Daniel Maurel

Chronic Restraint Enhances lnterleukin-1-Beta Release in the Basal State and after an Endotoxin Challenge, Independently of Adrenocorticotropin and Corticosterone Release

To explore the interactions between the hypothalamic-pituitary-adrenocortical axis and the immune system under stress conditions, we used an experimental rat model for chronic tail-restraint devised earlier for ground studies in space physiology. The system was used in two positions: (1) the orthostatic restraint position (OR) and (2) the antiorthostatic position (AOR) after the rat hind limbs had been raised by a head-down tilt. After 7 days of either restraint, sequential blood samples were taken via an indwelling aortic cannula, before and at various time intervals between 15 and 300 min after an intravascular infusion of 25 micrograms/kg lipopolysaccharide (LPS). The plasma titers of adrenocorticotropin (ACTH), corticosterone (CORT) and interleukin-1 beta (IL-1 beta) were assayed. Under basal conditions, both OR and AOR restraints induced a 5-fold increase in IL-1 beta with no significant changes in ACTH and CORT levels. A robust increase in all three variables was observed after LPS injection. However, the IL-1 beta response to LPS was significantly higher in both restrained groups than in controls. Both the amplitude and the percentage of individually restrained rats displaying elevated IL-1 beta levels were increased up to 5 h. In contrast, the ACTH and CORT post-LPS responses were normal in the OR group. They were unusually dissociated in the AOR rats, which displayed depressed ACTH levels associated with slightly increased CORT levels. Our results suggest that immune-neuroendocrine responses to chronic restraint stress may differ from those generally observed in acute stress.

Effects of acute tilt from orthostatic to head-down antiorthostatic restraint and of sustained restraint on the intra-cerebroventricular pressure in rats

The tail-cast suspension rat model was developed to explore in ground laboratories the physiological effects of some of the stresses prevailing during space flight including and among them those of the headwards body fluid shifts. We recently showed in rats that an acute head-down tilt (45 degrees) from tail-cast orthostatic (OR) to antiorthostatic restraint (AOR) induced within 30 min and for 2 to 4 h an acute stress-like surge in plasma ACTH and corticosterone levels. Considering the proximity of the CRF producing neurons with the 3rd ventricle, we decided to explore the acute and longer-term effects of the OR/AOR tilt on the intra-cerebroventricular pressure (Picv) measured with an indwelling sensor-transmitter catheter stereotaxically implanted in the 3rd ventricle. At 1- or 10-min intervals the unit sent radiotelemetric signals for both Picv and motor activity (MA) to a receiver coupled with an automatic data analyser. The acute AOR-tilt induced within 10 min and for 60 min a 2.5-fold rise in Picv which receded to baseline between 60 and 90 min. During this time, the normally close correlation between Picv and MA was lost, as assessed by Spearmans rank coefficient. In a long-term experimental series we explored the evolution of both Picv and MA in individual rats subjected successively to a 7 day control phase (C). 7 days OR, and 3 days AOR. After the 1-h-long post-tilt rise of the Picv, the mean Picv levels measured for the next 3 days decreased significantly vs. both the preceding OR phase (-30%) and the initial C Phase (-40%). The circadian pattern of the diurnal Picv profile was impaired, as evidenced by a significant fall (i) in the night/day ratio (-25% vs. C). and (ii) even more in the spectral power of the circadian 1 c/24 h frequency (-85% vs. C). The simultaneously recorded MA fluctuations similarly displayed an altered diurnal pattern with a spectral power of the circadian frequency reduced to 7% of controls. However, contrary to the short-term experiment, in the long-term study the large alterations to both Picv and MA were strongly correlated, as during the control phase. The mechanisms involved in the swift post-tilt rise in the Picv together with an aroused corticotropic axis, and in the impact of sustained head-down restraint on CNS-controlled adaptive regulations including their circadian rhythms remain unknown.

Ependymal and choroidal cells in culture: Characterization and functional differentiation

During the past 10 years, our teams developed long‐term primary cultures of ependymal cells derived from ventricular walls of telencephalon and hypothalamus or choroidal cells (modified ependymal cells) derived from plexuses dissected out of fetal or newborn mouse or rat brains. Cultures were established in serum‐supplemented or chemically defined media after seeding on serum‐, fibronectin‐, or collagen‐laminin‐coated plastic dishes or semipermeable inserts. To identify and characterize cell types growing in our cultures, we used morphological features provided by phase contrast, scanning, and transmission electron microscopy. We used antibodies against intermediate filament proteins (vimentin, glial fibrillary acidic protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin, and fibronectin in single or double immunostaining, and monoclonal antibodies against epitopes of ependymal or endothelial cells, to recognize ventricular wall cell types with immunological criteria. Ciliated or nonciliated ependymal cells in telencephalic cultures, tanycytes and ciliated and nonciliated ependymal cells in hypothalamic cultures always exceeded 75% of the cultured cells under the conditions used. These cells were characterized by their cell shape and epithelial organization, by their apical differentiations observed by scanning and transmission electron microscopy, and by specific markers (e.g., glial fibrillary acidic protein, ciliary rootlet proteins, DARPP 32) detected by immunofluorescence. All these cultured ependymal cell types remarkably resembled in vivo ependymocytes in terms of molecular markers and ultrastructural features. Choroidal cells were also maintained for several weeks in culture, and abundantly expressed markers were detected in both choroidal tissue and culture (Na+‐K+‐dependent ATPase, DARPP 32, G proteins, ANP receptors). In this review, the culture models we developed (defined in terms of biological material, media, substrates, duration, and subculturing) are also compared with those developed by other investigators during the last 10 years.

Different responses of plasma ACTH and corticosterone and of plasma interleukin-1 beta to single and recurrent endotoxin challenges.

In a parallel study in 10 individual rats, three time series of plasma concentrations of ACTH, corticosterone (CORT), and interleukin‐1β (IL‐1β) were measured before (time 0) and at intervals between 15 and 480 min following intra‐arterial (i.a.) infusions of 25μg/kg lipopolysaccharide (LPS). All LPS injections were given at 9 AM. The first time series was performed on naive rats (day 1). A sequence of six daily injections (days 3–8) of the same dose of LPS followed. The post‐LPS time course of the plasma ACTH, CORT and IL‐1β levels were studied on days 3 (second injection) and 8 (seventh injection). The first LPS injection induced a rapid (30 min) eightfold rise in plasma ACTH and CORT, culminating in concentrations 30 times the baseline at 60 min (ACTH) and 15 times baseline at 120 min (CORT). Both hormones receded back to the initial basal level at 480 min. On the other hand, IL‐1β increased slowly to peak at 13 times baseline 120 min before declining to minimal seven‐ to ninefold basal levels, 480 min and even 48 h post‐LPS. During the second phase of the experiment starting 48 h after the initial LPS priming sequence, the ACTH and CORT responses to daily recurrent LPS injections again differed from those of IL‐1β. The post‐LPS time courses of the ACTH and CORT reaction displayed a typical pattern of a progressive attenuation studied at days 3 and 8. The peak amplitudes at days 3 and 8 were reduced to 60 and 10%, respectively, for ACTH, and to 85 and 45% for CORT of those observed at the first LPS test. The duration of the response (both) was also shortened from 480 min (first LPS test) to 300 min at days 3 and 8. The post‐LPS patterns of the IL‐1β responses were characterized, first by basal levels seven to nine times higher than the initial baseline values (day 1), and by a rapid suppression of the post‐LPS response, with only a slight (30%) increase at day 3 and no increase at day 8. Thus, after both acute and recurrent LPS administration, ACTH/CORT and IL‐1β reacted differently to the endotoxin challenge. The two LPS reactive systems were not correlated. This is inconsistent with the often proposed role of increased plasma IL‐1β release as an intermediary factor in the LPS‐induced recruitment of the corticotropic axis in general infections.

Chronic orthostatic and antiorthostatic restraint induce neuroendocrine, immune and neurophysiological disorders in rats

The tail-cast suspension rat model has been developed in ground laboratories interested in space physiology for extensive study of mechanisms causing the pathophysiological syndrome associated with space flights. We used individually-caged male rats to explore the effects of acute and chronic (7d) orthostatic restraint (OR) and head-down anti-orthostatic restraint (AOR) on a series of physiological variables. The acute restraint study showed that (1) the installation of the OR device induced an acute reaction for 2 days, with a substantial rise in ACTH (x2) and CORT (x6), and that (2) the head-down tilt from OR to AOR induced (i) within 10 min and lasting 60 min a 2-fold rise in the intra-cerebro-ventricular pressure (Picv) monitored with an icv telemetric recording system, which receded to normal between 60 and 120 min; and (ii) within 30 min a short-lived 4-fold rise in plasma ACTH and CORT levels. Chronic OR induced (1) the suppression of the diurnal ACTH/CORT rhythm, with increased mean levels, especially for ACTH, (2) a degraded circadian locomotor activity rhythm manifested by a significant reduction in the spectral power of the 24h periodicity and a concomitant emergence of shorter (ultradian) periodicities, (3) an associated, but less pronounced alteration of the diurnal rhythm in body temperature; and (4) a marked increase in baseline plasma levels of IL-1 beta and an increased reactivity in cytokine release following an E. coli endotoxin (LPS) challenge. AOR induced (1) a similar obliteration of the circadian ACTH/CORT rhythm, (2) the loss of close correlation between ACTH and CORT, (3) a generalized increase in baseline plasma IL-1 beta levels and (4) more extensive degradation of the circadian periodicity for both locomotor activity and, to a lesser extent, body temperature, replaced by dominant spectral powers for ultradian periodicities (3 to 10h). In conclusion, both experimental paradigms--but AOR more than OR--caused a blockade of the circadian rhythmicity of major physiological variables, the loss of normal correlations between ACTH and CORT, and inflammatory-immune hyperreactivity. These pathophysiological disorders may all be parts of a complex chronic stress syndrome.

Suprachiasmatic nucleus lesions abolish photoperiod-induced changes in the testis function and GnRH immunoreactivity in the mink, a short-day breeder

Testicular activity (testis volume and plasma testosterone) and immunoreactive GnRH hypothalamic system were examined after suprachiasmatic nucleus (SCN) lesion in the mink, a short-day breeding mammal, whose sexual activity is inhibited by day lengths exceeding 10 h. In animals maintained under a natural photoperiod, SCN destruction performed during the period of maximum sexual activity (February) was shown to have no effect on onset of the testicular inactive period which begins at the end of winter and continues through spring. On the other hand, while gonadal activity began again at the end of autumn in intact animals, minks that had undergone SCN destruction remained sexually inactive until the end of the experiment period (February). The SCN could thus be crucial to the onset of sexual activity triggered by the reduction of day length, whereas onset of sexual inactivity is a spontaneous phenomenon. This was confirmed in a second experiment demonstrating that a short photoperiod (4 L:20 D), highly gonadostimulatory in intact animals, had no effect on testicular activity after SCN destruction. An immunocytochemical study of the hypothalamic GnRH system (staining intensity and number of labeled perikarya and immunoreactive endings in the external layer of the median eminence) also showed consistent by very low rates of immunoreactivity and number of labeled perikarya and endings in operated animals.

Effects of an 11-day spaceflight on the choroid plexus of developing rats

Cellular distributions of ezrin, a cytoskeletal protein involved in apical cell differentiation in choroid plexus, and carbonic anhydrase II, which is partly involved in the cerebrospinal fluid production, were studied by immunocytochemistry, at the level of choroidal epithelial cells from the lateral, third and fourth ventricles in normal or experimental fetuses, in parallel with the ultrastructure of apical microvilli, observed by transmission electron microscopy. We compared choroid plexuses from developing normal rats (gestational day 15 to birth) with choroid plexuses from 20-day-old rat fetuses, developed for 11 days in space, aboard a space shuttle (NASA STS-66 mission, NIH-R1 experiments), from gestational day 9 to day 20. The main changes observed in fetuses developed in space were demonstrated by immunocytochemistry and concerned the distribution of ezrin and carbonic anhydrase II. Thus, in fetuses developing in space, ezrin was strongly detected in the choroidal cytoplasm and weakly associated to the membrane in the apical domain of the choroid plexus from the fourth ventricle. Such alterations suggested that choroid plexus from rat fetal brain displays a delayed maturation under a micro-gravitational environment. In contrast, intense immunoreactions to anti-carbonic anhydrase II antibodies showed that this enzyme is very abundant in rats developed in space, compared to ground control fetuses.

Short-day stimulation of testicular activity and immunoreactivity of the hypothalamic GnRH system in mink following deafferentation of the pineal body by bilateral superior cervical ganglionectomy and melatonin replacement

The effects of superior cervical ganglionectomy on testicular function (testis volume and plasma testosterone levels) and the immunocytochemical activity of the GnRH hypothalamic system were studied in the mink, a short-day breeder. Animals reared in a natural photoperiod were (i) ganglionectomized at four different times during the period extending from the end of summer to the end of autumn (September 15, October 20, October 28, and December 1), and (ii) reared for 50 days in a short gonadostimulatory photoperiod (4L:20D). Lastly, an attempt was made to overcome the effects of superior cervical ganglion removal by administering melatonin to mink reared in a natural photoperiod. In mink reared in a natural photoperiod, deafferentation of the pineal on September 15 (L = 12.5 h) or October 20 (L = 10.5 h) resulted in consistently low values of testicular volume and plasma testosterone until the end of the experiment (February). When the operation was performed on October 28 (L = 10 h) testicular activity was initiated but only lasted a short time and did not allow maximal gonadal development. When superior cervical ganglionectomy was carried out on December 1 (L = 8.5 h), during the phase of renewed testicular activity, the increases in testicular volume and testosterone levels were not affected by the operation and the subsequent variation of these parameters was identical to that observed in intact animals. Similarly, in mink reared for 50 days in a photoperiod of 4L:20D before superior cervical ganglionectomy, deafferentation of the pineal did not prevent gonadostimulation induced by short days.(ABSTRACT TRUNCATED AT 250 WORDS)

Superior cervical ganglionectomy suppresses circadian corticotropic rhythms in male rats in the short term (5 days) and long term (10 days)

Superior cervical ganglionectomy (SCGx) has drastic effects on numerous hormonal circadian rhythms and particularly on pineal melatonin secretion. We investigated the hormonal consequences of ablation of the superior cervical ganglion on the corticotropic circadian rhythms in the male rat. Plasma were obtained by sampling blood every 4 h, using a chronic carotid cannula. Adreno-corticotropin hormone (ACTH) was assayed by radioimmunoassay (RIA) and corticosterone (B) by radiocompetition. Urinary 6-sulphatoxymelatonin (aMT6s), considered as an index of the pineal gland activity, was assayed by specific RIA: a decrease in the aMT6s concentration after ganglionectomy was taken as proof of adequate surgical operation. Control animals showed classical circadian rhythms for ACTH and B with basal values during the light phase and circadian peaks around the light/dark interface. Five and ten days after ganglionectomy, the circadian rhythms of ACTH and B were suppressed. In addition, the mean ACTH concentrations increased significantly 10 days after ganglionectomy compared to those in sham-operated rats and 5 days post-operation group. The mean plasma corticosterone levels were similar in those three groups of animals. This is the first study demonstrating the suppressive effect of superior cervical ganglionectomy on the circadian corticotropic hormonal cycle.

Diurnal variations of urinary 6‐sulphatoxymelatonin in male intact or ganglionectomized mink

Abstract: The existence of the major urinary metabolite of melatonin, 6‐sulphatoxymelatonin (aMT6s), was validated for mink and the 24 hr urinary excretion pattern was determined in intact and superior cervical ganglionectomized animals under different photoperiodic conditions. Within‐and between‐assay variations, parallelism between serially mid‐night pooled urine dilutions and standard curves in aMT6s free urine of mink at 1:125 dilution and recovery of aMT6s in mid‐day pooled urine at 1:125 dilution provided a good validation for the mink urinary a MT6s assay. In natural photoperiods (January, LD 9:15; April, LD 13:11) the diurnal rhythm was characterized by low aMT6s values during the day and high values at night. There were no differences in the nocturnal values measured under long‐ (April, 4.11 ± 0.40 ng/hr) or short‐day (January, 4.74 ± 0.36 ng/hr) conditions. In an experimental long photoperiod (LD 15:9), the same result was obtained on the 24 hr rhythm in intact animals, but in ganglionectomized mink the nocturnal rise in aMT6s was abolished and the nocturnal values were always low (0.88 ± 0.09 ng/hr). Our results agree with those obtained in other species concerning plasma melatonin rhythm and urinary aMT6s excretion; we thus conclude that this is an effective assay for measuring pineal activity in mink.