and by liquid scintillation counting. While substantial loss of 3H isotope into the embedding fluids (about 95%) was found, the scintillation counts and the autoradiograms showed good fixation of the isotope in situ, no evidence of isotope movement into the emulsion, and no gradients of diffusion in the sectioned material. Patterns of regional labeling were similar to those reported from brains prepared by conventional 2-[14C]deoxy-D-glucose (14C-2-DG) autoradiography; for instance, auditory and vestibular pathways in the brainstem were heavily and specifically labeled. Trigeminal structures associated with the intact (stimulated) whiskers were labeled relatively heavily, indicating that label uptake is specific with respect to neuronal activity. In the cortex, the patterns of label corresponded directly and precisely to those barrels known to receive inputs from the intact whiskers. Distribution of silver grains in the cortex and in the brainstem was correlated directly with neuronal profiles, including processes, some of which were identified by means of a Nissl counterstain. Clearly, this approach offers considerable technical advantages, in particular, the ease with which the histological material is prepared. The resolution of the autoradiograms and the quality of the histology are excellent.

Animals↗

PubMed1987

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents.

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.

Animals↗

PubMed1975

The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase.

Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory effect of iron. The inhibition of uroporphyrinogen decarboxylase by ferrous ion, coupled with its previously reported inhibitory effect on uroporphyrinogen III cosynthetase, provides a possible biochemical explanation for the pattern of urinary porphyrin excretion observed in patients with porphyria cutanea tarda and the clinical association with disordered iron metabolism.

Animals↗

PubMed1984

Glucocorticoids preferentially increase fetal alveolar beta-adrenoreceptors: autoradiographic evidence.

To localize fetal rabbit lung beta-adrenoreceptors before and after glucocorticoid treatment, light microscopic autoradiography was performed with the reversible radiolabeled beta-adrenergic antagonist, [3H]dihydroalprenolol, on day 26 of gestation. Autoradiograms of adult lung and fetal myocardium were also prepared. Examination of these autoradiograms showed densely labeled airways, alveoli, and myocardium. Specific labeling, defined as that prevented by incubation with l-propranolol (1 microM), was 90%. Analysis of grain counts in the fetus showed that airways were more densely labeled than alveoli (p less than 0.001), labeling was increased by treatment (p less than 0.001) and treatment increased alveolar (p less than 0.002) but not airway labeling. Adult lungs were much more densely labeled than fetal, and fetal myocardial labeling was not altered by treatment. Adult untreated lung showed the same pattern as fetal untreated lung with airways being more densely labeled than alveoli (p less than 0.001). To validate estimates of relative beta-adrenoreceptor concentration derived from autoradiograms, comparisons to determinations of beta-receptor concentration from scintillation counting of lung section digests and from previously performed radioligand binding studies, using membranes prepared from whole lung homogenates, were made. There is excellent agreement between estimates of relative receptor concentration and specific binding derived from the counting of autographic grains and both scintillation counting of lung section digests and previously performed radioligand binding of lung particulate. In all preparations, specific binding was (90%), increased with glucocorticoid treatment in fetal lung (50-100%), was greater in concentration in adult compared to fetal lung (7-10-fold), and did not increase in fetal myocardium with treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

Xylose-1-14C absorption test: the use of urine, serum and breath analysis, and comparison with a colorimetric assay.

The xylose absorption and excretion test has been reassessed in controls and patients with coeliac disease. Xylose has been assayed in both serum and urine colorimetrically and by liquid scintillation counting using xylose-1-14C. The excretion of 14CO2 in breath following administration of xylose-1-14C has been measured. Liquid scintillation counting is a simple and reliable method for the measurement of xylose absorption. Serum xylose levels, urinary xylose excretion, or breath 14CO2 in isolation were poor screening tests for mucosal disease. The use of serum levels and urinary excretion in combination improved the discriminatory value of the test though small gut biopsy was more reliable.

Carbon Dioxide↗

PubMed1999

The use of GAFchromic film to determine the absolute activity of beta emitters.

A method of using GAFchromic film to determine the absolute activity of beta emitters by immersing the film in a bath of activity is described. This includes calculations of the effect of part of the radioactive solution being displaced by the film that is performing the measurement. Calculations have been performed for 90Y, 32P and 188Re. It is shown that for the film used in this study, this effect will cause a correction of the order of 15-20% for the three radionuclides studied. The results of the GAFchromic film measurement are compared with results obtained from liquid scintillation counting and from a commercial dose calibrator. It is shown that for 32P all methods were in reasonable agreement, but for 188Re, although the GAFchromic film and the liquid scintillation counting methods give similar results, the dose calibrator measurement differed by more than 20%. The GAFchromic film was also used to perform a direct measurement of the radiation dose at a distance of 0.5 mm from the surface of a cylindrical source of 188Re activity. This gave good agreement with the value of the dose predicted from theoretical calculations.

Beta Particles↗

PubMedDate not supplied

Effects of exocrine pancreatic secretions on hyperthermic injury of rat small intestine.

The influence of the exocrine pancreatic secretions on development of small intestinal injury following localized hyperthermia was studied. In male Holtzman rats the excretory pancreatic ducts were occluded with metal hemostatic clips. An intraperitoneal injection of [3H]thymidine was given 3 weeks later. Three or 48 h after the injection a 10 cm segment of small intestine was exteriorized through a midline abdominal incision and heated at 38.0 degrees C, 42.5 degrees C, or 43.5 degrees C for 45 min. Intestinal damage was assessed 24 h after hyperthermia. The following four endpoints were used: histopathological injury score, the number of villi per intestinal circumference, the number of labelled epithelial cells in fixed areas of autoradiographic specimens, and incorporation of [3H]thymidine as determined by liquid scintillation counting. The correlation of results among the four methods of assessment was highly significant. The autoradiography data showed better correlation with both morphological parameters than the results of liquid scintillation counting. There was significantly less damage in heated segments from pancreatic duct-occluded animals than in segments from sham-operated controls. When hyperthermic injury was assessed morphologically the protection conferred by pancreatic duct occlusion was equivalent to lowering the temperature of heating by 1 degree C. It is concluded that morphological criteria may be superior to endpoints based on [3H]thymidine incorporation for assessment of hyperthermic injury in rat small intestine. Reducing the intraluminal pancreatic secretions appears to confer significant protection from small bowel injury after localized hyperthermia.

Animals↗

PubMed1986

Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.

Rates of protein turnover have been measured in three normal and three Duchenne muscular dystrophy (DMD) skin fibroblast cell lines. Cell populations were analyzed at identical states with regard to cell number, state of topoinhibition, and cumulative population doublings (CPD). Net protein synthesis measured by the incorporation of [35S]methionine in an 18-hr pulse was reduced by an average of 34%; degradation of total cellular protein measured after an 18-hr pulse with [35S]methionine and a 24-hr chase was enhanced by an average of 50% in DMD fibroblasts. Two-dimensional gel electrophoresis analyses revealed that the breakdown of the majority of [35S]methionine polypeptides was markedly increased in DMD fibroblasts. Quantitative determinations of the differential degradation rates of 10 selected proteins in the tropomyosin region of two-dimensional gels were undertaken by scintillation counting and computer analyses. In three series of experiments, the degradation of the 10 proteins in DMD fibroblasts was enhanced by individual polypeptides between 12.0% and 151.2% as measured by scintillation counting or between 0.8% and 128% as determined by computer analyses.

Autoradiography↗

PubMed1983

DNA synthesis in rat heart cells after injury and the regeneration of myocardia.

The regenerative responses of the myocardia of post-natal rats of different age groups (1, 2, 3 and 4 weeks old) to an injury made by a clinical electricator were studied. DNA synthesis and the ultrastructural organization of the cardiac myocytes of the injured myocardia were examined for an evaluation of the potential for regeneration of the developing myocardia. The maximum labeling index of cardiac myocytes was observed in 1-week-old rats showing 8% labeled myocytes 3 days after injury as opposed to 3.2, 2.2 and 0.2% indices in 2-, 3- and 4-week-old rats respectively, 3 days after injury . In subsequent days after injury the labeling indices declined considerably in all age group hearts, and attained values less than 1% labeled myocytes 30 days after injury with the lowest labeling index in the oldest age group heart. When DNA synthesis in uninjured myocardial tissue adjacent to the injured tissue was examined, it was found to be significantly lower than it was in the injured tissue. However, both injured and adjacent uninjured tissue attained a peak in the labeling indices 3 days after injury, with the exception of 3- and 4-week-old uninjured tissue. The overall incorporation of 3H-thymidine into the DNA of heart cells as revealed by scintillation counts showed that the rate of incorporation of the isotope in younger hearts was significantly higher than in the older hearts. Non-muscle cells contributed significantly to the rise of scintillation counts in hearts of all age groups. Ultrastructural analyses of 1- to 4-week-old hearts showed that 24 hr after injury, injured areas of myocardia were heavily crowded with macrophages that surrounded damaged myocytes. Later on, fibroblasts and other non-muscle cells predominated the injury sites along with fibrous connective tissue. Scattered regenerating cardiac myocytes were frequently observed in the injury sites of 1- and 2-week-old hearts 3 days after injury. Myocytes were rare in the corresponding regions of 3- and 4-week-old hearts. Instead abundant non-muscle cells and fibrous connective tissue were predominant. In the fourth and final week of this study, the repaired areas of myocardia in 1- and 2-week-old rats contained more myocytes than those of the 3- and 4-week-old rats, and the repaired zone of the 1-week-old heart contained more myocytes than the repaired areas of the other age groups. These findings suggest that the mammalian myocardia possess an age-dependent potential for regeneration that involves the healing of injury sites with contractile and connective tissues.

Animals↗