Localization of healing tracheal wounds using bromodeoxyuridine immunohistochemistry

Immunohistochemical demonstration of the thymidine analogue bromodeoxyuridine (BrdU) in regenerating cells was useful in determining the size and location of the wounded areas (epithelial and submucosal) during regeneration of the hamster tracheal epithelium, at times late in the healing process (72-144 hr postinjury) when the wound sites and their boundaries were not recognized with certainty in conventionally stained paraffin sections. Cells distant from the wound sites remained unlabelled. The success of this method resulted from prolonged exposure to BrdU released over several hours from a 25mg tablet implanted subcutaneously at 24 hr postwounding at the time when DNA synthesis and cell proliferation are maximal. This simple technique promises to be useful in determining the size and location of wound sites with application to a wide variety of organs and tissues in studies of repair and healing.     

Localization Of Healing Tracheal Wounds Using Bromodeoxyuridine Immunohistochemistry

Immunohistochemical demonstration of the thymidine analogue bromodeoxyuridine (BrdU) in regenerating cells was useful in determining the size and location of the wounded areas (epithelial and submucosal) during regeneration of the hamster tracheal epithelium, at times late in the healing process (72-144 hr postinjury) when the wound sites and their boundaries were not recognized with certainty in conventionally stained paraffin sections. Cells distant from the wound sites remained unlabelled. The success of this method resulted from prolonged exposure to BrdU released over several hours from a 25 mg tablet implanted subcutaneously at 24 hr postwounding at the time when DNA synthesis and cell proliferation are maximal. This simple technique promises to be useful in determining the size and location of wound sites with application to a wide variety of organs and tissues in studies of repair and healing.     

Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical, immunocytochemical and ultrastructural studies

All stages of regeneration in hamster tracheal epithelium were studied following a denuding mechanical injury. At 1 h all the cells had sloughed from the wound site leaving a bare and sometimes disrupted basal lamina. Viable cells at the wound margins rapidly changed shape, flattened and migrated to cover the denuded lesion by 12 h. In addition, epithelial cells that remained viable demonstrated sublethal changes that included the rapid discharge of mucous granules from secretory cells, internalization of cilia by ciliated cells and evidence of heterophagy in both cell types. By 24 h a wave of epithelial cell divisions occurred, primarily by secretory cells. This produced a multilayered epidermoid metaplasia that was best developed at 48 h. The metaplastic epithelium was largely composed of cells with both secretory (mucous granules) and epidermoid (tonofilament bundles and numerous desmosomes) characteristics. The peroxidase-antiperoxidase (PAP) method demonstrated a few keratin-positive cells in the wound as early as 12 h post-wounding and keratin was demonstrated in more cells by 24 h. All cells in the metaplastic wound epithelium were keratin-positive by 48 h. Following 48 h some of the most superficial keratinized cells sloughed from the epithelium and the keratin content of the remaining cells began to decline. At 72 h pre-ciliated and pre-secretory cells were seen in the wound. Pre-ciliated cells were characterized by an abundant electron-lucent cytoplasm, large pale nucleus, filiform apical microvilli and evidence of ciliogenesis, similar to that seen during fetal development. Pre-ciliated cells often contained apical mucous granules, apparently carried over from the parent secretory cells. With the appearance of these columnar cells the normal mucociliary morphology was restored in small wounds by 120 h, but some persistent epidermoid metaplasia remained in the large wounds through 168 h post-wounding. These data provide further evidence for the important role of secretory cells in the histogenesis of epidermoid metaplasia and the regeneration of normal morphology following injury. The implications of these findings in understanding the histogenesis of other lesions in the tracheo-bronchial epithelium are discussed.     

Vitamin A deficiency and inflammation: the pivotal role of secretory cells in the development of atrophic, hyperplastic and metaplastic change in the tracheal epithelium in vivo.

We showed previously that the proliferation of hamster airway secretory cells decreases during vitamin A deficiency (VAD) but later increases when submucosal inflammation develops (Virchows Arch [B] 59:231-242, 1990). This observation has important biological implications since two morphological extremes (atrophy and quiescence versus hyperplasia and hyperproliferation) are reported in the literature for VAD tracheal epithelium in vivo. In the present study, histological slides of tracheal rings from 35-day-old control and VAD hamsters (Virchows Arch [B] 45:197-219, 1984) were reviewed again. Rings from VAD hamsters were selected based on the absence or presence of a florid submucosal inflammation. Quantitative analyses were made on the cartilaginous part of rings from the anterior third of the trachea. When inflammation was absent, a mucociliary pseudostratified epithelium was, for the most part, maintained. The mitotic rate (MR, 6 h colchicine blockade) of secretory cells was markedly reduced (29-fold) but that of basal cells was not changed significantly. Moreover, cell density was not changed by VAD but ciliated cells and secretory cells were decreased and basal cells were increased, proportionally. We call this "minimal morphological change." Thinning (atrophy) of the minimally changed epithelium was associated with focal cell sloughing. Small scattered foci of epidermoid metaplasia (multiple layers of highly keratinized cells which were extremely flat, so that the epithelium was thin and attenuated) were also seen. We call this "atrophic epidermoid metaplasia." When inflammation was present, hyperplastic changes (stratification and epidermoid metaplasia) predominated and cells were in mitosis at all epithelial levels (low, middle, superficial) except in the most superficial (terminally differentiated) squames. The tracheal epithelium was thickened and hypercellular. The cells were piled up at the stratified lesions, and epithelial height, cell density and epithelial MR were significantly increased compared with the non-inflamed VAD epithelium. The effects of VAD and inflammation on cell proliferation were analyzed further by studying 7 h bromodeoxyuridine (BrdU) labelling patterns of cells in VAD tracheal epithelium, with and without submucosal inflammation. In addition, inflammation was induced in "minimally changed epithelium" by mild mechanical injury. The BrdU labelling patterns confirmed that DNA synthesis by secretory cells is reduced markedly by VAD. However, this suppression is overidden by the influx of inflammatory cells (the nature of the stimulus is unknown). The results indicate that the morphological contrasts (atrophy and hyperplasia) seen in the trachea during VAD in vivo are related to extremes in proliferation rates of tracheal secretory cells, regulated by VAD alone (minimal replication) and by inflammation (maximal replication).     

Effects of vitamin A-deficiency and inflammation on the conducting airway epithelium of Syrian golden hamsters

The effects of vitamin A-deficiency and inflammation were studied in the conducting airways of Syrian golden hamsters. An important goal of the study was to characterize epithelial changes that occur early in vitamin A-deficiency, that might precede yet predispose to infection, and precipitate inflammatory changes in the lungs. Age-matched vitamin A-replete control and vitamin A-deprived hamsters were killed at 33 days of age (preweight-plateau); at 41 days of age (weight plateau-early weight loss); and at 48-55 days of age (prolonged weight plateau followed by weight loss). A tablet containing bromodeoxyuridine (BrdU) was implanted subcutaneously into each hamster 7 h before it was killed. No changes were seen in the conducting airway epithelium of vitamin A-deprived hamsters in the preweight plateau. However, labelling of secretory cells for BrdU was reduced 6-7 fold in the epithelium lining the lobar bronchus (p less than 0.0002) and the bronchioles (p less than 0.0001), and the proportions of ciliated cells were decreased (p less than 0.0001) at both airway levels in vitamin A-deficient hamsters in the weight plateau-early weight loss stage. Changes in cellular morphology were minimal in the intrapulmonary airway epithelium at this time but a few small focal patches of epidermoid metaplasia were seen in the tracheal epithelium. Small foci of inflammation were closely associated with the airways in the weight plateau, and the inflammation became more widespread when the deficiency was prolonged. The results suggest that the defense of the lungs to infection was impaired initially in the vitamin A-deficient hamsters by a widespread reduction in the numbers of ciliated cells throughout the epithelium of the conducting airways (trachea, bronchi, bronchioles). At the foci of inflammation, labelling of epithelial secretory cells for BrdU was greatly increased at all airway levels. A highly stratified cornifying epidermoid metaplasia developed in the tracheal epithelium, and goblet cell metaplasia developed in the cranial portion of the lobar bronchus, in association with submucosal inflammation. Goblet cell metaplasia appeared to be the only abnormality that was not reversed when vitamin A was restored to the diet.     

Effects of vitamin A-deficiency and inflammation on the conducting airway epithelium of Syrian golden hamsters

The effects of vitamin A-deficiency and inflammation were studied in the conducting airways of Syrian golden hamsters. An important goal of the study was to characterize epithelial changes that occur early in vitamin A-deficiency, that might precede yet predispose to infection, and precipitate inflammatory changes in the lungs. Age-matched vitamin A-replete control and vitamin A-deprived hamsters were killed at 33 days of age (preweight-plateau); at 41 days of age (weight plateau-early weight loss); and at 48–55 days of age (prolonged weight plateau followed by weight loss). A tablet containing bromodeoxyuridine (BrdU) was implanted subcutaneously into each hamster 7 h before it was killed. No changes were seen in the conducting airway epithelium of vitamin A-deprived hamsters in the preweight plateau. However, labelling of secretory cells for BrdU was reduced 6–7 fold in the epithelium lining the lobar bronchus (p< 0.0002) and the bronchioles (p< 0.0001), and the proportions of ciliated cells were decreased (p<0.0001) at both airway levels in vitamin A-deficient hamsters in the weight plateau-early weight loss stage. Changes in cellular morphology were minimal in the intrapulmonary airway epithelium at this time but a few small focal patches of epidermoid metaplasia were seen in the tracheal epithelium. Small foci of inflammation were closely associated with the airways in the weight plateau, and the inflammation became more widespread when the deficiency was prolonged. The results suggest that the defense of the lungs to infection was impaired initially in the vitamin A-deficient hamsters by a widespread reduction in the numbers of ciliated cells throughout the epithelium of the conducting airways (trachea, bronchi, bronchioles). At the foci of inflammation, labelling of epithelial secretory cells for BrdU was greatly increased at all airway levels. A highly stratified cornifying epidermoid metaplasia developed in the tracheal epithelium, and goblet cell metaplasia developed in the cranial portion of the lobar bronchus, in association with submucosal inflammation. Goblet cell metaplasia appeared to be the only abnormality that wasnot reversed when vitamin A was restored to the diet. This is contribution no. 2911 from the Pathobiology Laboratory     

Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.     

Regeneration of the respiratory epithelium]

It is very important to clarify the mechanism of the regeneration of the respiratory epithelium in not only Oto-Naso-Laryngology but also in the field of chest medicine and surgery. Because of the experimental study carried out on dogs, involving the curing process of the tracheal anastomosis site, a scanning electron microscopic study showed that after two weeks, ciliated cells could be seen making a thin layer. It took nine weeks or more till the whole tracheal anastomosis site was completely covered by the ciliated epithelial cells. Having reviewed the experimental papers concerning the regeneration of the respiratory epithelium which have been published since 1953, a conclusion has been reached that after the injury of the respiratory mucosa, the existence of the basement membranes remains were covered by the migration of the traditional epithelium from the margin of the wound and four weeks later, the wound was covered by the normal epithelium. It has been stated up until now that the differentiation of the epithelium was covered from basement cells, but in fact, it seems to occur from the secretory cells. This has been demonstrated by the culture process of the respiratory epithelium. From these facts, the cultured respiratory epithelium cells are very important in elucidating carcinogenesis.     

Mechanical damage to rabbit tracheal epithelium from inhaling inert pyrite dust of needle - like structure. Part I.

The effect of pyrite dust inhalation on the ultrastructure of rabbit tracheal epithelium was studied as a model of mechanic damage of the epithelium from a chemically practically inert aerosol of needle-like structure. Rabbits were eposed to a mean pyrite dust concentration of 49.7 mg/m3 in a solid-aerosol inhalation chamber for intervals of 1/2,2 or 8 h. At each interval, tissue from three animals was examined. Dust particles were found to penetrate gradually into and affect epithelial cells but were not transported further by the cells or phagocytized. Dust particles especially cumulated in the secretory goblet cells. The ciliary border was also deranged and consequently the vital self-cleaning function of the respiratory passages epithelium was impaired.     

Histochemical methods for characterizing secretory and cell surface sialoglycoconjugates

Paraffin sections of trachea, sublingual gland, and pancreas from rats, mice, and hamsters were stained with peanut agglutinin (PNA) or Dolichos biflorus agglutinin (DBA) conjugated to horseradish peroxidase before or after enzymatic removal of sialic acid. Adjacent sections were oxidized with periodate prior to incubation with sialidase and staining with PNA and DBA. PNA binding demonstrated terminal beta-galactose in secretions, at the basolateral plasmalemma of mouse tracheal serous cells, in or at the surface of zymogen granules, and at the apical and basolateral surface of mouse and hamster pancreatic acinar cells. Sialidase digestion revealed PNA binding, demonstrative of penultimate beta-galactose, in secretions of mucous cells in tracheal and sublingual glands and at the apical glycocalyx of ciliated and secretory cells in the tracheal surface epithelium of all the rodents studied. Sialidase also imparted PNA affinity to endothelium in all three species and to secretions and the basolateral plasmalemma of tracheal serous cells and pancreatic acinar cells in the rat. Periodate oxidation blocked the enzymatic removal of N-acetylneuraminic acid as judged by prevention of staining with the sialidase-PNA procedure. Sites in which periodate prevented sialidase-PNA staining included pancreatic islet cells and at the luminal glycocalyx of ciliated and secretory cells in tracheal surface epithelium in all three rodents, most sublingual mucous cells in the hamster, pancreatic acinar cells in the rat, and endothelium, except that of the rat. Glycoconjugate in other sites remained positive with the periodate-sialidase-PNA sequence. Resistance to periodate was interpreted as evidence for the presence of terminal sialic acid with an O-acetylated polyhydroxyl side chain. DBA binding demonstrated terminal alpha-N-acetylgalactosamine in the secretion of all mucous cells in the hamster trachea and 50-90% of those in the rat, secretion and the basolateral plasmalemma of all glandular serous cells in the mouse trachea, at the apical surface of most secretory cells lining the lumen of the rat and hamster trachea, and cilia of 5-10% of ciliated cells in the rat trachea. Periodate oxidation and sialidase digestion demonstrated N-acetylneuraminic acid and penultimate alpha-N-acetylgalactosamine in cilia in the mouse trachea and sialic acid containing O-acetylated polyhydroxyl side chains subtended by N-acetylgalactosamine in the secretion of all mucous cells in the rat and hamster trachea and of 80-90% of mucous cells in the hamster sublingual gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo restitution of airway epithelium

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-m wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [³H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 m/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.     

Temporal distribution of 5BrdU-labelled cells suggests that most injured tissues contribute proliferating cells for the regeneration of the tail and limb in lizard

After tail and limb amputation in lizard, injection of 5BrdU for 6 days produces immunolabelled cells in most tissues of tail and limb stumps. After further 8 and 16 days, and 14 and 22 days of regeneration, numerous 5BrdU-labelled cells are detected in regenerating tail and limb, derived from most stump tissues. In tail blastema cone at 14 days, sparse-labelled cells remain in proximal dermis, muscles, cartilaginous tube and external layers of wound epidermis but are numerous in the blastema. In apical regions at 22 days of regeneration, labelled mesenchymal cells are sparse, while the apical wound epidermis contains numerous labelled cells in suprabasal and external layers, indicating cell accumulation from more proximal epidermis. Cell proliferation dilutes the label, and keratinocytes take 8 days to migrate into corneous layers. In healing limbs, labelled cells remain sparse from 14 to 22 days of regeneration in wound epidermis and repairing tissues and little labelling dilution occurs indicating low cell proliferation for local tissue repair but not distal growth. Labelled cells are present in epidermis, intermuscle and peri-nerve connectives, bone periosteum, cartilaginous callus and sparse fibroblasts, leading to the formation of a scarring outgrowth. Resident stem cells and dedifferentiation occur when stump tissues are damaged.     

大鼠体内气管损伤修复过程及气管干细胞的定位研究

目的观察大鼠体内气管损伤修复过程,进行气管干细胞的定位.方法应用氟尿嘧啶(5-FU)诱发在体气管上皮损伤,动态观察修复过程;对损伤后气管上皮细胞行Hoechst33342荧光染色,并用RT-PCR法检测ABC转运蛋白ABCG2/bcrp1基因.结果1.5-FU作用30min后大鼠气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,免疫组化检测增殖细胞核抗原阴性,证明为G0期细胞.其中部分细胞Hoechst33342染色阴性,为侧群(side population,SP)细胞.2.将5-FU 去除3-6h后,上皮细胞形态变为扁平,9-12h 后细胞变为立方,细胞数目逐渐增多,24h上皮细胞数更多,连接成片,可见纤毛,48h 接近恢复假复层纤毛柱状上皮.3.RT-PCR检测ABCG2/Bcrp1阳性反应产物长度为272bp.结论5-FU打击后,残余的G0期气管上皮细胞中含有干细胞.     

Vitamin A deprivation in hamsters. Correlations between tracheal epithelial morphology and serum/tissue levels of vitamin A

The effects of vitamin A deprivation on the tracheal epithelium of young hamsters were investigated. Colchicine was administered 6 h prior to death to induce metaphase arrest, thus making it possible to quantify the mitotic rates of basal cells and secretory (mucous) cells in the epithelium. Blood samples were taken from all hamsters, and liver samples from some, in order to measure serum and tissue levels of vitamin A. Age-matched controls were compared with the following groups of hamsters maintained on a vitamin A deficient diet: pre weight plateau animals (those gaining weight), weight plateau-early weight loss animals (those maintaining approximately the same weight for 3 or 4 days, followed in some cases by a loss of weight for 3 or 4 days), and prolonged weight loss animals (those showing a loss of weight for 5 or more days). Four week old hamsters in a pre weight plateau had undetectable amounts of vitamin A in their livers and declining levels in their serum, whereas 4 1/2 week old hamsters still gaining weight had barely detectable levels of vitamin A in their serum. Nevertheless, the tracheal epithelium of these animals was not different from controls in appearance, proportions of different cell types, mitotic rates of secretory and basal cells, or in the number of cells per millimeter of basement membrane (cell density). Vitamin A was undetectable in the serum and livers of hamsters in the weight plateau-early weight loss stage. At this time the tracheal epithelium showed minimal morphological change, with small focal areas of epidermoid metaplasia in some animals. The tracheas of animals in early weight loss were smaller than tracheas in the control group, and there was a trend towards an increase in the number of epithelial cells per millimeter basement membrane. Cell types in the minimally changed epithelium appeared nearly normal, but there was an increase in the proportion of basal cells, and an absence (or near absence) of division in both basal and secretory cells. Tracheal rings from hamsters in the prolonged weight loss stage were lined by a cornifying metaplastic epidermoid epithelium. Our findings demonstrate that barely detectable levels of vitamin A in the serum are sufficient to maintain normal growth and differentiation of hamster tracheal epithelium (late pre weight plateau stage). When vitamin A serum levels fall below detectable limits the animals enter the weight plateau-early weight loss stage. This stage is accompanied by an inhibition of tracheal epithelial cell growth, although nearly normal cellular differentiation is maintained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spheroids of granulosa cells provide an in vitro model for programmed cell death coupled to steroidogenesis

We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 μm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3β-HSD by immunoblot and immunohistochemistry, respectively).     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

Identification of late DNA-replicating regions in Chinese hamster chromosomes using a BrdU-giemsa technique

Asynchronous Chinese hamster cells were labelled with BrdU for 3 h prior to harvesting the metaphase cells. The late DNA replicating sites became unifilarly BrdU-substituted as compared to the earlier replicating sites having a normal DNA constitution. Those late replicating sites were identified by pale coloration or dot formation after treatment with 1.0 M Na-phosphate solution (adjusted to pH 9.0 with supersaturated amount of NaHCO₃ and at a temperature of 69–75° C) and staining with Giemsa dye. Using this technique, nuclei with incorporated BrdU could be distinguished from nuclei that had not incorporated BrdU. — One of the advantages of using this technique for identification of late DNA replicating sites is that cells are treated continuously with BrdU for a short period of time before harvesting and only one sampling, rather than a series of samplings, is required to achieve a clear-cut result.     

Histogenesis and morphogenesis of epidermoid metaplasia in hamster tracheal organ explant culture

The formation of epidermoid metaplasia was studied in hamster tracheal epithelium in long-term serum-free organ explant culture. Explants were cultured up to 5 weeks in CMRL 1066 with antibiotics and amphotericin B. At 3 weeks there were rare small foci of epidermoid metaplasia and they became larger and more numerous at 4 and 5 weeks. Three dimensional reconstructions from serial sections demonstrated that the small deep-seated foci were discrete and did not reach the epithelial surface, whereas the larger foci were expansive and involved the full thickness of the explant epithelium. Each small focus consisted of a few swollen electron-lucent basal cells attached to the basal lamina, covered by a layer of flattened electron-dense secretory cells which formed a tight-fitting cap over the basal cells. The altered secretory cells displayed moderately well-developed rough endoplasmic reticulum and tonofilament bundles. During the early stages of formation the deep-seated metaplastic foci were completely covered by a layer of normal appearing cuboidal to low-columnar secretory and ciliated cells. Expansion of the metaplastic foci occurred by addition of flattened, electron-dense secretory cells to the cap so that multiple layers of altered secretory cells covered a core of basal cells, analogous to the structure of an onion. The secretory cells became cornified and with time the foci broke through the columnar mucociliary surface layer. In well-advanced foci, the uppermost cornified squames (metaplastic secretory cells) exfoliated into the tracheal lumen. The study emphasizes similarities and differences between the morphogenesis and histogenesis of epidermoid metaplasia in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical comparative analysis of transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor in normal, hyperplastic and neoplastic human prostates.

Immunoreaction to TGF-alpha was limited to the basal epithelial cells of focal areas in the normal prostates. In benign prostatic hyperplasia (BPH) the immunostained areas were more widespread and immunolabelling was observed in both basal and columnar (secretory) cells of the epithelium. Some cells in the connective tissue stroma were also stained. In prostatic adenocarcinoma, epithelial immunostaining was even more extensive and intense than in BPH, and some stromal cells were also stained. Epidermal growth factor (EGF) immunostaining was only present in some basal cells in normal prostates. In BPH, this immunoreaction was strong in the basal cells and even stronger in the secretory cells. In prostatic cancer, the intensity of epithelial cell immunoreactivity was intermediate between that of normal prostates and that of BPH specimens. EGF-receptor immunostaining was focal and located in the basal cells in normal prostates. In BPH, labelling was also localized in basal cells but extended to wider areas. Some stromal cells appeared weakly labelled. In the prostatic carcinoma, both basal and columnar cells appeared stained and the number of immunolabelled stromal cells was higher than in BPH. The results presented suggest that, in normal conditions, EGF and TGF-alpha act as autocrine growth factors for the basal cells of the prostatic epithelium. In BPH this action is maintained and, in addition, the columnar cells start to secrete both factors which are bound by the basal cell receptors, giving rise to a paracrine regulation which probably overstimulates basal cell proliferation. In prostatic carcinoma, besides these regulatory mechanisms, the acquisition of EGF-receptors by the secretory cells develops an autocrine regulation which might induce their proliferation.