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.

     

Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.     

Changes of insulin in plasma and receptor for insulin in various tissues after the exposure of rats to space flights and hypokinesia

The explanation of the mechanism of the response to gravity changes is of great importance for the determination of the capacity of human subjects to adapt to the load of gravitational stress. Therefore several studies were performed to investigate the activity of endocrine system, since the hormones are involved in the regulation of physiological functions and metabolic processes. However the studies of endocrine system activity during altered gravity conditions, especially during the weightlessness are influenced by the several interventions in biomedical observations due to operational program of astronauts, wide variability in individual response and tolerance, use of extensive countermeasures, differences in the type of space missions and in the studies after landing also a hypergravity effect at landing and variability in postflight readaptation process. The significant changes of plasma insulin and glucose levels were observed in astronauts during space flights and in the first days of recovery period. In the first inflight period plasma insulin levels were increased, unchanged or decreased however after 4-5 weeks of exposure to weightlessness a decrease of insulin plasma levels were noted. After space flights an increase of plasma insulin levels were demonstrated in experimental animals and in human subjects. Since plasma insulin level is considered as most important factor involved in the regulation for insulin receptors in target tissues, an investigation of insulin receptors in various tissues was performed in rats exposed to space flight or to hypokinesia (model used for simulation of some effects of microgravity).     

Analysis of metopirone-induced hypertrophy of adrenals in fetal rats encephalectomized in utero

Pregnant rats were treated with 30 mg metopirone (M) each day for 2 days and autopsied on the third day in various gestational periods (Days 18-20, 19-21, and 20-22). Control rats were treated with saline alone (S). The adrenals of intact fetuses in M-treated dams were significantly heavier than those of intact fetuses in S-treated dams in every experimental period. In both M- and S-treated dams, the adrenals of encephalectomized (E) fetuses were lighter than those of intact littermates. However, in the experimental period of Days 18-20 and 19-21, the adrenals of E fetuses in M-treated dams were slightly but significantly heavier than those of similar E fetuses in S-treated dams. In contrast, in the experimental period Days 20-22, there was no significant difference in the weight of adrenals of E fetuses of M- and S-treated dams. These changes in fetal adrenal weight were reflected histologically in parallel changes in the size of adrenocortical cells. The observations suggest that the fetal adrenal hypertrophy following maternal treatment with metopirone can occur to some extent independent of the fetal brain, but that close to the end of gestation the hypertrophy occurs completely under the control of the fetal brain.     

Rat pregnancy and parturition survive spaceflight challenge: new considerations of developmental consequences

Results of the NASA-NIH.R1 and NASA-NIH.R2 pregnant rat studies are reported and compared with results of Cosmos-1514 study. Similarities and differences between the Cosmos and STS flights are reviewed. STS rats were videotaped so that in-flight and post-flight behavior could be observed. Rats were observed during readaptation to 1-g and labor and delivery. Results indicate that pregnancy can proceed after exposure to microgravity and that vaginal delivery can occur despite readaptation to 1-g. Analysis of videotape revealed that flight dams experienced almost twice as many labor contractions as controls.     

Developmental toxicity evaluation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in Wistar rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a genotoxic chlorination by-product in drinking water. There is some evidence that it has developmental toxic effects in vitro but its potential to cause developmental effects in vivo is not known. The developmental effects were evaluated in Wistar rats. Rats (22-26 dams per dose group) were administered MX by gavage at the dose levels of 3, 30, or 60 mg/kg in water on gestation days 6-19. Control animals received plain water. Clinical signs, body weight, and food and water consumption were recorded for the dams. On gestation day 20, a cesarean section was performed and the ovaries anduterine contents of the dams were examined and the liver, kidneys, spleen, and thyroid glands weighed. The fetuses of all dose groups were weighed, sexed, and observed for external and skeletal malformations and the fetuses of the two highest dose groups were evaluated for visceral malformations. The highest dose, 60 mg/kg of MX, was slightly toxic to the dams. It decreased the corrected body weight gain of dams by 32% and the water consumption by 16-17%. Kidney and liver weights were slightly increased. MX did not affect the number of implantations nor did it cause any resorptions. The body weights of fetuses were not significantly affected. MX did not cause external malformations or skeletal anomalies. Two fetuses at 60 mg/kg and one fetus at 30 mg/kg had major visceral malformations (persistent truncus arteriosus, diaphragmatic hernia, dilated aorta with a stenosis of pulmonary arteries) and two minor artery abnormalities were observed in those animals. The frequency of unilateral displaced testis was slightly higher (9.2%) in the 60-mg/kg dose group than in controls (1.6%). Since the abnormalities did not form a consistent pattern and occurred most at maternally toxic dose, we conclude that MX can be regarded as non-teratogenic.     

Simultaneous measurement of atrial natriuretic polypeptide (ANP) messenger RNA and ANP in rat heart--evidence for a preferentially increased synthesis and secretion of ANP in left atrium of spontaneously hypertensive rats (SHR)

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the rat heart were measured simultaneously. In Wistar rats, ANPmRNA of the same size (approximately 0.95 kbp) was detected in all four chambers of the rat heart. The ANPmRNA level was the highest in the right atrium, and the left atrial level was slightly lower than the right atrial level. Ventricular levels were more than two orders of magnitude lower than atrial levels. Tissue ANP concentrations of four chambers were roughly parallel to ANPmRNA levels. In spontaneously hypertensive rats (SHR) with the elevated plasma ANP level, the ANPmRNA level in the left atrium was substantially increased. The left/right ratio of atrial ANPmRNA level in SHR (150%) was higher than that in control Wistar Kyoto rats (WKY) (90%). In contrast, the left/right ratio of atrial ANP concentration was decreased in SHR (44%) compared with that in WKY (84%). The ratio of ANP to ANPmRNA levels in the left atrium of SHR was about three times smaller than that in the right atrium of SHR, and those in bilateral atria of WKY. These results indicate that the biosynthesis and secretion of ANP from the left atrium is preferentially increased in SHR. Thus, simultaneous determination of ANPmRNA and ANP levels is a refined strategy of investigation for the biosynthesis, storage and secretion of ANP.     

Atrial natriuretic polypeptide (ANP) in the development of spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP)

In order to investigate the pathophysiological role of atrial natriuretic polypeptide (ANP) in genetic hypertensive rats, the atrial content and plasma concentration of ANP were measured by a sensitive radioimmunoassay (RIA) for rat ANP in 5-, 10- and 20-week-old spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) and compared to age-matched Wistar Kyoto rats (WKY). Atrial content of immunoreactive ANP (ir-ANP) tended to be higher in SHR and was already significantly higher in SHRSP than in WKY at 5 weeks of age. Atrial content in the hypertensive strains became significantly higher than in WKY when hypertension developed at 10 and 20 weeks. On the other hand, plasma ir-ANP in SHR was significantly lower than in WKY at 5 weeks, however, it became significantly higher in both SHR and SHRSP than in WKY at 10 and 20 weeks. These findings suggest that ANP release may increase to compensate for the elevation of blood pressure in SHR and SHRSP and that biosynthesis of ANP may be concomitantly stimulated, resulting in an increase in atrial ANP.     

The influence of phenobarbitone on maternal and perinatal hepatic drug-metabolizing enzymes in the rat.

Phenobarbitone (PB) (75 mg/kg) was administered orally for three consecutive days to pregnant or lactating rats at different pre- and postnatal stages in order that the perinatal animals would receive the agent either by transplacental passage or via the milk. Control animals received equivalent volumes of saline. The dams, fetuses, and pups were killed 24 h after the last dose. Hepatic p-nitroanisole O-demetnylase (OD), carboxylesterase (CE), and bromosulfophthalein-glutathione (BSP-GSH) conjugating enzyme activities in a 12 100 g - 20 min supernatant of a 20% w/v homogenate were measured. The morphology of the developing rat liver in the absence and presence of PB was examined by electron microscopy. The results demonstrated that the transplacental passage of PB to rat fetuses at term or 3 days prepartum had no effect on either the hepatic drug-metabolizing enzyme activities or on the ultrastructural appearance of the liver. Increased hepatic OD activity was observed in the pregnant animal but no effect was observed in the lactating dam. Phenobarbitone received by the suckling rat had two distinct effects. Compared to control activities, twofold increases in hepatic OD activity were observed in rat pups as early as 4 days after birth, associated with a marked proliferation in hepatic smooth endoplasmic reticulum. In contrast, PB-related significant increases in neonatal hepatic CE and BSP-GSH conjugating enzyme activities were not observed until 21 days of age. In the 4-day-old treated pups, characteristic morphological changes included numerous small membrane whorls in addition to increased smooth endoplasmic reticulum and microbodies in the liver.     

Influence of doxycycline administration on rat embryonic development during organogenesis

This experiment was performed to evaluate the possible embryotoxic and teratogenic effects of doxycycline during rat development. Twenty‐one female rats were used and distributed into three groups equally (seven animals/group). The low dose group received doxycycline at a dose of 5 mg/kg bw/day orally from the 6th to 14th day of gestation. The high dose group received 10 mg/kg bw/day orally for the same period, the Control group received 1 mL distilled water orally for the same period. The dams were dissected on the 20th day of gestation and their fetuses were subjected to morphological, skeletal, and histological examination. Moreover, DNA damage analysis of liver cells of pregnant rats and their fetuses or fetal skull was assessed by Comet assay. The obtained results showed a significant decrease in fetal body weight, several morphological anomalies, and severe lack of ossification on the skull bones, phalanges, and sternum bone as well as shortness in the ulna and radius bones. Histological studies of pregnant rats revealed congestion and dilatation of the central vein of the liver lobules and fatty degeneration of the hepatocytes. In addition, 20 day‐fetuses showed a marked increase of necrotic hepatocytes associated with an increased average of megakaryocytes and periportal leukocytic infiltration. Moreover, doxycycline induced a significant increase in the percentage of DNA damage and tail length of examined samples. Conclusively, doxycycline caused certain fetal abnormalities, so it is advisable to avoid using this drug during pregnancy.     

Effects of Anoectochilus formosanus Hayata extract and glucocorticoid on lung maturation in preterm rats.

We investigated the effects of maternal administration of Anoectochilus formosanus extract and dexamethasone on lung maturation in preterm rats. A. formosanus group mothers were tube-fed A. formosanus extract (300 mg/kg body wt./day) for 7 days from days 12-18 of gestation. Dexamethasone group mothers were injected intraperitoneally with dexamethasone (0.2 mg/kg body wt.) in saline on day 18 of gestation. Control group mothers were similarly injected with saline alone. On day 19 of gestation, fetuses were delivered by cesarean section. A. formosanus treatment significantly increased the fetal lung/body weight ratio, as compared to dexamethasone treatment. Saturated phosphatidylcholine levels in fetal lung tissue and growth hormone levels in maternal serum were significantly increased in the A. formosanus- and dexamethasone-treated groups as compared to controls. The histological appearance of preterm rat lungs revealed extensive branching of intermediate airways, denser mesenchyme, and more epithelial tubules in the dexamethasone and A. formosanus groups as compared with the control group. These results suggest that antenatal A. formosanus treatment may play a role in accelerating fetal rat lung maturation.     

Effect of L-propargylglycine on metabolism of sulfur-containing amino acids in pregnant rats and their fetuses

Cystathionine accumulated in several tissues of dams and fetuses by a single intraperitoneal administration of L-proparglyglycine to pregnant rats. Cystathionine in the liver of dams reached its maximal level at about 15 hrs after L-proparglyglycine injection (10 mg/300g), while that in the kidney and brain of dams, and in the liver, kidney, and brain of fetuses reached a maximum at about 21 hrs. The content of cystine in the liver of fetuses decreased gradually in proportion to the amount of L-proparglyglycine administered. Cystathionine gamma-lyase activity in the liver of dams and fetuses decreased to about 2-4% of that of control rats at 15 hrs after L-proparglyglycine injection, and that in the kidney and pancreas of dams to about 10-20% of that of control rats. On the other hand, cystathionine beta-synthase activity did not show significant changes from that of control rats.     

Co-operation between particulate and soluble guanylyl cyclase systems in the rat renal glomeruli.

ANP and NO act via different receptors, although inducing the common intracellular messenger - cyclic GMP. However, interaction between both factors remains unclear. Our observations suggested that in the rat kidney glomeruli, activities of the ANP- and NO-dependent guanylyl cyclase systems may be mutually compensated. To check this, we have tested effects of ANP and sodium nitroprusside (SNP) on cGMP synthesis and relaxation of glomeruli contracted with angiotensin II. The glomeruli were isolated from Wistar rats receiving saline (Control), dexamethasone (DEX), deoxycorticosterone (DOCA) or N-c-nitro-L-arginine methyl ester (NAME) for 1 or 2 days. In the DEX glomeruli exposed to 100 microM SNP, rate of cGMP synthesis was significantly higher then in the Control (26.3 vs 16.0 pmol/mg.prot./2 min., P<0.05), while 1 microM ANP was markedly less effective (2.8 vs 16.7 pmol/mg.prot./2 min in Control, P<0.01). On the contrary, in NAME group 1 microM ANP stimulated cGMP synthesis up to 35.6 pmol/mg.prot./2 min whereas efficacy of SNP was slightly suppressed. High correlation coefficient (r = 0.979, p<0.01) indicates interrelationship between NO- and ANP-dependent cGMP synthesis. Ability of the glomeruli to relax in response to ANP or SNP was in accord to their ability to cGMP generation. This was confirmed by high correlation (r = 0.845, p<0.001) between degree of relaxation and rate of cGMP synthesis. Our results support strongly the hypothesis that both, ANP and NO dependent systems co-operate in regulation of the function of kidney glomeruli.     

Characterization of the molecular forms of ANP released by perfused neonatal rat heart

Although cultures of neonatal rat atria and ventricles have been widely used to study ANP biosynthesis and secretion, little is known regarding the circulating form of ANP in neonatal animals. To begin to address this issue, we have developed a method for perfusing isolated neonatal rat hearts. Reversed phase-HPLC analysis of the heart effluents coupled with ANP RIA demonstrated that the predominant form of ANP released was chromatographically identical to ANP(99-126). Size exclusion-HPLC confirmed that the secreted ANP was indistinguishable from ANP(99-126). This demonstrated that the neonatal rat heart can efficiently generate and secrete a peptide similar to the circulating form of ANP found in adult rats, and further justifies the use of neonatal rat atria as a source of primary cells for studies of ANP biosynthesis and secretion.     

Permanent depression of plasma cGMP during long-term space flight

The purpose of this study was to investigate plasma concentrations of cyclic guanosine monophosphate (cGMP) and atrial natriuretic peptide (ANP) during and after real and simulated space flight. Venous blood was obtained 3 min after the beginning and 2 min after the lower body negative pressure maneuver in two cosmonauts preflight (supine), inflight, and postflight (supine) and in five other subjects before, at the end, and 4 days after a 5-day head-down tilt (-6 degrees) bed rest. In cosmonaut 1 (10 days in space), plasma cGMP fell from preflight 4.3 to 1.4 nM on flight day 6, and was 3.0 nM on the fourth day after landing. In cosmonaut 2 (438 days in space), it fell from preflight 4.9 to 0.5 nM on on flight day 3, and stayed <0.1 nM with 5, 9, and 14 months in space, as well as on the fourth day after landing. Three months after the flight his plasma cGMP was back to normal (6.3 nM). Cosmonaut 2 also displayed relatively low inflight ANP values but returned to preflight level immediately after landing. In a ground-based simulation on five other persons, supine plasma cGMP was reduced by an average of 30% within 5 days of 6 degrees head-down tilt bed rest. The data consistently demonstrate lowered plasma cGMP with real and simulated weightlessness, and a complete disappearance of cGMP from plasma during, and shortly after long-duration space flight.     

Effects of the spaceflight on organ-development in the neonatal rats: results in the Neurolab (STS-90)]

In the Neurolab mission, we found that spaceflight affects the development of the aortic baroreflex system and the body weight of the flight rats was significantly lighter [correction of lightess] than that of the control group. The aim of this study is to examine the structural and functional development in various tissues and organs. One hundred and eighteen nine-day old rats and seven fifteen-day old rats, which were launched at these ages and nursed by their dams in the space shuttle Columbia for 16 days, were served for this study. Two hundred and twenty one neonates were used as the ground controls (VIV: vivarium and AGC: asynchronous ground controls). On the landing day after they returned to the earth, the rats were perfused with a fixative under deep urethane anesthesia, and the organs were weighed and the ratio of the organ weight to the body weight was calculated. Six animals of the nine-day old group were reared on the ground for 30 more days after landing and also examined in the same protocol as the landing-day-examination. The organs obtained to examine were heart, lung, spleen, thymus, adrenal glands, kidney, liver, small intestine, large intestine, mesentery, pancreas, testis and ovary. Paraffin sections were made from some organ tissues and prepared for HE staining and immunohistochemistry. We compared these organs in the flight rat with those in the ground controls. All organs except the lung of nine-day old group were significantly smaller. In the ratio of organ weight to body weight, the lung and heart were significantly larger. The weight and ratio of the liver showed no significant difference. The thymus, spleen, mesentery and pancreas were smaller in the weight and the ratio. There were no differences in the body weight among 30-day reared groups, but the lung in the flight group is significantly heavier than the control groups and thymus also tends to be relatively heavy. In flight rats of the fifteen-day group, the kidney was heavy and the ovary was light as compared to the controls. The adipose tissue was macroscopically little found around the thoracic and abdominal organs in all rats of the flight group. These results suggest that the organs related to oxygen supply like as the lung and heart have priority in development over the mesentery and immune system organs even during spaceflight. Lightness of the mesentery in space rats is due to small contents of adipose tissues, and may reflect amounts of the food taken by the flight dams. Lightness of the organs like as the thymus, spleen and pancreas suggests that spaceflight may affect the immune system and also affect continuously the lung and thymus development even after landing.     

Experimental production of myeloschisis associated with hindbrain crowding in the central nervous system of rat fetuses by a single intragastric administration of ethylenethiourea

66 pregnant rats were divided into 9 groups according to gestation day 11 to 19. These pregnant rats were subjected to a single intragastric administration of ethylenethiourea (ETU) and cesarian sectioned on day 20. No dam died following the ETU treatment, but the rate of fetal death was as high as 21.2% on day 11, followed by a gradual decrease in the fetal death rate thereafter. The rate of production of various types of externally visible malformations was 100% except in the fetuses of dams treated with ETU on gestation day 19. The important results were as follows. (I) Fetuses of dams treated with ETU from gestation day 11 were found to suffer from a high incidence of myeloschisis associated with hindbrain crowding. (II) Exencephaly and an abnormally enlarged head with occipital bossing due to herniation of the mesencephalic tectum, with and without dilatation of the mesencephalic and 4th ventricle, were induced among the fetuses of dams given ETU at gestation day 12 and 13. (III) Various degrees of hydranencephaly and dysgenic hydrocephalus were found among the fetuses of dams treated with ETU from gestation days 14 to 18. The above results suggest that ETU may be a useful agent for the production of congenital malformations in the rat.     

Effects of Hyperthyroidism on Expression of Vascular Endothelial Growth Factor (VEGF) and Apoptosis in Fetal Adrenal Glands

This study investigated the expression of vascular endothelial growth factor (VEGF), vascular density, and apoptosis in fetal rat adrenal glands with hyperthyroidism in late gestation. Twelve mature female Wistar albino rats with the same biological and physiological features were used for this study. Rats were divided into two groups: control and hyperthyroidism. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (250 µg/kg) before pregnancy for 21 days and during pregnancy. Rats in the control and hyperthyroidism groups were caged according to the number of male rats. Zero day of pregnancy (Day 0) was indicated when the animals were observed to have microscopic sperm in vaginal smears. Pregnant rats were sacrificed on the 20th day of pregnancy; blood from each animal was collected to determine the concentrations of maternal adrenocorticotropic hormone and thyroxine. Rat fetuses were then quickly removed from the uterus, and the adrenal glands of the fetuses were dissected. VEGF expression, vascular density, and apoptosis were analyzed in fetal rat adrenal glands. Maternal serum levels of the ACTH and free thyroxine were significantly higher in the hyperthyroidism group than in the control group. Immunohistochemistry revealed that the number of VEGF positive cells and vessel density significantly increased in the hyperthyroidism rat fetal adrenal group compared with the control group. Hyperthyroidism did not change the fetal and placental weights and the number of fetuses. This study demonstrates that hyperthyroidism may have an effect on the development of rat adrenal glands mediated by VEGF expression, angiogenesis, and apoptosis.