Rat In Organ Culture Research Articles

The optical properties of rat, porcine and human lenses in organ culture treated with dexamethasone

Organ culturing of lenses is useful for testing the direct effect of moieties such as cations, glucose, oxygen, antioxidants or drugs on the lens. Lens opacity changes in organ cultured lenses have never been measured quantitatively. Our aims were twofold: to establish the time course of lens opacity changes in organ cultured rat, porcine and human lenses and to test the utility of our system to measure opacity changes in lenses treated with dexamethasone. Human, porcine and rat lenses were incubated with or without 5 μM dexamethasone in minimum essential medium for 13 days. Lens absorbance and light scattering were measured using a spectrometer. Lens opacity was graded from lens photographs. Sixteen human lenses were obtained from 10 donors. Light scattering accounted for all of the absorbance of the human lens above 550 nm, 72% of the absorbance at 500 nm and 50% of the absorbance at 450 nm. Similarly, for the porcine lens, light scattering accounted for all of the absorbance above 400 nm and 40% of the absorbance at 350 nm. For the rat lens, scattering accounted for all of the absorbance above 325 nm, and 37% of the absorbance at 300 nm. In the rat lens spectrum, the broad absorbance peak near 600 nm was unique to the rat lens. After day five in organ culture, the level of light scattering, absorbance and optical grade of human lenses were higher in dexamethasone treated lenses compared with those without dexamethasone. Similar differences were observed for rat lenses after one day in organ culture. The opacity of porcine lenses did not change with dexamethasone treatment. In conclusion, this study showed that dexamethasone-induced opacity in human and rat lenses could be measured which is important to future studies to test the positive or negative efficacy of moieties that cause or ameliorate cataract.

Effect of far infrared radiation-ceramics (YYT-1 and YYT-2) on alkaline and acid phosphatase activities in the first lower molar tooth germs of neo-natal rats in organ culture

Effect of far infrared radiation-ceramics (YYT-1 and YYT-2) on alkaline and acid phosphatase activities in the first lower molar tooth germs of neo-natal rats in organ culture

Enhanced Gene Transfer to Rabbit Jugular Veins by an Adenovirus Containing a Cyclic RGD Motif in the HI Loop of the Fiber Knob

Gene therapy using recombinant adenoviral vectors represents a promising therapeutic tool to prevent vein graft stenosis, the main complication of coronary artery bypass grafting. However, the low transduction efficiency of vascular smooth muscle cells and endothelial cells (EC) is a potential limitation, presumably due to the low levels of functional adenovirus receptor (coxsackie:adenovirus receptor; CAR). Designing vectors specifically targeted to α_v integrins is a strategy that might overcome the poor expression of CAR in vascular smooth muscle cells and EC. RGD, a receptor-binding motif that can interact with α_v integrins, was inserted into the HI loop and at the C-terminus of the adenoviral fiber protein in two separate adenovirus vectors encoding a β-galactosidase reporter gene. Av1nBgCRGD (C-terminus) and Av1nBgHIRGD (HI loop) were evaluated in EC in culture and in jugular vein organ culture. Transduction of primary rat and rabbit EC with Av1nBgHIRGD was significantly more efficient when compared to Av1nBgCRGD or Av1nBg. Transduction of mouse, rat and rabbit jugular veins in organ culture using Av1nBg showed that adenovirus-mediated gene expression was greatest in rabbit jugular veins compared to rat and mouse veins. Av1nBgHIRGD augmented gene expression approximately four-fold in rabbit jugular veins when compared to Av1nBg. Histochemical analysis showed that numerous EC but few smooth muscle cells were transduced at all vector concentrations. A substantial number of adventitial fibroblasts were transduced only at the highest vector concentrations of Av1nBgHIRGD. These findings demonstrate that integrin-targeted vectors allow for enhanced gene delivery to veins and strengthen the viability of adenoviral-mediated gene transfer of therapeutic transgenes to human veins prior to vein grafting.

Regulation of TRH release by the cultured neonate rat pancreas

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic β-cells.

Suckling Rat Colon Synthesizes and Processes Active Lactase-Phlorizin Hydrolase Immunologically Identical to that from Jejunum

To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.

Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.

Effect of thyrotropin-releasing hormone on development of the pituitary-thyroid system in fetal rats in organ culture.

Six groups of thyroid glands from 18-day fetal rats were explanted to organ culture for 2 days. In one group, thyroid was cultured alone and in the remaining five groups thyroid was cocultured with pituitaries from fetuses ranging in age from 17 to 21 days. In each of the groups, half of the cultures had thyrotropin-releasing hormone (TRH) added to the medium. Histometric parameters of the thyroid follicle such as diameter and cell height were used as indicators of development of the thyroid gland. When 18-day thyroid was cultured alone, addition of TRH did not accelerate development. When either one 18-day or two 17-day pituitaries were cocultured with thyroid, a significant increase in diameter and cell height was seen. Addition of TRH to the medium induced little or no further change. When the thyroid was cultured with 19- to 21-day pituitaries, a marked increase in thyroid development was observed; and the addition of TRH caused further acceleration in thyroid development. These results suggest that, in organ culture, 17- to 18-day pituitary glands can release some thyrotropin (TSH) with or without additional TRH. Older pituitaries (19- to 21-day) apparently can release an amount of TSH in the presence of TRH that is greater than their own spontaneous TSH secretion.

Corticosteroid binding by fetal rat and rabbit lung in organ culture

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1 2 for dissociation of nuclear-bound [ 3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [ 3H]dexamethasone was approx 5-fold slower by both procedures. The K D for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6nM, respectively. In rat lung, the k d for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the K D values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or K D. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.

Effect of glucocorticoids on alpha-2 adrenoceptors in vas deferens of reserpinized rat in organ culture

The changes in alpha adrenoceptors in vasa deferentia of reserpinized rats in organ culture were examined by binding studies with 3H-clonidine, 3H-yohimbine and 3H-prazosin. On organ culture for 1 day, the 3H-clonidine binding sites decreased by 1.07 pmol/g tissue and after 2 days no binding sites were detectable. 3H-Yohimbine binding sites also decreased during culture, but in smaller extent than 3H-clonidine binding sites. On the other hand, 3H-prazosin binding sites showed no apparent change in amount during culture. Addition of 0.1 mM hydrocortisone or dexamethasone to the culture medium inhibited the decrease in 3H-clonidine binding sites (1.7 pmol/g tissue), and the effect of glucocorticoid was blocked by the inhibitors of protein synthesis, cycloheximide and puromycin. However, hydrocortisone showed no effect on the 3H-yohimbine and the 3H-prazosin binding sites. An anti-endocytotic agent, an anti-microtubular agent and protease inhibitors had no effects on the decrease of 3H-clonidine binding sites during culture. These results suggest that the amount of alpha-2 adrenoceptors can change rapidly while alpha-1 adrenoceptors are stable and that glucocorticoids are important in regulation of conformation of alpha-2 adrenoceptor through synthesis of certain protein(s).

Growth of the mandibular condylar cartilage of the rat in serum-free organ culture

To evaluate the intrinsic capacity for growth, the mandibular condylar cartilage of 4-day-old rats was cultured in a serum-free, chemically-defined medium for 28 days and compared with the condylar cartilage cultured in a serum-supplemented medium and with the normal growth in vivo. Under the serum-free culture conditions, cell proliferation, differentiation into functional chondroblasts and matrix formation continued, but chondroclasia and osteogenesis were absent. This resulted, after 28 days of culture, in an enlarged condylar cartilage with altered proportions, a severe reduction of the prechondroblastic and the transitional zone and a considerable increase of the hypertrophie zone. It appears that the serum-free culture system is unfavourable for endochondral osteogenesis and lacks factors modulating the rate of proliferation, differentiation and maturation of the prechondroblasts. In the functional environment of the condyle, humoral or mechanical factors seem to exert this essential modulating influence on the condylar-cartilage growth.

Epithelial-mesenchymal interaction in differentiation of duodenal epithelium of fetal rats in organ culture.

Epithelial-mesenchymal interaction in the differentiation of duodenal epithelium of fetal rats was investigated by recombination experiments in vitro. The proportion of goblet cells in duodenal epithelium was significantly greater on recombination of developing duodenal epithelium with mesenchyme of the glandular stomach than on recombination with that of the duodenum. Mesenchyme of the glandular stomach or forestomach was better than duodenal mesenchyme in supporting morphogenesis of duodenal epithelium. Treatment of tissues with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) did not affect these tissue interactions.

Some ultrastructural effects of testosterone and insulin on the ventral prostate of rats in organ culture.

The fine structure of the secretory epithelial cells of rat's ventral prostate has been studied following organ culture. Culturing with either testosterone or insulin alone, and with the two hormones combined, were carried out to investigate how insulin modifies the action of testosterone on the maintenance of cellular integrity. After 4 days in hormone-free culture, the secretory epithelial cells showed signs of cellular atrophy and regression, involving loss of the apical microvilli, absence of the apical secretory vacuoles, atrophy of the Golgi apparatus, decrease in rough endoplasmic reticulum and the appearance of autophagic vacuoles. The presence in the medium of either testosterone or insulin alone, or combined, prevented cellular atrophy and regression. The best maintenance of cellular integrity was obtained in a culture containing both hormones. The effects of insulin was approximately equivalent to those of testosterone in the maintenance of cellular integrity.

Responsiveness of the ovary to gonadotrophins in pre- and perinatal life: oestrogen secretion in tissue and organ cultures.

Secretion of oestrogen by the ovaries of foetal (15-19 days of gestation) and newborn rats in organ and tissue culture was not detectable by fluorometry when the ovary was taken from foetuses before folliculogenesis had occurred. In organ cultures of ovaries, the time of folliculogenesis corresponded with the normal timing of folliculogenesis in vivo. In tissue cultures the process of formation of follicles was delayed. Oestrogens were present in the medium when folliculogensis was fully established in the cultured foetal ovaries. Secretion began spontaneously and did nto depend on the addition of gonadotrophins to the medium. The addition of gonadotrophins the the culture medium did not effect the level of oestrogen secreted by the foetal and newborn rat ovaries during the period of incubation (2-3 weeks).

Effect of estrogens on growth and differentiation of the salivary glands of albino rats in organ culture

Salivary glands of albino rats in organ culture were investigated after the addition of 17β-estradiol to the medium. After addition of estrogens characteristic glandular tubes with secretion containing acid and neutral mucopolysaccharides were found in the zone of growth. Necrobiotic changes occurred in explants of the control group in the early stages of culture and no marked differentiation of the epithelium was observed. Addition of estrogens in a physiological dose led to differentiation of the epithelium of the salivary glands with the formation of the specific secretion and it improved the changes of survival of the cultured tissue.

Insulin release from isolated islets of Langerhans of the rat in organ culture.

Inseln adulter Wistarratten wurden auf Glasfaservlies auf Metallgitter unter den Bedingungen der Organkultur gezuchtet und der Einfluss der Inselzahl unter basalen und stimulierten Bedingungen auf die Insulinsekretion in 24 h-Perioden wahrend der Kultivierung untersucht.

Some biochemical effects of insulin and steroid hormones on the rat prostate in organ culture.

The effect of various steroids and insulin on expiants of the ventral prostate of adult rats in organ culture was investigated. Testosterone activated the incorporation of labeled precursors into RNA and protein and the formation of 14CO2 from 14C-glucose by expiants. All these effects were mimiced by insulin. The testosterone action was suppressed by cyproterone in vitro. In addition to androgens, glucocorticoids activated the incorporation of 3H-uridine into RNA. Simultaneous addition of testosterone with hydrocortisone or insulin produced an augmented effect on the incorporation of 3H-uridine into RNA which exceeded that resulting from a simple summation of the individual hormone responses. Insulin facilitated likewise the action of testosterone on the incorporation of 14C-amino acids into protein. When all three hormones were added simultaneously to the culture medium the stimulation of the three biochemical parameters was maximal. It was therefore concluded that all three hormones are necessary for an adequate maintenance of the ventral prostate of the rat.

Differentiation of lingual filiform papillae of the rat in organ culture

Explants of 14-day rat foetal tongue were grown in organ culture for periods up to 6 days. Filiform papillae similar to those that develop in vivo developed in the cultures. It is concluded that development of these complex structures can occur in the absence of nervous or vascular influences and is probably dependent on interaction between epithelium and mesenchyme.

Pharmacological and kinetic properties of uptake of [ 3H]-norepinephrine by superior cervical ganglia of rats in organ culture

The increase in uptake of [ 3H]-norepinephrine ([ 3H]-NE) by superior cervical ganglia grown in organ culture was used as an index of the uptake of norepinephrine (NE) by axonal sprouts formed during organ culture. The kinetic and pharmacologie properties of NE uptake by such axonal sprouts were examined. The K m (1.9 × 10 −6M) for [ 3H]- dl-NE uptake by axonal sprouts was similar to that found in sympathetic nerve endings; the K m in fresh ganglia was 8.0 × 10 −6M. The intensity of inhibition by cocaine, metaraminol and phenoxybenzamine of the [ 3H]-NE uptake into axonal sprouts also resembled that found for nerve endings and was significantly different from that of fresh ganglion. Our observations indicate that the uptake mechanism for NE into axonal sprouts is pharmacologically and kinetically similar to the uptake mechanism in sympathetic nerve endings.

Induction of Tyrosine-α-Ketoglutarate Transaminase in Fetal Rat and Fetal Human Liver in Organ Culture

Extract: Explants of fetal rat (13 days to term) and fetal human liver (crown to rump length = 9–20 cm) were grown in organ culture. The survival of the explants was examined by histological appearance, mitotic index, and incorporation of precursors of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein. Tissues adjusted to the culture system by the 24th hr and remained so throughout the duration of the experiments. A slight increase in tyrosine transaminase activity occurred in control explants during the 3-day period in culture. Eighteen-hour incubation with hydrocortisone (10-6 M), glucagon (5–25 μg/ml), and insulin (0.05–0.5 U/ml) increased tyrosine transaminase levels, 3.1–6.5, 2.5, and 2.3 times the control values, respectively, in 18-day to term fetal rat liver explants. Actinomycin D and cycloheximide completely inhibited the corticosteroid stimulation. Thirteen-day fetal rat liver exhibited no increase in tyrosine transaminase activity after hydrocortisone incubation for 24 hr, whereas glucagon plus dibutyryl cyclic adenosine monophosphate (AMP) did stimulate tyrosine transaminase activity twofold at this developmental stage. No increase in human fetal liver tyrosine transaminase occurred with any of the agents tested.Speculation: These studies support the hypothesis that mammalian liver is incompetent to respond to hormone-induced enzyme synthesis before a certain developmental stage is reached.

Mechanism of thyrotropin secretion from the pituitary gland of the rat in organ culture

Mechanism of thyrotropin secretion from the pituitary gland of the rat in organ culture