冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响 精灵论文.docx

1、冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响 精灵论文冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响 精灵论文Hibernation effects on muscle morphological characteristics and fiber type composition in dauria ground squirrels (Spermophilus dauricus) 12111GAO Yunfang, WANG Jun, WANG Huiping, FENG Ban, DANG Kai,

2、13WANG Qi, Helmut G. Hinghofer-Szalkay (1. Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education, Xian 710069, China; 2. Department of Obstetrics and Gynecology, Tangdu Hospital, Forth Military Medical University, Xian 710038, China; 3. I

3、nstitute of Adaptive and Spaceflight Physiology, Wormgasse 9, A-8010 Graz, Austria) Abstract: Aim: We studied effects of hibernation on extensor digitorum longus (EDL) weight, fiber cross-sectional area (CSA), fiber type distribution, and myosin ATPase activity in dauria ground squirrels (Spermophil

4、us dauricus). Methods: Muscle mass, fiber CSA (video analysis), fiber type, and myosin ATPase activity (Ca2+-ATPase) were measured before and after 1 and 2 months of hibernation, and 2-4 days after arousal in different groups of ground squirrels. Results: Compared with pre-hibernation, body weight d

5、ecreased by about 10% per hibernation month and muscle wet weight was not altered. However, muscle-to-body weight ratio increased significantly. Both type I and type II fiber CSA increased by about 8% during hibernation, with no obvious influence of hibernation duration. Hibernation caused a slight

6、but significant decrease in both ATPase activity and the percentage of type II fibers. 2-4 days after arousal, both fiber CSA and fiber type distribution returned to pre-hibernation levels. No type II fiber sub-type can be identified in dauria ground squirrel EDL samples. Conclusion: We report first

7、 data on EDL fiber type distribution and hibernation effects in dauria ground squirrels. EDL did not show any sign of atrophy over the 3-month winter dormancy. These results suggest that a potential mechanism exists in hibernators that would allow them to prevent muscle atrophy for the prolonged dis

8、use. Key words: Dauria ground squirrels; extensor digitorum longus; myosin ATPase; hibernation 0 Introduction Decreased skeletal muscle activation as with inactivity, bed rest, or spaceflight leads to disuse atrophy. Astronaut muscular performance diminishes both in-and postflight, and rehabilitatio

9、n from long time bed rest is delayed in patients. In non-hibernating animals, this atrophy is associated with reduced cross-sectional muscle area (CSA) and decreased oxidative capacity. A shift from slow- to fast-twitch fiber is seen after ?7 days disuse in antigravity muscles that are rich in slow

10、twitch fibers, and after ?2 weeks disuse in muscles with a high percentage of fast twitch fiber (1, 7, 12, 23). If such changes occurred in hibernating animals, both locomotor and thermogenic function would be compromised. This would be dangerous or even deadly for hibernating animals because the re

11、covery would be slowly despite the quick demand as an emergency appeared. (3, 6). As animals being hibernating, its skeletal muscle is undoubted in a long time disused status. Now, available evidence on muscle physiology in hibernation is limited and partly controversial (2, 9, 16). Some have observ

12、ed atrophy effects associated with hibernation (5, 18, 22), some did not (8, 10, 11, 19). We studied changes of muscle weight, fiber type distribution, cross-sectional area (CSA), and myosin ATPase activity in dauria ground squirrel extensor digitorum longus (EDL) samples after various duration of h

13、ibernation, and compared data from before and during hibernation, and after Foundations: National Nature Science Foundation of China (No. 30770273), Key Laboratory foundation of Resource Biology and Biotechnology in Western China (KH09026), and Science Foundation of Northwest University (No.NH0918).

14、 Brief author introduction:GAO Yunfang(1958-),female,Prof. PhD,physiology. E-mail: gaoyunf arousal. We report an unexpected increase in fiber CSA and believe to be first to present details on these adaptive changes in ground squirrel muscle tissue during hibernation. 1 Procedures and methods 1.1 Ani

15、mals and experimental procedures 32 dauria ground squirrels from the Weinan region, Shaanxi province of China, of either sex were matched for body mass and randomly assigned to four group equally: 1. Pre-hibernation group (Pre-H) 2. After 1 and 2 months of hibernation group, respectively. (H-1mon an

16、d H-2mon) 3. Post-hibernation group (Pos-H, i.e. 2-4 days after arousing from 3 months hibernation.) The animals were raised individually in conventional plastic cages and provided with standard laboratory rat chow and water ad libitum, and were housed in an environment with natural light, following

17、 international guidelines for the care and use of laboratory animals. The Laboratory Animal Care Committee of the P.R. China Ministry of Health approved all procedures. The ground squirrels were brought to a dark, hibernation room (4-6?C) at a time in year when animals usually start to hibernate. 1.

18、2 Sample Preparation Animals were anaesthetized with 90-mg/kg sodium pentobarbital i.p., one extensor digitorum longus (EDL) muscle was removed and weighed to 0.1 mg accuracy (Sartorius, BS210S, Germany). 5 mm samples from the EDL mid-belly were prepared and put into cold 30% sucrose solution, embed

19、ded vertically with an Optimal Cutting Temperature gum (Sakura Finetek USA Inc., Torrance, CA,USA), and 10 m slices cut in a cryostat (Leica, Wetzlar, CM1850, Germany) at -25C. Tissue sections were put on microscope slides, which were pre-treated with Poly-L-Lysine (10 g/L). At the end of the surgic

20、al intervention, the animals were sacrificed by overdose injection of sodium pentobarbital. 1.3 Histochemical Analyses After 5 minutes acid preincubation at pH 4.6 and 30 seconds alkaline preincubation at pH 10.4 (0.02 M sodium barbital, 0.036 M calcium chloride), muscle samples were incubated for 4

21、5 minutes in an ATPase reaction solution (0.02 M sodium barbital, 0.018 M calcium chloride, 0.3% Adenosine-5-triphosphoric acid disodium salt - Genview, Houston, Texas, USA, 0.06% 2,4-dinitrophenol, at pH 9.4 and 37?C). We followed Dubowitz-Brooke fiber classification - slow fiber (type I), fast oxi

22、dative glycolytic (type IIA) and fast glycolytic (type IIB) (4). It is generally assumed that preincubation at pH 4.4 induces dark staining of type I, light or negative staining of type IIB, and intermediate staining of type IIA. Type II fibers in rat EDL can be classified into type IIA and type IIB

23、 fibers with acid pre-incubated m-ATPase staining methods (17, 20). In this study, we made a differentiation into slow fiber (type I) and fast fiber (type II) only, since no type II subtypes can be identified in dauria ground squirrel EDL. However, there was a clear distinction between the dark stai

24、ned Type I and lighter stained Type II fibers. The fiber type distribution is expressed as a ratio between the number of fibers of each type and the total number of fibers. Measurements were made for all fibers in each section. The CSA of no less than 100 fibers of each type was measured with a Quan

25、timet-570 (Leica, Cambridge, UK) video image analyzer and color digital video enhancement. 1.4 Statistical Analysis SPSS 10.0 was used for all statistical analyses. Data are indicated as Mean ? SD. A one-way ANOVA was used to determine overall differences; Fishers LSD post hoc test was used to deter

26、mine group differences. ANOVA-Dunnetts T3 method was used when no homogeneity was detected. Paired-samples T test was applied to test for body weight loss. Significance was assumed with alpha error p3 months), respectively (Table 1). Tab 1 Body weight, EDL muscle wet weight and muscle-to-body weight

27、 ratio before, during and after hibernation (Mean?SD, n=8) EDL MWW at EDL MWW/BW at BW loss BW before BW at experiment Group experiment time experiment time hibernation (g) time (g) (%) (mg) (mg/g) Pre-H 345.38?17.96 345.38?17.96 0.00 132.48?12.61 0.385?0.045 H-1mon 362.50?35.66 314.88?30.10* 13.05?

28、3.31 127.33?11.15 0.406?0.043 #H-2mon 332.75?30.54 253.88?22.29* 23.46?6.13 122.31?13.18 0.483?0.042 #Pos-H 349.38?32.89 219.50?18.99* 36.54?9.40 115.85?14.79 0.529?0.061 Cross-sectional fiber area (CSA) CSA of type I and II fibers in extensor digitorum longus (EDL) samples both increased during hib

29、ernation compared with that of pre-hibernation. Type I fiber CSA increased 11.3% and 9.6% for 1 and 2 months hibernation (0.05p0.1), respectively, and type II fiber CSA 8.9% and 8.4%, respectively. 2-4 days after arousal, type I and II fiber CSA resumed to pre-hibernation levels (Figure 1). 2800 Typ

30、e I Type II 2400 ) 2 ? 2000 1600 1200 800 EDL muscle fibers cross sectional area ( m400 0 Pre-H H-1 mon H-2 mon Post-H Group Figure 1. EDL muscle fiber cross sectional area before, during and after hibernation. CSA = cross sectional area.?0.05p0.1, compared with Pre-H group (ANOVA-LSD); 2.2 Myosin A

31、TPase activity and fiber type distribution Compared with pre-hibernation group, we observed a shift of fiber type from type II to type I during hibernation (Figure 2). Figure 2. EDL muscle fiber myosin ATPase activity before, during and after hibernation I = type I fiber, II = type II fiber. Magnification x 200. Panel A: Pre-hibernation. 2.3 Panel B: During hibernation. Panel C: 2 days after arousal. The percentage of type I fiber increased significantly by 42.49% and 47.28% after 1 and 2 months hibernation, respectively. The percentage of ty