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Acta Astronautica 54 (2004) 737–747www.elsevier.com/locate/actaastro

Electrophysiological, histochemical, and hormonal adaptationof rat muscle after prolonged hindlimb suspensionChrysoula Kourtidou-Papadelia ;∗, Antonios Kyparosa, Maria Albania,

Athanasios Frossinisa, Christos L. Papadelisb, Panagiotis Bamidisb;c, Ana Vivasc,Olympia Guiba-Tziampiria

aAristotle University of Thessaloniki, School of Medicine, Laboratory of Experimental Physiology, University Campus,54006 Thessaloniki, Greece

bAristotle University of Thessaloniki, School of Medicine, Laboratory of Medical Informatics, University Campus,54006 Thessaloniki, Greece

cCITY Liberal Studies, A(liated Institution of the University of She(eld, 13 Tsimiski Str., 54624 Thessaloniki, Greece

Received 14 May 2002; received in revised form 11 September 2003; accepted 2 October 2003

Abstract

The perspective of long-duration 5ights for future exploration, imply more research in the 7eld of human adaptation.Previous studies in rat muscles hindlimb suspension (HLS), indicated muscle atrophy and a change of 7bre composition fromslow-to-fast twitch types. However, the contractile responses to long-term unloading is still unclear. Fifteen adult Wistarrats were studied in 45 and 70 days of muscle unweighting and soleus (SOL) muscle as well as extensor digitorum longus(EDL) were prepared for electrophysiological recordings (single, twitch, tetanic contraction and fatigue) and histochemicalstainings. The loss of muscle mass observed was greater in the soleus muscle. The analysis of electrophysiological propertiesof both EDL and SOL showed signi7cant main e;ects of group, of number of unweighting days and fatigue properties. Singlecontraction for soleus muscle remained unchanged but there was statistically signi7cant di;erence for tetanic contraction andfatigue. Fatigue index showed a decrease for the control rats, but increase for the HLS rats. According to the histochemical7ndings there was a shift from oxidative to glycolytic metabolism during HLS. The data suggested that muscles atrophied,but they presented an adaptation pattern, while their endurance in fatigue was decreased.c© 2003 Elsevier Ltd. All rights reserved.

1. Introduction

The unique environment of microgravity causesmany physiological changes in the human body, whichis used to live and function in 1 g environments. The

∗ Corresponding author. Erythreas 2, Kalamaria 55132 Thessa-loniki, Greece. Tel./fax: +30-2310-435-331.

E-mail addresses: papadeli@koz.forthnet.gr, cpapad@med.auth.gr (C. Kourtidou-Papadeli).

perspective of long-duration 5ights for future explo-ration, imply that characteristics and origin of mus-cle modi7cations, especially muscle atrophy shouldbe well understood, so that countermeasures can bedeveloped for long-term space 5ight. Muscle atrophyand decalci7cation of bone are characteristics of pro-longed limited muscular function and immobilization[1–4].

It has been shown that in rats subjected to �g con-ditions, there is a transformation of slow muscle 7bre

0094-5765/$ - see front matter c© 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.actaastro.2003.10.001

738 C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747

(type I) expression to fast muscle 7bre (type II) ex-pression. Most of the changes occur in muscles thatare important to posture and have a high proportion of7bres slow myosin as soleus and gastrocnemius mus-cle [5–7]. Animal studies have shown that chronicperiods of unloading result in a shift from oxidative toglycolytic metabolic pro7le [8–10], transition of 7bretypes from slow to fast [11–14], increase in shorteningvelocity [15–17], decrease in force-generation capac-ity [15,17,18], and alteration in excitation–contractioncoupling [19–24], such as increased rate of Ca2+ up-take and leakage to and from sarcoplasmic reticulum[24], in hindlimb muscles, as for the soleus, tibialisanterior, and vastus lateralis. However, the durationof muscle unloading period was relatively short inmost of these studies. Further evidence shows thatin postural muscles of rats, there is also an increase inthe number of muscle 7bres expressing both types Iand II isoforms [5,7]. It has been shown that as shortas 14 days, there may be no pure type I 7bres, whichcould represent a stepwise transformation process [7].The study of Zhou et al. on 7ve shuttle astronautshas shown evidence that this transformation alsooccurs in humans. Most of the information on un-loaded muscle function in humans has been obtainedfrom ground-based simulation of weightlessness[25–27].

There are a few reports regarding the e;ects ofdecreased activity on the contractile properties ofhuman soleus muscle 7bres [11,28,22,29–31]. Forexample, an increase in unloaded shortening velocity(V0) and/or a decrease in force production associatedwith 7bre atrophy was observed after 17 days of bedrest [29,30]. Goto et al. [26] examined the e;ects of2 and 4 months bed rest on the contractile propertiesof slow 7bres in the human soleus muscle and theyobserved a reduction of 7bre size, force generationcapacity, and Ca2+ sensitivity, as well as enhance-ment of shortening velocity in slow 7bres of thesoleus muscle. However, the contractile responses tolong-term unloading are still unclear.

We used animal model to study the e;ects of sim-ulated microgravity on muscle atrophy. Unweightingthe hind limbs by tail suspension of adult rats, espe-cially long-term unloading leads not only to atrophy ofthe soleus muscle, but also to a shift in the metabolicand mechanical properties of the soleus muscle, a pre-dominately slow postural muscle, towards those ob-

served in faster muscles. Previous studies [32–34]have also reported that hindlimb suspension (HLS)leads to signi7cant alterations in muscle contractileproperties and 7bre type distribution.

Clinical laboratory data from blood samples in hu-mans revealed signi7cant biochemical and hormonalchanges after exposure to microgravity. More speci7-cally serum calcium was not elevated after short-termspace 5ights [35], a 3-week rat tail suspension study[36] did not induce testosterone changes, but consid-eration was given to the hormonal e;ects after longerperiods of time. Vasques et al. [37] showed a decreasein plasma testosterone and thyroxin levels of ratsfollowing exposure to HLS compared to respectivecontrols.

The objective of this study was to assess rat muscleadaptability and fatigability, as well as plasma chem-istry for longer periods of HLS and determine thisprocess from the histochemical, hormonal and elec-trophysiological point of view.

2. Materials and methods

After approval was received from the UniversityAnimal care Committee, 7fteen adults Wistar ratsweighting between 275 and 300 g were obtainedfrom the University rat pool and assigned each oneto a cage located in the University operated centralanimal care facility. They were divided into twogroups, the experimental group, which underwentHLS of 45 and 70 days and the control group withno HLS with the same characteristics of body weight,age and sex, as the experimental group. The roomwas maintained between 22 and 24◦C, with 12 h oflight (6:00 a:m to 6:00 p:m:) and darkness, respec-tively. The animals were fed daily and after theirarrival, they were weighted daily and inspected twicea day by one or more of the researchers and oncedaily by the Veterinarian of the Department. Thetail suspension method used to simulate microgravityconditions was a modi7cation of the one developedby Overton–Timpton–Stump [35] for longer durationsuspension. The animal was suspended in a 45◦ headdown position with the hind legs being non-weightbearing. Since forelimbs were allowed contact withthe 5oor, the animals were able to move around toobtain food and water. The animal was able to use its

C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747 739

forelimbs to manoeuvre, within an 180◦ arc, to obtainfood and water and to permit limited grooming of theforequarters.

The animals from both groups (control and HLS)were anaesthetized with 1 ml=100 g body weightaqueous solution of 4.5% chloral hydrate and pre-pared for tension recording of the soleus (SOL) andthe extensor digitorum longus (EDL) muscle of bothlegs. The preparation procedure for both musclesconsisted of dissecting muscles free from surround-ing tissue in order to protect nerve and blood vessels.The distal tendons were dissected free and attachedto strain gauges, and the exposed parts of the muscleswere kept moist with warm oxygenated Krebs salinesolution. The preparation for isometric tension record-ing consisted of hindlimb immobilization, contactingstimulating bipolar electrode to the sciatic nerve, con-necting muscle to the tip of the isometric force trans-ducer, aligning the longitudinal axis of the transducerand begin irrigation of the muscle preparation with ratringer solution, allowing 10 min for thermal equilibra-tion. The tendon of the EDL muscle was connected toa strain gauge and the tension elicited by nerve stimu-lation was displayed on the screen of an oscilloscope.Muscle length was adjusted the way to produce maxi-mal twitch tensions. Whole muscle tension was mon-itored frequently and the force of tetanic contractions,elicited by stimulating the sciatic nerve at 40, 80, and100 Hz was recorded before the isolation of the motorunits.

Immediately after tension recording, the muscleswere weighted and freezed at −15◦C to −25◦C. Eachpair of muscles was subsequently handled as a singleblock of tissue. A 10 �m thick transverse cryostatsections from the middle third of each muscle werecut. Serial sections of the muscle were processedfor succinic dehydrogenase (SDH) by the methodof Nemeth et al. [38] and for myosin ATPase atpH 4.6 [52,51].

3. Results

Muscle weight, single, tetanic contractions and fa-tigue of slow-twitch soleus and fast-twitch EDL mus-cles of the rats were evaluated after 45 days and 70days of HLS.

Table 1Average EDL and SOL muscle weight and standard deviation(within parenthesis) after 45 and 70 days of testing for both theHLS group and the control group

Group SOL muscle EDL muscle

45 days 70 days 45 days 70 days

HLS 61.5 (7.7) 86.5 (0.7) 98.5(2.12) 104.5 (2.12)Controls 93 (7.0) 118.6 (6.0) 116 (10.4) 125 (8.4)

3.1. Muscle weight

There was a statistically signi7cant decrease ofweight for the soleus muscle (p¡ 0:05) and a de-crease for EDL, but not statistically signi7cant.Speci7cally there was a 33.87% decrease of the SOLmuscle and 15.08% decrease of the EDL after 45 daysas well as 6.98% and 9.91% after 70 days of HLSrespectively, compared to controls (see Table 1).

3.2. Electrophysiological data

A low-frequency muscle fatigue protocol (stimu-lation for 5 min) was applied to test for fatigabilityof the EDL (see Table 2) and the SOL muscle (seeTable 3). Tension recording data of the EDL musclewere submitted to a 2 × 2 × 3 analysis of variance(ANOVA) with group (control and HLS) as betweensubject factor and testing phase (45 and 70 days) andfatigue (Fatigue 1, 2, 3, 4, and 5) as within subjectfactors. The results showed signi7cant main e;ectsof group and fatigue, F(1; 8) = 50:95, p¡ 0:001 andF(1; 32) = 312:55, p¡ 0:001 respectively. Post hoccomparisons showed signi7cant di;erences betweenall the conditions, p′s ¡ 0:05.

Also there was a signi7cant interaction betweengroup and fatigue, F(1; 32) = 5:35, p¡ 0:05.

To further analyse the interaction we collapsed thevariable day and conducted independent sample t-testsfor all the fatigue condition. The result showed sig-ni7cant di;erences between the control group and theHLS group for all the fatigue conditions, t(8) = 3:40,p¡ 0:05, t(8)=3:82,p¡ 0:05, t(8)=6:14,p¡ 0:05,t(8)=11:23, p¡ 0:05, t(8)=13:12, p¡ 0:05, respec-tively. No other interaction reached statistical signi7-cance, p¿ 0:05.

740 C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747

Table 2Tension recording data of EDL muscle for both the control and the HLS group as function of fatigue (F0, F1, F 2, F 3, F 4 and F 5)and testing phase (45 and 70 days)

EDL Fatigue 1 Fatigue 2 Fatigue 3 Fatigue 4 Fatigue 5

Day Control HLS Control HLS Control HLS Control HLS Control HLS

45 511.7 573.4 429.5 466.4 367.6 432.3 332.76 418.6 307.83 394.970 513.7 585.3 430.6 509 367.5 464.1 332.69 442.7 309.23 427.5

Table 3Tension recording data of SOL muscle for both the control and the HLS group as function of fatigue (F0, F1, F 2, F 3, F 4 and F 5)and testing phase (45 and 70 days)

SOL Fatigue 1 Fatigue 2 Fatigue 3 Fatigue 4 Fatigue 5

Day Control HLS Control HLS Control HLS Control HLS Control HLS

45 195.0 252.6 219.6 249.1 235.5 263 242.2 266 247.5 267.970 195 214.8 216.2 218.6 237.6 226.6 241.4 232.3 246.2 238

Tension recording data of the soleus muscle weresubmitted to a 2× 2× 3 ANOVA with group (controland HLS) as between subject factor and testing phase(45 and 70 days) and fatigue (fatigue 1, 2, 3, 4, and5) as within subject factors. The results showed maine;ects of day and fatigue, F(1; 8)=35:04, p¡ 0:001,and F(4; 32) = 34:29, p¡ 0:001, respectively. Thefollowing interactions were also signi7cant betweengroup and day, as well as group and fatigue, F(1; 8)=32:23, p¡ 0:001, and F(4; 32) = 7:31, p¡ 0:001,respectively.

In order to analyse the interaction between groupand days of unloading, we collapsed the data for thevariable fatigue. t-Tests showed marginally signi7cantdi;erences between the control and the experimen-tal group only for the day 45 (227.96 vs. 259.664),t(8) = 2:083, p= 0:07. Also, we conducted separatedANOVAs for each group with day as within subjectfactor. The results showed a signi7cant simple e;ectof day only for the HLS group (day 45 = 259:71 andday 70=226:07), F(1; 4)=57:60, p¡ 0:05 (see Figs.1a and 2a). There were no signi7cant di;erences be-tween day 45 (227.97) and day 70 (227.26) for thecontrol group (see Figs. 1b and 2b). In order to anal-yse the interaction between group and fatigue, we col-lapsed the data for the variable day. t-Tests showedsigni7cant di;erences between the control and the

HLS group only for the fatigue 1 condition (194.97 vs.233.69), t(8) = 4:85 p¡ 0:005, no other comparisonsreached statistical signi7cance, p′s ¿ 0:05. Also weconducted two separated ANOVA for each group withfatigue as within subject factor. The analysis showedsigni7cant simple e;ect of fatigue for both groups thecontrol and HLS group, F(4; 16) = 19:38, p¡ 0:001and F(1; 4)=36:24, p¡ 0:001. Post hoc comparisonsfor fatigue in the control group showed signi7cant dif-ferences between fatigue 1 and fatigue 3, 4 and 5,between fatigue 2 and fatigue 3, 4 and 5, and betweenfatigue 3 and 5, no other comparisons di;ered signi7-cantly. Post hoc comparisons for fatigue in the exper-imental group showed signi7cant di;erences betweenfatigue 1 and all the other fatigue conditions, fatigue2 and all the other fatigue conditions, no other com-parisons reached statistical signi7cance.

The fatigue properties of HLS muscles were eval-uated during a 5 min test, using two fatigue indexes,FI2 and FI5, which expressed the relative capacity ofHLS muscles to generate tension after 2 and 5 min,respectively. The fatigue index (Fin − F7n=Fin) × 100in 5 min and 4 Hz=s was measured and revealed a, de-crease for the control group, whereas for the 45-d and70-d for the HLS group was signi7cantly increased(p¡ 0:05). The FI 2 was not di;erent for both EDLand SOL muscle, whereas FI 1 (0–1 × 100) was not

C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747 741

150

200

250

300

F1 F2 F3 F4 F5

Day 45 Day 70

Ten

sion

rec

ordi

ng o

f SO

L m

uscl

e

100

150

200

250

300

FI 1 FI 2 FI 3 F4 F5

Day 45 Day 70

Ten

sion

rec

ordi

ng o

f SO

L m

uscl

e

(a)

(b)

Fig. 1. (a) Muscle fatigue protocol of the SOL muscle of HLSgroup comparing muscle capacity to generate tension after 2–5 minof stimulation time in 45 and 70 days after HLS. (b) Musclefatigue protocol of the SOL muscle of the control group comparingmuscle capacity to generate tension after 2–5 min of stimulationtime in 45 and 70 days with no HLS.

statistically signi7cant for EDL, but it was signi7cantfor SOL muscle.

Isometric twitch force (Pt), time-to-peak tension(TPT), half-relaxation time (1/2 RT), and tetanicforce at stimulation frequencies of 40, 80, and 100 Hzwere recorded. Time-to-peak tension (TPT) andhalf-relaxation time (1/2 RT) seem to be greater inthe HLS group for EDL as well as SOL compared tocontrols. Single twitches for both fast EDL and slowSOL did not reveal any statistically signi7cant di;er-ences compared to controls. Single 2 of soleus muscleincreased in HLS group compared to control groupafter 3’ recover followed by fatigue either expressedin (g) or (g/g muscle) (see Table 4).

Direct tetanic stimulation at a frequency of 40,80 and 100 Hz was exerted on both EDL and SOLmuscle. Only soleus muscle tension was signi7cantly

300

350

400

450

500

550

600

FI 1 FI 2 FI 3 F4 F5

Day 45 Day 70

Ten

sion

rec

ordi

ng o

f E

DL

mus

cle

Ten

sion

rec

ordi

ng o

f E

DL

mus

cle

250

300

350

400

450

500

550

F1 F2 F3 F4 F5

Day 45 Day 70

(a)

(b)

Fig. 2. (a) Muscle fatigue protocol of the EDL muscle of HLSgroup comparing muscle capacity to generate tension after 2–5 minof stimulation time in 45 and 70 days after HLS. (b) Musclefatigue protocol of the EDL muscle of the control group comparingmuscle capacity to generate tension after 2–5 min of stimulationtime in 45 and 70 days with no HLS.

reduced after 70 days HLS (p¡ 0:05), and reducedbut not reached signi7cance after 45 days. For EDLmuscle a reduction of the muscle tension was presentonly after 70 days, but it was not statistically signi7-cant (see Table 2).

3.3. Histological and histochemical evaluation

For the histological evaluation eosin–hematoxylinstaining procedure was used (see Fig. 3).

For the histochemical evaluation myosin ATPase(acid and alkaline) as well as succinate dehydroge-nase (SDH) were used. The measurements were basedon the acid-preincubated mATPase reaction by whichthe IIa 7bres were clearly distinguished from transi-tional and type I 7bres. In alkaline preincubation typeII 7bres were dark coloured and type I had lightercolour, whereas in acidic preincubation it was the op-posite. Both type I and IIa were more atrophied after70-d HLS. There was a relative increase in type II 7-bres from 20% to 30% suggesting muscle 7bre type

742 C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747

Table 4Recordings of isometric twitch force (Pt), time-to-peak tension (TPT), half-relaxation time (1=2 RT), fatigue index and tetanic force ofEDL and SOL muscle in the control group as well as in the experimental group after 45 and 70 days of HLS

Muscle and Single TTP 1=2 RT Sciatic nerve Sciatic nerve Sciatic nerve Single 2 TTP 1=2 RT Fatiguedays of (g/g mus) Single Single stimulation at stimulation at stimulation at (g/g mus) Single2 Single2 Index 1-5study 40 Hz Tet 80 Hz Tet 100 Hz Tet

(g/g mus) (g/g mus) (g/g mus)

EDL control 515.7 26.7 18.3 1307 1865 1971 450 20 18 39.7EDL 45 618.9 30 21 1502 1808 1867 653 28 22 30.9EDL 70 579.5 29 23 1262 1674 1800 549 25 29 26.8SOL control 255.6 58 57.3 1120 1304 1339 306 57 55 −25.9SOL 45 301.4 62 92.5 905 1084 1139 346 65 84 −6.1SOL 70 276.4 58 58 836 982 1023 322 60 67 −10.8

Fig. 3. Micrographs from transverse sections of rat soleus muscle: (a) normal appearance (control muscle), (b) central nucleation-myopathicappearance (suspended muscle). Eosin–hematoxylin (calibration: 50 �m).

transformation from type I to II (see Table 5). Trans-verse sections of soleus muscle (see Fig. 3) and EDL(see Fig. 4) were photographed.

SDH participates in the Krebs cycle and its histo-chemical detection is a useful index of the activity ofthis cycle. The percentage of dark blew 7bres, fatigueresistant was 59.5%, and the percentage of lightercoloured 7bres (less fatigue resistant) was 40.5%.

3.4. Hematological evaluation

From plasma chemistry and hormones when con-trol rats were compared to 45-d and 70-d of HLS rats,

all plasma constituents except for calcium levels, werestatistically lower in rats after 45-d HLS and all weresigni7cantly lower in rats after 70-d HLS. When wecompared 45-d to 70-d HLS although T4, Testosteroneand Ca++ continued decreasing, total protein and al-bumin remained unchanged (see Table 6).

4. Discussion

Prolonged HLS led to a marked decline in mus-cle mass. Consistent with previous studies, our resultswere comparable with those reported in other mod-els of muscle atrophy. Such studies as analysed af-ter 1 week (suspension hypokinesia by 37%) [39],

C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747 743

Table 5Fibre distribution of EDL and SOL muscle and standard deviation (within parenthesis) of both groups after 45 and 70 days of HLS

Muscle Fibre type Control % (SD) 45-d HLS % (SD) 70-d HLS % (SD)

EDL Type I 8.2 (2.0) 10 (1.9) 12.5 (3.0)Type II 93.4 (3.2) 90 (4.1) 87.5 (4.5)

SOL Type I 78.2 (3.5) 71.4 (5.1) 57 (5.2)Type II 21.8 (1.2) 28.6 (2.5) 43 (4.1)

(a) (b)

Fig. 4. Micrographs of transverse sections of rat EDL muscle after prolonged hindlimb suspension. (a) Atrophic 7bres (arrows) SDH. (b)Predominance of dark (type I) 7bres. Myosin-ATPase, acid preincubation (calibration: 50 �m).

immobilization in the shortened position by 27% [40],nerve crush by 30% [41], after 10 days (suspensionhypokinesia by 26%) [42], or after 14 days (suspen-sion hypokinesia by 33%) [43]. Hindlimb unload-ing caused a progressive decrease in soleus musclemass as a result of muscle 7bre atrophy. The degreeof atrophy was di;erent among the muscles, e.g. thesoleus being the most atrophic and the EDL the leastatrophic. This di;erential response among muscles toadaptive perturbations has been a common 7nding inmany studies [44] and is similar to the responses toHLS [45]. In rats, antigravity slow muscles atrophymore than fast-twitch muscles [46,47].

The weight loss can mainly be explained by themarked reduction in mean 7bre area of both types Iand IIa 7bres and partly by a decrease in total numberof 7bres. When the suspension was extended to longerperiod of time, 70 days of HLS, we noticed that therewas a lighter loss of muscle weight than in the 7rstdays after HLS, which partially recovered after 70days and reached a plateau. This is indicative of an

adaptive process due to the muscle 7bre alterationstowards a shift of their roles. The slow twitch muscle,soleus (SOL), containing predominantly slow twitch,oxidative 7bres (type I) was atrophied to a greater ex-tend than the EDL containing fast twitch, glycolytic7bres (II). The mATPase procedure stains slowtype I 7bres strongly after acid preincubations andlightly after alkaline preincubation, whereas type IIa7bres exhibit opposite staining characteristics. TypeII b 7bres that, unlike IIa 7bres, exhibit moderatemyosin-ATPase activity after preincubation at pH4.6 are not present in normal soleus muscles [39,48].These glycolytic enzyme data are consistent withother studies [49] in that slow muscles tend to de-velop characteristics of fast muscles. This metabolicadaptation seems to be related to the shift in con-tractile proteins towards “faster” isoforms [50]. SDHand ATPase histochemical staining was applied inthe present study as also successfully used by otherauthors [38,51,52] to show a rough and qualitativeshift of the muscle 7bre enzyme content since we did

744 C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747

Table 6Plasma chemistry and hormones, with standard deviation (within parenthesis) of both groups and comparison of 45-d and 70-d of HLSwith the control group (∗ = statistically signi7cant compared to control with p¡ 0:01, NS = Non-signi7cant di;erence), as well as 45-dto 70-d of HLS

Variable Control (n = 5) 45-d HLS (n = 5) 70-d HLS (n = 5) 45-d/70-dAve (SD) Ave (SD) Ave (SD)

T4 2.8 (0.04) 1.4 (0)∗ 1.12 (0.01)∗ P¡ 0:001Testosterone 91.2 (14,9) 69.8 (0.80)∗ 31.20 (5.44)∗ P¡ 0:001Calcium 5.50 (0.45) 5.20 (0.22) 4.58 (0.13)∗ P¡ 0:001Tot. proteine 6.64(0.33) 4.92 (0.08)∗ 4.88 (0.13)∗ NSAlbumine 3.90 (0.20) 2.71 (0.08)∗ 2.79 (0.13)∗ NS

not have the availability of myosin heavy chain iso-forms and cytophotometric optical density techniquesystem in our laboratory.

Most researchers have attributed the di;erencesbetween slow and fast muscles regarding contrac-tile properties, structure of myosin and musclemetabolism, mainly to di;erent patterns of activity[53–55].

From the contractile properties we revealed a de-cline in the peak force of limb skeletal muscle, whichwas also demonstrated by Fitts et al. [57] from thedata they collected from Skylab and MIR missions.According to Baldwin et al. the decline in peak forcecan be attributed both to muscle atrophy and to a se-lective loss of contractile protein [58]. Single twitchesof SOL increased in HLS group only when expressedin g/g muscle, whereas in “absolute” power (g) therewas no statistically signi7cant di;erence between thetwo groups. Single (g/g) of EDL muscle for the HLSgroup increased compared to the control group. Thiswas due to the fact that the muscle weights of the HLSgroup were smaller than those of the control group.Comparing single (g) there was no statistically sig-ni7cant di;erences between the two groups. Althoughthere is a change in muscle weight (decrease) for theHLS group, the electrophysiological data are similarbetween the two groups (single g).

Tetanic tensions of the slow SOL were found sig-ni7cantly weaker (p¡ 0:05) whereas there was nostatistically signi7cant di;erences for tetanic contrac-tions of EDL muscle in 45 days HLS and 70 days HLS.This reduction could be partly explained by muscleatrophy and 7bre type.

The twitch time to peak (TTP) and (1=2 RT) seemto be greater in the HLS group for EDL as well as

SOL compared to controls, which means that HLSmuscle needs more time to peak tension and to reachhalf relaxation time. Similar behaviour is revealedfor EDL muscle and the tetanic tension (not com-plete) in 40 Hz. In the contrary in 80 and 100 Hz thetetanic tensions of control group are higher when ex-pressed in g and relatively high even when expressedin g/muscle, indicating a possible change in the func-tional properties during the maximum distress (e.g.complete tetanus). Tetanic force for soleus muscle atstimulation frequencies of 40, 80, and 100 Hz eitherexpressed in g or g/g muscle was signi7cantly reducedcompared to controls, indicating a higher in5uence onthe slow muscle 7bres.

It is known that muscles contract faster and becomemore susceptible to fatigue when subjected to micro-gravity conditions [5,6,56]. The fatigue index (FI) 1(0–1 · 100) was not statistically signi7cant for EDL,but it was signi7cant for SOL muscle. That was at-tributed to the fact that the SOL muscle of HLS groupin 1′ (250 contractions, 4 Hz=s) was more fatiguedthan the control group, indicating that slow twitch 7-bres were more in5uenced by weight unloading. Theincrease in fatigue index for the HLS group representsconsequently decrease in muscle performance. Thoseresults are in accordance with the histochemical 7nd-ings of increased glycolytic muscle 7bres of soleusmuscle, whereas EDL muscle 7bres were not changed.

Thyroxin levels were lower in the HLS rats com-pared to controls, indicating that thyroid function wasinhibited in simulated microgravity, but the reasonfor decreased thyroid function is still unclear. Thedecreased plasma testosterone levels of rats followingexposure to HLS may have contributed to the mus-cle wasting. Total protein levels and albumin levels

C. Kourtidou-Papadeli et al. / Acta Astronautica 54 (2004) 737–747 745

signi7cantly decreased after 45 days and especially af-ter 70 days of HLS compared to controls (p¡ 0:05).The present 7nding suggest further that the mag-nitude of enzymatic and cell volume changes thatoccur in response to muscle unloading depend onseveral factors, as the muscle, its 7bre-type composi-tion, and the initial size of those 7bres. It appears thatto associate physiological relevance to the observedenzymatic changes, cell volume also should be con-sidered. Although the triggering mechanism of thoserapid changes after HLS is not well clari7ed someresearchers suggest that the reduction in mechanicalloading of muscle may be more important than thetotal amount of activation over a 24-h period [59].

The data suggested that although muscles atro-phied after long-term-simulated microgravity, theypresented an adaptive process, while their endurancein fatigue was decreased.

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