IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

Conditioning to time: evidence for a role of hippocampus from unit recording.

Unit activity of the dorsal hippocampus was recorded in partially restrained but awake and undrugged rats during a "conditioning to time". In this type of conditioning, only one stimulus, the equivalent of the unconditioned stimulus of the usual procedures, is used. It is delivered at a constant interval which, in principle, is the conditioned stimulus. In our experiments, the unconditioned stimulus was a mechanical stimulation of a vibrissa; two successive unconditioned stimuli were separated by a 24-s interval. In 11/18 rats, anticipatory movements of a "trained" vibrissa developed at the end of the interstimulus interval. In a number of cases, in parallel to this conditioned behavior, there was a significant change in unit activity, either an increase or a decrease, during the last third of the interstimulus interval. Controls showed that these changes in unit activity did not merely reflect modifications of arousal state or of vibrissa and body movements. From autocorrelograms, it appeared that anticipatory increases in unit activity were associated with the development of a bursting mode of discharge. These data constitute one of the rare examples of a neurophysiological correlate of a "conditioning to time" at the unit level and the first recorded from the dorsal hippocampus of rats.

"Learned" changes in the responses of the rat barrel field neurons.

The effect of pairing two vibrissa stimulations on unit responses of the barrel field of the somatosensory cortex were studied in partially restrained but awake and undrugged rats. Before pairing, one of the stimulations (S2) evoked a stable, short-latency and excitatory response from the recorded unit. Depending on the neuron, the other stimulation (S1), preceding S2 by 500 ms, did or did not have an effect before pairing. In a number of cases, the S1-S2 association produced significant changes in the unit responses: (1) the appearance of an excitatory response to S1 when that stimulus was ineffective before pairing; (2) the modification of pre-existing responses to S1 and/or S2. In all instances these modifications consisted in the decrease or disappearance of the "afferent inhibition" and/or the appearance of long-latency excitatory components. These effects appeared after some 30-100 trials and persisted in some cases up to 20 min after interruption of pairing. Our observations provide the first physiological data on the plasticity of the vibrissa projections in the chronic adult rodent. Though the underlying plastic neural elements and mechanisms remain to be specified, these phenomena suggest that "learned" changes in unit activity may occur in sensory systems and not only in "non-specific" ones.

Modifications of the responses of barrel field neurons to vibrissal stimulation during theta in the awake and undrugged rat.

In partially restrained but awake and undrugged rats, excitatory unit responses of the somatic cortex barrel field to vibrissal stimulation, were recorded in two conditions: during spontaneous episodes of theta and in the absence of this rhythm. Two main variables were considered: a signal-to-noise ratio and an index of the "afferent inhibition". Both measures were extracted from peristimulus time histograms. "Theta effects" were characterized by an increase in signal-to-noise ratio and afferent inhibition. They were most important in neurons located in infragranular layers of the cortex; they went in the same direction but only approached significance in supragranular neurons; neurons of the granular layer were not affected. Spontaneous unit activity and latencies were not modified in any group. These data were obtained during a preliminary step of a sensory-sensory conditioning procedure which in some cases modified the receptive field of the neurons. Theta effects were less marked in future "conditioned" than in future non-conditioned neurons but this was probably due to the fact that conditioned neurons had significantly higher signal-to-noise ratio and afferent inhibition. The origin of these "theta effects", hippocampal versus non-hippocampal, and their functional significance, relation to selective attention, are discussed.

Evidence for a cholinergic mechanism of "learned" changes in the responses of barrel field neurons of the awake and undrugged rat.

Due to its functional importance and its large and highly differentiated central projections, the vibrissal system of rodents is a prime object for the study of sensory plasticity, especially at the cortical level: the representation of vibrissae in the "barrel field", a part of the somatic cortex, is exceptionally precise and is susceptible to experience-induced changes. In a previous series of experiments, we found that a sensory-sensory conditioning procedure, pairing two vibrissal stimulations, produces significant changes in responses of single neurons of the barrel field in the chronic awake and undrugged rat: (1) the appearance of an excitatory response to a stimulus that was ineffective before pairing ("conditioned response"); (2) the modifications of pre-existing responses consisting of the suppression of afferent inhibition and the appearance of long-latency excitatory components. We report here that the micro-iontophoretic application of atropine abolishes "conditioned responses" and restores afferent inhibition. Acetylcholine facilitates an enlargement of the receptive field and induces a sustained mode of discharge to stimuli. These data provide a new and direct support to the hypothesis that cholinergic mechanisms are involved in the sensory cortex plasticity.

Effect of mineral particles containing iron on primary cultures of rabbit tracheal epithelial cells: possible implication of oxidative stress.

Environmental mineral particles such as asbestos are responsible for numerous respiratory diseases. In addition to effects related to their geometry, particles are now assumed to act by triggering an oxidative stress process. Iron-containing particles, in particular, can produce oxygen-activated species by oxidizing their iron. To evaluate the involvement of iron-containing particles in respiratory diseases, three mineral particles (chrysotile, nemalite, and hematite) were tested in primary cultures of tracheal epithelium. Because of the ciliary beat, the three mineral particles were quickly concentrated at the periphery of the mucociliary epithelium, reconstituted in vitro where they induced cellular lesions. Endocytosis of the three types of particles was observed. Cytotoxicity studies have indicated that among the tested particles, the most cytostatic after 24 hr of treatment was the one that contained more Fe2+ available on the surface, nemalite. Moreover, the effect of nemalite was reduced by pretreatment with desferrioxamine. As mineral particles, especially asbestos, are suspected to induce squamous metaplasia, we chose to study two specific transformations of the epithelium: the expression of cytokeratin-13 and the formation of cross-linked envelopes. Under our culture conditions, nemalite and chrysotile increased the expression of the cytokeratin-13, a specific marker of squamous metaplasia, whereas nemalite was the only particle able to strongly induce the formation of cross-linked envelopes. Nemalite was the most cytostatic particle and the most efficient at inducing squamous metaplasia. Measures of oxidizing power by electron-spin resonance revealed that nemalite produced the most oxygen-activated species.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of transient cerebral ischemia on the hippocampal dentate theta (theta) profile in the acute rat: a study 4-5 months following recirculation.

This study mainly describes the long-term effects of 20 min of cerebral ischemia on the profile of the presumed cholinergic theta rhythm in the rat dorsal hippocampal formation during ether anesthesia and injection of the muscarinic agonist agent arecoline. The experimental data were collected 4-5 months after ischemia. They show that ischemia results in a statistically significant reduction in both superficial and deep theta recorded from the CA1 area of the hippocampus and the dentate gyrus, respectively. Amplitude reduction is similar for both rhythms and co-varies positively with the extent of CA1 stratum pyramidale damage which, from light microscope observation, appeared to be the major neuroanatomical consequence of ischemic insult in the dorsal hippocampal formation. The medial septal nucleus-diagonal band of Broca complex involved in theta generation did not suffer visible anatomical damage. Moreover, no significant alteration in the spatial distribution and the density of hippocampal dentate acetylcholinesterase reaction product was seen in ischemic animals. These histological data were statistically confirmed by computerized image analysis. Finally, this is the first investigation to show that transient interruption of cerebral blood flow results in a long-lasting alteration of theta rhythm which is probably the major aspect of the basic activity of the hippocampal formation. Thus, the present findings obtained in the acute rat at 4-5 months postischemia confirm and extend, in most respects, our previous results collected in the chronic animal 2-29 days following 4-vessel occlusion. Possible significance of these findings for the hypothesis of the dependent generation sites of superficial and deep thetas in the hippocampus assumed to be crucial in learning and memory, is discussed.

Effects of selective lesions of fimbria-fornix on learning set in the rat.

The effects of selective partial lesions of the Fimbria-Fornix (FiFx) on reversal and place learning sets were investigated in rats by using a T-maze and a semi-circular multiple discrimination apparatus. Lesions restricted to the Fimbria (Fi) produced a significant deficit in reversal and place learning set, whereas lesions to the Fornix (Fx) only disturbed the learning set based on a reversal procedure. Combined Fi + Fx lesions resulted in impairment in the retention of spatial discrimination tested in the two mazes. Ventral Hippocampal Commissure (vhc) had no significant effect on reversal learning set. These results confirm previous data that the hippocampal formation is involved in learning transfer, and suggest that the Fi and the Fx may play a role in learning set. Our data also confirm previous demonstrations of the ability of rats to rapidly acquire place learning set.

In vitro exposure of rabbit tracheal epithelium to SO(2): Effects on morphology and ciliary beating.

The aim of this in vitro study was to characterize the direct effects of short-term exposure to low concentrations of sulfur dioxide (SO(2)) on both the morphology and the physiology of rabbit tracheal primary cultures. Scanning electron microscopy (SEM) studies revealed that ciliated cells exposed for 1 hr to 10 ppm or 30 ppm SO(2) exhibited aggregated cilia. Transmission electron microscopy revealed numerous swollen mitochondria in cells exposed to 30 ppm SO(2) for 1 hr. This morphological damage to cells was coupled with physiological alterations. A 25% decrease in ciliary beat frequency (CBF) was measured in cells exposed to 30 ppm SO(2). This inhibition was partially reversible within 24 hr. This SO(2) concentration also induced a significant depletion of cellular ATP content which was completely restored after a 24-hr recovery period. A correlation was found between cellular ATP level depletion and CBF decrease.

Responses of the rabbit tracheal epithelium in vitro to H(2)O(2)-induced oxidative stress.

A model of rabbit tracheal epithelial (RTE) cells in primary culture was used to characterize specific and repair responses of airway epithelial cells to oxidative stress. Two well-known reactive oxygen species (ROS) generating systems were used: H(2)O(2) alone or in combination with Fe(2+) to produce the hydroxyl radical. RTE cells exhibited lipid peroxidation when exposed to H(2)O(2) + Fe(2+). Moreover, catalase (CAT) activity decreased after a 1-hour treatment in 3-day-old cultures but increased in 7-day-old cultures which have higher antioxidant enzyme activities. Superoxide dismutase (SOD) activity was never affected. In addition, RTE cells displayed a repair response leading to squamous metaplasia. H(2)O(2) + Fe(2+) treatment resulted in a time-dependent increase in the steady-state level of c-myc mRNA while c-jun and c-fos were not activated. Moreover, a chronic exposure induced the expression of the squamous phenotype characterized by the expression of the cytokeratin 13 confirmed both at the message and protein levels. RTE cells in primary culture react early to H(2)O(2) + Fe(2+) exposure by an increase in c-myc expression and by modifications in CAT activity. Further, a lipid peroxidation occurs and the tracheal epithelium evolves to squamous metaplasia.

Similar cellular effects induced by diesel exhaust particles from a representative diesel vehicle recovered from filters and Standard Reference Material 1650.

Standard reference diesel exhaust particles (DEP) SRM 1650 are often used to evaluate the toxicity of DEP. However, these particles did not necessarily reflect the effects of DEP representative of present diesel automobiles. This study was designed to compare the effects of SRM 1650 to DEP from representative cars (RC-DEP) on airway epithelial cells. Therefore we established a method to recover RC-DEP impacted on filters after emission from diesel automobiles on test beds. Electron microscopy and flow cytometry showed that these two types of particles were phagocytosed to the same extent by epithelial cells. This phagocytosis is not dependent on the adsorbed organic compounds in contrast to the cytotoxic effect evaluated by measurements of LDH release. This is emphasized by the fact that RC-DEP equipped with an oxidation catalyst are less cytotoxic than particles from a non-equipped vehicle or SRM 1650. This type of catalyst also reduces significantly the release of GM-CSF by bronchial epithelial cells. We have shown in the present paper that SRM 1650 may be used as a surrogate of DEP. However, exhaust gas post-treatment devices of current diesel automobiles reduce the cytotoxicity as well as the inflammatory response of these particles.

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture.

The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.

[Statistical relationship between "slow-wave sleep" and "paradoxical sleep" in rats]. / Relations statistiques entre phases de "sommeil lent" et phases de "sommeil paradoxal" chez le Rat

In the Rat, statistical analysis of slow sleep (SS) and paradoxical sleep (PS) episodes disclosed two significant (p less than 0,01) positive correlations: (1) Over a four-hour period, the mean duration of PS episodes was correlated to the mean of the just preceding "light" SS episodes; (2) On the contrary, from cycle to cycle of sleep-wakefulness, the duration of each PS episode was correlated to that of the "light" SS episode of the next cycle.

[Neocortex in the human embryo and fetus. Electron microscopic and Golgi staining study]. / Le néo-cortex chez l'embryon et le foetus humain. Apport du microscope électronique et du Golgi.

The telencephalic vesicle (or hemisphere) of human embryos and fetuses has been studied by electron microscopy and Golgi impregnations. Our observations confirm recent ones on various laboratory animals and revise some concepts of corticogenesis. At 4/6 weeks, the neural tube was a single pseudostratified epithelium in which mitoses occurred. At 7 weeks the telencephalic vesicle was made up of two layers, the ventricular layer or matrix and the outer plexiform or marginal layer. The first cells, appearing in the plexiform layer before any cortical plate could be identified, had the morphological attributes of Cajal-Retzius cells. In this primitive layer we found the earliest synapses yet described in human embryos. From 8 weeks on, the cortical plate was formed by successive waves of cells, migrating from inside-out. The next synapses found were in the subcortical layer. On Golgi impregnations the Cajal-Retzius cells were already visible at 15 weeks, and from 20 weeks on they presented the usual morphological characteristics.

[Experimental dissociation between hippocampal theta activity in wakefulness and hippocampal theta activity in paradoxal sleep in the rat]. / Dissociation expérimentale entre l'activité thêta hippocampique de veille et l'activité thêta hippocampique du sommeil paradoxal chez le rat.

The effects of electrolytic lesion of the septum on the theta activity of the dorsal hippocampus were studied in the chronically implanted Rat during wakefulness and paradoxical sleep. The experimental results show that depending on its localization, septal lesion can either: (1) eliminate the wakefulness theta rhythm without suppressing that of paradoxical sleep; (2) eliminate the paradoxical sleep theta rhythm without suppressing that of wakefulness. These results suggest that there are two kinds of theta activity having different anatomophysiological bases and a different functional significance: one associated with wakefulness and the other with paradoxical sleep.

Specificity of avoidance deficits produced by 6-hydroxydopamine lesions of the nigrostriatal system of the rat.

Microinjections of 6-hydroxydopamine (6-OHDA) into the dopaminergic nigrostriatal system (NS) of the rat impaired acquisition of a two-way avoidance response. This effect was independent of nutritional deficiencies since it was observed even when a special postoperative treatment ensured a comparable state of nutrition in control and experimental rats. It was likewise independent of locomotor disturbances. A third question remains open: Doses of 6-OHDA that impaired acquisition of an active avoidance response produced lesions that were, to a great extent, nonspecific. Therefore, behavioral effects of these doses cannot be entirely ascribed to selective destruction of the NS.

Toxic effects of mechlorethamine on mammalian respiratory mucociliary epithelium in primary culture.

Mechlorethamine (HN2) is an alkylating agent usually used in cancer chemotherapy. Nevertheless, HN2 is extremely toxic and its use is accompanied by severe side-effects that may cause lung complications. Many studies report the morphological and biochemical modifications induced by sulfur mustard (SM) but no report has been published concerning the toxic effects of HN2 on the ultrastructural and functional activity of surface respiratory epithelial cells. This study was performed on rabbit tracheal epithelium (RTE) cells in primary culture. The functional activity of the culture was evaluated by measuring the ciliary beating frequency (CBF) of the ciliated cells using a videomicroscopic method, and the culture growth was determined by an image analysis system. The morphological aspects of the cells were analyzed by light, scanning electron, and transmission electron microscopy. An important inhibition of cell growth was observed associated with a detachment of the outgrowth cells. Morphological changes were expressed by vacuolization, increases in the intercellular spaces, and by disorganization of the cytoskeleton associated with a specific attack of the ciliated cells that show ciliary blebbing. The sudden CBF inhibition is more likely due to the detachment and the death of the ciliated cells than to a specific ciliotoxic effect of HN2. All these observations demonstrated the high sensitivity of respiratory epithelial cells to HN2 and showed that HN2-induced injuries were irreversible, and time- and dose-dependent.