The reaggregation of adult rat liver cells maintained in vitro

The reaggregation of dissociated adult rat liver cells maintained in vitro for up to 96 h is described. Cultures were examined by dark-ground fluorescence and electron microscopy. Spaces resembling bile canaliculi were formed between reaggregated hepatocytes. Desmosomes and ‘tight’ junctions were formed between hepatocytes but ‘gap’ junctions were not detected. In older cultures structural damage was observed in many hepatocytes and some of them ingested cell debris by phagocytosis. Structures resembling bile ducts and sinusoids were also formed but complex association between all three types of cell aggregate was not observed.     

A protocol for rat in vitro fertilization during conventional laboratory working hours

In vitro fertilization (IVF) is a valuable technique for the propagation of experimental animals. IVF has typically been used in mice to rapidly expand breeding colonies and create large numbers of embryos. However, applications of IVF in rat breeding experiments have stalled due to the inconvenient laboratory work schedules imposed by current IVF protocols for this species. Here, we developed a new rat IVF protocol that consists of experimental steps performed during common laboratory working hours. Our protocol can be completed within 12 h by shortening the period of sperm capacitation from 5 to 1 h and the fertilization time from 10 to 8 h in human tubal fluid (HTF) medium. This new protocol generated an excellent birth rate and was applicable not only to closed colony rat strains, such as Wistar, Long-Evans, and Sprague–Dawley (SD), but also to the inbred Lewis strain. Moreover, Wistar and Long-Evans embryos prepared by this protocol were successfully frozen by vitrification and later successfully thawed and resuscitated. This protocol is practical and can be easily adopted by laboratory workers.     

Comparative rates of protein synthesis of rat kidney medulla and cortex in vitro

The $\text{in}\text{vitro}$ rate of ¹⁴C-leucine incorporation into protein has been examined in rat kidney tissue. The presence of a marked gradient was observed. Thus, the white medulla was the most active in this respect followed by, in descending order, red medulla and cortex. ¹⁴C-Leucine incorporation into protein was completely abolished in the presence of cycloheximide. The distribution of labeled protein between the medium and slice suggests a high degree of cellular integrity and little secretion of labeled protein from slice to medium. The pattern of ¹⁴C-leucine incorporation amongst the different zones of kidney of hypophysectomized rats was similar to that noted in normal rats.     

乙酰胆碱和A23187对离体大鼠肾上腺髓质细胞肾上腺素分泌的作用

通过胆碱能激动剂乙酰胆碱及离子诱导剂A23187（以下简称激动剂）作用于分离的肾上腺髓质细胞,以引起离体细胞的刺激－分泌耦联过程,运用细胞立体形态计量法计算分泌过程中的嗜铬颗粒数目的变化,运用电镜X射线显微分析法测量分泌过程中嗜铬颗粒内钙含量的变化,并运用高 液相色谱分析法测定离体细胞在激动剂作用后的肾上腺素分泌情况,结果发现,分离的肾上腺髓质细胞嗜铬颗粒内钙含量在激动剂作用10min时有明显下降,颗粒数目在激动剂作用过程中呈缓慢下降趋势,而细胞悬液中的肾上腺素含量在激动剂作用20min以后有明显的升高,激动剂作用引起的离体肾上腺髓质的细胞分泌时颗粒内钙含量的下降早于颗粒数目减少或肾上腺素升高,提示颗粒释放的Ca^2 可能是引起细胞分泌的原因之一。     

Antioxidant responses to chronic hypoxia in the rat cerebellum and pons

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH) and sleep fragmentation and deprivation. Exposure to CIH results in oxidative stress in the cortex, hippocampus and basal forebrain of rats and mice. We show that sustained and intermittent hypoxia induces antioxidant responses, an indicator of oxidative stress, in the rat cerebellum and pons. Increased glutathione reductase (GR) activity and thiobarbituric acid reactive substance (TBARS) levels were observed in the pons and cerebellum of rats exposed to CIH or chronic sustained hypoxia (CSH) compared with room air (RA) controls. Exposure to CIH or CSH increased GR activity in the pons, while exposure to CSH increased the level of TBARS in the cerebellum. The level of TBARS was increased to a greater extent after exposure to CSH than to CIH in the cerebellum and pons. Increased superoxide dismutase activity (SOD) and decreased total glutathione (GSHt) levels were observed after exposure to CIH compared with CSH only in the pons. We have previously shown that prolonged sleep deprivation decreased SOD activity in the rat hippocampus and brainstem, without affecting the cerebellum, cortex or hypothalamus. We therefore conclude that sleep deprivation and hypoxia differentially affect antioxidant responses in different brain regions.     

Fine structure of equine oocytes matured in vitro for 15 hours

Transmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microsocopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 ± 0.2°C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero-time oocytes contained few cortical granules, and these were scattered throughout the cytoplasm, whereas 15 hr oocytes contained numerous cortical granules primarily found in very close proximity to the oolemma. Further ultrastructural analysis of both groups revealed organelle structure similar to that previously described for in vivo matured equine oocytes. Evaluation of nucelar maturity (lacmoid stain) showed that 15 hr of culture resulted in significant numbers of oocytes at metaphase II (8/17; 47%). These data demonstrate that oocytes cultured for 15 hr in modified TCM 199 with 15% mare serum (day of ovulation) are mature with respect to nuclear configuration and cortical granule migration and, therefore, would be appropriate candidates for in vitro fertilization. © 1994 Wiley-Liss, Inc.     

The effects of 6 hours of hypoxia on protein synthesis in rat tissues in vivo and in vitro.

Rates of protein synthesis were measured in vivo in several tissues (heart, skeletal muscles, liver, tibia, skin, brain, kidney, lung) of fed rats exposed to O2/N2 (1:9) for 6 h starting at 08:00-11:00 h. Protein synthesis rates were depressed by 15-35% compared with normoxic controls in all of the tissues studied. The decreases were greatest in the brain and the skin. Although hypoxia inhibited gastric emptying, its effects on protein synthesis could probably not be attributed to its induction of a starved state, because protein-synthesis rates in brain and skin were not decreased by a 15-18 h period of starvation initiated at 23:00 h. Furthermore, we showed that protein synthesis was inhibited by hypoxia in the rat heart perfused in vitro, suggesting a direct effect. The role of hypoxia in perturbing tissue nitrogen balance in various physiological and pathological states is discussed.     

Neuronal properties of monkey adrenal medulla in vitro

Summary Chromaffin cells from the monkey adrenal medulla were maintained in vitro in the presence of nerve growth factor (NGF) and the neuronal properties of these cells were assessed. Single-cell preparations were obtained by collagenase-trypsin treatment of the minced adrenal medulla tissue. Cells assumed a glandular to epithelioid morphology after twenty-four hours of culture. Twelve percent of these cells were shown to extend neurites spontaneously after five days. NGF-stimulated neuritic outgrowth from most cells after five days of culture and these neurites remained for at least three weeks. Cells exhibited intense histofluorescence for catecholamines even after three weeks in vitro in the presence of NGF and positive staining for tyrosine hydroxylase and dopamine beta hydroxylase could be detected by immunocytochemistry. Moreover, the chromaffin cells were shown to bind tetanus toxin, which is a specific marker for neurons. Tetanus toxin labelling was not dependent upon the presence of neurites on these cells. Transmission electron microscopy indicated that cultured cells contained numerous dense-core vesicles similar to noncultured medulla cells. Many of the neurites possessed the morphological features of axons; long varicose processes resembling noradrenergic fibers were identified by catecholamine histofluorescence and tyrosine hydroxylase immunocytochemistry. Microtubular arrays, in an axonal-like organization pattern, were seen ultrastructurally along with the presence of many dense-core vesicles. These data support the potential of adult primate chromaffin cells as a source of sympathetic neuronal tissue for neural transplantation.Supported in part by a Grant from the Alzheimer's Disease and Related Disorders Association, Inc.     

Metronidazole induces gametocytogenesis in gregarine associations maintained in vitro

Gametocytogenesis was induced in mature associations of Protomagalhaensia wolfi and Protomagalhaensia blaberae maintained in vitro by inclusion of metronidazole in the culture medium. The response was neither strictly dosage dependent nor uniform across gregarine species. We hypothesize that metronidazole induces gregarine gametocytogenesis by disrupting PUF2 proteins responsible for the translational control of sexual development and gametocytogenesis in apicomplexans.     

Amino acid pharmacology in a mature rat spinal cord preparation in vitro

1. It has been shown that a relatively large intact piece of mature mammalian spinal cord can be maintained in vitro if suitable experimental conditions are employed. 2. The preparation as described gives robust and reproduceable reflex responses in both dorsal and ventral horns (ventral horn activity has been maintained for over 36 hr in vitro). 3. The action of antagonists at both excitatory and inhibitory amino acid receptors give qualitatively predictable results from known in vivo experiments with the added advantage of fine quantitative control. 4. A significant advantage of such an adult preparation over more common immature spinal cord preparation is the ability to clearly separate low threshold reflexes, such as those described in this article, from reflexes evoked by small diameter non-myelinated afferents.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

Tissue-type plasminogen activator in rat adrenal medulla

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Tissue-type plasminogen activator in rat adrenal medulla

Summary Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenalin. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

High-affinity angiotensin receptors in rat adrenal medulla

Angiotensin II receptors have been quantitated in single rat adrenal medullas by incubation of tissue sections with 125I-[Sar1]-AII, autoradiography with exposure to 3H-sensitive Ultrofilm, computerized densitometry and comparison with 125I-labelled standards. Rat adrenal medulla contains a single class of high affinity AII receptors with a Ka of 0.84 +/- 0.02 X 10(9) M-1 and a Bmax of 3259 +/- 502 fmol/mg protein, one of the highest densities in AII receptors found in rat tissues. These observations provide evidence for a local site of action of AII in the release of adrenal medullary catecholamines.