Induction of atypical squamous metaplasia with benzo[a]pyrene in cultured hamster tracheas

Areas of hyperplasia were produced in hamster tracheal epithelium maintained in vitro by exposure to a suspension of benzo[a]pyrene (BP) in gelatin. Typical and atypical epiodermoid metaplasia were seen by 2 weeks. In atypical areas, cell nuclei were enlarged with prominent nucleoli, the cytoplasm contained dense bundles of tonofilaments and the cells were joined by numerous desmosomes. The peak response to the carcinogen was reached 4 weeks after the application of BP and consisted of extensive atypical epidermoid metaplasia. Tracheas treated with gelatin alone maintained a columnar epithelium for 6 weeks of culture. The characteristics of the metaplastic changes in vitro are essentially identical to those described after exposure of the hamster tracheobronchial epithelium to benzo[a]pyrene-ferric oxide in vivo.     

Carcinogen-induced changes in tracheal epithelium cultured in serum-free, chemically defined medium

Rat tracheal tissue was cultured for periods up to 2 months in medium containing benzo[a]pyrene, and the epithelium was studied for the histologic effects of deletion of serum from the medium. Nodular hyperplasia occurred in 3 days in the absence of serum, but was not seen until several weeks of culture in media containing the same concentrations of carcinogen with 10% calf serum. In serum-free culture, hyperplasia was induced in 2 weeks with one-tenth of the smallest concentration of benzo[a]pyrene that yielded this change in serum-containing medium. The cells of the hyperplastic epithelium in both serum-containing and serum-free culture exhibited ultrastructural changes described in carcinogenesis in vivo. In the absence of serum, squamous hyperplasia was uniformly seen, a feature that distinguished serum-free culture from culture in the presence of calf serum. No frank intraepithelial or invasive malignant lesions were produced in either medium. It was concluded that exposure of organ cultures to carcinogen in the absence of serum is the more promising method for bioassay because the response to carcinogen was more rapid, more sensitive, and more reproducible than that seen during exposure in media that contained serum.     

Effect of soya bean saponins on azoxymethane-induced preneoplastic lesions in the colon of mice

The effect of saponins isolated from soya bean flour on the incidence of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the colonic wall of CF1 mice was investigated. Four weekly injections of AOM, a known colon carcinogen, were administered to mice. One week after the last injection, mice were placed on an AIN-76 diet supplemented with 3% soya bean saponins or continued on the basal AIN-76 diet. Another group of mice was placed on the saponin diet without AOM initiation to observe the effect of saponins on the growth characteristics of mice. Dietary intake of soya saponins significantly reduced the incidence of ACF at the end of 14 weeks (postinitiation). Noninitiated mice maintained on a similar soya bean saponin-supplemented diet did not show any adverse effects on the growth and overall health of the animals. These findings suggest that soya bean saponins can play an important role in inhibiting the incidence of ACF in the colon of mice.     

Is hypothalamic GABA involved in immune function in relation to dietary protein during aging?

Hypothalamic GABAergic activity and immune response in spleen were not significantly changed with the increase of age from 3 to 6 months in adult male albino rats. Further increase of age from 6 to 9 months increase the GABAergic activity and decreased the cell viability in spleen without any change in its T-lymphocyte cytotoxicity. Consumption of low protein diet (LPD) for a short-term period (STP; 7 consecutive days) increased the hypothalamic GABAergic activity without changing the immune response in 3 months old rats. When supplemented for a long-term period (LTP; 30 consecutive days) to 3 months old rats, a reduction of hypothalamic GABAergic activity and the immune response was observed. Intake of high protein diet (HPD) for both STP and LTP increased the GABAergic activity and immune response, but the increase of GABAergic activity in hypothalamus under STP was greater than that observed under LTP. In 6 months old rats consumption of LPD for STP reduced the GABAergic activity without any alteration of its immune response. Long-term supplementation of this LPD to the same age group increased GABAergic activity and the mitotic activity of spleen cells without any alteration of the functional activity of the T-cells in spleen. Consumption of HPD for STP failed to produce any change in hypothalamic GABAergic activity and the immune response of 6 months old rats. Supplementation of HPD for LTP reduced the hypothalamic GABAergic activity and the immune response of the same age group. The reduction in hypothalamic GABAergic activity without any change in the immune response was observed following the supplementation of low protein diet to 9 months old rat for STP. Intake of the LPD for LTP also reduced the hypothalamic GABAergic activity and the mitotic activity of the spleen cells without any alteration of the functional activity of the T-cells in spleen of 9 months old rats. Supplementation of HPD for STP to this aged rat, on the other hand, failed to produced any change in hypothalamic GABAergic activity and the immune response. Intake of HPD for LTP by this aged rats increased the hypothalamic GABAergic activity along with the immune response. The results of this study, thus, suggest that hypothalamic GABAergic activity during aging is an index of immune response and it is modulated following the short- and long-term consumption of protein poor and protein rich diet.     

Characterization of the growth factor activity of amniotic fluid on cells from hematopoietic and lymphoid organs of different life stages

We investigated the proliferation-promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation-promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells, liver cells or peritoneal cells from 2-week-old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1-year-old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti-mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti-mouse SCF antibody revealed a band at 30-32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type-dependently antagonized by TNF-alpha and TGF-beta. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.     

The effects of hyperoxia upon bone in organ culture

Hyperoxia has been reported to stimulate both resorption and synthesis of bone in vitro. The effects of increased oxygen tension were re-investigated using calvaria from infant mice maintained in a stationary grid culture system for 48 48 hours with an unsupplemented chemically defined medium. Resting resorption due to osteoclastic activity was demonstrated in the explants in air by Von Kossa staining, histology, and 45 Ca release. Resorption was inhibited by exposure to 95 per cent oxygen or hyperbaric oxygenation at 2 atmospheres pressure. Hyperoxia also depressed new bone formatin by osteoblasts although the production of a new collagen, as measured by the incorporation of 3H-proline, was greater in calvaria cultured in hyperbaric oxygen than in paired explants in 95 per cent oxygen. Thus hyperoxia was toxic for both synthetic and resorptive activity of bone cells; these effects may stem from the loss of vital factors present in natural MEDIA supplements.     

Mesencephalic artery syndrome

The diurnal changes of mitosis and DNA-synthesizing cells count in the tongue epithelium of intact and adrenalectomized mice were studied. A partial desynchronization of mitotic division, as well as diminution of mitotic index variation amplitude and prolongation of high mitotic activity were observed in the operated animals in the course of 24 hours. The average 24-hour mitotic index in the basal layer of the tongue epithelium was the same in the intact and the operated animals. The rhythm of the DNA synthesis failed to differe from control. Thus, adrenalectomy in mice led to distrubances of the diurnal mitotic rhythm in the tongue epithelium, but had no effect on the rhythm of the DNA synthesis and the level of the proliferative activity.     

In-vitro models of B-lineage commitment

There is substantial evidence that growth hormone (GH) is particularly important in the control of the age-related decline of thymus function. It was therefore of interest: (a) to assess the overall capacity of tissue extracts from mediobasal hypothalamus (MBH), anterior pituitary (AP) and testis, obtained from young (3 months, Yc), middle-aged (13 months, MAc) and old (18 months, Oc) intact C57BL/6 mice to stimulate in vitro the release of thymulin, a Zn-bound immunoregulatory thymic peptide, from pure cultures of mouse thymic epithelial cells (TEC); (b) to perform the same evaluation utilizing MBH, AP and testicular extracts from mice of the same age-range but treated for 45 days with a sc dose of ovine GH (2 micrograms/g body wt) known to stimulate thymulin secretion in vivo. Pituitary hormones were measured by heterologous rat RIAs, whereas thymulin release was estimated by a rosette assay. Untreated animals showed a significant age-dependent increase in the AP content of follicle stimulating hormone but not in other AP hormones. In both control and treated animals, pituitary GH content decreased significantly with age. MBH extracts from C57BL/6 males evidenced thymulin-releasing activity on mouse TEC lines. This activity was maximal in the MBH from young animals and declined with the age of the MBH donors. The thymulin-releasing activity of MBHs from GH-treated mice was higher than that of the control animals and showed a less pronounced decline with age. AP extracts from the same animals showed a higher thymulin-releasing activity than did MBH preparations. This activity showed a progressive age-associated reduction in the APs from untreated mice, whereas in the GH-treated group, an age-related decline was only seen in the old donors. Control testicular extracts had little effect on thymulin release whereas GH treatment induced a definite thymulin-release inhibiting activity in the testicular homogenates of our animals which increased progressively with the age of the testis donors. We conclude that the MBH, AP and testis of the young mouse contain factors able to affect directly the endocrine activity of the thymic epithelium. The amount of these substances declines with age and seems to be modulated by GH.     

Paradoxical action of activin A on folliculogenesis in immature and adult mice

Activin A is a gonadal protein originally isolated from follicular fluid and is recognized as a local regulator of granulosa cell differentiation. Whether activin A promotes folliculogenesis, however, still remains unclarified. The present study was designed to elucidate the effect of activin A on follicular growth in in vitro follicle culture systems. Preantral follicles, 100-120 microm in diameter, were mechanically isolated from BDF1 hybrid immature mice (11 days old) and adult mice (8 weeks old), then cultured for 4 days in a serum-free medium supplemented with activin A (100 ng/ml), FSH (100 mIU/ml), and a combination of both. Follicular diameter was measured daily, and the amount of estradiol and inhibin released at day 4 was determined by RIA. Preantral follicles collected from immature mice showed a significant increase in diameter when cultured with activin A or both activin A and FSH. FSH alone showed no significant effect on the diameter of follicles from immature mice. In contrast, the diameter of preantral follicles from adult mice significantly increased in response to FSH. Activin A did not stimulate growth of follicles from adult mice, and more interestingly, blocked the effect of FSH. The inhibitory action of activin A was in part restored by co-culture with follistatin (100 ng/ml). These results indicate that activin A is folliculogenetic in the prepubertal mouse, but not in adults.     

Molecular approaches to the diagnosis of male infertility

We investigated the effects of aging and/or swimming training on the antioxidant enzyme system in diaphragm of mice. Young (2 months old) and old (26 months old) male mice were swimming-trained for 6 weeks (1 h/day, 5 days/week). Cu,Zn-Superoxide dismutase (Cu,Zn-SOD) activity was significantly upregulated with aging, and swimming training definitely enhanced the activity only in young mice. Neither aging nor swimming training had overt effect on Mn-SOD activity. Glutathione peroxidase activity in young mice was significantly increased after training, but not in old mice. Both of immunoreactive Cu,Zn-SOD and Mn-SOD were significantly increased with aging but were unaffected by swimming training. Consequently, physical training significantly enhanced the specific activity of Cu,Zn-SOD in young mice, but not in old mice. Meanwhile, swimming training significantly increased xanthine oxidase activity in both age groups, the extent of the increase being greater in old mice than in young mice. We concluded that the antioxidant enzyme system in mouse diaphragm trends to be upregulated with aging, but that swimming training improved the system only in young mouse diaphragm.     

Tissue culture of middle ear epithelium using fibroblast-reorganized collagen gels

The middle ear epithelium of the guinea pig has been cultured on a modified floating collagen matrix. Fibroblasts harvested from abdominal skin dermis of allogenic animals were used to reorganize hydrated collagen gels into a dermal-like matrix. Within two days, the explants placed on the surface of these matrices showed proliferation of polygonal "outgrowth cells" which could be maintained for up to one month. During the first two weeks of the culture period, dividing cells were frequently identified. These cells were especially abundant in the marginal area of the explants and consisted of pseudostratified columnar cells. The reorganized collagen gel matrix was stable and did not dissolve even after the "outgrowth cells" became confluent. The present system should allow further elucidation of the growth and differentiation mechanisms of middle ear epithelial cells.     

New techniques for biopsy and culture of human olfactory epithelial neurons

OBJECTIVE: To improve the success of culturing olfactory neurons from human nasal mucosa by investigating the intranasal distribution of the olfactory epithelium and devising new techniques for growing human olfactory epithelium in vitro. DESIGN: Ninety-seven biopsy specimens were obtained from 33 individuals, aged 21 to 74 years, collected from 6 regions of the nasal cavity. Each biopsy specimen was bisected, and 1 piece was processed for immunohistochemistry or electron microscopy while the other piece was dissected further for explant culture. Four culture techniques were performed, including whole explants and explanted biopsy slices. Five days after plating, neuronal differentiation was induced by means of a medium that contained basic fibroblast growth factor. After another 5 days, cultures were processed for immunocytochemical analysis. RESULTS: The probability of finding olfactory epithelium in a biopsy specimen ranged from 30% to 76%, depending on its location. The dorsoposterior regions of the nasal septum and the superior turbinate provided the highest probability, but, surprisingly, olfactory epithelium was also found anteriorly and ventrally on both septum and turbinates. A new method of culturing the olfactory epithelium was devised. This slice culture technique improved the success rate for generating olfactory neurons from 10% to 90%. CONCLUSIONS: This study explains and overcomes most of the variability in the success in observing neurogenesis in cultures of adult human olfactory epithelium. The techniques presented here make the human olfactory epithelium a useful model for clinical research into certain olfactory dysfunctions and a model for the causes of neurodevelopmental and neurodegenerative diseases.     

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis

PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.     

Modification of an in vitro technique in order to grow adult rat anterior pituitary in organ culture

The study of adult rat anterior pituitary maintained in vitro was made possible by a modification of the suspension system used in the watch-glass method of organ culture. The integrity and health of the tissue was observed with the electron microscope. Somatotrophs and gonadotrophs were seen routinely. A cell with large (400-750 nm) granules and thought to be a luteotroph (mammotroph) was also seen. These results support the use of this method for the growth of adult rat anterior pituitary, or other large explants, for short periods of time in organ culture.     

Restoration of sexual behavior and dopaminergic neurotransmission by long term exogenous testosterone replacement in aged male rats

PURPOSE: We investigated the effects of long-term testosterone replacement on copulatory behavior and dopaminergic neurotransmission in the medial preoptic area of aged male rats. MATERIALS AND METHODS: The rats were divided into 3 groups depending on testosterone replacement. Those in the long-term replacement group were castrated at the age of 12 months and received testosterone replacement thereafter for 12 months. In the short-term replacement group, rats were castrated at the age of 22 months and high or low dose testosterone replacement was done for 2 months. The control group consisted of aged rats 24 months old and young rats 12 weeks old, neither of which had been castrated or received testosterone replacement. We observed sexual behavior in rats of these groups. After a behavioral test, we measured the tissue concentration of dopamine in the MPOA and the change rate of the extracellular dopamine level induced by infusion of N-methyl-D-aspartic acid (NMDA) in the MPOA and compared the long-term replacement and no-replacement groups. RESULTS: The rats in the long-term replacement group showed a mount rate at the same level as that of young rats at 6 weeks after starting replacement and it was maintained to 24 months of age. Their mount rate was significantly higher than that of the rats with the short-term replacement. A significantly higher change rate of dopamine release was recognized in the long-term group; however, no significant difference in the concentration of dopamine was recognized between aged rats with long-term replacement and those without replacement. CONCLUSIONS: Aged rats (24 months old) with long-term testosterone replacement maintained almost the same level of mount behavior as young rats (12 weeks old). The results imply that long-term testosterone replacement may favorably alter the decline in the process of sexual activity with aging. The restoration by testosterone replacement of dopaminergic activity in the MPOA may be involved in the maintenance of sexual function in aged rats.     

Capability of neurite regeneration of retinal explant from adult rat after optic nerve injury

Axons of the central nervous system in adult mammals do not regenerate spontaneously after axotomy. To understand whether the optic nerve of adult mammals loses the intrinsic capability to regenerate after injury, we have studied the capability of neurite regeneration of retinal explant from adult rat after optic nerve axotomy in vitro. After experimental blunt damage to the optic nerve of adult Wistar rat, the retinal explants from three days, one week, two weeks and three weeks after axotomy were put in tissue culture to observe the neurite growth after four days' incubation. The neurites were identified as retinal neuron origin by immunocytochemical staining using monoclonal antibody to neurofilament. The results demonstrate that retinal explants from adult rat after optic nerve damage have the capability of neurite regeneration; the capability is strongest in the group of one week after axotomy of optic nerve, but it decreases with passage of the time. On the other hand, the retinal explant from the control group of uninjured eye does not regenerate neurite in tissue culture. These results indicate that the retinal explant of adult rat has intrinsic capability to regenerate after optic nerve injury in vitro, and the capability of neurite regeneration decreases after one week post-trauma.     

The role of apoptosis in the modulation of colon carcinogenesis by dietary fat and by the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate

Studies in laboratory animals have demonstrated that dietary supplements of organoselenium, 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibit colon carcinogenesis. Diverse chemopreventive agents and clinically used anticancer drugs have been shown to induce apoptosis in colonic tumors. Inducing apoptosis is a key mechanism for the effectiveness of some chemopreventive agents; however, failure of apoptosis is now believed to contribute to the development of human cancer. In this study, we determined the number of apoptotic bodies in the colon tumors of rats fed a low-fat (LF) or a high-fat (HF) diet with or without p-XSC treatment. At 5 weeks of age, male F344 rats were divided into four groups, which were then maintained on one of the following diets: LF, 5% corn oil; HF, 23.5% corn oil; and LF and HF supplemented with 20 ppm p-XSC. In addition, the LF or HF diet with p-XSC supplements was administered either during the initiation stage or postinitiation. At 7 weeks of age, all rats except those intended for vehicle (normal saline) treatment were given 15 mg/kg of body weight of azoxymethane once weekly for 2 weeks. The animals were sacrificed 38 weeks after carcinogen treatment, and their colonic tumors were examined for appearance of apoptosis. The LF diet significantly increased the percentage of apoptosis as compared to the HF diet; the percentage of apoptosis in LF and HF diets were 12.4 and 2.9. The colon tumors that were present in the groups fed p-XSC together with a LF or a HF diet after carcinogen administration (postinitiation period) had a higher number of apoptotic bodies than those that were present in the animals fed p-XSC before carcinogen treatment (initiation period). The extent of apoptosis was weak when p-XSC was given with a HF diet (4.4%) during the initiation phase, but it was high significant when p-XSC was administered with LF diet (25.2%). Taken together, our data suggest that administration of LF diet supplemented with p-XSC increases apoptosis as compared to a HF diet alone.     

Changes with ageing in the modulation of murine lymphocyte chemotaxis by CCK-8S, GRP and NPY

The general immunodepression found in ageing organisms may be related to changes in the neuroimmune network. In the present study, the migration capacity of lymphocytes from BALB/c mice of three different ages: young (12 +/- 2 weeks), adult (24 +/- 2 weeks) and old (72 +/- 2 weeks), has been assayed in vitro in response to three neuropeptides: sulfated cholecystokinin octapeptide (CCK-8s), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY) in a physiological range of concentrations (10(-8)-10(-12) M). The capacity of migration to a chemical gradient or chemotaxis was studied by the Boyden's technique using f-met-leu-phe at 10(-8) M as chemoattractant. The results show a different response of lymphocytes to the different neuropeptides, as wells as to age, concentrations and locations studied. However, some similarities were found, for instance the three neuropeptides inhibited chemotaxis in thymus. The stimulatory effects that GRP and NPY exerted in young and adult mice were not observed in old animals. CCK-8s inhibited the chemotaxis in every organ studied, with the effect being more striking in old mice. Our conclusion is that stimulatory effects of the neuropeptides disappear or become inhibitory with ageing.