Upsala J Med Sci 81: 175-178, 1976 Dimensions of the Rabbit Tenuissimus Muscle C. MICHAEL CHILDS' and KARL-E. ARFORS From the Department of Experimental Medicine, Pharmacia AB, Uppsala, Sweden ABSTRACT A study of the physical dimensions of the rabbit tenuissimus muscle is described and compared with previous work on the same muscle from the cat. The muscle in the rabbit was found to be larger than in the cat, but was consistent in its dimensions and suitable for use as a model for microvascu- lar research. INTRODUCTION The cat tenuissimus muscle has been analysed by Eriksson & Myrhage (2). A similar study is de- scribed of the rabbit tenuissimus muscle. Data are presented which show the rabbit muscle to be more substantial than its counterpart in the cat, and reasons are suggested for differences found in muscle fibre diameter. MATERIAL AND METHODS New Zealand White rabbits fed on a standard diet (Tekno- san pellets, Al3 Ferrosan, Malmo, Sweden) were used in this study. Tenuissimus muscle from the legs of 40 rabbits (mean weight 1.0 kgk0.3 kg s.d.) (Table I) were ex- amined, after the rabbits were anaesthetised with urethane and the tenuissimus muscle exposed. Examination of the muscle was camed out in several ways. The muscle length from origin to insertion and its width were measured in vivo with vernier calipers and its thickness with a micrometer eyepiece x 16 on a stereo- microscope (Leitz). Before removal from the rabbit, some muscles were fixed either by bathing with 3 % glutaraldehyde (buffered with 0.075 % sodium cacodylate at pH 7.2) or by cannulation of the aorta and intra-arterial perfusion of the muscle with this solution. Other muscles were perfused with Indian ink (Pelikan) after cannulation of the main artery supplying the muscle 1 cm before it entered the muscle. In this way all sections of the vascular bed were filled with carbon particles, making possible the measurement of vascular lengths and diameters by microscopy. Attempts were made to ensure maximal vas- ' Present address: Department of Surgery, University of Aberdeen, Aberdeen, Scotland. odilatation in some of these muscles by electrical stimula- tion or by adding papaverine to the ink. After removal from the rabbit, each muscle was pre- pared for measurement of vascular dimensions by fixation for a further 15 hours in 3 % glutaraldehyde and dehydra- tion with ethanol. Some of the muscles which had been perfused with ink were then placed in a mixture of 85% benzylbenzoate and 15% wintergreen oil (6, 8). By this procedure the muscle becomes quite transparent and ink- filled vessels can be visualised. The diameters of ink-filled vessels, including capillaries, were measured under a microscope using a measuring eyepiece (X 12.5, Leitz) and X6.5 X12.5 or x 2 3 objective lenses. The eyepiece was calibrated by viewing a micrometer slide. The lengths of vessels were measured by moving the microscope table under a cross-wire eyepiece, the table being connected to a potentiometer calibrated over a micrometer slide so that the distance the table moved was indicated as vascular length. The rest of the ink-perfused muscles, and those which had not been perfused, were embedded in epoxy resin (Shell Epon 812) for sectioning (4). 3 pm thick sections were cut with a glass microtome knife and stained with toluidine blue. Wax embedding was found to give inade- quate support to the muscle which disintegrated when sec- tioned. For estimation of the distribution of capillaries relative to muscle fibres, photomicrographs (Leitz Ortho- mat) were taken of these sections through x2.5 or X10 (Leitz) microscope objectives. Areas of muscle sections were measured from enlargements of these photographs with a digitizer (HD9107 A, Hewlett Packard). RESULTS The muscle was found to be 6.4 cm long in the left leg (k0.9 cm S.D.) with a variation in length per kg body weight of from 4.0 cm to 9.3 cm. Its greatest thickness was found to be 1.1 mm (k0.2 mm S.D.) and the muscle was shaped like an aerofoil with its narrower angle anterior (Fig. 1). The central vessels were located in the thickest part of the muscle run- ning parallel to its edges (Fig. 2). The width of the muscle was found to vary, being greater at the distal end of the muscle (mean 4.4 mmk0.8 mm S.D.) than a t its proximal end (mean 4.2 mmk0.7 mm S.D.) or mid-way along it (mean 4.2 Vpsala J Med Sci 81 176 C . M . Childs and K . - E . Arfors Table I. Summary of experimentalfindings Measurements of length, width and thickness of the mus- cle were made before removal from the rabbit No. of muscles Mean value Rabbit body weight, kg Tenuissimus length Left leg, mm 34 Left leg, mm/kg b.wt. Right leg, mm 26 Right leg, mm/kg b.wt. 26 Left leg, Proximal, mm 26 Left leg, Mid-level 46 Left leg, Distal 42 Right leg, Proximal 25 Tenuissimus length Muscle width Right leg, Mid-level 37 Right leg, Distal 34 Muscle width, mm/kg b.wt. Left leg, Proximal 25 Left leg, Mid-level 46 Left leg, Distal 42 Right leg, Proximal 25 Right leg, Mid-level 37 Right leg, Distal 35 1.050.3 S.D. 64.4f8.8 S.D. 65.1f 8.9 S.D. 65.1f11.9 S.D. 4.2k0.8 S.D. 4.250.8 S.D. 4.4k1.1 S.D. 4.3k0.8 S.D. 4 4k0.7 S.D. 4.5k0.8 S.D. 4.32 1.0 S.D. 4.35 1 .O S.D. 4.5k1.1 S.D. 4.4f1.1 S.D. 4.5f 1.1 S.D. 4.521.1 S.D. mmf0.8 mm S.D.) The width of the muscle was not significantly related to the weight of the rabbit, varying from 2.1-8.0 mmlkg body weight. There was no significant difference between the dimen- sions of muscles from left and right legs. The ratio of the part of the muscle lying anterior to the central vessels to the part lying posterior to them was calculated and found to be 0.58 at its upper end, 0.59 at mid-level and 0.60 at the distal end of the muscle. The density of 10 muscles was calculated after weighing each and determining its volume by dis- placement of saline in a 2 ml pipette. The mean density was found t o be 1.20 g/cc ( f O . l S.D.). Areas of muscles from 2 rabbits were measured from photographs, as described above, to determine the cross-sectional area of muscle fibres (Table 11). The cross-sectional area of each fibre was calcu- lated to be 1022 pm2 (k.50 S.D.) corresponding to 978 muscle fibres per mm2 and a fibre diameter of about 36 pm. COMMENT Distance between arterior muscle In all its physical dimensions, the rabbit tenuis- Left leg, Proximal 25 2.5f0.7 S.D. simus muscle was found to be more substantial than Left leg, Mid-level 46 its counterpart in the cat. At its thickest part it was nearly twice as thick (0.8-1.4 mm compared with Left leg, Distal 42 0.3-0.6 mm). Like the cat muscle, however, it had Right leg, Proximal 25 Right leg, Mid-level 37 Right leg, Distal 35 2.6f0.7 S.D. an antenor part, accounting for just over half its edge and central vessels, mm 2.4kO.6 S.D. 2.750.7 S.D. 2.5f0.7 S.D. 2.6f0.6 S.D. Ratio of anterior section to whole muscle width Left leg, Proximal Left leg, Mid-level Left leg, Distal Right leg, Proximal Right leg, Mid-level Right leg, Distal Tenuissimus thickness, mm Cross-sectional area of each Calculated muscle fibre muscle fibre, pm2 diameter=36pm 25 46 42 25 37 33 14 4 0.58f0.1 S.D. 0.59k0.1 S.D. 0.60f0.08 S.D. 0.60f0.07 S.D. 0.60f0.08 S.D. 0.58f0.08 S.D. 1.1 f0.2S.D. 1 022f50 S.D. width, which tapered to become less than 0.1 mm thick at the anterior edge. This was clearly the most suitable area for blood flow studies a s transillumina- tion of the muscle would be unlikely t o be satisfactory towards the thickest part. An illustra- tion of the shape of the muscle cross section is given in Fig. 1. The width of the muscle was found t o vary along its length between 4.2 and 4.4 mm (mean values) compared with 3-5 mm for the cat. Measurements of length, thickness and width central outline Fig. 1. Diagram of a cross-section of the tenuissimus muscle. In calculations the area of this cross-section was approximated to that of the two triangles (shown as dotted vessels lines). Upsala J Med Sci 81 Muscle dimensions 177 Table 11. Data f r o m 2 rabbits f o r calculation of cross-sectional area of musclejibres and calculation of the area of muscle cross-section served by each capillary Calculated cross sectional area of muscle Muscle area measured Number of Number of per fibre per capillary Cum3 muscle fibres capillaries (pm') (I*.m2) 53 187 54 29 985 1 8 3 4 37 225 38 17 980 2 190 23 893 22 14 1 0 8 6 1 707 13 436 13 9 1035 1 493 Mean 1 022 (f50 S.D.) 1 806 ( f 2 9 2 S.D.) were made in unfixed muscles in situ so that no distortion occurred. Some difficulty was experienced in obtaining satisfactory measurements for muscle fibre diame- ter. It is reported by Brhnemark & Eriksson (1) that shrinkage or swelling of muscle fixed in 3% glutaraldehyde and embedded in epoxy resin is less than 5 % , but this was not the experience of this study. The change in muscle width during prepara- tion vaned from a decrease of 1.7 mm t o an increase of 1.2 mm, representing an average change of width of 22% (Table 111). A further problem was that there was sometimes shrinkage of muscle fibres leading to their separation along connective tissue planes. These changes did not occur with every muscle, however, and it was possible to calculate fibre central vessels running parallel to muscle fibres muscle insertion Fig. 2. Diagram showing the main artery and vein supply- ing the muscle at about the mid-point in its length. diameter from muscles that showed no change in overall dimensions during fixation and whose fibres showed no separation because of shrinkage. Calcu- lation of average fibre diameter showed this to be 36 pm. Eriksson & Myrhage (2) found the diameter of Type A fibres in the cat tenuissimus to be 55 p m compared with 41 p m for Type B fibres and 26 for Type C fibres. The mean diameter, taking the pro- portion of each fibre type into account was found to be 44 pm. This value is higher than that found for the rabbit in this study and probably represents a species difference in fibre size as well as in the proportion of each fibre type present. No attempt was made in this study to differentiate between muscle fibres types. It has been suggested (2, 3 , 5) that connective tissue makes up between 20 and 30% of the bulk of skeletal muscle. Photographs of sections of muscle in this study suggested, however, that connective tissue in the tenuissimus formed a much smaller proportion of the total bulk than this. If muscle fibre diameter is calculated, from the data obtained from the rabbit, on the assumption of an 8 : 2 ratio be- tween muscle tissue and connective tissue plus vas- cular tissue, the diameter of each fibre decreases to 32 pm. In their study of skeletal muscle fibre density in the gastrocnemius muscle (Table IV), Schmidt- Nielsen & Pennycuik ( 7 ) found that this muscle contained only 14% of Type B fibres, with a total fibre density of 518 fibres mm-2. Eriksson & Myr- hage (2) found that the cat tenuissimus contained 48% of the smaller Type B fibres but did not calcu- late the density per square millimeter. In this study muscle fibre density was found to be 978 mm-2 and suggests that the relatively high percentage of Type B fibres found by Eriksson & Myrhage ( 2 ) in the cat 12 -762853 Upsala J Med Sci 81 178 C. M . Childs and K . - E . Arfors Table Ill. Mid-/eve/ muscle width changes with preparation Each pair of values is from a separate rabbit ~ Muscle width Before After Change in preparation preparation width (m) (Pm) (w) 4.1 2.4 2.8 3.7 4.1 4.9 5.7 3.2 4.2 3.6 4.5 4.4 3.6 4.8 4.4 3.9 4.1 4.2 4.0 3.8 5.0 3.3 5.1 3.1 3.7 4.0 4.2 2.8 Mean, 4.3 y m -1.7 +0.9 +0.8 -1.5 -0.6 -0.1 +l.2 -0.5 +o. 1 -0.2 -1.7 -2.0 +0.3 -1.4 Mean, 0.93 y m tenuissimus is also present in the rabbit and ac- counts for the greater number of muscle fibres per unit area. It is also likely that a species difference also accounts for the lower value for fibre diameter of 36 p m found in this study. It is not likely that fibre shrinkage during preparation is responsible because great care was taken to ensure that the overall dimensions of the muscles actually used was unchanged by preparation and that there was no microscopic evidence of muscle fibre separation in the areas measured. In the aerofoil shape of the rabbit tenuissimus muscle, cross section is treated as two triangles joined at their bases, its area can be estimated. Calculated from the mean values found for width and thickness (Table I) the cross-sectional area is 2.31 mm2. With a muscle fibre density of 978 mm-2 a cross section would contain 2 259 fibres reflecting the rabbit muscle’s greater size than the cat tenuis- simus where Eriksson & Myrhage found 1 375 fibres in a cross section, CONCLUSION This study of the rabbit tenuissimus muscle pro- vides information for comparison with already published data for the cat. No attempt has been made at fibre-typing the muscle, although the re- sults suggest it to be of a similar mixed type to that Table IV. Distribution o f f i b r e types in the tenuis- simus muscle of the cat (Eriksson & Myrhage, 1972) ( 2 ) Mean Type of muscle fibre A B C value Distribution of fibres, Muscle fibre diameter, Number of capillaries % 38 48 14 P m 55 41 26 44 surrounding one muscle fibre 3.5 3.6 3.8 3.6 in the cat but with a different distribution of fibre types. Although physically more substantial than its counterpart in the cat, the rabbit tenuissimus is confirmed as a muscle of sufficiently small dimen- sions to be of value in microvascular research. ACKNOWLEDGEMENT We thank Mr Ove Forsberg for skilful technical as- sistance. REFERENCES 1. Brlnemark, P. I. & Eriksson, E.: Method for studying qualitative and quantitative changes of blood flow in skeletal muscle. Acta Physiol Scand84: 284-288, 1972. 2. Eriksson, E. & Myrhage, R.: Microvascular dimen- sions and blood flow in skeletal muscle. Acta Physiol 3. Hammersen, F.: The pattern of the terminal vascular bed and the ultrastructure of capillaries in skeletal muscle. In Oxygen Transport in Blood and Tissue (ed. D. W. Lubbers eta].), pp. 184-195, 1968. 4. Luft, J. H.: Improvements in epoxy resin embedding methods. Biophys Biochem Cytol J 9:409414, 1961. 5. Pfaff, G. H.: A quantitative study of the capillary sup- ply in certain mammalian skeletal muscles. Anat Rec 46: 401406, 1930. 6. Romeis, B.: Mikroskopische Technik, p. 200, Old- enbourg, Miinchen, 1948. 7. Schmidt-Nielsen, K. & Pennycuik, P.: Capillary den- sity in mammals in relation to body size and oxygen consumption. Am J Physiol2W: 746-750, 1961. 8. Spalteholz, W.: Die Verteilung der Blutgefasse im Muskel. K. S. Ges d Wiss 24: 507-532, 1888. Scmd86: 211-222, 1972. Received August 5, 1976 Address for reprints: Karl-E. Arfors, Ph.D. Pharmacia AB Department of Experimental Medicine Box 181 S-75104 Uppsala Sweden Upsala J Med Sci 81