Does the observation of immunopositive cells after application of cell cycle markers always indicate that these cells proliferate?
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Animals↗
Biomedical subjects
H Korr
Publications and source records attributed to H Korr.
Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo.
It is generally accepted that mitochondria are able to proliferate even in postmitotic cells due to their natural turnover and also to satisfy increased cell energy requirements. However, no detailed studies are available, particularly with respect to specific cell types. Since [3H]-thymidine is incorporated not only into nuclear (n) DNA but also into the DNA of cytoplasmic mitochondria, an autoradiographic approach was developed at the light microscopy level in order to study basic questions of mitochondrial (mt) proliferation in organs of rodents in situ via the cytoplasmic incorporation of [3H]-thymidine injected into the animals 1 h before sacrifice. Experiments carried out on mice after X-irradiation showed that cytoplasmic labeling was not due to a process such as unscheduled nuclear DNA synthesis (nUDS). Furthermore, half-lives of mitochondria between 8-23 days were deduced specifically in relation to cell types. The phase of mtDNA synthesis was about 75 min. Finally, mt proliferation was measured in brain cells of mice as a function of age. While all neurons showed a decreasing extent of mtDNA synthesis during old age, nUDS decreased only in distinct cell types of the cortex and hippocampus. We conclude that the leading theories explaining the phenomenon of aging are closely related, i.e., aging is due to a decreasing capacity of nDNA repair, which leads to unrepaired nDNA damage, or to an accumulation of mitochondria with damaged mtDNA, which leads to a deficit of cellular energy production.
Aging↗
Unscheduled DNA synthesis and mitochondrial DNA synthetic rate following injury of the facial nerve.
Unscheduled DNA synthesis (UDS) of nuclear DNA and mitochondrial (mt) DNA synthetic rates were determined autoradiographically in different cell types of the rodent brain 14 days after unilateral facial nerve transection. In addition to an increased synthetic rate of mtDNA in facial motoneurons 12 h after axotomy, a significant increase of UDS, i.e., DNA repair, and mtDNA synthesis were found in the regenerating facial nucleus 4 days after axotomy. Specificity of the observed labeling was confirmed by injection of 3H2O instead of [3H]thymidine. Using electron microscopic autoradiography, it was further shown that cytoplasmic labeling of neurons was mainly due to incorporation of radioactive label into mitochondria, indicating their subsequent multiplication by division. The observation that Northern blot signals for O6-alkylguanine-DNA-alkyltransferase mRNA from homogenized facial nuclei of both the axotomized and normal side remained unchanged over 14 days after axotomy indicated that the observed DNA-repair activity was not caused by endogenously produced alkylating agents. The combined presence of transiently increased UDS, enhanced mtDNA synthesis and elevated protein synthetic rates of regenerating motoneurons (as shown in the literature) suggests that free radicals produced by mitochondria in injured nerve cells could cause unspecific DNA damage followed by immediate repair.
Animals↗
Correction factors of 3H-beta-self-absorption for quantitative autoradiography of different cell types in the brain of pre- and postnatal mice.
Interferometric and morphometric studies were performed on seven types of brain cells of deparaffinised and Feulgen-stained brain sections of pre- and postnatal mice in order to find out to what extent 3H-beta-self-absorption factors (c.f.s) of karyoplasm and perikaryal cytoplasm change as the animals' age increases. This is important when comparing grain numbers of different cell types among mice of different age in quantitative autoradiographic studies after application of tritium-labelled substances. While optical path differences (o.p.d.s) of euchromatin and cytoplasm decreased in large neurons postnatally, heterochromatic o.p.d.s remained more or less unchanged. The fraction of euchromatin (fEu) and heterochromatin (fHe) changed specifically in relation to cell type with increasing age, i.e. mostly heterochromatin increased postnatally. Within a given age, c.f.s, calculated from o.p.d.s, fEu and fHe, varied up to a maximum factor of 2.16 for karyoplasm and a factor of 1.19 for cytoplasm between different cell types. A significant decrease of c.f.s postnatally was found only for the cytoplasm of cortical and hippocampal pyramidal cells and for cerebellar Purkinje cells. The c.f.s of karyoplasm remained more or less unchanged with increasing age of the mice. The calculation of relative c.f.s makes these c.f.s applicable for every other autoradiographic study using 3H-labelled substances.
Animals↗
Do multilineage potential neural stem cells really exist in the brain of adult mammals.
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Animals↗
Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro.
Pulse labelling experiments with [3H] thymidine (dT) and double labelling experiments with [3H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 +/- 2.7% (mean +/- SEM) and Ts = 7.2 +/- 0.7 h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (Tc) of 11.2-14.2 h. The longer Tc values (18-20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G1/S and S/G2 borders of in vivo vs. in vitro studies.
Animals↗
Problems encountered when immunocytochemistry is used for quantitative glial cell identification in autoradiographic studies of cell proliferation in the brain of the unlesioned adult mouse.
We have used sections of adult mouse brain to determine whether antibodies specific for oligodendroglia (anti-carbonic anhydrase II, CA II; anti-galactocerebroside, GC; anti-myelin basic protein, MBP) and astroglia (anti-glial fibrillary acidic protein, GFAP; anti-S 100 protein) are suitable for quantitative studies of the proliferation and subsequent differentiation of these cells. Unlesioned adult mice received a single injection of 3H-thymidine (TdR) and were killed between 1 h and 70 days later. Quantitative evaluations of autoradiographs of 2-microns-thick serial sections stained immunocytochemically with the antibodies mentioned above or with Richardson's method for histological control led to the following conclusions. Anti-GC and anti-MBP stained only the oligodendrocytic processes and, thus, cannot be used in well-myelinated brain areas. Anti-CA II stained only a portion of the differentiated oligodendrocytes, but no proliferating cells. Anti-S 100 protein recognized all the astrocytes, but also many (interfascicular) oligodendrocytes. Anti-GFAP stained only a few astrocytes in the unlesioned mouse; all astrocytes may become GFAP-immunopositive only after wounding the brain. Thus, in contrast to in vitro studies, immunocytochemical studies with these antibodies on sections of adult animals cannot be recommended for the quantitative analysis of cell proliferation. In addition, our results show that differentiated glial cells proliferate in adult mice. Astro- and oligodendrocytes divide with the same cell cycle parameters and mode of proliferation up to about 1 month after 3H-TdR injection. In contrast to oligodendrocytes, some astrocytes might re-enter the cycle after a few weeks of quiescence.
Animals↗
RCA-I lectin histochemistry after trypsinisation enables the identification of microglial cells in thin paraffin sections of the mouse brain.
A biotinylated lectin from Ricinus communis (RCA-I) and avidin-biotin-horseradish peroxidase complex (ABC) were used to identify microglial cells in 3-microns-thick sections of formalin-fixed paraffin-embedded brains of adult mice required for quantitative cell kinetic studies. In 3-microns-thick sections of the mouse brain the staining intensity of RCA-I-positive cells compared to background staining was too low for evaluation, quite in contrast to rat brain. However, perikarya and cytoplasmic processes of microglial cells were clearly stained in 10- and 20-microns-thick sections. The low contrast characteristic of thin mouse brain sections could be enhanced by pre-incubating the sections with trypsin before application of the lectin. We assume that different densities of RCA-I binding sites among microglial cells of rats and mice, respectively, are responsible for the different staining intensities observed. Our protocol of lectin staining did not influence subsequent autoradiography for studies of cell proliferation after [3H]thymidine application.
Animals↗
Autoradiographic studies of rat astroglial cell proliferation in vitro with and without treatment with basic fibroblast growth factor.
Using specific autoradiographic methods, cell cycle parameters of untreated and basic fibroblast growth factor (bFGF)-treated astroglial cells from newborn rats grown in primary culture were directly measured. The mode of proliferation was also analysed. In untreated cultures, S phase duration (Ts = 6.9-13.1 h) and cell cycle time (Tc = 10-18 h) can be modified by about a factor of 2 depending on the culture conditions (serum-supplemented or defined medium, thyroid hormone concentration). However, growth fraction (GF = 0.15) and the ratio Ts/Tc remain stable. With increasing days in vitro (DIV) (DIV 7-DIV 20), Ts (7.8-10.6 h) and Tc (10-21 h) are prolonged and GF (0.14-0.06) decreases, probably due to cell maturation. In general, astroglial cells proliferate exponentially with a GF < 1, but stop proliferating about 30-36 h after the last feeding, probably caused by exhaustion of the medium. However, after refeeding they continue to proliferate. As opposed to in vivo, no transition of non-proliferating cells into the GF occurs. After addition of bFGF, GF increases (e.g. GF at DIV 7 = 0.43), but Ts and Tc are not influenced at DIV 7 and 12. At DIV 20, bFGF additionally shortens Ts and Tc, thereby producing values of Ts, Tc and GF like 'younger' cultures. However, the revitalizing effect on 'mature' cells is only transitory. In general, bFGF leads to a single re-entry of G0 cells into the GF. Thereafter, bFGF does not affect the mode of proliferation.
Animals↗
Characterization of astroglial cell proliferation in vitro and in vivo.
On the basis of experimental set-ups in vitro and in vivo and by making use of specific autoradiographical techniques, the following data on the proliferation of astrocytes from newborn rats in vitro and unpretreated rats and mice in vivo could be obtained: (i) The commonly employed immunohistochemical staining techniques in vitro are not applicable in tissue sections. (ii) In vivo, astrocytes show increasing durations of cell cycle (tc) as well as S phase (ts) prenatally until about birth. A similar trend can be observed in vitro. However, the absolute values for ts and tc can be substantially modified depending on the culture conditions. (iii) As regards the mode of proliferation, astrocytes in vitro grow exponentially and without transition of quiescent cells from the non-growth fraction into the growth fraction (GF). In contrast, astrocytes in vivo exhibit steady-state growth and continuous recruitment of proliferating cells from the non-GF. These differences show that there is a need for further in vivo-experiments when studying new strategies in the treatment of gliomas.
Animals↗
Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin-6 by fibroblasts transfected with human interleukin-6-receptor cDNA.
A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.
Autoradiography↗
Autoradiographic studies of cell proliferation in the brain of rodents: a review of methods and results.
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Animals↗
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.
Animals↗
X-ray dose-effect relationship on unscheduled DNA synthesis and spontaneous unscheduled DNA synthesis in mouse brain cells studied in vivo.
X-irradiation of the head of adult mice leads to DNA repair synthesis (unscheduled DNA synthesis, UDS) in non-proliferating cells of the brain as shown autoradiographically after injection of 3H-thymidine and subsequent irradiation. The extent of UDS induced by one and the same X-ray dose varies between different cell types and also between different brain areas. Within the range of X-ray doses studied (2 to 100 Gy) a linear dose effect relationship was observed. No evidence of a saturation effect was found. The slopes of the regression lines for the dose effect relationship differ considerably for the different cell types. Two interesting correlations were found, if the present results were compared with other data in the literature: (i) There seems to be a correlation between the extent of UDS and radiosensitivity of the different cell types, the cells with low DNA repair synthetic rates being more radiosensitive. (ii) The extent of UDS of the different cell types correlates well with the extent of protein synthesis of the corresponding cell types. Apart from radiation induced UDS, spontaneous UDS was found to occur in sham-irradiated animals. The extent of spontaneous UDS also differs considerably between different cell types as well as between different brain areas. The increase of spontaneous UDS with increasing duration of immobilization of the animals during sham irradiation suggests a relationship between spontaneous UDS and stress.
Animals↗
A new procedure for correcting background in quantitative autoradiographic studies.
A computer aided procedure is explained of how to get to the percentage as well as the mean grain count of specifically labelled cells or cell structures in autoradiographs, especially in the case of weak labelling.
Autoradiography↗
An improved procedure for background correction in autoradiography.
In the event of weak autoradiographic labelling, the proportion of truly labelled cells or structures can be calculated from the frequency distributions of grains per area or cell structure for i = 0, 1,..., n grains using the results obtained for an experimental group after the application of a radioactively labelled substance and those obtained for a control group without radioactivity. The principle of this computer-aided method is also applicable when the grain counts are related to varying areas in histological sections.
Animals↗
[Histochemical, fluorescent autoradiographic study of paraganglion cells in the superior cervical ganglion of the newborn rat following maternal stress].
Catecholamine storing paraganglionic cells in the neonatal rat superior cervical ganglion were investigated after maternal stress exposure. Paraganglionic cells as fluorescence-microscopically identified were significantly increased in number when compared with controls. The autoradiographically recorded labelling index of these cells, however, was unaltered. Fluorescent cell clusters were apparently composed of different cell types. The results support the suggestion that the stress-dependent increase in the number of catecholamine storing cells is due to differentiation of premature cell forms rather than to enhanced mitotic activity.
Animals↗
Determination of correction factors of 3H-beta-self-absorption for quantitative evaluation of grain number in autoradiographic studies. Interferometric studies of different cell types in the mouse brain.
Deparaffinized and Feulgen-stained sagittal sections of the mouse brain were studied interferometrically in order to measure optical path differences of euchromatin and heterochromatin of various cell types. Furthermore, the ratio eu-: heterochromatin of each cell type was determined. From these data mass densities of karyoplasm and, finally, correction factors of 3H-beta-self-absorption were calculated for comparing grain numbers of different cell types in quantitative autoradiographic studies after application of tritium-labelled substances. Remarkable differences of correction factors up to a factor of 2.18 were found. Furthermore, the actual section thickness was determined interferometrically. A reduction to about 0.60 of the microtome setting was measured in two different areas of the brain. Using mass densities together with actual section thickness correction factors for a thickness of 1 micron were calculated. This was done also for cell types outside the brain the data of which were taken from literature. Thus, differences in correction factors up to about a factor of 4 were found pointing out the importance of considering 3H-beta-self-absorption in quantitative autoradiographic studies.
Animals↗