Epidermal proliferation characteristics are similar in the pilary canal of mouse hair follicles and in interfollicular epidermis

Proliferation characteristics of basal cells in the pilary canal of resting hair follicles were investigated and compared with corresponding parameters in interfollicular epidermis of hairless mice. The mitotic rates had similar 24-h means at both locations. Distinct circadian rhythms which showed phasing and amplitudes similar to that in interfollicular epidermis, were demonstrated by the 3H-TdR labelling index, the mitotic rate and the mitotic index. Influx of cells to and efflux of cells from the S phase were measured in the early morning and in the evening by a 3H-TdR double labelling method. The influx values were similar at both times of both locations. The efflux values recorded in the morning were more than twice the values seen in the evening in both the pilary canal and in interfollicular epidermis. The epidermal motitic rate in the pilary canal was depressed by epidermal extracts, and increased after adhesive tape stripping in the same way as in interfollicular epidermis. The results indicate no heterogeneity in cell proliferation characteristics between the two locations, and suggest that similar mechanisms are responsible for maintainance of growth equilibrium at both sites.     

Cellular proliferation during cicatrization of the skin of 7-day chick embryos cultured in vitro

A cell proliferation study during in vitro wound healing of dorsal thoraco-lumbar skin of 7-day chick embryos was performed by pulse labelling using a single isotope (tritiated thymidine). The unoperated (controls) and operated explants were incubated in the radioactive medium (1 microCi/ml tritiated thymidine) 1 h prior to fixation and where fixed 1 h (start control), 48, 72 and 96 h after the excision. Mean labelling and mitotic indices of the unwounded epidermis were, respectively, 18.22% and 2.66% at 7 days, and 7.03% and 0.88% at 11 days (7 days + 96 h). 72 h after the excision, the labelling and mitotic indices of wounded epidermis increased, on average, respectively to 212,5% and 220% with respect to those of the controls, in the proximal zones near the inner edge on the wound. The labelling and mitotic indices in the dermis were, respectively, 27.95% and 3.63% at 7 days and 7.65% and 1.30% at 11 days. 72 h after the excision, the labelling and mitotic indices of the operated dermis increased, on average, respectively to 220% and 130% with respect to those of the controls in the centre and the proximal zones of the wound. The increase of the labelling index of the operated integument persisted for a maximum of 24 h, between 48 to 72 hours after the excision.     

EPIDERMAL REGENERATION AFTER CELLOPHANE TAPE STRIPPING OF HAIRLESS MOUSE SKIN

After repeated applications of cellophane tape to the dorsal skin of hairless mice, the proliferative response in the treated epidermis was estimated by three different methods. The mitotic rate was determined in the interfollicular epidermis using the Colcemid technique, and the DNA synthetic activity was estimated after ³H-thymidine injection by counting labelled interfollicular cells in autoradiographs and by determining the specific activity of epidermal DNA. An initial 40–50% inhibition of DNA synthesis and mitosis was followed by an increase in the labelling index and the mitotic rate 8–10 hr after tape stripping. By 24 hr, peak values 5–6 times the controls were attained for both parameters. The labelling index and the mitotic rate were nearly normal at 3–4 days, but a second small peak was seen on day 5. Normal values were found on days 6 and 8. A similar pattern of response was found biochemically, but the peak of DNA specific activity was much broader and the extent of the increase was only about half as great as the increase in the labelling index. Possible reasons for these differences are discussed.     

Studies of the effects of aminopterin on the crypts of Lieberkühn in rats

Summary Studies have been made on the effects of an intramuscular injection of aminopterin on the crypts of Lieberkühn in rats. A decrease in the mitotic counts was accompanied by a rapid increase in the number of abnormal cells present in the epithelium of the crypt. Three hours after administration of the aminopterin, an almost complete absence of true metaphase chromosoms was found. By 24 hours, a partial return towards normal mitotic activity was observed but the number of abnormal cells present was still very high. It is suggested that the mitotic changes are in keeping with the conclusion of Grampa and Dustin (1952) of an arrest at interphase but that a secondary arrest at metaphase cannot be excluded.A morphological feature of some of the abnormal cells was the presence of a Feulgen positive granule in the cytoplasm, which by electron microscopy was also shown to contain many cytoplasmic constituents. It is suggested that material is lost from the nucleus and incorporated into a granule in the cytoplasm. A possible explanation of the purpose and function of the granule, as a means of disposing of unwanted or aberrant material, is put forward. Acknowledgements. I am grateful to Professor R. J. Brocklehurst for his continued interest and support of this work, and to the Stage II, B. Sc. students (1964) who counted the cells in many of the specimens as a laboratory exercise. My thanks are also due to Mr. J. Clements for technical assistance.     

Changes in the microstructure of the human epidermis following local vacuum exfoliation]

Structural rearrangements of the human epidermis have been studied after its local vacuum exfoliation. Blisters have been formed in 48 men-volunteers by means of negative pressure up to 0.7 kg/cm2 and during the following 72 h structure of the exfoliated epidermis has been investigated. Immediately after the blister formation the epidermal basal layer is traumatized, a part of its cells die in some time after the lesion. In the center of most of the cells of the spinous layer there is a large vacuole which presses back the nucleus. However, the whole epidermis is not ruined, and during 24 h actively regenerates. The remaining viable cells into the blister lumen. By the end of the first 24 h span they practically cover from below the whole surface of the exfoliated epidermis. In the cells of the spinous layer amount and size of vacuoles decrease, the nuclei return to the central position. In 48 h in the spinous layer keratohyalin granules are revealed, moreover, in the cells, arranging on the border with the basal layer. By 72 h within the epidermis of the number of necrotic areas sharply increases. All the arrangements in the epidermal structure occur at the absence of mitotic division of cells.     

Cicatrization of the skin of the 7-day-old chick embryo cultured in vitro

A morphological study of in vitro wound healing has been performed by light, transmission and scanning electron microscopy in dorsal thoraco-lumbar skin of 7-day chick embryos. A circular wound, 750 microns in diameter, was punched out of dorsal skin, removing epidermis and the underlying dense dermis. Wound closure was completed within 96 to 120 hours. Feather bud development was not observed at the wound site. The epidermis began to migrate some 24 h after the wounding; the migration of peridermal cells preceded that of basal epidermal cells by some 12 hours. Mechanisms of the epidermal migration were similar to those observed in situ during wound healing of the integument in 5-day chick embryos (THEVENET, 1981), Superficial epithelization of bare dermis occurred as soon as 12 h after the injury. Cytoplasm of dermal cells exhibited many microtubules and a dilated rough endoplasmic reticulum. During the first 48 h, the epidermal cells established direct contacts and zones of close parallel apposition with epithelized dermal cell processes. The basement membrane lamina densa was maintained at the edges of the wound without retraction or ruffling. It was reconstituted concomitantly with the epidermal migration within 72 h. Cytoplasm of migratory epidermal and epithelized dermal cells exhibited many cytoskeleton structures.     

Hypothalamic control of the pars intermedia in Xenopus laevis tadpoles

Summary In Xenopus laevis tadpoles the relation between a paired nucleus of bio-amine producing neurons in the caudal hypothalamus and the pars intermedia of the hypophysis was studied.Treatment of the animals (stage 49 to 50 of Nieuwkoop and Faber's normal table) with reserpine caused aggregation of the skin melanophores within one hour, followed by redispersion five to six hours after the beginning of the experiment. This was at exactly the same time as the bio-amines in the caudal hypothalamus disappeared. However, the drug was ineffective if the nuclei had been removed. This indicates that reserpine acts via these nuclei and does not influence the skin melanophores directly.It was concluded that the initial aggregation of the melanophores may be the result of a reduced extrusion of MSH from the pars intermedia, caused by an increased output of a MIF by the bio-amine producing nuclei. The redispersion was explained by assuming that the bio-amines were depleted and the nuclei stopped with the extrusion of the MIF. This does not mean that the production of a MIF is the only function of the paired bio-amine producing nucleus in the caudal hypothalamus.The author thanks Prof. Dr. P. G. W. J. van Oordt for his helpful comments and criticism. Mr. J. H. I. J. M. ten Berge and Mr. E. W. A. Kamperdijk provided great assistance during the course of the experiments. Mr. H. van Kooten made the diagram and the photograph.     

Cellular responses in the skin of the trout (Oncorhynchus mykiss) exposed to temperature elevation

The skin of rainbow trout was examined at the ultrastructural and cytochemical level after a 3–h exposure to an elevation of the water temperature, from 15 to 22° C. Within 3 h, the thickness of the epidermis had significantly ( P <0·05) decreased when compared to control fish. After 24 h it was restored, and from day 4 onwards even increased above control levels. The thickening of the epidermis was associated with appearance of many mitotic cells, not observed in control fish. Within 24 h many apoptotic epidermal cells were found, indicating enhanced ageing of the cells. Filament cells from the outer epidermal layers synthesized vesicles with peroxidasc activity within 3 h after temperature elevation. This enzyme was found also in apoptotic as well as in necrotic filament cells. Mucous cells became elongated and their mucosomes displayed peroxidase activity. Occasionally electrondense, probably serous, mucosomes appeared. In the epidermis rodlet cells were found. Both epideimis and dermis, became invaded by many lymphocytes and macrophages. The latter contained vesicles with peroxidase activity. Pigment–containing cytoplasmic extensions of melanocytes penetrated the epidermis while iridocytes disappeared from the dermis. The synthetic activity of dermal fibroblasts was stimulated. These results show that a moderate temperature elevation has pronounced and prolonged effects on the skin of the exposed fish. The effects are to a high extent comparable with those of stressors such as heavy metals, acid water or wounding.     

Mitotic and Labelling Activity In Normal Human Epidermis In Vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours > 09.00 hours by a factor of 2.6, P < 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.     

Mitotic and labelling activity in normal human epidermis in vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours greater than 09.00 hours by a factor of 2.6, P less than 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.     

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.     

The effect of bleomycin on rapidly proliferating epidermis. A comparative investigation using micro-flow fluorometry, H3Tdr incorporation and a stathmokinetic method (colcemid).

At different time intervals after injection of Bleomycin (BLM) th effect on several kinetic parameters of the hairless mouse epidermis stimulated to proliferate by previous adhesive tape stripping was measured. Micro-flow fluorometry was used to determine the relative number of cells in the various phases of the cell cycle (G1, S and G2). Tritiated thymidine was used to determine labelling indices and grain counts. Colcemid was used to observe the mitotic rate. An initial decrease followed by a subsequent significant increase compared to the non-BLM-treated controls was observed in all parameters studied except the mitotic rate, which remained lower than in the control animals during all 48 hours. The transit time of the cells through the S-phase was initially slightly prolonged, but thereafter it seemed to be shorter than that of the controls. BLM seems to provoke a partial blocking of cells in the G1 phase. When the block is released, a greater number of cells pass through the S phase in partial synchrony at a higher than normal speed. BLM induced a low mitotic rate which remained below the level of that of the normal animals after stripping, even though there obviously was a considerably higher influx of cells from the S phase to the G2 phase. This resulted in a subsequent accumulation of cells in the G2-phase. Thus, BLM has also a blocking effect on the G2-M boundary of the cell cycle. This inhibitory effect of BLM on the mitotic rate was shown to be independent of the effect of BLM on the DNA synthesis. BLM therefore seems to have complex influence on epidermal cell kinetics in vivo. Cells in G1-phase are partially and transiently blocked, but this block is soon released. These cells thereafter pass through the S-phase and pile up in the G2-phase, because BLM also blocks the passage of cells from the G2-phase to mitosis. The overall reduction in cell proliferation seen after BLM in vivo seems mainly to be due to the effect on the G2-M boundray of the cell cycle.     

The effect of the epidermal G2 chalone on the mitotic duration in nude mouse epidermis and in a transplanted squamous cell carcinoma.

Balb/c/nu nude mice transplanted with a moderately differentiated squamous cell carcinoma were injected intraperitoneally with different doses of aqueous skin extracts containing the epidermal G2 chalone. The mitotic counts and the mitotic rates were determined in histological sections using a stathmokinetic method with vinblastine sulphate. The mitotic duration was calculated from the mitotic rates and counts. Skin extracts containing epidermal G2 chalone increased the mitotic duration in the epidermis, and a similar trend was seen in the tumour. The higher the dose of chalone, the longer the mitotic duration tended to be. A straight line of best fit used to indicate the dose/response relationship was steeper for the epidermis than for the tumour. The study thus shows that the epidermal G2 chalone not only prevents epidermal cells from entering mitosis, it also prolongs the mitotic duration. Further, the results do not contradict the theory that tumour cells may be less sensitive to chalone than normal cells.     

Changes in the skin epithelium and nerve elements of the rat sole with directed regeneration of the sciatic nerve

The work has been performed on Wistar rats and non-inbred animals. Their ischiatic nerves have been dissected at the femoral superior third under nembutal narcosis. The end of the sectioned nerve are connected by a fragment of an aorta from rats of the same age. The state of nervous elements and dermal epithelium of the hind extremity sole in the animals is studied by means of general histological and neurohistological techniques. Mitotic activity of cells in the plantar epidermis, thickness as a whole and its separate layers are estimated, keratinization coefficient and correlation of thickness of separate sheaths in the whole layer are calculated. Use of the arterial vessels for connecting the end of the cut ischiatic nerve, trophic ulcers, that usually take place after the nerve section, do not develop. At early stages after the operation mitotic activity in the epidermis decreases by 70%, and the layer thickness--by 40%. Restoration of both indices proceeds slowly. As soon as the regenerating nerve fibers reach the distal part of the ischiatic nerve, the state of the epidermis improves: the mitotic activity differs from the normal by 20-30%, and thickness of the epithelium--by 28-30%. Coordination of thickness of separate layers in the epidermis is not nearly disturbed. It remains in the same state up to complete restoration of receptory structures in the rat plantar skin (during 9-9.5 months after the operation).     

Cytochemical evidence for the neural crest origin of mammalian ultimobranchial C cells

Summary The cells of the neural crest have APUD properties at an early stage of devel opment (72 hours in the chick embryo). The FIF procedure provides a cytochemical means for their distinction.Using mouse embryos from mothers injected, intraperitoneally, 1 hr before removal, with l-DOPA (100 mg/kg), the peripheral stream of neural crest cells was clearly identifiable at the 7-somite stage (7–8 days). At the 10-somite stage (8–9 days) the cells were observed to invade the lateral processes of the foregut, and the foregut itself. A particularly high concentration of fluorescent APUD cells was observed in the anterior portion of the IVth pharyngeal pouch, destined to become the ultimobranchial body.At the 14-somite stage (11–12 days) the developing ultimobranchial body still contains fluorescent cells of neural crest origin.The implications of these findings on the question of the origin of the entire APUD series of endocrine polypeptide cells is discussed.     

Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA)

Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m(2) (95 mW/cm(2)). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of gamma radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 +/- 0.3 degrees C. Immediately after the exposures, lymphocytes were cultured at 37 +/- 1 degrees C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of gamma-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.     

Adult precursor cells in the tail epidermis of Xenopus tadpoles

Polyclonal antibodies were raised against Xenopus larva-specific 58 kDa keratin (PAK58) and adult-specific 63 kDa keratin (PAK63), in order to examine the origin of 63 kDa-keratin-producing cells in the tail skin. By immunofluorescent staining of the tail skin, the 58 kDa keratin was recognized in almost all of the larval epidermal cells, although a small number of PAK58-negative cells were detected at stage 64. In contrast, 63 kDa keratin was immunohistochemically recognized at stage 58, but the signal was very weak. The number of epidermal layers in the tail epidermis increased during a period from stage 58 to stage 64. At stage 64, a small number of PAK63-positive cells was clearly identified in the multilayered tail epidermis. Comparative analysis of successive sections showed that PAK63-positive cells are derived from a cell group differing from PAK58-positive cells. Immunohistochemical studies using cultured epidermal cells demonstrated that 58 kDa keratin is localized in the cytoskeletal bundles of skein cells, whereas 63 kDa keratin is produced not by skein cells but by basal cells and their descendants. These results suggest that basal cells are the adult precursor cells within the larval epidermis even in the tail area.     

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

SPECIFICITY of EPIDERMAL CHALONE EXTRACTS FOR HUMAN EPIDERMOID TUMOUR CELLS IN VITRO: A PRELIMINARY REPORT

In order to test the mitosis-inhibiting effect and the tissue specificity of the epidermal G₂ chalone for tumour cells, extracts from hairless mouse epidermis were tested in short-term tissue cultures of cells from human respiratory tract epidermoid carcinomas and adenocarcinomas. the chalone inhibited strongly the mitotic activity in two cases of histologically proven epidermoid carcinoma, and had no effect in two cases of adenocarcinoma. In one case of a supposed epidermoid carcinoma, the chalone had no effect. Revision of the histology, and the result of autopsy 11 months later, showed that in this case the lesion in the lung had been a poorly differentiated metastasis from an adenocarcinoma of the ovary. Liver extracts produced in the same way as the epidermal extracts showed no mitotic inhibition in any of the cultures. These results indicate that epidermal G₂ chalone produced from mouse skin is tissue specific for human epidermoid tumour cells, and also indicate that a chalone test might be used as a diagnostic tool for poorly differentiated carcinomas to see whether they are of epidermoid origin or not.