Muscle Fibre Types and Fibre Morphometry in the Tibialis Posterior and Anterior of the Rat: a Comparative Study

FACTA UNIVERSITATIS Series: Medicine and Biology Vol.10, No 1, 2003, pp. 16 - 21 UC 611.018.6:599.323.4 577.15 591.86

MUSCLE FIBRE TYPES AND FIBRE MORPHOMETRY IN THE TIBIALIS POSTERIOR AND ANTERIOR OF THE RAT: A COMPARATIVE STUDY

Desanka Tasić, Dragan Dimov, Jasmina Gligorijević, Ljubinka Veličković, Katarina Katić, Miljan Krstić, Irena Dimov

Institute of Pathology, Faculty of Medicine, University of Niš, Serbia

Summary. Muscle fibre-type composition, fibre cross-sectional area and volume density were investigated in the rat tibialis posterior (TP) and compared to those in the tibialis anterior (TA), using histochemical and stereological methods. The muscle fibres were defined according to their myosin-ATPase (with preincubation at pH 10.4; 9.4; 4.5 and 4.3) activity. Succinate dehydrogenase and α-glycerophosphate dehydrogenase activities were also determined in the fibres. In whole muscle cross-section twenty fields were chosen for measurements (from superficial to deep and lateral to medial regions). The deep regions of TP muscle contained a higher proportion of type IIA and I fibres, the latter being restricted in distribution similar to those previously reported for the TA. The overall fibre-type proportions in the TP were 51.5%, 42.6% and 5.8% types IIA, IIB and I, respectively. The proportion of type I fibres was significantly higher than that in the TA. The estimated volume density values of fibre types were in the order IIB, IIA and I, for both muscles, indicating that the former constituted the main mass volume. This parameter is related to the size (area) and number of a given fibre type. The volume density of type I fibres was significantly higher in TP, while this parameter for type IIB fibres was significantly higher in TA. Differences in estimated parameters were noted also within muscles from various animals. These findings indicate that the rat TP is also regionalised muscle and suggest that sample sizes should be taken into consideration for the validity of quantitative results. Key words: Muscle, fibres, rats, enzymology, morphometry

Introduction sition (11,12). For example, the slow soleus muscle in the adult rats is composed of approximately 80% slow- Locomotory muscles of the rat hindlimb, such as twitch (type I) fibres and 20% fast-twitch (type IIA and tibialis anterior, are composed of three major fibre types small number of undifferentiated fibres – type IIC) fi- designated as type I or slow-twitch oxidative (SO), IIA bres, whereas the fast tibialis anterior muscle of the rat or fast-twitch oxidative glycolytic (FOG) and IIB or possess about 95% fast-twitch (types IIA and IIB) fibres fast-twitch glycolytic (FG) fibres. The proportions of and only 4% to 5% slow-twitch (type I) fibres (2-6). each fibre type vary between individual muscles within It is known that certain human neuromuscular disor- an animal and between homologous muscles of different ders selectively affect various muscles or are associated species (1-7). The proportion of fibre types in a given with pathologic variation in the proportion of fibre types muscle varies in people, as well. The samples of similar (9, 13). It is also known that, for example, cross-inner- muscles from different subjects may show considerable vation experiments have demonstrated changes in the variation in fibre-type composition, a difference that histochemical and other properties of fibre types in a probably reflects the inherent histochemical mutability given muscle under the influence of foreign innervation of muscle fibres and their capacity to adapt their prop- (8). Therefore, the first pre- requisite for quantitative erties to the range of functional loads (8). However, assessement of the extent of these changes is access to many investigators tend to believe that differences in the appropriate data on normal muscle fibre size and fibre- fibre-type composition, which are marked between type proportions in the given muscles. These parameters sprinters and long-distance runners, are apparently ge- as well as the regional distribution of muscle fibre types netically determined and that fibre-type composition in the rat tibialis anterior have been investigated in nu- does not change significantly during the training (9,10). merous studies (2,5,6,14,15). In contrast, little attention Muscle fibre types defined by myosin-ATPase and oxi- has been paid to the rat tibialis posterior muscle, as well dative enzyme histochemistry, such as succinate dehy- as to volume density of fibre types in both of these mus- drogenase, are related to two distinct physiological pa- cles. Therefore, the purpose of the present study was to rameters, speed of contraction and resistance to fatigue determine proportions, sizes (areas) and volume densi- respectively (11). Hence, the contractile properties of a ties of histochemically characterized fibre types in the whole muscle are dependent upon its fibre-type compo- adult rat tibialis posterior (TP) and to compare them MUSCLE FIBRE TYPES AND FIBRE MORPHOMETRY IN THE TIBIALIS POSTERIOR AND ANTERIOR OF THE RAT 17 with the same parameters in the tibialis anterior (TA), and IIB fibre types stained darkly for α-GPDH, which is using stereological methods. indicative of a high concentration of enzymes for gly- colysis, whereas type I fibres were stained weakly (Fig. 4). Material and Methods The tibialis posterior and anterior muscles were ex- cised from seven adult male rats of Wistar strain under ether anaesthesia and prepared for freezing in isopen- tane cooled by liquid nitrogen. Serial cross-sections 10µm thick were cut in a cryostat at -20oC and stained for demonstration the activity of following enzymes: succinate dehydrogenase (SDH), NADH-tetrazolium reductase (NADH-TR), α-glycerophosphate dehydrogenase (α-GPDH), and myosin-ATPase (mATPase) with preincubation at pH 10.4; 9.4; 4.5 and 4.3 (13,16). The remaining sections were stained with hematoxylin-eosin (HE), van Gieson, periodic acid-Schiff (PAS) for glyco- gen and Sudan black B (for lipids) techniques. Preliminary studies showed a variation in the re- Fig. 1a.Cross-section of the superficial regions of the rat gional fibre-type composition in the TP muscle, similar tibialis posterior muscle stained for NADH- to those in the TA (5). According to this observation, in tetrazolium reductase (NADH-TR, obj.×20). the present study, morphometric parameters were estimated from analysis of 20 fields of whole both muscle cross-section stained for mATPase after preincubation at pH 4.5 (from superficial to deep and lateral to medial regions), using stereological methodology, with test system M42 and objective ×40 (17).The obtained data of proportions of three major fibre types, their cross- sectional areas and volume densities, were used to cal- culate both standard deviations and standard errors. Sta- tistical comparisons were made with Student's t test. The level of significance was set at p<0.05.

Results Histochemical techniques for demonstration of SDH, NADH-TR, α-GPDH and mATPase (pH 10.4; Fig. 1b.Cross-section of the superficial regions of the rat 9.4; 4.5 and 4.3) activites showed that TP of the rat pos- tibialis posterior muscle stained for myosin-ATPase sess three basic fibre types (type I, IIA and IIB), with after preincubation at pH 4.3: mATPase darkly stained × considerable variation in the regional fibre-type compo- fibres of type I are absent (mATPase, obj. 20). sition.The deep regions of the muscle contained a higher proportion of type IIA and type I fibres, whereas the superficial regions contained a higher proportion of type IIB than type IIA fibres (type I fibres were absent) (Fig. 1). The three fibre types observed in the TP were indis- tinguishable from those previously seen in the TA. Type IIA (FOG) fibres possessed the high activity of SDH (and NADH-TR), with prominent subsarcolemmal ac- cumulations of reaction product (Fig. 2). This fibre type exhibited a high mATPase activity (Fig. 3) and it was inactivated by acid preincubation at pH 4.5 (Fig. 3). Type I (SO) fibres generally contained a moderately high oxidative enzyme activity (SDH, NADH-TR) and low activity of mATPase (pH 10.4; 9.4) being acid sta- ble, so it was activated by pretreatment with acid (pH 4.5 and 4.3) (Fig. 2 and 3). Type IIB (FG) fibres were Fig. 2a.Cross-section of the deep regions of the rat tibialis characterized by low activities of SDH and NADH-TR, posterior muscle stained for succinate dehydrogenase high activity of mATPase and by complete inhibition of (SDH, obj.×20). their mATPase after preincubation at pH 4.3. Both IIA 18 D. Tasić, D. Dimov, J. Gligorijević, et al.

Fig. 2b.Three major fibre types (I, IIA and IIB) are seen Fig. 4. Cross-section of the deep regions of the rat tibialis at higher magnification (SDH, obj.×60). posterior muscle stained for α-glycerophosphate dehydrogenase (α-GPDH, obj.×20).

Table 1. The proportions of three major fibre types in the rat tibialis posterior and anterior muscles Fibre Tibialis posterior Tibialis anterior type Mean S. D. S. E. Mean S. D. S. E. I 0.0583 ± 0.0118* 0.0048 0.0444 ± 0.0097 0.0040 IIA 0.5155 ± 0.0320 0.0131 0.5001 ± 0.0299 0.0122 IIB 0.4262 ± 0.0340 0.0139 0.4555 ± 0.0280 0.0114 Values of fibre-type proportions from the superficial to deep and lateral to medial regions of both muscles are pooled for the overall proportions of fibre types. Data are given as overall means ±standard deviation (S.D.) and standard error (S.E.) of means. (a) Significantly higher from tibialis anterior: * p < 0.05 The predominating fibres of type IIA, with high activity of mATPase and oxidative enzymes, showed the lowest cross-sectional areas of all fibre types measured (Table 2). The proportions of type IIA fibres as well as the values for their cross-sectional area were larger in the TP than in TA, but differences were not statistically significant. However, the volume density (Vv) values of these fibres were significantly (p<0.05) higher in the former.

Table 2. Mean fibre cross-sectional areas for three major fibre types in the rat tibialis posterior and anterior muscles Tibialis posterior Tibialis anterior (b) Fibre Mean area S. D. Mean area S. D. N type (µm2) (µm2) Fig. 3. Serial cross-sections of the deep regions of the rat 140 I 1156.62 ± 358.50 1197.54 ± 325.05 tibialis posterior muscle stained for myosin-ATPase. S.E.=30.30 S.E.=27.47 (a) Myosin-ATPase reaction after preincubation at pH 140 IIA 1107.03 ± 303.00 1046.34 ± 288.91 9.4 (obj.×20). (b) Modified mATPase reaction × S.E.=25.61 S.E.=24.42 following preincubation at pH 4.5 (obj. 20). (c) 140 IIB 2305.64 ± 727.40 2400.25 ± 689.06 Modified mATPase reaction following preincubation S.E.=61.48 S.E.=58.24 × at pH 4.3 (obj. 20). Values are quoted together with ± standard deviation (S.D.) and standard error (S.E.) of means The results of morphometric analysis showed that the overall fibre-type proportions in the TP were: 51.55% for The type IIB fibres, the next most frequent type of type IIA, 42.62% for type IIB and only 5.83% for type I fibres, were found to have the highest value for the fibres (Table 1). The proportion of type I fibres in the TA cross-sectional area (Table 2). These fibres also had the was 4.44% (Table 1), indicating the significant prepon- highest value of volume density (Table 3) – this pa- derance of fast-twitch types of fibres in both analyzed rameter strongly depends on the size and number of muscles. given fibre type. There was difference, although not MUSCLE FIBRE TYPES AND FIBRE MORPHOMETRY IN THE TIBIALIS POSTERIOR AND ANTERIOR OF THE RAT 19 significant, in the mean area of type IIB fibres between TP (Table 1) are similar to those in study of Ariano et al the TP and TA, being larger in the latter. This is also the (1), in which FOG fibres constituted 51%, FG 44% and case with relative proportion of these fibres. The vol- SO fibres only 5% of the whole population of them. ume density values for type IIB fibres were, however, The regional variation of fibre types that slow-twitch significantly (p<0.05) higher in the TA in comparison fibres usually lie in deeply and fast-twitch fibres super- with those for their counterpart in the TP. ficially, is a common observation for limb muscles. However, the degree of regionalisation varies for ho- Table 3. Morphometric estimates of volume density (Vv) of mologous muscles of different species (7,15). The re- three major fibre types in the rat tibialis posterior gional variation in fibre-type composition, in which type and anterion muscles I or SO fibres were restricted to the deep regions of Tibialis posterior Tibialis anterior muscle, has been demonstrated for numerous muscles of Fibre type Mean S. D. S. E. Mean S. D. S. E. rat including sternomastoideus, tibialis anterior, exten- I 0.0398 ± 0.0091* 0.00371 0.0302 ± 0.0068 0.00278 sor digitorum longus, biceps brachii (long head), triceps IIA 0.3294 ± 0.0330* 0.01347 0.2854 ± 0.0355 0.01449 brachii (long head), gastrocnemius medialis, peroneus IIB 0.5663 ± 0.0510** 0.02082 0.6220 ± 0.0422 0.01723 longus, flexor digitorum and hallucis longus (2,3,5,6,7, Interstitium 0.0645 ± 0.0120 0.00490 0.0624 ± 0.0099 0.00404 14,15). In this study it was found that the tibialis pos- Vv in mm0 Values are expressed as mean ± standard deviation (S.D.) and terior of the rat is also regionalised muscle. standard error (S.E.) of means The size of muscle fibres, their contractile and other Significantly higher from tibialis anterior: * p < 0.05 properties can be influenced by a variety of factors such Significantly lower from tibialis anterior: ** p < 0.05 as changes in motor neuron activity, alterations in hormone levels or changes in physical activity, what was The type I fibres, which constitute a minor popula- documented in numerous experimental studies (21-26). tion of fibres, had a relatively low values for their cross- Although the molecular pathways by which enviromental sectional areas (Table 2). The volume density values of factors affect gene expression remain largely unknown, these fibres were the lowest in comparison with those of experimental data suggest that myogenin, as well as the types IIA and IIB (Table 3), which is due to a small other basic/helix-loop-helix muscle regulatory factors number of them as well as their low sizes (areas). The (MRFs), referred to as MyoD, Myf-5, and MRF4, may be obtained data of relative proportion and of volume den- involved in both the initial establishment as well as sity for these fibres in the TP were significantly maintenance of fibre-type diversity in the developing (p<0.05) higher than those in the TA. organism (23). Recent evidence have suggested that MRFs and MEF2 (myocyte enhancer factor-2) act within Discussion a regulatory network that established the differentiated phenotype of skeletal muscle (27). The muscle fibres can be categorized as slow- or The determination of size and distribution of fibre fast-twitch based upon several physiological and mo- types in normal muscles, as well as the other morpho- lecular criteria, including contractile protein isoforms metric parameters (i.e. muscle fibre volume density), is and metabolic enzyme activity. Mammalian skeletal pre- requisite in detection of their alterations in various muscle fibres have been routinely categorized into three animal experimental models and human diseases. major types (SO or type I, FOG or type IIA and FG or Hence, morphometric analysis of normal muscles pro- type IIB) on the basis of their histochemical staining vide a quantitative basis for comparison and evaluation properties. The fibre types, identified using myofibrillar of lesions severity induced by the same factors (i.e. actomyosin ATPase histochemistry, correlate with the myotoxin, denervation) in different muscles; it can also distribution of myosin isoforms in the fibres (18, 19). be helpful in establishing the pathogenetic mechanisms The limb and trunk muscles of adult rats express four of arised lesions. myosin heavy chain (MCH) isoforms: one slow (MHCI) Among the parameters proposed as a descriptor of and three fast (MHCIIa, MHCIId or IIx, and MHCIIb) muscle fibre size, fibre cross-sectional area appears to (19,20). Thus, for example, type I fibres express MHCI offer the greatest accuracy in that this measurement is and fibre types IIA, IID or IIX, and IIB express the least susceptible to the effects of oblique sectioning MHCIIa, MHCIId or IIx, and MHCIIb, respectively. or kinking of muscle fibres (9). In the present study, in Histochemically, type IIX fibres resemble to IIB in their both fast muscles examined, the three major fibre types acid-mATPase activity, but have much higher oxidative had mean area values in the order: IIA, I and IIB, the (SDH) activity (18). The fibres which contain two MHC latter being the largest. The type IIB fibres had the isoforms, a population of "hybrid" fibres, also exist in highest values for the cross-sectional area and volume rat skeletal muscle (19). density, indicating that they were most prominent in The tibialis posterior muscle examined was found to mass, although not in number, in both tibialis posterior contain approximately 94% histochemically fast-twitch and anterior muscles. There were significant differences fibres and about 6% slow-twitch fibres, indicating the between the volume densities of these fibres in the TP significant preponderance of the fast-twitch types. Our and TA; differences between the mean areas and data regarding the muscle fibre-type composition in the proportions were, however, not statistically significant. 20 D. Tasić, D. Dimov, J. Gligorijević, et al.

The predominating fibres of type IIA possessed the and was approximately two times higher that one for lowest cross-sectional areas in both rat muscles investi- type IIA. Considerable variation in the proportions and gated. The mean value of diameter for type IIA fibres in sizes of muscle fibre types between the reported esti- the TA was also the smallest of all the fibre types meas- mates (1,2,5,6,14) may be the consequence of the rats ured (5). The volume density of these fibres, however, strain differences, age and sex, the level of sections of was relatively high and was found to be significantly muscle investigated and also of differences between the higher in the TP than in the TA muscle. The type IIA methods of quantitative analysis used by the various fibres constituted 51.55% in TP and 50.01% in TA of authors. Regarding the sizes of fast-twitch fibre types the whole population of muscle fibres. one has to take into consideration the conditions of lim- The values of cross-sectional areas for type I fibres ited movement of experimental animals, which may, and were larger than for type IIA, but much smaller than certainly are different, in various laboratories. those for type IIB fibres. These fibres, which were in minority and had the lowest value of volume density, constituted the minor mass volume in both rat muscles. Conclusion Significant differences in the volume density and pro- In conclusion, based upon histochemically catego- portion of these fibres have been observed among the rized muscle fibres of the tibialis posterior muscle, the TP and TA muscles. considerable variation in this regional fibre-type compo- The estimated data regarding volume density in this sition, was found. Type I fibres were confined to the study showed that the relative volume of muscle fibre deep regions of muscle similar to those previously re- types was 93.55%, whereas the relative volume of ported for the tibialis anterior. These finding indicates interstitium was 6.45% in TP, and 93.76% and 6.24% in that the rat tibialis posterior is also regionalised muscle. TA, respectively. Interstitium represents the interstitial The tibialis posterior have shown to contain overall space and component tissue, including blood vessels 51.55% type IIA, 42.62% type IIB and 5.83% type I and nerve bundles. In the extensive study of Kilarski fibres. The proportion and volume density values of and Sjöström (28), in which they analyzed rat and rabbit type I fibres, being in minority, were significantly diaphragm, among other morphometric parameters, was higher in comparison to those of their counterpart in the estimated the volume density of muscle fibre types, but tibialis anterior. not volume density of interstitium. In an other study The estimated values for the mean cross-sectional (29), the areal density for both muscle fibre types and area were in the order: types IIB, I and IIA fibres, while interstitium in the rat extensor digitorum longus (EDL) the values of volume density were in the order: types was examined. The obtained value for areal density of IIB, IIA and I fibres, indicating that type IIB fibres con- interstitium was low, only 2.1% (29). stituted the main mass volume in both rat analyzed mus- The results of Pullen (2) regarding the mean cross- cles. The volume density of type IIB fibres was signifi- sectional area for type IIB fibres in the TA are higher cantly higher in the tibialis anterior, while this parame- than those found in the present study. In this study the ter for type IIA fibres was significantly higher in the mean cross-sectional area of type IIB fibres was 2 tibialis posterior. Differences in estimated parameters 3391.88µm , which is approximately three times higher were noted also within muscles from various animals. than value for type IIA fibres. The mean cross-sectional area of type IIB fibres in this study was 2400.25 µm2,

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TIPOVI MIŠIĆNIH VLAKANA I MORFOMETRIJA VLAKANA U MUSCULUS TIBIALIS POSTERIORU I MUSCULUS TIBIALIS ANTERIORU PACOVA: KOMPARATIVNA STUDIJA

Desanka Tasić, Dragan Dimov, Jasmina Gligorijević, Ljubinka Veličković, Katarina Katić, Miljan Krstić, Irena Dimov

Institut za patologiju Medicinskog fakulteta u Nišu

Kratak sadržaj: Ispitivana je kompozicija tipova mišićnih vlakana, njihova area (površina poprečno presečenih vlakana) i volumenska gustina u m. tibialis posterioru pacova i komparirana sa parametrima u m. tibialis anterioru, primenom histohemijskih i stereoloških metoda. Mišićna vlakna su tipizirana na bazi aktivnosti njihove miozinske ATPaze sa preinkubacijom pri pH 10,4; 9,4; 4,5; i 4,3. U vlaknima su takođe određivane aktivnosti sukcinat dehidrogenaze (SDH) i α-glicerofosfat dehidrogenaze (α-GPDH). Merenja su vršena na dvadeset polja poprečnog preseka oba mišića (od superficijalne do duboke i od lateralne do medijalne regije mišića). Utvrđeno je da duboke regije m. tibialis posteriora sadrže veću proporciju vlakana tipa IIA i I, pri čemu su poslednja bila ograničena u distribuciji na ove regije, slično nalazima koji su prethodno saopšteni za m. tibialis antrior pacova. Nađeno je da ukupna proporcija tipova vlakana u m. tibialis posterioru iznosi 51,5% za vlakna tipa IIA, 42,6% tipa IIB i 5,8% za vlakna I tipa. Proporcija vlakana I tipa bila ja značajno veća u poređenju sa vrednostima za m. tibialis anterior. Dobijeni rezultati ispitivanja volumenske gustine tipova vlakana pokazuju opadanje vrednosti od vlakana tipa IIB, preko IIA do vlakana I tipa, što ukazuje da vlakna tipa IIB čine glavnu volumensku masu u oba ispitivana mišića. Ovaj parametar je u direktnoj korelaciji sa veličinom (areom) i brojem datog tipa vlakana. Volumenska gustina vlakana I tipa bila je značajno veća u m. tibialis posterioru, dok je volumenska gustina vlakana tipa IIB bila značajno veća u m. tibialis anterioru. Razlike u ispitivanim parametrima su takođe zapažene unutar mišića koji potiču od različitih životinja. Zaključeno je da je m. tibialis posterior pacova takođe regionalizovan mišić i sugerisano da pri proceni validnosti rezultata kvantitativne analize treba uzeti u obzir veličinu uzorka. Ključne reči: Mišić, vlakna, pacovi, enzimohistohemija, morfometrija