Whole organ culture of the postnatal sensory inner ear in simulated microgravity

Among the three major biological in vitro models, cell culture, tissue culture, and organ culture, the latter provides the closest approximation to the in vivo situation, but also requires the most demanding culture conditions. Due to its small size and complex tissue architecture, the mammalian inner ear provides a particular challenge to the development of whole organ culture. Using a rotating bioreactor system with simulated microgravity conditions, the entire mouse inner ear organ can be maintained in culture for up to seven days with preservation of sensory organ morphology and robust marker protein expression in sensory hair cells. Controlled sensory cell lesions can be induced by the ototoxic agent, neomycin sulphate, as a toxicologic model of hair cell degeneration and hair cell loss. The results demonstrate that simulated microgravity organ culture of the inner ear affords an in vitro model for the investigation of developmental, regulatory, and differentiation processes, as well as toxicological, biotechnological, and pharmaceutical screening applications within the normal and pathologic sensory hearing organ.     

Development of the gabaergic phenotype in murine spinal cord-dorsal root ganglion cultures

Murine spinal cord and dorsal root ganglion GABAergic neurons, derived from 12-day-old fetuses, were examined autoradiographically, biochemically and immunocytochemically in vitro to determine the timecourse of appearance and maturation of this phenotype and the extent and mode of its innervation of target neurons. Specific ³H-GABA uptake into spinal cord neurons was the first property to develop and was present at the earliest time studied, one day in vitro. Immunocytochemical localization of glutamic acid decarboxylase (GAD) revealed positively stained neurons beginning at four days. At five days in vitro, electron microscopic immunocytochemistry revealed GAD-immunoreactive (GAD-IMR) boutons investing neuronal perikarya as well as neuronal processes. By one week in vitro, GAD-IMR neurons constituted 27% of the total population and GAD-IMR boutons could be seen contacting every cell with a neuronal morphology. The mode of investment of target neurons by GAD-IMR boutons was not circumscribed to either soma or dendrites but usually involved the entire neuronal perimeter and did not change with time in culture. Three morphologically distinct types of GAD-IMR neurons were evident: a small, bipolar type; a medium-sized multipolar neuron which was the most common and a large, multipolar type, resembling a motoneuron. A small population (8%) of dorsal root ganglion neurons was found to contain GAD both biochemicaly and immunocytochemically but was never invested by GAD-IMR boutons. GAD activity in vitro paralleled in vivo levels with maximal activity being reached at four weeks in vitro and 10 days postnatally in the intact mouse spinal cord. Murine spinal cord GABAergic neurons are a morphologically diverse and abundant neuronal population with extensive, precocious innervation of all other neuronal phenotypes in vitro suggesting that GABA has a widespread influence over other developing neuronal systems in the murine spinal cord.     

Role of acetylcholinesterase in the development of axon tracts within the embryonic vertebrate brain

In the developing vertebrate brain, acetylcholinesterase (AChE) expression coincides temporally with axon tract formation. Although AChE promotes neurite outgrowth in vitro, the role of this molecule in the development of axon tracts in vivo is unknown. To address this question, we examined the effects of the AChE inhibitor, BW284C51, on the formation of the early scaffold of axon tracts in the embryonic Xenopus brain. In exposed Xenopus brain preparations, axons elongate and establish a normal topography of axon tracts. However, when brains were exposed to BW284C51, the thickness of the major longitudinal axon tract, the tract of the post-optic commissure decreased in a dose-dependent manner. When BW284C51 was removed from the culture media axon tract development returned to normal within 5 h. These findings provide the first evidence for a non-classical role of AChE in the initial formation of axon tracts within the developing vertebrate brain.     

Development of phenylethanolamine N-methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata.

The adrenergic phenotypic marker, phenylethanolamine N-methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons.

Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT-immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT-IR neurons were observed in cultures prepared only from embryos of 46–51 somites (embryonic day E13–13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16. In cultures of the E13 rat, PNMT was found to be catalytically active at 4 days in vitro and the levels of PNMT activity per neuron, as estimated from the number of PNMT-IR neurons in sister cultures, increased 2-fold in cultures grown for 8 days in the presence of 10% fetal bovine serum and 1.5-fold in defined, serum-free conditions. Treatment of cultures with either corticosterone or dexamethasone had no effect on PNMT activity, supporting previous studies suggesting that PNMT in central neurons is not regulated by glucocorticoids.

These studies provide a convenient and defined in vitro system for studying factors that influence the development of central adrenergic neurons. The differential development of PNMT-IR and TH-IR neurons in these cultures suggests that the factors involved in adrenergic neuron survival and/or differentiation are not common to catecholaminergic neurons in general.     

Survival of chick embryo sympathetic neurons in cell culture

Changes in neuronal numbers during the development of the chick embryo paravertebral sympathetic nervous system have been examined using cell culture techniques. Early sympathetic ganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequently there is a large decrease in the number of neurons and an increase in the population of non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-day or older embryos than those from earlier stages of development.     

Latex beads phagocytosis capacity and ecto-NAD+ glycohydrolase activity of rat brain microglia cells in vitro

Conditions are described which allow the preparation in vitro of pure (> 95%) microglial cell cultures isolated from newborn rat brain. Such ameboid cells cultivated in vitro can efficiently phagocytize opsonized latex beads and are capable of ingesting more (100–200 beads of 1.1 μm diameter per cell) and larger (6.4 μm) particles than other nerve cells, such as oligodendrocytes and astroglia. The microglial cells also show an important ecto-NAD⁺ glycohydrolase activity which is characteristic of phagocytic cells. We noted that the phagocytic capacity and ecto-NAD⁺ glycohydrolase of these cells were correlated and increased notably during the in vitro culture.

Microglia cultivated in vitro appear to be a good model to study the activation of phagocytic properties in the central nervous system and corresponding modulation by natural or pharmacological immunomodulators.     

Brain filament proteins in primary cultures derived from chick embryos early in development

Brain filament expression and neurofilament post-translational modifications (phosphorylations) were studied in primary cultures derived from whole 3–4 day chick embryos. After 2–3 days in culture, neurofilament-positive cells formed neuronal aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the flat cells surrounding the neuronal aggregates remained constantly GFAP-negative throughout the whole experimental period (10 days). GFAP-positive cells were first observed within the neuronal aggregates on day 8 in vitro.     

Motoneuron survival factors: Biological roles and therapeutic potential

The existence of factors that promote motoneuron survival in the spinal cord at critical stages of development was first deduced 50 yr ago. The large amount of work that has been put into characterizing such factors reflects both their biological importance and the hope that such molecules may be used therapeutically to slow motoneuron death in pathologies such as the spinal muscular atrophies and amyotrophic lateral sclerosis. Since 1990, several factors have been shown to have in vitro and/or in vivo activities that suggest they play a role in regulating motoneuron survival. Their physiological functions during motoneuron development are probably different and complementary. Several of them seem reasonable candidates for preclinical development, but many crucial experiments remain to be done.     

An organotellurium compound with antioxidant activity against excitotoxic agents without neurotoxic effects in brain of rats

The glutamatergic system is an important target in many neurodegenerative diseases and for several neurotoxic drugs. Organotellurium compounds are often very good free radical scavengers’ agents. Recently, we reported that diethyl-2-phenyl-2-tellurophenyl vinylphosphonate is a compound with low toxicity in vitro and in vivo, as well as also possesses antioxidant activity against iron-induced lipid peroxidation. The aim of this study was to evaluate in vitro the antioxidant and mitochondrial protective effect of this organotellurium compound against quinolinic acid (QA) and sodium nitroprusside (SNP), and to evaluate the in vitro actions of this organotellurium compound in the glutamatergic system in brain of rats. We observed that the telluro vinylphosphonate possess an antioxidant activity against QA and SNP at micromolar concentrations. When tested at antioxidant concentrations (from 2 to 10 μM), the compound does not affect the mitochondrial viability and [³H]glutamate uptake in slices from cerebral cortex, hippocampus and striatum, [³H]glutamate release from synaptosomal preparations and [³H]glutamate binding in membrane preparation. Our data suggest that the telluro vinylphosphonate act as an antioxidant in the central nervous system in vitro with no effects on the glutamatergic system; nevertheless more studies in different models of brain injury must be performed in order to corroborate our findings.     

Increased phosphate content of fibrinogen in vivo correlates with alteration in fibrinogen behaviour.

Fibrinogen was purified from five patients admitted for hip-replacement surgery the day before (day 0), the day after (day 2) and and one week after the operation (day 8). The behaviour of each patient's three fibrinogens was compared in thrombin gelation assays and plasmin degradation experiments to investigate whether the reported increase in protein-bound phosphate at day 2 and day 8 had any effecton the functional behaviour of fibrinogen as has been demonstrated in vitro. It was found that the thickness of the fibrin fibres produced by thrombin increased markedly at day 2 and declined thereafter. Susceptibility to plasmin appeared to decrease post-operatively by 50% and remained at that level on day 8 despite the phosphate content returning to normal. This has also been shown for fibrinogen phosphorylated in vitro. We conclude, after testing the fibrinogens with and without alkaline phosphatase pretreatment, that our data most resemble the published findings for in vitro phosphorylation of fibrinogen by casein kinase II.     

Chronic Suppression of Bioelectric Activity and Cell Survival in Primary Cultures of Rat Cerebral Cortex: Biochemical Observations

Chronic suppression of spontaneously occurring bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but may also affect astrocytes. We investigated this process in primary cultures of rat cerebral cortex by measuring the levels of NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) in relation to general tissue markers, including measurements for cell death and proliferation. In electrically active (control) cultures, the content of DNA, protein, and NSE became maximal between 21 and 28 days in vitro (DIV) and thereafter decreased, whereas the content of GFAP rose continuously up to 43 DIV. Chronic suppression of BEA by tetrodotoxin (TTX; from 6 DIV) decreased the content of DNA, total protein, and especially NSE. The content of GFAP was decreased in all culture series investigated, but with great temporal variations among culture series. Chronic TTX treatment (started at 6 DIV) increased the efflux of lactate dehydrogenase, a marker for cell lysis, between 12 and 21 DIV, but this efflux was mainly derived from the supporting glial cells with which the cerebral cortex cultures were cocultured. Chronic, but not acute (7 h) TTX treatment decreased total [3H]thymidine incorporation into DNA from 14 DIV; this appeared to be due to a reduced number of astrocytes. Chronic suppression of BEA with xylocaine from 6 DIV had similar effects on DNA-, protein-, and NSE-content as TTX, but led to an increased content of GFAP at 21 DIV. Chronic suppression of synaptic transmission with 10 mM Mg2+ and 0.2 mM Ca2+, starting at 6 DIV, increased the content of DNA, protein, and GFAP at 21 DIV, but NSE was still decreased. We conclude that chronic suppression of BEA in cerebral cortex cultures enhances neuronal cell death, whereas astrocytes are differentially affected, depending on the suppressing agent. As astrocytes may have a modulating effect on neuronal survival, their involvement should be regarded when studying the effects of chronic suppression of BEA on neuronal development.     

Transient loss of serum protective activity following short-term stress: A possible biochemical link between stress and atherosclerosis

Very low density lipoproteins (VLDL) are toxic to aortic endothelial cells in vitro, and toxicity preventing activity (TxPA) inhibits this toxic effect of VLDL. Stress, an established arteriosclerosis risk factor, was examined for its effect on TxPA and on the ability of serum to protect endothelial cells from in vitro injury by VLDL. A standardized mirror tracing task with noise was administered to four healthy subjects. Blood samples were obtained at 0, 30, (stressor) 35, 50 and 80 min. Cortisol and non-esterified fatty acids increased during the stress period. TxPA significantly decreased following the stressor and had recovered by 80 min. When the ratio of non-TxPA/TxPA rose above 2, serum was no longer able to protect the cells from VLDL injury. If endothelial cells in vivo respond similarly to the endothelial cells in culture, the effect of stress on atherosclerosis may be mediated through these transient decreases in TxPA.     

Cognin distribution during differentiation of embryonic chick retinal cells in vitro

Differentiation of individual retina neurons is closely linked to development of retina function. This differentiation may be intrinsic to the cell or determined by the position of the cell within the developing tissue. Retina cognin, a cell-cell recognition protein, which may itself mediate positiondependent cell interactions in vivo exhibits a characteristic change in distribution during embryonic chick development. Cognin is progressively lost from the outer retina in a manner which appears position-dependent. ¹⁰ We asked if this change in cognin distribution was actually position-dependent or intrinsic to the retina cells. Neural retina cells from 8-day-old chick embryos were cultured in vitro. Continued differentiation of the cultured cells was demonstrated by neurite outgrowth and characteristic increases in choline acetyltransferase and glutamic acid decarboxylase activity. In such cultures, the characteristic developmentally related disappearance of retina cognin occurred as in vivo. This indicated that this aspect of retina neuronal differentiation was independent of position within the tissue and likely intrinsic to individual cells after 8 days of embryonic development.     

Neuronal sprouting and synapse formation in response to injury in the mouse organ of corti in culture

The effect of mechanical injury on induction of regenerative phenomena within the neurosensory epithelium was investigated in cultures of neonatal mouse cochlea. The oldest examined culture in which new neuronal growth followed insult, was injured at 13 days in vitro and fixed 24 h later. By far, the most vigorous regenerative reaction was observed in a 3-day culture 4 h post-injury. The reaction included sprouting of nerve fibers injured directly, synapse formation between the surviving hair cells and sprouting neuronal growth cones, wrapping of growing nerve fibers by extending processes of hair cell cytoplasm, and collateral sprouting of synaptically-engaged nerve endings and of nerve fibers in passage.     

The effects of apolipoprotein e deficiency on braincholinergic neurons

Previous studies utilizing apolipoprotein E (apoE)-deficient mice revealed distinctdecreases in the levels of cholinergic synaptic markers of projecting basal forebrain cholinergicneurons and no such alterations in other brain cholinergic systems. In order to investigate themechanisms underlying these neuron-specific cholinergic effects, primary neuronal cultures fromapoE-deficient and control mice were prepared and characterized. These include basal forebraincultures, which are enriched in projecting cholinergic neurons, and cortical cultures, which containcholinergic interneurons. The levels of cholinergic nerve terminals in these cultures were assessedby ligand binding measurements of the levels of the vesicular acetylcholine transporter (VAChT).This revealed that basal forebrain cultures of apoE-deficient mice contain markedly lower VAChTlevels (50%) than do control cultures, but that VAChT levels of the corresponding corticalcultures of the apoE-deficient and control mice were the same. Time course studies revealed thatVAChT levels of the basal forebrain cultures increased with culture age, but that the relativereduction in VAChT levels of the apoE-deficient cholinergic neurons was unaltered and was thesame for freshly prepared and for 96 h old cultures. These in vitro observations are inaccordance with the in vivo findings and suggest that projecting basal forebraincholinergic neurons, but not cholinergic interneurons, are markedly dependent on apoE and thatsimilar mechanisms mediate the in vivo and in vitro effects of apoE deficiencyon cholinergic function.     

Modulation of parkin gene expression in noradrenergic neuronal cells

Typical autosomal recessive juvenile Parkinsonism (AR-JP) is resulted from the loss of function mutation in the parkin gene. In an effort to learn more about the cell type-specific functional role of parkin, we used in vitro model such as locus coeruleus (LC) noradrenergic (NA) neuronal progenitor cell line, LC3541. Employing this in vitro model, we revealed that parkin plays a crucial role in phenotypic differentiation of NA neurons. Our results showed that parkin mRNA was expressed during the differentiation of NA neuronal progenitor cell line and that the level of the parkin mRNA was down-regulated by oxidative stress in the NA neuronal cells. Parkin protein overexpression in LC3541 cells induced expression of NA markers (TH, phox2a and DBH). Small interfering RNA (SiRNA) for parkin supressed NA differentiation and DBH expression. Preventing protein kinase A (PKA) activation with PKI attenuated expression of DBH in parkin overexpressed LC3541 cells. These findings suggest that the relative abundance of parkin enhances differentiation of NA phenotypes via a PKA-dependent pathway.     

The human desmin gene: A specific regulatory programme in skeletal muscle both in vitro and in transgenic mice

Desmin synthesis is restricted to cardiac, skeletal and smooth muscles. In several familial myopathies involving fibre disorganization, filamentous aggregation of desmin has been characterized. During the development of the mouse embryo, desmin is one of the first muscle proteins detected in both the heart and the somites. To identify the DNA sequences involved in the regulation of desmin gene expression a 4.5 kb 5′-flanking region of the human desmin gene has been isolated. Different mutants were used to characterize specific enhancers in vitro and in vivo. The results obtained with transgenic mice provide evidence that the 1 kb cis-regulatory sequences, functional in skeletal muscle cells in vitro, confer specific developmental control for skeletal muscles. Furthermore, distinct programmes for cardiac and skeletal muscle-specific expression of the desmin gene are revealed.     

N-cadherin and N-CAM-mediated adhesion in development and regeneration of skeletal muscle

In this review, the experimental evidence supporting the fact that the cell adhesion molecules N-CAM and N-cadherin are involved in myogenesis has been surveyed. In order to give access to the function of these molecules, a strategy of in vivo localization and in vitro perturbation of their adhesive function by interfering antibodies and peptides was applied. Both molecules are expressed at the surface of myogenic cells during myogenesis in vivo and in vitro. The blockade of the N-CAM adhesion function leads to a mild reduction of the rate of myoblast fusion, while the inhibition of the N-cadherin function induces a drastic inhibition of fusion suggesting that N-cadherin-mediated adhesion is a critical step in the process of myoblast fusion. Both molecules are re-expressed during muscle regeneration suggesting that adult myogenesis is under the control of the same adhesive systems as embryonic and foetal myogenesis.     

Depressed RNA synthesis in the brains and livers of thyroidectomized, normal and hormone injected rats

Depression of rat brain and liver RNA synthesis was seen at 22 and 90 days subsequent to neonatal radiothyroidectomy. Cerebral RNA synthesis (per unit DNA) was depressed by 16% (P < 0.001) while hepatic RNA synthesis was reduced by 43% (P < 0.01). Nuclear protein synthesis was not detectably altered. RNA and protein synthesis determinations were conducted, using isolated nuclei in standardized in vitro assay systems which circumvented complications (often seen in vivo approaches) such as pool size changes, cell membrane transport alterations and mediation of blood-brain barrier related effects.

Chronic daily administration in vivo of l-thyroxine (10 μg/100 g body weight) restored the depressed cerebral synthesis of RNA to normal levels while daily administration of bovine growth hormone (100 μg per rat) did not.     

Sex-specific schedule in steroid response of rhombencephalic catecholaminergic neurons in vitro.

Morphological differentiation of tyrosine hydroxylase-immunoreactive neurons was investigated in dissociated cell cultures of rhombencephalon of male and female day 14 rat embryos grown in the presence or absence of sex steroids. Numbers of cells were counted and morphometrical measurements carried out of soma size and length of tyrosine hydroxylase-immunoreactive neurites (processes). Subtle sex differences in length of stained neurites, which were not yet present after 3 days in vitro, were observed after 6 days in cultures grown in the absence of sex steroids. Female tyrosine hydroxylase-immunoreactive neurites could be traced over longer distances than male ones. Daily treatment of cultures with testosterone or 17β-estradiol resulted in an increase of lengths of stained neurites of female neurons after 3 days and of male neurons after 6 days in vitro. Regarding cell numbers or soma size, there were no differences between genders or between controls and hormone-treated cultures.

It is concluded that sex steroids promote the outgrowth of neurites from noradrenergic neurons within a gender-specific time frame. It appears that the critical period for developmental effects of sex steroids differs between males and females.