Expression of argyrophilic nucleolar organizer regions (AgNORs) can be used to assess cellular proliferation as shown in rat thymic sections

Summary Silver-stained nucleolar organizer regions (AgNORs) were studied in thymic sections from 4- and 30-day-old rats. By direct examination under the light microscope cells with a low or high content of AgNORs (type I and type II cells, respectively) were identified and their relative numbers calculated. The mean area of AgNORs per cell was calculated for each type of cell and age group. Additionally, the proportion of cells labelled with bromodeoxyuridine was calculated in sections from the same animals. Visual identification of type I and type II cells was confirmed by a significant (p < 0.01) difference in the mean AgNOR area in both types of cell. Both the proportion of cells with a high expression of AgNORs (type II cells) and that of bromodeoxyuridine-labelled cells were significantly greater (p < 0.01) in 4-day-old rats than in 30-day-old rats. A significant correlation was found between both variables (R² = 0.45;p = 0.002), the relation being best between both variables and age (R² = 0.91;p = < 0.001). These data offer support for an easy interpretation of the AgNOR reaction in which the proportion of cells with a high expression of AgNORs can be used as an index of proliferative activity in a tissue sample.     

Effects of experimental hypothyroidism on the development of the hypothalamo-pituitary-adrenal axis in the rat

Hypothyroid pups were obtained by adding methimazole in the mother's drinking water from day 15 of gestation and sacrificed at 4, 8 or 15 days. Circulating corticosterone decreased at all ages, while CBG concentrations diminished at day 4, increased at day 8 and did not change at day 15 in hypothyroid rats. As opposed to controls, plasma ACTH concentrations decreased steadily with age while there was an accumulation of ACTH in the anterior pituitary of hypothyroid 15-day-old rats. Anterior pituitary POMC contents were unaffected by the treatment. In the hypothalamic PVN, CRF mRNA levels in the total population of CRF-synthesizing cells and in the CRF+/AVP+ subpopulation were below those of controls whatever the age considered while AVP mRNA in the CRF+/AVP+ subpopulation did not change at day 4 and decreased at day 8 and 15 in hypothyroid animals. Both the number of cell bodies expressing detectable levels of CRF mRNA and the percentage of CRF and AVP colocalization decreased at day 4 and were unchanged thereafter. CRF and AVP immunoreactivity in the zona externa of the median eminence increased with age but was not affected by methimazole treatment. The concentration of AVP mRNA in the magnocellular cell bodies of the PVN and the SON as well as AVP immunoreactivity in the zona interna of the median eminence were not changed by the treatment at days 4 and 8. In hypothyroid 15-day-old rats, SON AVP mRNA increased, AVP immunoreactivity decreased while plasma osmolality was enhanced. In conclusion, our data demonstrate that experimental hypothyroidism impairs specifically the maturation of hypothalamic parvocellular CRF and AVP gene expression during the stress hyporesponsive period. These observations suggest that the physiological peak in plasma thyroxine concentrations that occur between day 8-12 may participate in the maturation of hypothalamic CRF- and AVP-synthesizing cells.     

Relationship of intracerebral pituitary grafts to central neuropeptide systems

Variable amounts of pituitary tissue from neonatal or 30-day-old donor rats were implanted in the recessus triangularis or third ventricle of hypophysectomized male host rats. The pituitary tissue was implanted either immediately or 30 days after hypophysectomy of the host rat. Grafts from donors of either age were capable of maintaining a significant degree of testicular weight in one-third of the implanted animals. Neonatal grafts were not capable of restoring testicular weight when implanted 30 days after hypophysectomy. Final body weights of all graft-bearing animals were greater than those of hypophysectomized controls. The pars distalis of all grafts contained large numbers of cells immunore-active for LH, GH, and ACTH; TSH-immunoreactive cells were sparse. Prolactin-positive cells were extensive in grafts of animals in which the testes were maintained, and virtually absent in grafts of animals with atrophic testes. The fiber systems of three central neuropeptides, LRF, SRIF, and ACTH, were stained and found not to enter the graft. The results suggest that pituitary grafts in the third ventricle may receive their hypophysiotropic neuropeptides from the CSF.     

Pituitary cell activities in gonadectomized rats treated with estrogen

Summary The incorporation of ¹⁴C-leucine into LTH and STH, the uptake of ¹⁴H-estradiol into the pituitary and the appearance of the LTH and LH cells were studied in male and female rats gonadectomized at the age of 30 days and chronically treated with estradiol (E).The biosynthesis of LTH in the pituitary of ovariectomized rats was decreased 15 and 60 days after the operation to the level of intact males. This decrease is followed by the reduction of the number of immunochemically stained LTH producing cells. Chronical administration of estradiol stimulated the LTH synthesis and maximal incorporation of ¹⁴C-leucine was obtained in ovariectomized rats. Maximal relative increase of labeled LTH was noticed in the pituitaries of intact male rats treated with E.STH synthesis is inhibited by treatment with E and maximal decrease was obtained in intact males.The luteinizing LH cells were still hypertrophic in the pituitaries of gonadectomized E treated rats, but the number of castration cells was reduced.On the basis of these results we can conclude that the castration of 30-day-old rats of both sexes does not alter the sex difference in the reaction of LTH and STH cells to estradiol.Supported by Serbian Academy of Sciences.We wish to thank Dr. Claud Robyn for the generous supplies of antiserum for ovine LTH and LH.     

Developmental changes in the inhibition of cultured rat uterine cell proliferation by opioid peptides

Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.     

CATIONIC COMPOSITION OF RAT CEREBRAL CORTEX SLICES. COMPARATIVE STUDY DURING DEVELOPMENT

Abstract—

• 1 Slices of cerebral cortex isolated from 20-day-old rats, when incubated in an appropriate oxygenated saline, show only a slight increase in weight (± 0.4 per cent of initial fresh wt.). At 30 days, under the same conditions, this increase in weight is 12.4 per cent. This is similar to the value observed in adult animals (13.6 per cent).
• 2 Modifications of the cationic content of slices in the case of 20-day-old animals are very slight while in 30-day-old animals they are greater and similar to those observed with adults.
• 3 The inulin space of 20- and 30-day-old rats are the same. The swelling exhibited by the slices isolated from 30-day-old animals may reasonably be attributed to a cellular uptake of extracellular fluid. This is accompanied by an increase of the Naf concentration and by a decrease of the K⁺ concentration. The values observed are similar to those observed with adult rats.
• 4 Comparing our results with previous histological and histochemical data, it may be suggested that the modifications of cationic content, as well as the swelling of slices isolated from 30-day-old rats or adult animals, are solely attributable to changes affecting glial cells and more specifically the astrocytes.

     

Neonatal phenobarbital imprinting of rat hepatic microsomal testosterone hydroxylations

The effects of neonatally administered phenobarbital (PB) on adult rat hepatic microsomal metabolism of testosterone were examined in 60-, 90-, and 120-day-old animals. Phenobarbital-induced imprinting was evident at all ages; however, female rats appeared to be more susceptible to the neonatal effects of phenobarbital than did male rats. In 60-day-old female rats, increased testosterone 2α-hydroxylase activity was observed in microsomes from noninduced rats, whereas decreased testosterone oxidation at all positions except 2α and 15β was observed in microsomes from Aroclor 1254-induced rats. The decreased oxidation of testosterone at specific sites in response to Aroclor 1254 induction was quite dramatic, decreasing the activities to near or below control levels. By contrast, phenobarbital-treated 60-day-old males exhibited approximately a twofold increase in Aroclor 1254-induced 16α and 2α-hydroxylase activities. The pattern of changes in testosterone metabolism observed in phenobarbital-treated animals was different at both 90 and 120 days from that at 60 days. Only minor alterations in the oxidation of testosterone were observed in 90-day-old animals of either sex. In 120-day-old animals the greatest effects of neonatal phenobarbital exposure were on Aroclor 1254–induced 16β-hydroxylase activities. In induced female rats 16β-hydroxylase activity was again decreased to noninduced levels, while in induced male rats a fourfold increase in this activity was observed. These results demonstrate that neonatal exposure to phenobarbital can alter both constitutive and Aroclor 1254–induced testosterone metabolism in adult rats and that the effects of neonatal phenobarbital exposure are age and sex differentiated.     

Onset of compensatory hypertrophy of interstitial tissue and Leydig cells in rats hemicastrated around the time of puberty

When rats were unilaterally castrated at 20, 30, and 40 days of age, only those rats hemicastrated at 40 days showed compensatory hypertrophy of the interstitial tissue and Leydig cells when killed 30 days after hemicastration. At the time of death, volume densities of interstitial tissue, Leydig cells, and vascular components were greater in 70-day-old hemicastrated rats than in intact rats of the same age. The total number of Leydig cells per testis in hemicastrated and intact rats was always the same at any age. Estimated Leydig cell volume in 70-day-old rats was twice that in intact rats. By contrast, the testes of 50- and 60-day-old rats at the time of death displayed essentially the same morphological features, regardless of whether animals were hemicastrated. The concentration of plasma testosterone was higher in 50-day-old controls than in hemicastrated rats. Seventy-day-old hemicastrated rats showed higher levels of plasma testosterone than controls. The level of plasma dihydrotestosterone in 60- and 70-day-old hemicastrated rats exceeded that in the controls. A significant increase in follicle-stimulating hormone was noted in 50- and 70-day-old hemicastrated rats compared to normal rats, while levels of luteinizing hormone were basically the same. The increase in Leydig cell volume, interstitial tissue volume, vascular component volume, and plasma testosterone level caused by hemicastration at 40 days of age differed from that at 20 and 30 days of age.     

DNA distribution of mast cell populations in growing rats

The proliferation of rat peritoneal mast cells was examined under normal conditions in vivo. DNA content of individual mast cells was measured by cytofluorometry after staining with the bibenzimidazole dye Hoechst 33258. Diploid non mast cells from each rat were used as a biological standard, which resulted in small long-term variations in the method. The proportion of mast cells in the S + G2 region of the DNA distribution was about 4% for young rats (24 days old, body-weights about 60 g). It decreased in relation to body-weight, and was less than 1% for 105-day-old rats weighing 400 g. During the same growth period the total number of mast cells in the peritoneal cavity increased about 8-fold. The total number of proliferating cells, about 30,000, remained constant throughout the observation period. No evidence of polyploidization or accumulation in G2 of mast cell nuclei was found. It is concluded that peritoneal mast cells increase in number by mitotic proliferation of differentiated cells.     

Evidence that Leydig precursors localize in immature band two cells isolated on Percoll gradients

The present studies examined responses to hCG and/or insulin of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase activity (3 beta-HSD) in cultured Band 2 and Band 3 cells from 25- to 40-day-old rats isolated on Percoll gradients. In Band 2 cells, from 25-day-old rats enzyme activity increased about 3- and 2.5-fold, after 6 days of exposure to hCG or insulin, respectively. However, hCG did not stimulate enzyme activity in Band 2 cells from 30-, 35- and 40-day-old animals, and responses to insulin alone or insulin plus hCG declined with age. In Band 3 cells only insulin increased enzyme activity at each age. Neither hCG or insulin altered DNA levels in Band 2 or Band 3 cells, suggesting that increased activity in Band 2 cells from 25-day-old rats was not due to cellular replication. However, hCG increased the number of cells staining positive for 3 beta-HSD about 4-fold in Band 2 cells from 25-day-old rats. Insulin did not increase the number of positive staining cells in Band 2 and Band 3 cells from 25-day-old rats, suggesting that its major effect was to increase enzyme activity in existing cells. These results suggest that during a limited period of maturation precursor cells in Band 2, which are undetected by histochemical staining for 3 beta-HSD, can be converted to Leydig cells in culture by hCG.     

Gluconeogenesis from lactate in the developing rat. Studies in vivo

1. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose, liver glycogen and skeletal-muscle glycogen were measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into 2-, 10- and 30-day-old rats. 2. Between 15 and 60min after the injection of the l-[U-(14)C]lactate, the specific radioactivity of plasma lactate decreased with a half-life of 20-33min in animals at all three ages. 3. At all times after injection examined, the specific radioactivity of plasma glucose of the 2- and 10-day-old rats was at least fourfold greater than that of the 30-day-old rats. 4. Although (14)C was incorporated into liver glycogen the amount incorporated was always less than 5% of that present in plasma glucose. 5. The results are discussed with reference to the factors that may influence the rate of incorporation of (14)C into plasma glucose, and it is concluded that the rate of gluconeogenesis in the 2- and 10-day-old suckling rat is at least twice that of the weaned 30-day-old animal.     

Changes in the dynamics of luteinizing hormone-releasing hormone-stimulated secretion of luteinizing hormone during sexual maturation of female rats

Our aim was to identify age-related changes in the dynamics of luteinizing hormone (LH) release that may contribute to the decline in pituitary sensitivity to luteinizing hormone-releasing hormone (LHRH) during sexual maturation of female rats. We studied LHRH-stimulated LH secretion curves of superfused pituitaries from rats ranging in age from 10 days to the first estrous cycle. Pituitary fragments were exposed for 10 min to medium alone or to medium plus LHRH; incubation continued in medium alone for 130 min and effluent was collected for LH analysis. Secretion curves were compared on the basis of total secretion (area under the curve), maximal change in LH secretion rate, and rates of rise and decay of the curves. The data show that total LH secretion in response to LHRH is greatest in 15-, 20-day-old and first-proestrus animals. Also, the maximal change in LH secretion rate was greater, and the increase in LH secretion rate faster in younger animals than in 30-day-old animals. Analysis of secretory granules in LH-containing gonadotropes of 15- and 30-day-old animals revealed changes in he granule population with age. We conclude that younger animals respond faster with a greater LH secretion response to LHRH than do 30-day-old or first-estrus animals, and that these age-related changes in the dynamics of LH secretion may be due in part to maturation of the LH secretory granules.     

Age dependence of hepatic transport in control and phenobarbital-pretreated rats.

Eosine is excreted in rat bile unchanged, which makes it suitable for the study of age dependent changes in hepatic uptake and excretion. Bile flow was approximately 40 μl/kg/min in 20-day-old rats and twice as high in 30-day-old animals. In 60- and 120-day-old rats the bile volume was decreased, moreover in 220-day-old ones it fell to the level of 20-day-old rats. The biliary excretion of eosine (150 μmol/kg i.v.) was highest in 60-day-old rats, however, the biliary flow reached its peak in 30-day-old rats. After phenobarbital (PB) pretreatment (75 mg/kg i.p. daily for five days) each age group showed enhancement in liver weight and bile volume. On the other hand, the hepatic concentration of eosine did not change after PB pretreatment caused an increase in the biliary excretion of eosine in 30-, 60-, 120- and 220-day-old rats but no significant change in 20-day-old animals. Our results indicate that the hepatic transport in young rats was immature and was not induced by PB. However, PB can increase the low excretion rate in old rats.     

The pubertal-related decline in cellular proliferation and neurogenesis in the dentate gyrus of male rats is independent of the pubertal rise in gonadal hormones

Pubertal development is marked by significant decreases in cellular proliferation and neurogenesis in the dentate gyrus of the hippocampal formation. Although it is unclear what mediates these developmental changes in the dentate gyrus, gonadal hormones have been implicated in modulating many neurobiological processes during puberty and various parameters of neurogenesis in adulthood. Thus, it is possible that the gradual and sustained increase in gonadal hormones experienced during puberty plays a role in these changes in neurogenesis. In this experiments, we first quantified cellular proliferation and neurogenesis using 5-bromo-2'-deoxyuridine (BrdU) and doublecortin (DCX) immunohistochemistry, respectively, in the dentate gyrus of prepubertal (30 d), midpubertal (45 d), and adult (90 d) male rats. We found the decline in BrdU and DCX cell numbers throughout these ages was coincident with increases in their plasma testosterone levels. We next tested whether exposure to the pubertal rise in gonadal hormones was necessary for this decrease in hippocampal neurogenesis to occur. Thus, we examined cellular proliferation and neurogenesis in intact 30 day (prepubertal) and 60-day-old (late-pubertal) rats, as well as 60-day-old rats that had previously been gonadectomized or sham-gonadectomized at 30 days of age. Although we again found the expected decline in BrdU and DCX cell numbers between 30 and 60 days of age in the intact groups, there were no differences among the 60-day-old animals, regardless of gonadal status. These data indicate that the pubertal-related decline in hippocampal cellular proliferation and neurogenesis is independent of the pubertal change in gonadal hormones.     

Developmental and lactational changes in the rat anterior pituitary VIP receptor

The regulation of female rat anterior pituitary vasoactive intestinal peptide (VIP) receptors was examined during postnatal development and lactation. VIP receptor binding to anterior pituitary membranes from 1- to 60-week-old rats and lactating rats was examined using HPLC purified [Tyr(125I)10]VIP. Nonlinear regression of competitive binding studies indicated the presence of 2 VIP binding sites in 3-week and older animals, whereas only 1 site was identified in 1- and 2-week-old rats. The single site did not differ significantly in affinity or number when compared to the low affinity site of older animals. The guanine nucleotide, GTP-gamma-S, decreased the specific binding of VIP by 60-80% in 3-week and older animals, but not in younger animals. Compared with adult nonlactating animals, the number of high affinity binding sites decreased significantly during lactation, with no change in receptor binding affinity.     

An immunohistochemical study of adenohypophyseal cells containing follicle-stimulating hormone and luteinizing hormone during the phase of selective follicle-stimulating hormone release in postnatal female rats

Summary Serum concentration of follicle-stimulating hormone (FSH) in the juvenile female rat increases independently from that of luteinizing hormone (LH). The objective of this study was to determine whether this increase in serum FSH is accompanied by a proliferation of FSH-cells greater than the proliferation of LH-cells. Thus, we measured circulating FSH and LH in female rats on days 3, 10, 13, 17, and 20, calculated the percentages of adenohypophyseal cells that contained FSH or LH on days 3, 10, and 20, and determined whether cells containing only FSH existed on day 10. Serum FSH concentrations on days 10 and 13 were significantly greater than those on days 3, 17, or 20. No differences existed in serum LH concentrations. Cells containing FSH or LH were distributed throughout the entire adenohypophyses of 3, 10, and 20-day-old females. Clusters of these cells were observed in the ventral regions of adenohypophyses of 3-day-old females. The percentages of adenohypophyseal cells containing FSH increased significantly from 9% in 3-day-old rats to 17% in 10-day-old rats and then decreased to 14% in 20-day-old animals. At all ages the percentages of adenohypophyseal cells containing FSH were similar to the percentages of cells containing LH. At 10 days of age, all cells containing FSH also contained LH and all cells containing LH also contained FSH. These data suggest that the increase in serum FSH in the juvenile female rat is associated with an increase in the percentage of adenohypophyseal cells containing FSH and that at this time all cells containing FSH also contain LH.     

Binding of intravenously injected antibodies against laminin to developing and mature endocrine glands

Summary To determine whether circulating antibodies against laminin can bind in vivo to basement membranes within endocrine glands, affinity-purified sheep or rabbit anti-laminin IgG was intravenously injected into rats. One to five hours after injection, anti-laminin IgG was bound to all basement membranes of adrenal and anterior pituitary glands of mature as well as 2-day-old newborn rats as shown by immunofluorescence microscopy. After the injection of anti-laminin conjugated directly to horseradish peroxidase (HRP), HRP reaction product was also present throughout adrenal and pituitary basement membranes in mature and immature glands 1–5 h post-injection. Ultrathin Lowicryl sections from rats that received unconjugated rabbit anti-laminin IgG 1 h prior to fixation with paraformaldehyde were labeled directly with anti-rabbit IgG-colloidal gold. In these cases, gold also bound specifically over the lamina densa and lamina rara. When adrenal or pituitary glands from mature rats were examined by immunofluorescence 1 week after the injection of sheep anti-laminin IgG, the patterns and amounts of bound sheep IgG were indistinguishable from those observed 1 h after injection. In contrast, significantly less fluorescence was present in glands from 7-day-old rat pups that had received anti-laminin IgG 5 days earlier. In addition, when anti-laminin IgG-HRP was injected into newborns and glands were fixed 5 days later, lengths of labeled endothelial and epithelial basement membranes were often interspersed with unlabeled lengths in zones of cellular proliferation in the outer adrenal cortex and throughout the pituitary gland. These results indicated that unlabeled basement membranes in these regions were probably assembled after the injection of anti-laminin IgG, which would also explain diminished labeling of basement membranes in these animals. Despite the continued presence of heterologous anti-laminin IgG within endocrine basement membranes, however, rat IgG, rat C3, inflammatory cells, or histologic abnormalities were observed in neither newborn nor adult glands under the conditions examined here. Sections from rats injected with control IgG or control IgG-HRP were entirely negative by immunofluorescence, immunoperoxidase, and immunogold techniques. We therefore conclude that (1) apparently large amounts of circulating anti-laminin IgG can bind to adrenal and pituitary basement membranes, and (2) at least some of these basement membranes are assembled during development by progressive splicing of newly synthesized matrix into that already present.     

Neonatal treatment with monosodium glutamate (MSG): structure of the TSH-immunoreactive pituitary cells

Glutamic acid represents the most abundant stimulatory neurotransmitter in the central nervous system. Monosodium glutamate (MSG), subcutaneously administered to newborn rats in the perinatal period, induces lesions in 80 to 90% of the neurocytes of arcuate nuclei in the hypothalamus. These nuclei are the site of production of numerous stimulatory and inhibitory hormones including growth hormone releasing hormone (GHRH). The present studies were performed on male Wistar strain rats, subcutaneously injected on days 2, 4, 6, 8, and 10 of postnatal life with MSG at a dose of 4 mg/g body weight. Eighteen-month-old rats were additionally treated with Ambinon. When the animals reached the ages of 6 or 12 months, their body weight, body length and weight of pituitary were determined. On paraffin sections, using immunohistochemical techniques, TSH-immunoreactive cells were detected and characterised by computerised image analysis. The results were subjected to statistical analysis using Student's t test. The rats which were perinatally treated with MSG and examined after 6 or 12 months of life were obese and shorter than control rats by 7% and 10% respectively. They also exhibited a reduction in the weight of the pituitary of 30% and 40% respectively in the two age groups. The proportion of TSH-immunoreactive cells in the pituitary remained unchanged and amounted to 4.5% in the 6-month-old and 5.4% in the 12-month-old rats, respectively. The number of TSH-positive cells per mm2 area remained unchanged. The area and circumference of the cells in the 12-month-old rats were reduced by 22% and 18%, respectively. Perinatal injury to hypophyseal arcuate nuclei induced by monosodium glutamate injection, was not associated with any significant alterations in pituitary structure, as defined by the proportion of pituitary volume occupied by TSH-immunoreactive cells.     

Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth

Neuronal number in the mature CNS is determined by the balance of cell proliferation and death. The effects of ethanol on cell proliferation and death were examined in primary cultures of neocortical neurons derived from 16-day-old rat fetuses. The cells were treated with ethanol (0 or 400 mg/dl) and examined for (1) immunohistochemical identity, (2) cell cycle kinetics using a cumulative bromodeoxyuridine labeling technique, (3) viable cell number via a trypan blue assay, and (4) the incidence of cell death with terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 immunhistochemistry. After two days in culture, most (>85%) cells expressed a neuron-specific antigen(s) whether or not ethanol was added to the culture medium. Ethanol affected the proliferation of the cultured cells, e.g., the length of the cell cycle was greater in the ethanol-treated cells than in controls. The number of trypan blue-negative (viable) cells was profoundly decreased by ethanol exposure. This decrease was accompanied by increases in the frequencies of TUNEL- and caspase 3-positive cells and of cells exhibiting nuclear condensations. Thus, ethanol decreases the number of viable cells in vitro by slowing cell proliferation and increasing the incidence of cell death. The expression of the death indices in untreated cultures is most consistent with a single (apoptotic) pathway of cell death, rather than simultaneous apoptotic and necrotic modes of death. Furthermore, it appears that ethanol initiates an apoptotic death among cultured cortical neurons.     

Age-related immunosenescence in Fischer 344 rats: influence of exercise training.

The present investigation examined the extent to which 15 wk of endurance training could influence immune function in young, middle-aged, and older animals. Forty-eight male Fischer 344 rats were divided into trained and untrained groups. Training consisted of treadmill running at 75% maximal running capacity for 1 h/day, 5 days/wk, for 15 wk. Animals were killed at 8, 17, and 27 mo, at which time splenocytes were isolated. The capacity for lymphocyte proliferation in response to mitogen (concanavalin A, ConA), interleukin-2 (IL-2) production, and cytolytic activity against YAC-1 target cells was determined. ConA-induced proliferation declined significantly with age. Training suppressed the proliferative response in the young (-41%) and middle-aged animals (-27%) compared with the age-matched controls; however, training improved this response (+58%) in the older group. IL-2 production followed a pattern similar to that for mitogen-induced proliferation, such that production declined with age and was reduced with training in young and middle-aged animals but was significantly more improved in the older animals than in age-matched controls. The ability to lyse target cells, measured as percent cytotoxicity, declined steadily with advancing age at all effector-to-target cell ratios tested: 52, 14, and -16% for 8-, 17-, and 27-mo-old rats, respectively. It was concluded that the capacity for ConA-induced splenocyte proliferation, IL-2 production, and cytolytic activity declines significantly with advancing age. Furthermore, 15 wk of endurance training suppressed proliferation and IL-2 production in young animals but improved these responses in older animals. Training had no effect on cytolytic activity.