Choroidal readaptation to gravity in rats after spaceflight and head-down tilt

Davet, Julien, Benoit Clavel, Lucien Datas, LaurenceMani-Ponset, Daniel Maurel, Serge Herbuté, Michel Viso, WilliamHinds, Joellen Jarvi, and Jacqueline Gabrion.Choroidal readaptation to gravity in rats after spaceflight andhead-down tilt. J. Appl. Physiol.84(1): 19-29, 1998.To determine when choroidal structures wererestored after readaptation to Earth gravity or orthostatic position,fine structure and protein distribution were studied in rat choroidplexus dissected either 6 h [Space Life Sciences-2 (SLS-2)experiments] or 2 days [National Institutes ofHealth-Rodent 1 (NIH-R1) experiments] after a spaceflight, or 6 hafter head-down tilt (HDT) experiments. Apical alterations were notedin choroidal cells from SLS-2 and HDT animals, confirming thatweightlessness impaired choroidal structures and functions. However,the presence of small apical microvilli and kinocilia and the absenceof vesicle accumulations showed that the apical organization began tobe restored rapidly after landing. Very enlarged apical microvilli appeared after 2 days on Earth, suggesting increased choroidal activity. However, as distributions of ezrin and carbonic anhydrase IIremained altered in both flight and suspended animals after readaptation to Earth gravity, it was concluded that choroidal structures and functions were not completely restored, even after 2 days in Earth's gravity.

     

Persistence of tight junctions and changes in apical structures and protein expression in choroid plexus epithelium of rats after short-term head-down tilt

Major alterations of choroidal cell polarity and protein expression were previously shown to be induced in rats by long-term adaptation to space flight (14 days aboard a space shuttle) or anti-orthostatic suspension (14 and 28 days) performed by tilting rats head-down (i.e. using a ground-based model known to simulate several effects of weightlessness). In rabbits, it was hypothesized that the blood-CSF barrier was opened in choroid plexus, after a short head-down suspension. To understand the early responses to fluid shifts induced by head-down tilts and evaluate the tightness of the choroidal junctions, we have investigated the effects of acute adaptations to anti-orthostatic restraints, using hindlimb-suspended Sprague-Dawley and Wistar rats. Ultrastructural and immunocytochemical studies were performed on choroid plexuses from lateral, third and fourth ventricles, after 30, 90 and 180 minutes of head-down tilt. Alterations were not perceptible at the level of choroidal tight junctions, as shown by freeze-fracture, claudin-1 and ZO-1 immunolocalizations and conventional electron microscopy, after intravenous injection of cytochrome C. The apical surface of choroidal cells was clearly more affected. Microvilli were longer and thinner and ezrin was over-expressed during all the periods of time considered, showing an early cytoskeletal response. Several proteins involved in the choroidal production of cerebrospinal fluid (sodium-potassium ATPase, carbonic anhydrase II, aquaporin 1) appeared first increased (30 minutes after the tilt), and then, returned to the control level or were lowered (after a 3-hour head-down suspension). Although head-down tilts do not seem to damage the blood-cerebrospinal fluid barrier in choroid plexus, it seemed that the expression of several apical proteins is affected very early.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Altered clearance of beta-amyloid from the cerebrospinal fluid following subchronic lead exposure in rats: Roles of RAGE and LRP1 in the choroid plexus

Formation of amyloid plaques is the hallmark of Alzheimer’s disease. Our early studies show that lead (Pb) exposure in PDAPP transgenic mice increases β-amyloid (Aβ) levels in the cerebrospinal fluid (CSF) and hippocampus, leading to the formation of amyloid plaques in mouse brain. Aβ in the CSF is regulated by the blood-CSF barrier (BCB) in the choroid plexus. However, the questions as to whether and how Pb exposure affected the influx and efflux of Aβ in BCB remained unknown. This study was conducted to investigate whether Pb exposure altered the Aβ efflux in the choroid plexus from the CSF to blood, and how Pb may affect the expression and subcellular translocation of two major Aβ transporters, i.e., the receptor for advanced glycation end-products (RAGE) and the low density lipoprotein receptor protein-1 (LRP1) in the choroid plexus. Sprague-Dawley rats received daily oral gavage at doses of 0, 14 (low-dose), and 27 (high-dose) mg Pb/kg as Pb acetate, 5 d/wk, for 4 or 8 wks. At the end of Pb exposure, a solution containing Aβ₄₀ (2.5 μg/mL) was infused to rat brain via a cannulated internal carotid artery. Subchronic Pb exposure at both dose levels significantly increased Aβ levels in the CSF and choroid plexus (p < 0.05) by ELISA. Confocal data showed that 4-wk Pb exposures prompted subcellular translocation of RAGE from the choroidal cytoplasm toward apical microvilli. Furthermore, it increased the RAGE expression in the choroid plexus by 34.1 % and 25.1 % over the controls (p < 0.05) in the low- and high- dose groups, respectfully. Subchronic Pb exposure did not significantly affect the expression of LRP1; yet the high-dose group showed LRP1 concentrated along the basal lamina. The data from the ventriculo-cisternal perfusion revealed a significantly decreased efflux of Aβ₄₀ from the CSF to blood via the blood-CSF barrier. Incubation of freshly dissected plexus tissues with Pb in artificial CSF supported a Pb effect on increased RAGE expression. Taken together, these data suggest that Pb accumulation in the choroid plexus after subchronic exposure reduces the clearance of Aβ from the CSF to blood by the choroid plexus, which, in turn, leads to an increase of Aβ in the CSF. Interaction of Pb with RAGE and LRP1 in choroidal epithelial cells may contribute to the altered Aβ transport by the blood-CSF barrier in brain ventricles.     

Body mass change during altered gravity: spaceflight, centrifugation, and return to 1 G

To assess the effect of gravity on growth, immature rats (130-200 g) were studied during chronic altered gravity exposure and while transitioning between gravity fields. Body mass gain of rats (n = 12) exposed to 14 days of microgravity (spaceflight) was evaluated and compared to mass gain of 1 G controls. Spaceflight did not affect mass gain. Six rats exposed to 1 G following spaceflight, when compared to controls, experienced a significant (0 < 0.05) post-flight mass loss over 48 h of 13 g. Over subsequent days, however, this loss was compensated for, and no difference from 1 G controls was noted after 5 days. Exposure to hypergravity (2 G) for 16 days was evaluated [(n = 6/group): Centrifuge (C); On Center Control (OCC); Centrifuge Control (CC)]. Body mass of centrifuged and OCC rats was reduced within 24 h, with OCCs regaining control mass within 13 days. The mass difference (44 g) in centrifuged animals persisted, however, with no subsequent difference in rate of mass gain between centrifuged animals and controls over Days 3-16 (3.7 +/- 0.1 vs. 3.9 +/- 0.1 g/day, respectively). Transitioning from 2 G to 1 G resulted in a mass increase within 48 hours for centrifuged animals. Over Days 3-16 at 1 G, the rate of gain for centrifuged animals continued to increase (3.1 +/- 0.1 g/day compared to 2.1 +/- 0.1 g/day for controls); differences from control, however, were still noted on Day 16. Transitioning to an increase in a gravity field causes acute losses in body mass. In hypergravity, the acute reduction in body mass persists but the rate of mass gain is normal. Animals returning to 1 G, after acute changes, adjust to attain control mass.     

Ultrastructure of the choroid plexus epithelium of pigeons treated with drugs: I. Effect of Thyradin, 2,4-dinitrophenol, and cycloheximide

Possible changes in the epithelial cells of the pigeon choroid plexus induced by administration of thyroid powder (Thyradin), 2,4-dinitrophenol, and cycloheximide were studied by scanning and transmission electron microscopy. A marked increase in the number of large bulbous and bleblike protrusions on the apical end of the epithelial cells was observed after oral administration of Thyradin for a month. The endoplasmic content of the protrusions consisted mainly of electron-lucent material. These results provide morphological evidence for the stimulatory effect of Thyradin. Intramuscular injections of 2,4-dinitrophenol for 15 days caused the collapse or deformation of the mitochondria and bleblike or bulbous protrusions. This indicates that changes in the surface configuration of the choroid plexus are controlled by an energy-dependent mechanism. The decrease of protrusions and polyribosomes and increase of the tubular saccules of varying electron density, size, and shape were noted in cells after 15 days of intramuscular cycloheximide injection. The electron density of the protrusions is lower than that of the control pigeons. The results of this study suggest that a curious pleomorphic structure on the apical surface of the choroid epithelial cell of pigeon is closely related to the functional state of choroidal cells. The study also demonstrates that a secondary ultrastructural response due to diverse physiologic effects is reflected in the architecture of the choroid plexus cells.     

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4⁺CD8⁺ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5⁺K8⁺) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5⁺K8⁺ TEC persisted. Interestingly, a surgical lesion of the inner ear’s vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.     

Ultrastructural localization of adhesion molecules in the healthy

The functional expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and MAdCAM-1 in the choroid plexus is indicative of a role of this structure in the communication of the immune system with the central nervous system (CNS). In order to gain further insight into the possible functions of adhesion molecules expressed in the choroid plexus, we investigated the exact ultrastructural localization of VCAM-1, ICAM-1 and MAdCAM-1 on semithin and ultrathin cryosections of the choroid plexus of healthy mice and of mice suffering from experimental autoimmune encephalomyelitis (EAE). In the healthy choroid plexus VCAM-1 and ICAM-1, but not MAdCAM-1, could be detected on the apical surface of the choroid plexus epithelial cells. During EAE, immunoreactivity for VCAM-1 and ICAM-1 was dramatically increased. Additionally, apical expression of MAdCAM-1 was observed on individual choroid plexus epithelial cells during EAE. At the same time, VCAM-1, ICAM-1 or MAdCAM-1 were never present on the endothelial cells of the fenestrated capillaries within the choroid plexus. The polar expression of VCAM-1, ICAM-1 and MAdCAM-1 on the apical surface of choroid plexus epithelial cells, which form the blood-cerebrospinal fluid barrier, implies a previously unappreciated function of this barrier in the immunosurveillance of the CNS.     

Complexity and developmental changes in the expression pattern of claudins at the blood–CSF barrier

The choroid plexus epithelium controls the movement of solutes between the blood and the cerebrospinal fluid. It has been considered as a functionally more immature interface during brain development than in adult. The anatomical basis of this barrier is the interepithelial choroidal junction whose tightness has been attributed to the presence of claudins. We used quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry to identify different claudins in the choroid plexuses of developing and adult rats. Claudin-1, -2, and -3 were highly and selectively expressed in the choroid plexus as compared to brain or parenchyma microvessels and were localized at epithelial junctions. Claudin-6, -9, -19, and -22 also displayed a previously undescribed choroidal selectivity, while claudin-4, -5, and -16 were enriched in the cerebral microvessels. The choroidal pattern of tight junction protein expression in prenatal brains was already complex and included occludin and zonula occludens proteins. It differed from the adult pattern in that the pore-forming claudin-2, claudin-9, and claudin-22 increased during development, while claudin-3 and claudin-6 decreased. Claudin-2 and claudin-11 presented a mirror image of abundance between lateral ventricle and fourth ventricle choroid plexuses. Imunohistochemical analysis of human fetal and postnatal brains for claudin-1, -2, and -3 demonstrated their early presence and localization at the apico-lateral border of the choroid plexus epithelial cells. Overall, choroidal epithelial tight junctions are already complex in developing brain. The observed differences in claudin expression between developing and adult choroid plexuses may indicate developmental differences in selective blood–cerebrospinal fluid transport functions.     

Expression of the immediate-early gene cFos in brain of the rats exposed to repeated 2 G influence

Early gene c-Fos expression was studied by means of c-Fos protein immunostaining in brain locus coeruleus (LC) of the rats exposed to primary and repeated hypergravity. One-hour 2 G influence on rats induced in LC cells expression of c-Fos protein, pointing out early gene c-Fos expression and synaptic activation of LC neurons. After repeated 1-hour 2 G, postponed for 35 days after primary 30-day 2 G influence, expression of c-Fos protein in LC neurons was not found. This fact is considered as a sign of memorizing of primary hypergravity influence.     

Pancreatic cell responses to primary and repeated 2 G influence

The pancreas of the rats exposed to primary and repeated prolonged hypergravity was studied by means of cytological methods. The rats were rotated on centrifuge at 2 G for 19 days and then after 30-day adaptation to 1 G were repeatedly exposed to 2 G for 5 days simultaneously with the rats first subjected to 5-day 2 G. After 5-day and 19-day hypergravity in pancreatic beta-cells the signs of decrease in insulin production were found. Adaptation of rats to 1 G for 30 days restored this process. Repeated 2 G for 5 days induced in beta-cells the changes of structure and hormonal product content indicating the pronounced increase in insulin synthesis and secretion. Response of alpha-cells to repeated 5-day 2 G was in parallel with beta-cell reactions.     

Quantitative changes in mRNA expression of glutamate receptors in the rat peripheral and central vestibular systems following hypergravity

In order to investigate the mechanisms responsible for adaptation to altered gravity, we assessed the changes in mRNA expression of glutamate receptors in vestibular ganglion cells, medial vestibular nucleus, spinal vestibular nucleus/lateral vestibular nucleus, cerebellar flocculus, and uvula/nodulus from rats exposed to hypergravity for 2 h to 1 week using real-time quantitative RT-PCR methods. The mRNA expression of GluR2 and NR1 receptors in the uvula/nodulus and NR1 receptors in the medial vestibular nucleus increased in animals exposed to 2 h of hypergravity, and it decreased gradually to the control level. The mRNA expression of GluR2 receptors in vestibular ganglion cells decreased in animals exposed to 1 week of hypergravity. Neither the metabotropic glutamate receptor 1 nor delta2 glutamate receptor in flocculus and uvula/nodulus was affected by a hypergravity load for 2 h to 1 week. It is suggested that the animals adapted to the hypergravity by enhancing the cerebellar inhibition of the vestibular nucleus neurons through activation of the NR1 and GluR2 receptors on the Purkinje cells in uvula/nodulus especially at the early phase following hypergravity. In the later phase following hypergravity, the animals adapted to the hypergravity by reducing the neurotransmission between the vestibular hair cells and the primary vestibular neurons via down-regulation of the postsynaptic GluR2 receptors in the vestibular periphery.     

Effects of hypergravity on mammary metabolic function: gravity acts as a continuum.

Mammary metabolic activity in pregnant rats is significantly increased in response to spaceflight. To determine whether changes in mammary metabolism are related to gravity load, we exposed pregnant rats to hypergravity and measured mammary metabolic activity. From days 11-20 of gestation (G), animals were centrifuged (20 rpm; 1.5, 1.75, or 2.0 x gravity) or were maintained at 1 G. On G20, five rats from each group were removed from the centrifuge and euthanized. The remaining dams (n = 5/treatment) were housed at 1 G until parturition. After 2 h of nursing by the pups, the postpartum dams were euthanized (G22). Glucose oxidation to CO2 and incorporation into lipids was measured. Mammary glands from dams euthanized on G20 revealed a strong negative correlation between metabolic rate and increased G load. Approximately 98% of the variation in glucose oxidation and 94% of the variation in glucose incorporation into lipids can be accounted for by differences in G load. Differences in metabolic activity disappeared in the postpartum dams. When we combined previous data from the microgravity with hypergravity environments and plotted the ratio of mammary metabolic rate vs. G load, there was a significant exponential relationship (r2 = 0.99). These data demonstrate a remarkable continuum of response across the microgravity and hypergravity environments and support the concept that gravitational load influences mammary tissue metabolism.     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.     

Hypergravity speeds up the development of T-lymphocyte motility

The effect of altered gravity on single cells has been reported in a number of studies. From the investigation of the immune system response to spaceflight conditions, interest has focused on the influence of gravity on single lymphocytes. Microgravity has been shown to decrease lymphocyte activation and to influence motility. On the other hand, the effect of hypergravity on lymphocyte motility has not been explored. We studied the migration of human peripheral blood T lymphocytes cultured in vitro in a hypergravity environment (10g). After hypergravity culture for 1–11 days, T cells were seeded on a fibronectin-coated glass surface, observed by time-lapse bright-field microscopy, and tracked by a computer program. We found that T cells, activated and then cultured in hypergravity, become motile earlier than cells cultured at normal gravity. These results suggest that hypergravity stimulates T cell migration.     

Maternal reproductive experience enhances early postnatal outcome following gestation and birth of rats in hypergravity

A major goal of space life sciences research is to broaden scientific knowledge of the influence of gravity on living systems. Recent spaceflight and centrifugation studies demonstrate that reproduction and ontogenesis in mammals are amenable to study under gravitational conditions that deviate considerably from those typically experienced on Earth (1 x g). In the present study, we tested the hypothesis that maternal reproductive experience determines neonatal outcome following gestation and birth under increased (hyper) gravity. Primigravid and bigravid female rats and their offspring were exposed to 1.5 x g centrifugation from Gestational Day 11 either through birth or through the first postnatal week. On the day of birth, litter sizes were identical across gravity and parity conditions, although significantly fewer live neonates were observed among hypergravity-reared litters born to primigravid dams than among those born to bigravid dams (82% and 94%, respectively; 1.0 x g controls, 99%). Within the hypergravity groups, neonatal mortality was comparable across parity conditions from Postnatal Day 1 through Day 7, at which time litter sizes stabilized. Maternal reproductive experience ameliorated neonatal losses during the first 24 h after birth but not on subsequent days, and neonatal mortality was associated with changes in maternal care patterns. These results indicate that repeated maternal reproductive experience affords protection against neonatal losses during exposure to increased gravity. Differential mortality of neonates born to primigravid versus bigravid dams denotes gravitational load as one environmental mechanism enabling the expression of parity-related variations in birth outcome.