We have previously shown that, during hindlimb unloading in rodents (the model of hypogravity) [6] the reduction of the volume of the lumbar region of the.
ISSN 00124966, Doklady Biological Sciences, 2013, Vol. 452, pp. 266–268. © Pleiades Publishing, Ltd., 2013. Original Russian Text © R.R. Islamov, N.I. Lannik, G.F. Shaimardanova, P.N. Rezvyakov, O.V. Tyapkina, A.A. Rizvanov, Yu.A. Chelyshev, I.B. Kozlovskaya, E.E. Nikolskii, 2013, published in Doklady Akademii Nauk, 2013, Vol. 452, No. 3, pp. 339–341.
PHYSIOLOGY
Effect of Hindlimb Unloading on Myelinated Fibers in the Mouse Lumbar Spinal Cord R. R. Islamova, N. I. Lannikb, G. F. Shaimardanovac, P. N. Rezvyakova, O. V. Tyapkinac, A. A. Rizvanova, b, Yu. A. Chelysheva, Corresponding Member of the RAS I. B. Kozlovskayad, and Academician E. E. Nikolskiia, c Received December 15, 2012
DOI: 10.1134/S0012496613050086
The evolution of living systems occurred under the conditions of earth gravity. Prolonged absence of grav ity disturbs the function of almost all organs and sys tems of living organisms at the molecular, cellular and tissue levels. Skeletal musculature is most sensitive to the state of weightlessness. For example, as soon as the first day under the conditions of microgravity, the dis turbances that underlie the hypogravitational motor syndrome are developed in the skeletal muscles main taining posture [1–4]. In consideration of the fact that motor neurons are directly involved in the control of functional and morphological characteristics of skele tal muscles [5], their key role in the pathogenesis of hypogravitational motor syndrome seems to be quite likely. However, the effects of microgravity on the spinal cord as a whole and the structural and functional char acteristics of motor neurons, glial cells, and elements of microvasculature remain insufficiently explored. We have previously shown that, during hindlimb unloading in rodents (the model of hypogravity) [6] the reduction of the volume of the lumbar region of the spinal cord occurred. Crosssectional analysis of the spinal cord showed that this phenomenon was associ ated with a decrease in the amount of white matter [7]. In addition, a decrease by 21% in the total protein content of the lumbar region of the spinal cord of experimental animals was found [8]. A fullgenome analysis of tissue of the lumbar enlargement of the
a
Kazan State Medical University, ul. Butlerova 49, Kazan, 420012 Tatarstan, Russia b Kazan (Volga Region) Federal University, ul. Kremlevskaya 18, Kazan, 420012 Tatarstan, Russia c Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, ul. Lobachevskogo 2/31, Kazan, 420111 Tatarstan, Russia d Institute of Biomedical Problems, Russian Academy of Sciences, Khoroshovskoe sh.76a, Moscow, 123007 Tatarstan, Russia
mice spinal cord after 30day antiorthostatic suspen sion of the hindlimbs revealed inhibition of the expres sion of the genes encoding proteins of the myelin sheath of the central nervous system (pmp2, pmp22) [9]. In this study performed using electronic and fluo rescence microscopy and realtime polymerase chain reaction, we obtained data indicating that disturbance of the process of myelination of motor nerve fibers play a role in the formation of function disorders of skeletal musculature under the conditions of hypogravity. The study was performed on male c57bL/6 mice weighing 25 ± 3 g (n = 24). All experiments with ani mals were performed in accordance with the recom mendations of the Physiological Section of the Rus sian National Committee on Bioethics [10]. The con sequences of hypogravity were modeled using the method of antiorthostatic suspension of hindlimbs of animals (n = 12) [6]. The control group of mice (n = 12) was housed under the standard vivarium conditions. Thirty days later, the animals of both groups were anesthetized and lumbar regions of the spinal cord, processed for molecular, immunofluorescence, and electron microscopy assays were dissected. Electron microscopy assay. The lumbar regions of spinal cord of animals from control (n = 4) and exper imental (n = 4) groups were fixed in 2.5% glutaralde hyde solution and then in l% osmium tetroxide solution and, after dehydratation, infiltrated with a mixture of Epon and Araldite. Transverse ultrathin sections from segments L4–L5 were examined with an electron microscope (JEM–1200 EX). On the electrondiffraction pattern in the area of the lateral funiculi of the spinal cord, severe destruc tion of myelin is observed, along with preserved fibers (Fig. 1). This destruction is manifested as a significant increase in the electron density of myelin, disturbance of its regular membrane structure, presence of extended regions of the membrane delamination, separation of the myelin sheath from the axial cylinder, and the
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(a)
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Fig. 1. A fragment of the lateral funiculus of mouse spinal cord in the region of lumbar enlargement: (a) intact animals (control); (b) 30 days after the beginning of antiorthostatic suspension (experiment). Magnification 12K; scale bar, 500 nm.
appearance of vacuoles, foci of complete destruction of the myelin sheath, and its unstructured dense frag ments in the vicinity of axons. In the area explored, one can sometimes find relatively large axons that are completely devoid of the myelin sheath. In the over whelming majority of axons except the cases with pro nounced deformation related, apparently, to the viola tion in the structure of sheath; the indicators of destruction, such as opacification and induration of contents, the appearance of vacuoles with compact electrondense matter are also observed. Immunofluorescence study. On the transverse free floating sections of the lumbar region of the spinal cord (20 µm), using immunofluorescence technique, the oligodendrocytes were detected by the specific marker OSP (oligodendrocyte specific protein), also known as claudin11, which is a representative of the family of the same name that is part of myelin of the CNS and regulates proliferation and migration of oli godendrocytes [11]. Polyclonal antibodies against OSP (Abcam) were used at a dilution of 1 : 1000. Rab bit Ig conjugated to fluorochrome Alexa 488 (Invitro gen) at a dilution of 1 : 1000 was used as a secondary antibody. Analysis of transverse sections of the lumbar region of the spinal cord at the level of segments L4– L5 by confocal scanning microscope LSM 510 Meta (Carl Zeiss) revealed differences in the fluorescence intensity in mice from the control (n = 4) and experi mental (n = 4) groups. In the white matter of the spinal cords of mice, 30 days after the beginning of antio rthostatic suspension, the specific fluorescence was significantly weaker than in the control group (Fig. 2). The result obtained indicates a decrease in the expres sion of the protein OSP/claudin11 under the condi tions of antiorthostatic suspension of animals, which is consistent with the data obtained using electron microscopy, which demonstrate destructive changes in oligodendrocytes of the white matter and a reduction in the amount of mRNA of myelinspecific genes in these cells. DOKLADY BIOLOGICAL SCIENCES
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Analysis of the expression of genes encoding proteins of CNS myelin by realtime polymerase chain reaction (RT–PCR). RNA was extracted from the spinal cords of mice of the experimental (n = 4) and the control (n = 4) groups using an RNeasy Kit in accordance with the manufacturer’s instructions (Qiagen). The cDNA synthesis was performed using sixnucleotide random hexamer primers (Random 6) (Synthol), dNTP, buffers and highprecision reverse transcriptase Revertaid (Fermentas). Quantitative analysis of the mRNA expression level was performed using an iCy cler iQ RealTime PCR Detection System (BioRad). Each reaction involved a Universal PCR Master Mix forward and reverse primers specific to the genes of myelin proteins (pmp2, pmp22) (Syntol), a fluorescent probe TagMan (Synthol), and cDNA. The amount of RNA was normalized to the 18S gene. Statistical anal ysis of the results was carried out using Student’s t test in the Microsoft Excel 2007 software. Comparative analysis of the pmp2 and pmp22 gene expression in the lumbar region of the spinal cords of mice revealed significant differences in the amount of mRNA between the animals of the experimental and control groups. In experimental mice, the content of mRNA genes, pmp2 and pmp22, reduced, respectively, by 96.5% and 86.5% (p < 0.05). Data on the expression level of myelinspecific genes are consistent with the results of fullgenome analysis performed by us earlier [9]. However, the difference in the level of pmp2 and pmp22 gene expression revealed by RT–PCR was more pronounced as compared with the data obtained using a microarray. It is known that the protein PMP22 is involved in the initial stages of myelin formation, as well as in maintaining of its stable state. The changes in its expression lead to disturbance of myelination of nerve fibers [12]. In turn, the protein PMP2 is required for the stabilization of the myelin sheath [13, 14], and claudin11 is essential in the organization of the radial component of myelin in the CNS [15].
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Fig. 2. The weakening of the intensity of immunofluorescence in the reaction with the antibodies against OSP on the transverse sections of the lumbar region of the mouse spinal cord 30 days after the beginning of antiorthostatic suspension (on the right). An intact animal (on the left).
Thus, the consistency of our data on changes in the lumbar region of the spinal cord of mice after 30 days of antiorthostatic suspension (reduction of the area occupied by the white matter, the amount of total pro tein, the content of mRNA genes of the proteins of the myelin sheath, the level of immunoexpression of oli godendrocyte cell marker OSP, the data obtained using electron microscopy) give us reason to believe that the disturbance of myelination in the spinal cord may be important for the pathogenesis of hypogravita tional motor syndrome. ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (project nos. 130400310a (R.R. Islamov) and 120400294a (Yu.A. Chely shev)), the grant of the President of the Russian Fed eration no. NS2669.2012.7 (E.E. Nikolskii), and Program of Presidium RAS no. 7 (E.E. Nikolskii). REFERENCES 1. Grigor’ev, A.I., Kozlovskaya, I.B., and Shenkman, B.S., in Ross. Fiziol. Zh. im. I.M. Sechenova, 2004, vol. 90, no. 5, pp. 508–521. 2. Fitts, R.H., Trappe, S.W., Costill, D.L., et al., J. Phys iol., 2010, vol. 88, no. 18, pp. 3567–3592.
3. Caiozzo, V.J., Haddad, F., Baker, M.J., et al., J. Appl. Physiol., 1996, vol. 81, no. 1, pp. 123–132. 4. Ohira, Y., Nomura, T., Kawano, F., et al., J. Gravit. Physiol., 2002, vol. 9, no. 2, pp. 49–59. 5. Rezvyakov, N.P. and Nikol’skii, E.E., Fiziol. Zh. SSSR im. I.M. Sechenova, 1978, vol. 64, no. 8, pp. 1117– 1123. 6. MoreyHolton, E.R. and Globus, R.K., J. Appl. Phys iol., 2002, vol. 92, pp. 1367–1377. 7. Tyapkina, O.V., Nurullin, L.F., Rezvyakov, P.N., et al., Biofizika, 2012, vol. 57, no. 5, pp. 876–879. 8. Islamov, R.R., Mishagina, E.A., Tyapkina, O.V., et al., Acta Astronaut., 2011, vol. 68, pp. 1469–1477. 9. Islamov, R.R., Rizvanov, A.A., Tyapkina, O.V., et al., Dokl. Biol. Sci., 2011, vol. 439, no. 3, pp. 197–200. 10. Genin, A.M., Il’in, A.E., Kaplanskii, A.S., et al., Avia kosm. Ekol. Med., 2001, vol. 35, no. 4, pp. 14–20. 11. De Gasperi, R., Gama Sosa, M.A., Naumowicz, Z., et al., Transl. Neurosci., 2010, vol. 1, no. 4, pp. 282– 285. 12. Snipes, G.J. and Suter, U., G. J. Anat., 1995, vol. 186, pp. 483–494. 13. Majava, V., Polverini, E., Mazzini, A., et al., PLoS ONE, 2010, vol. 5, no. 4, pp. 1–10. 14. Marchesi, C., Milani, M., Morbin, M., et al., Neurol ogy, 2010, vol. 75, pp. 1830–1838. 15. Gow, A., Southwood, C.M., and Li, J.S., et al., Cell, 1999, vol. 99, no. 6, pp. 649–659.
Translated by V. Kovalyova
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