The effects of simulated microgravity on skeletal muscle of japanese quail: transmission electron microscopic study

The aim of the present study was to investigate the effects of simulated microgravity (hypodynamia) on the structure of the skeletal muscle (m. gastrocnemius) in developing Japanese quail by transmission electron microscopy. Samples of muscle tissues from experimental (n = 28) and control (n = 28) birds were collected at day 7, 14, 28, 42 and 56 of age. The structure of m. gastrocnenmius was changed depending on hypodynamia length. The first extensive structural changes were found on day 14 of age. The mitochondria were enlarged and the spaces between the myofibrils were slightly extended compared to control. The sarcomeres were irregular and lipid droplets occurred in the sarcoplasm. Further developmental changes occurred on day 28 of age. Mitochondria fused into the giant mitochondria which frequently exceeded the length of one sarcomere. Moreover, at 42 days of age, beside the above mentioned changes, sarcoplasmic reticulum was dilated and the number of mitochondrial cristae was reduced. However, the structure of m. gastrocnemius on day 56 was less damaged compared to the damage observed on day 42 of age. Presented results indicate that the continuous stay of male Japanese quail under simulated microgravity has a negative impact on the structure of m. gastrocnemius, but also the ability of muscle tissue to cope with these specific conditions. Hypodynamia, m. gastrocnemius, mitochondria, sarcoplasmic reticulum Simulated and real microgravity affect considerably the skeletal muscles and bones. With regard to the considerable mass of skeletal muscles, even relatively slight damage inducing changes to the permeability of cellular membranes results in marked physiological and biochemical changes. Muscle atrophy is an accompanying feature related to a long-term stay of an organism under conditions of weightlessness (Riley et al. 2005; Mazzatti et al. 2008; Tesch et al. 2008). Animal muscles show distinct signs of atrophy, and in the final stage, destruction of muscle proteins inducing decline in muscle volume can be observed (Lebedeva et al. 1998; Litvinova et al. 2007). While during the first stage of exposure to simulated weightlessness, the ion transport through sarcolemma in the rat hind limb is impaired, during the later stages an increased content of proteolytic myofibrilsdamaging enzymes can be observed (Reznicka et al. 1995). Changes could be seen even in the capillary network of immobilized muscles. Vascular endothelium is very thin and discontinuous nuclei exhibit an irregular shape with condensed chromatin (Oki et al. 1995). The density of capillary network is reduced and capillary lumina diameter is also smaller (Fujino et al. 2005). In future long-term space missions, Japanese quail is a suitable animal model representing higher heterotrophic link of the proposed autonomous closed ecosystem at the spaceship or space stations. Low body weight, high productive and reproductive abilities, short individual development as well as a high ability to cope with crowded conditions aboard orbital stations and planetary bases are some of the many reasons for this choice (Boďa 1993). The first space experiments showed that microgravity does not have a dramatic ACTA VET. BRNO 2011, 80: 119–124; doi:10.2754/avb201180010119 Address for correspondence: MVDr. Katarína Holovská, PhD. Department of Anatomy, Histology and Physiology University of Veterinary Medicine and Pharmacy Komenského 73, 041 81 Košice, Slovak Republic Phone: +421 55 6429587 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm impact on embryogenesis of Japanese quail (Guryeva et al. 1993). Nevertheless, the question how microgravity affects further development after hatching remains still open. Ground-based experiments with simulated microgravity can contribute to answering this question. The aim of the present study was to investigate the effects of simulated microgravity on the structure of skeletal muscle (m. gastrocnemius) in Japanese quail males from day 2 post-hatch to 56 days of age. Materials and Methods Hypodynamia is a method to simulate weightlessness in the laboratory conditions on the Earth. The experiment was carried out at the Institute of Animal Biochemistry and Genetics of the Slovak Academy of Sciences in Ivanka pri Dunaji. Fifty-six newly hatched Japanese quail males were used in the present study. On the second day after hatching, 28 chicks of the experimental group were exposed to hypodynamia as described by Škrobánek et al. (2004). At the same time 28 chicks (control group) were placed in a rearing box. Birds from both experimental and control groups were kept under these conditions until day 56 of age in a windowless poultry room with controlled ventilation and electrical heating by infrared lamps. The temperature was adjusted to 35-36 °C for the first few days after hatching and gradually declined to 20 °C at 4 weeks and remained at this level until the end of the experiment. A commercial started mash HYD-13 and water were available ad libitum. The diet was granular and contained 260 g·kg-1 protein and 11.5 MJ metabolisable energy·kg-1. The lighting in the rearing room was continuous. The care and use of animals were in accordance with laws and regulations of the Slovak Republic and approved by the Ethics Committee of the Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji and the State Veterinary and Food Agency (ŠVPS SR Č. K. Ro-7879/04-220/3). Four randomly selected birds of each group were euthanized by cervical dislocation on day 7, 14, 28, 42 and 56 of age. Tissue samples of m. gastrocnemius were collected by small excision. Thereafter, the samples were processed for transmission electron microscopy (TEM). Samples of m. gastrocnemius were fixed by immersion in 3% glutaraldehyde in cacodylate buffer, pH 7.2, and post-fixed in 1% OsO4, dehydrated in acetone and embedded in the Durcupan. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Photographs were taken using the TESLA BS 500 electron microscope.