Genesis of rhythmic respiratory activity in pons independent of medulla

We hypothesized that rhythmic respiratory-related activity could be generated in pons independent of medullary mechanisms. In decerebrate, cerebellectomized, vagotomized, paralyzed, and ventilated cats, we recorded efferent activities of the phrenic nerve and mylohyoid branch of the trigeminal nerve. Following transections of the brain stem at the pontomedullary junction, the phrenic and trigeminal nerves discharged with independent rhythms. Spontaneous trigeminal discharges eventually ceased but were reestablished after strychnine, doxapram, and/or protriptyline were administered. In some animals having no spontaneous trigeminal discharges after transection, these discharges appeared, with a rhythm different from the phrenic, following administration of these agents. In other cats having no transections between pons and medulla, these pharmacological agents induced trigeminal and phrenic discharges after kainic acid had been injected into the entire dorsal and ventral medullary respiratory nuclei. Phrenic and trigeminal discharges were linked, indicating survival of bulbospinal neurons or presence of pontospinal units. We conclude that rhythms, similar to respiratory rhythm, can occur by mechanisms in isolated pons. Such mechanisms are hypothesized to be within the pneumotaxic center and may underlie the neurogenesis of eupnea.     

Characterization of the rat adrenal medulla cultured in vitro

Summary A wide variety of experimental animal models have been used to investigate the mechanisms of synthesis, storage, and release of catecholamines. Whereas in vivo experimental models are situated at one end of the spectrum, cell culture models are situated at the other end. In the present study, we have characterized various aspects of the rat adrenal medulla cultured in vitro as a whole tissue, aiming to establish a new experimental model in between in vivo animal models and cell culture models. We adapted a bottle rotator system commonly used for culturing rodent whole embryos. Changes in histology, activities and mRNA levels of catecholamine-synthesizing enzymes, and concentrations of catecholamines in the adrenal medulla were studied. In addition, the effects of cholinergic stimulation on catecholamine release from the adrenal medulla were examined. Overall the results indicate that various aspects of the adrenal medulla become stable after 4 d of culture and the adrenal medulla at this stage releases catecholamines in response to cholinergic stimulation. The whole adrenal medulla culture system may be a useful tool for investigating catecholamine-related functions dependent on intercellular reactions or communications.     

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.     

Survival characteristics of normal differentiated rat thyroid cells maintained in vitro

The radiation response of a cultured line of differentiated rat thyroid epithelial cells (FRTL-5) was determined by clonogenic assay in vitro and compared to the previously reported response curve for primary rat thyroid cells assayed by transplantation in vivo. The beta components (or D0 estimates) for the two sets of data were not significantly different although the response of the FRTL-5 cells was displaced to the right as a consequence of differences in the shoulder of the survival curves. These results indicate that this in vitro model can be used to study the radiation response of thyroid epithelial cells and by extrapolation perhaps other hormonally responsive, slowly proliferative parenchyma. The cell age response for FRTL-5 cells was determined after separation of the cells into various phases of the cell cycle by centrifugal elutriation. Resistant peaks in mid-G1 and G2M phases of the cell cycle were detected. The cell age response data for other portions of the cell cycle should be interpreted cautiously because less than satisfactory enrichments were achieved.     

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.     

Hypoxia and acidosis increase the secretion of catecholamines in the neonatal rat adrenal medulla: an in vitro study

Hypoxia elicits catecholamine (CA) secretion from the adrenal medulla (AM) in perinatal animals by acting directly on chromaffin cells. However, whether innervation of the AM, which in the rat occurs in the second postnatal week, suppresses this direct hypoxic response is the subject of debate. Opioid peptides have been proposed as mediators of this suppression. To resolve these controversies, we have compared CA-secretory responses with high external concentrations of K⁺ ([K⁺]_e) and hypoxia in the AM of neonatal (1- to 2-day-old) and juvenile (14- or 15- and 30-day-old) rats subjected to superfusion in vitro. In addition, we studied the effect of hypercapnic acidosis on the CA-secretory responses in the AM during postnatal development and the possible interaction between acidic and hypoxic stimuli. Responses to high [K⁺]_e were comparable at all ages, but responses to hypoxia and hypercapnic acidosis were maximal in neonatal animals. Suppression of the hypoxic response in the rat AM was not mediated by opioids, because their agonists did not affect the hypoxic CA response. The association of hypercapnic acidosis and hypoxia, mimicking the episodes of asphyxia occurring during delivery, generates a more than additive secretory response in the neonatal rat AM. Our data confirm the loss of the direct sensitivity to hypoxia of the AM in the initial weeks of life and demonstrate a direct response of neonatal AM to hypercapnic acidosis. The synergistic effect of hypoxia and acidosis would explain the CA outburst crucial for adaptation to extrauterine life observed in naturally delivered mammals. hypercapnia; chemoreceptors; chromaffin cells     

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.     

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.