Characterization of spinal cord glial cells in a model of hindlimb unloading in mice.

Exposure to microgravity has been shown to result in damaging alterations to skeletal muscle, bones, and inner organs. In this study, we investigated the effects of microgravity by using a hindlimb unloading model (HUM) in mice. The characteristics of the lumbar spinal cords of HUM mice 30 days after hindlimb unloading were examined. Morphometric analysis showed reductions of the total area, gray matter, and white matter by 17%, 20%, and 12%, respectively. Myelinated fibers in the white matter showed prominent myelin destruction. Analysis of the number of glial fibrillary acidic protein (GFAP+)/S100 calcium-binding protein B (S100B-), GFAP+/S100B+, and GFAP-/S100B+ astrocytes in the ventral horn (VH), central channel area (CC), dorsal root entry zone (DREZ), main corticospinal tract (CST), and ventral funiculi (VF) showed that the number of GFAP+/S100B- astrocytes was increased in the DREZ and CST of HUM mice. Additionally, GFAP+/S100B+ cell numbers were significantly decreased in the VH and CST but did not differ in the CC or DREZ of HUM mice, as compared with the control. The numbers of GFAP-/S100B+ cells were significantly reduced only in the VH of HUM mice. Moreover, the number of ionized calcium-binding adaptor molecule 1 (Iba1+) microglia cells was significantly increased in the CC and DREZ of HUM mice. In control mice, homeobox protein HoxB8 (HoxB8+) cells were found only in the CC; in contrast, HoxB8+ cells were observed in all studied areas in HUM mice, with the greatest number found in the CC. Genome-wide transcriptome analysis of the lumbar spinal cords of HUM mice showed decreased expression of genes encoding myelin, extracellular matrix, cytoskeleton, and cell adhesion proteins. Real-time polymerase chain reaction (PCR) confirmed reductions in the expression of mpz, pmp2, pmp22, and prx genes, which are involved in myelination, as well as decreases in the levels of genes encoding extracellular matrix molecules, including glycoproteins (matrix gla protein (MGP), osteoglycin (OGN), microfibrillar associated protein 5 (MFAP), and collagen, type IV, alpha 1 (COL4A)), proteoglycans (perlecan (heparan sulfate proteoglycan) (HSPG)), and metalloproteinases (lysyl oxidase (LOX)). Thus, our results showed that hindlimb unloading caused decreases in gray and white matter areas, changes in gene expression, alterations in myelination, and phenotypic modifications in glial cells in the lumbar spinal cords of mice.

[Influence of modeling of gravitational unloading on the postsynaptic acetylcholine receptor organization and acetylcholinesterase activity in neuromuscular synapses of rat fast and slow muscles].

Using immunofluorescent techniques, we have revealed that, after 35 days of rats hindlimb unloading, neuromuscular synapses of fast and slow muscles show enhanced fluorescence intensity and decreased area of fluorescent staining of acetylcholine receptors; increased fluorescent intensity and area of fluorescent staining for acetylcholinesterase. The ratio of the number of postsynaptic acetylcholine receptors and the amount of acetylcholinesterase changed as well as their spatial position in relation to each other. These rearrangements correspond to electrophysiological data on the reduction of the amplitude of the miniature endplate currents in both muscles. Identified synapses restructuring accompanied by a decrease in the volume of muscle fibers. Hindlimb unloading (simulation of hypogravity) leads to an increase in functional activity of acetylcholinesterase on the background of reduced postsynaptic membrane area occupied by acetylcholine receptors. This leads to a decrease in the amplitude of excitatory postsynaptic potentials thereby reducing the nerve-muscle excitation transmission safety factor.

[The role of spinal motoneurons in the mechanisms of hypogravitational motor syndrome development].

Studies results of gravity unloading influence on spinal control system of muscle structure and functions are summarized. It was shown that demyelization of axons due to reduction of genes expression responsible for myelin proteins synthesis, decrease in one of the key enzymes of cholinergic system--cholineacetyltransferase activity, alteration of normal kinetics of quantal and non-quantal neurotransmitter secretion, impaired autoregulation of acetylcholine secretion from motor nerve endings through presynaptic cholinergic receptors, slowing of axonal transport of substances in motor neurons that innervate postural muscles played the important role in the development of hypogravitational motor symptoms. At the same time, the evidences of neuroprotective mechanisms enclosing (increase in heat shock proteins Hsp25 and Hsp70 expression), that hinder apoptosis development in motor neurons and glial cells in the spinal cord under conditions of model hypogravity, were revealed.

[Myelination disorders in mechanism of hypogravity motor syndrome development].

When modeling effects of hypogravitation by the method of hindlimb unloading in rats the area of cross-section in lumbar part of a spinal cord was found to reduce. The analysis of spinal cord slides showed that these changes are associated with a decrease in the area of white substance of a spinal cord. Data obtained are consistent with our previous observation of a decrease in expression of the genes encoding myelin proteins. Results of our researches give the good reasons to believe that miyelinization failure in CNS is one of the factors that underlie the development of hypogravitational motor syndrome.

[About possible specialization of different compartments of a motoneuron axon for synthesis of specific proteins].

The spontaneous quantum secretion of neurotransmitter and its regulation through the system of presynaptic acetylcholine receptors have been studied on a neuromuscular preparation of rat m. soleus of intact animals and animals in which the axonal transport was blocked via the application of colchicine to the sciatic nerve. It was shown that, after six days of colchicine application, the spontaneous quantum secretion, the reaction of presynaptic membrane, and the reaction of neurosecretory apparatus to the depolarization of nerve endings via increase of the content of potassium ions in the environment and to the activation of presynaptic receptors by carbachol are not disturbed. Keeping in mind a rather short half-life of proteins that take part in the exocytosis and its regulation, it may be concluded that their functioning does not depend on the state of the axonal transport. These data correspond to the hypothesis put forward earlier that the synthesis of some proteins performing their function in nerve terminals occurs directly at the site of their utilization but not in the perikaryon, as it has been traditionally assumed.

[Electrogenic activity of Na-K-ATPase and calcium ions in m. soleus fibers of rats and Mongolian gerbil during simulation of gravitational unloading].

Some of the electrophysiological parameters of m. soleus of rat and Mongolian gerbil, and Ca ions content in fiber myoplasm were compared in different periods of gravitational unloading simulated by tail-suspension. No difference was found between the control animals as for membrane potential at rest, electrogenic activities of Na-K-ATPase and its isoforms, and input resistance of m. soleus fibers. At the same time, unlike rats, gerbils exhibited a substantial Ca decrease in myoplasm. From day one to 14 of gravitational unloading the pace of electrophysiological changes in gerbil's m. soleus was noticeably slower than of rat's, whereas Ca ions depositing in myoplasm was observed in both species already at the beginning ofsuspension. Analysis of the results suggests that adaptive changes in m. soleus of Mongolian gerbil and rat during simulated gravitational unloading are fundamentally different due to, probably, peculiar water-electrolyte metabolism, type of locomotion, and other factors which are still unclear.

[The influence of hindlimb unloading on the effectiveness of modulation of the mediator secretion via the autoreceptor system].

In experiments on neuromuscular synapses of rat fast (m. Extensor digitorum longus, EDL) and slow (m. soleus) skeletal muscles, changes in the intensity of spontaneous quantal mediator secretion in response to the activation of presynaptic cholinoreceptors by the nonhydrolyzable acetylcholine analogue carbachol and to an increase in K+ concentration in the control group of animals and in animals subjected to different terms of unloading of hindlimbs have been compared. The intensity of spontaneous secretion of mediator quanta was evaluated from the mean frequency of miniature endplate potentials. In the control group of animals, the frequency of miniature endplate potentials by the action of carbachol increased by 363% in m. EDL and by 62% in m. soleus. The frequency of miniature endplate potentials in the synapses of m. EDL was more sensitive to K(+)-induced depolarization too. The bearing unloading of hindlimbs abolished the sensitivity of spontaneous secretion to carbachol in the synapses of m. EDL, whereas in m. soleus it was unchanged. However, the preservation of sensitivity of nerve endings of fast muscle to K(+)-induced depolarization allows one to assume that the hindlimb unloading leads to a decrease in the number of functioning presynaptic receptors.