Analysis of respiratory movements in a mouse model of late Parkinson's disease submitted to stress.

Parkinson's disease (PD) is known to cause tremor and rigidity, but other symptoms such as respiratory and autonomic dysfunctions are a major cause of disability and mortality in patients. In this study, we examined respiratory movements by using cineradiography on a murine model of late/advanced PD. Under surgical anesthesia, C57BL/6J mice received an injection of either 6-OHDA or vehicle solution to the right striatum. Two weeks after surgery, the animals had their respiratory movements recorded by video X-ray without any restraint. During recordings the animals were submitted to a mild acute-stress challenge. Behavioral tests were performed to assess the severity of the 6-OHDA lesion. As a result, behavioral tests confirmed severe motor impairments in 6-OHDA mice as compared to controls. 6-OHDA mice showed a predominantly thoracic respiratory pattern with reduced diaphragmatic excursion, and reduced respiratory frequency after stress. These results suggest that advanced nigrostrial degeneration may cause respiratory alterations with the features of obstructive-type respiratory disorders.

Cineradiographic analysis of respiratory movements in a mouse model for early Parkinson's disease.

Parkinson's disease (PD) is a progressive degenerative disorder of the central nervous system known to cause a typical pattern of motor symptoms. In its later stages, PD is known to cause respiratory alterations including shortening of operational volumes and reduced velocity of respiratory-muscle contraction. It has been proposed that such changes are secondary to changes in posture and osteoarticular degeneration, leading to an alteration in the spinal axis that in turn could affect breathing mechanics. In this study, we examined respiratory movements by using cineradiography on a murine (C57BL/6J) model of mild hemi-PD. Under surgical anesthesia, PD mice received an injection of 6-OHDA solution to the right striatum, and were compared to control mice, which received an injection of saline solution. Two weeks after surgery, all mice had their respiratory movements recorded by video X-ray without any restraint. Behavioral tests were performed to assess the severity of the 6-OHDA lesion. As a result, behavioral tests confirmed mild motor impairments in PD mice as compared to controls. Parameters of respiratory function showed mild alterations in the PD group, suggestive of a restrictive-type respiratory disorder. These results suggest that respiratory alterations in PD may emerge simultaneously to other motor symptoms, and not as a consequence of the latter.

Measuring biting behavior induced by acute stress in the rat.

Previous reports have indicated that biting behavior is enhanced in rats that are subject to acute stress. Several methods have been proposed for studying this phenomenon, one of which is the electromyography (EMG) of the jaw muscles. In this study, we compared total EMG activity with the EMG activity related to biting behavior, as determined by video monitoring, before and after restraint stress. Wistar albino rats (150 g) were subject to surgery for electrode placement 48 h before measurements. The EMG activity of the masseter muscle on one side was recorded for 10 min before and immediately after 30 min of restraint stress. Restraint stress increased jaw muscle EMG activity, although some of the activity was related to behaviors other than biting. Recording the EMG activity of jaw muscles is useful for measuring the stress response in rats, but careful video monitoring is needed if biting behavior is to be studied in particular.

Hypergravity modulates behavioral nociceptive responses in rats.

Hypergravity (2G) exposure elevated the nociceptive threshold (pain suppression) concomitantly with evoked neuronal activity in the hypothalamus. Young Wistar male rats were exposed to 2G by centrifugal rotation for 10 min. Before and after 2G exposure, the nociceptive threshold was measured as the withdrawal reflex by using the von Frey type needle at a total of 8 sites of each rat (nose, four quarters, upper and lower back, tail), and then rats were sacrificed. Fos expression was examined immunohistochemically in the hypothalamic slices of the 2G-treated rats. When rats were exposed to 2G hypergravity, the nociceptive threshold was significantly elevated to approximately 150 to 250% of the 1G baseline control levels in all the examination sites. The 2G hypergravity remarkably induced Fos expression in the paraventricular and arcuate nuclei of the hypothalamus. The analgesic effects of 2G hypergravity were attenuated by naloxone pretreatment. Data indicate that hypergravity induces analgesic effects in rats, mediated through hypothalamic neuronal activity in the endogenous opioid system and hypothalamo-pituitary-adrenal axis.

Effects of neurectomy and tenotomy on the bone mineral density and strength of tibiae.

The right hindlimbs of 5 or 6-week old Wistar male rats were sciatic/femoral neurectomized, tenotomized or sham operated. The rats were sacrificed 2 weeks after the surgery and the tibiae were removed. pQCT measurement was performed on total, cortical, and trabecular bone separately at different regions. Reduction of the bone mineral density by unloading was observed more significantly at metaphysis than at diaphysis due to histological heterogeneity between metaphysis and diaphysis; metaphysis is rich in trabecular bone and diaphysis is abundant in cortical bone. Trabecular bone might be more sensitive to unloading because the reduction rate of volumetric bone mineral density in trabecular bone was approximately 10 times and 3 times larger than that of cortical bone in both neurectomy and tenotomy rats, respectively, Unloading also reduced the cross-sectional area and stress strain index at metaphysis.

Gravity stress elevates the nociceptive threshold level with immunohistochemical changes in the rat brain.

Young Wistar male rats were exposed to 2G hypergravity by continuous centrifugation for 15 minutes. The nociceptive threshold was measured by using the von Frey type filament on the rat skin surfaces after hypergravity exposure. Following the hypergravity exposure, rats were sacrificed with anesthesia, then perfused and fixed for immunohistochemical examination. The 2G hypergravity elevated the nociceptive threshold up to 2-fold and induced analgesic effects on rats that remained for 2 hours after termination of centrifugation. Expression of Fos-immunoreactive proteins was prominently induced by 2G hypergravity in the arcuate nucleus and the paraventricular nucleus of the hypothalamus. The 15-minute flash exposure to 2G hypergravity induced pain suppression in rats, which might be attributed to change of neuronal activity in rat hypothalamus.

Hypergravity and opioid-mediated pain suppression in rats.

It is known that pain suppression in animals is induced by certain environmental stimulus. However, little is known about the effects of gravitational alteration on the nociceptive responses in rats. A recent study indicated that Fos protein expression was strongly induced in the vestibular-related brainstem regions of rats that were exposed to 2 G hypergravity (Gustave Dit Duflo et al., 2000). A number of studies indicate that Fos expression is induced in the brain by various kinds of stress. We showed that either long-term exposure or short-term exposure to 2 G hypergravity elevated the nociceptive threshold in the rat skin surfaces, in concomitant with Fos induction in the hypothalamus including the arcuate nucleus and paraventricular nucleus (Kumei et al., 2000). We have examined the possible involvement of beta-endorphin, an endogenous opioid, in the hypergravity-induced analgesic effects on rats and its counteraction by naloxone, an opioid receptor antagonist.

Studies on the effects of microgravity on the ultrastructure and function of cultured mammalian cells.

Four cultures of monkey kidney cell line, JTC-12, were flown on the Spacelab-J (SL-J) mission during 8 days. The results of the present study showed that the space flight gave no essential effect on morphology, cell cycle, glucose consumption and urokinase production of the mammalian culture cell. However, the cell proliferation slightly decreased under microgravity. Moreover, the lack of gravity induced the trypsin-treated dissociated cells to keep floating in the culture medium. Therefore, the attachment of the cells onto the substratum was delayed, and that caused difficulties in subculturing the cells. The present research also offered some important information on techniques for establishment of cell cultures in space laboratories.

Nociceptive responses and immunohistochemical changes in the rat brain under gravity stress.

It is well known that exposure to various stresses leads to pain suppression in animals. However, there is no report about the effects of gravitational alteration to serve as a kind of stress. The purpose of the present study is to clarify the effect of hypergravity (2 G) on the nociceptive responses and histochemical changes in rats. We examined the level of the threshold of withdrawal reflex against the noxious [correction of noxicious] stimulation in rats that were exposed to 2 G. Data show that the 2 G exposure elevates the nociceptive threshold. We have demonstrated for the first time that gravity change induces analgesic effects on rats in concomitant with c-fos induction in the arcuate, and paraventricular nuclei of rat hypothalamus. Gravity change acts as a kind of stress in rats.

Expression of PDGF-beta receptor, EGF receptor, and receptor adaptor protein Shc in rat osteoblasts during spaceflight.

A number of studies have indicated that microgravity induces osteopenia and modulates functions of mammalian cells. However, the molecular mechanisms underlying these effects of microgravity are still unknown. Rat osteoblasts were cultured for 4 and 5 days during Shuttle-Spacelab flight, and fixed by guanidine isothiocyanate solution on board after treatment with 1alpha, 25 (OH)2 vitamin D3. The mRNA levels for platelet-derived growth factor (PDGF)-beta receptor, epidermal growth factor (EGF) receptor, the growth factor receptor adaptor protein Shc, and c-fos were determined using the method of quantitative reverse transcription-polymerase chain reaction. The mRNA levels for EGF receptor were not altered by microgravity. However, the mRNA levels for PDGF-beta receptor, Shc, and c-fos were decreased to 62, 55 and 25% on the 4th day of flight, and 47, 40, and 43% on the 5th day, respectively, as compared to the corresponding ground controls. Expression of the growth factor receptor and the receptor adaptor protein was modulated in rat osteoblasts during spaceflight. Data suggest that signal transduction via growth factor receptors in rat osteoblasts is impaired by microgravity. Dysfunction of osteoblasts might be involved in spaceflight-induced osteopenia.

Space flight modulates signal transduction pathway of growth factor receptors in rat osteoblasts.

Bone metabolism is regulated by the balance of bone formation and resorption. Osteoblasts serves primarily for bone formation. Microgravity deteriorates osteoblastic function and inhibit bone formation. Growth factors regulate osteoblast function via receptors. Binding of EGF and PDGF to the receptors activates receptor tyrosine kinase and rapid association of adapter proteins Shc and Grb2, and evokes the Ras/MAP kinase cascade. Signals from various chemical and physical stimuli are transmitted to the nucleus, and induce c-fos and c-jun gene expression. However, effects of microgravity on the molecular events in osteoblasts remain unknown. The purpose of this study is to investigate the mRNA levels for PDGF-beta receptor, EGF receptor, Shc, and c-fos in rat osteoblasts during space flight.

Spaceflight modulates insulin-like growth factor binding proteins and glucocorticoid receptor in osteoblasts.

Rat osteoblasts were cultured for 4 or 5 days during a Space Shuttle mission. After 20-h treatment with 1alpha,25-dihydroxyvitamin D3, conditioned media were harvested and cellular DNA and/or RNA were fixed on board. The insulin-like growth factor binding protein (IGF BP)-3 levels in the media were three- and tenfold higher than in ground controls on the fourth and fifth flight days, as quantitated by Western ligand blotting and radioimmunoassay, respectively. The increased IGF BP-3 protein levels correlated with two- to threefold elevation of IGF BP-3 mRNA levels, obtained by reverse transcription-polymerase chain reaction. The IGF BP-5 mRNA levels in flight cultures were 33-69% lower than in ground controls. The IGF BP-4 mRNA levels in flight cultures were 75% lower than in ground controls on the fifth day but were not different on the fourth day. The glucocorticoid receptor mRNA levels in flight cultures were increased by three- to eightfold on the fourth and fifth days compared with levels in ground controls. These data suggest potential mechanisms underlying spaceflight-induced osteopenia.

Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization.

It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5- to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3- to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4- to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.

An improved quantitative RT-PCR fluorescent method for analysis of gene transcripts in the STS-65 space shuttle experiment.

We describe a reverse transcription polymerase chain reaction (RT-PCR) technique using fluorescent dUTP to examine changes in mRNA level in samples. In this procedure, the amplified product is identified by the fluorescent detection system in an automated DNA sequencer, and if the MW of the DNA/RNA or the fluorescent dye is different, several samples can be analyzed in a single lane. The basis for this technique is similar to that of radiolabeled methods, and we applied this technique for the comparison of the expression level of the rat c-myc gene in osteoblasts exposed to microgravity and unit gravity conditions. Using the fluorescent- and radiolabeled methods, the level of rat c-myc mRNA were compared quantitatively and the results demonstrated that the c-myc expression level was not altered by microgravity. Therefore, this fluorescent RT-PCR technique is useful for gene expression analysis particularly when the samples are limited, such as in space flight experiments. The method also allows for rapid assessment of mRNA changes in many samples simultaneously.

Hypergravity signal transduction and gene expression in cultured mammalian cells.

A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.

Hypergravity signal transduction in HeLa cells with concomitant phosphorylation of proteins immunoprecipitated with anti-microtubule-associated protein antibodies.

We have shown that hypergravity (35g) stimulates production of inositol 1,4,5-trisphosphate (IP3) and decreases adenosine 3',5'-cyclic monophosphate (cAMP) levels in HeLa cells. IP3 production rapidly increased 1.5- and 2.1-fold greater (P less than 0.05) than the control after 2- and 5-min exposures to 35g, respectively. The intracellular cAMP levels, determined in the presence of isobutylmethylxanthine, were decreased by 11% (P less than 0.05) and 16% (P less than 0.01) relative to the control after 10- and 20-min exposures to 35g, respectively. The phosphorylation of proteins which were immunoprecipitated by antibodies recognizing microtubule-associated proteins (ipMAPs) was also apparent after exposure of these cells to hypergravity. In the detergent-insoluble fraction, phosphorylation of a 115-kDa protein was significantly enhanced compared to the control after a 5-min exposure to 35g. In the detergent-soluble fraction, phosphorylation of a 200-kDa protein was observed served after a 20-min exposure to 35g. Our study suggests that IP3 and cAMP may act as second messengers in hypergravity signal transduction. Phosphorylation of ipMAPs in both the detergent-soluble and -insoluble fractions suggests that cytoskeletal structures may be influenced by gravity.

Indomethacin reverses interleukin-1-induced hyperinsulinemia in conscious and freely moving rats.

With the aim of studying the mechanism by which peripherally injected interleukin-1 increases the serum levels of insulin in conscious rats, the effects of indomethacin, a cyclooxygenase inhibitor, on interleukin-1-induced hyperinsulinemia were examined using male Wistar rats implanted with intra-atrial cannulas. The i.v. injection of interleukin-1 (1 microgram/rat) into conscious rats resulted in a significant increase in serum insulin levels with the peak response being observed 10 min after injection. It was also found that this insulin response to interleukin-1 was dose-related. Pretreatment with indomethacin (3 mg/kg body weight), however, completely blocked the interleukin-1-induced stimulation of insulin secretion. These findings suggest that i.v. injected interleukin-1 stimulates insulin release from the endocrine pancreas by a mechanism involving the prostaglandin system.