MORPHOMETRIC ANALYSIS OF MUSCLE FIBERS OF THE PSOAS MUSCLE IN INDIVIDUALS OF BOTH GENDERS WITH AGEING

Background/Aim. In this paper, we analyzed type I and type II muscle fibers of the iliopsoas muscle in persons of both genders with ageing. The aim of this study was to detect the presence and distribution of types I and II muscle fibers in the human psoas muscle using the hematoxylin and eosin method in individuals of different ages and genders. Methods. The material consisted of tissue samples of the right iliopsoas muscle taken from 30 adult cadavers (18 males and 12 females), aged from 30 to 90 years, divided into three age groups. The material was obtained from the Institute of Forensic Medicine, Faculty of Medicine University of Niš. Results. The values of astereological parameters (area, perimeter and Feret's diameter) of type I and type II muscle fibers were higher in male cases, although without any statistical significance. Conclusion. Based on the histochemical and morphometric analysis, the conclusion was drawn that after 70 years of life there occurred a loss of type II muscle fibers, more conspicuous in female cases.


Introduction
The iliopsoas muscle (m. iliopsoas) belongs to the group of inner hip muscles. It consists of the iliacus (m. iliacus) and psoas muscles (m. psoas) and it functions as the chief flexor of the hip joint.
This muscle is specific since it represents a sort of connection between the trunk and lower extremity 1 , and since it attaches to the vertebrae, it is responsible together with other muscles for the upright posture and ambulation of humans. Some of the studies 2 in which the authors used computerized tomography imaging to examine the transversal section of the psoas muscle reported a reduction of its size with ageing, and histopathological analysis of the muscle on postmortem material also showed significant changes in the muscle with advancing age. Imamura et al. 3 had similar results in 1983 studying the quadriceps femoris and plantar flexors of the foot. Morphometric analysis of the muscle was performed mostly on animal models 4,5 .
Skeletal muscles consist of connective tissue sheaths enveloping them from the outside (epimysium) and numerous fascicles, enveloped and separated one from another by a connective tissue layer called perimysium. Muscle fascicles consist of a large number of myofibrils containing contractile proteins, actin and myosin, arranged in a parallel fashion 6,7 .
A muscle fiber is a multinuclear, syncytial unit, shaped as an elongated, narrow and slender cylinder. The nuclei are subcapsular and there are 4-6 of them per cell on the transversal section 8 .
Red muscles, with a greater mitochondrial and lipid content and greater capillary density, are by function intended to maintain posture or to be engaged during longer activities. The color of red muscles is the consequence of a relatively higher content of myoglobin compared to white muscles which contain fewer mitochondria but abundant glycogen, which makes them better suited for anaerobic respiration and sudden and occasional contractions.
Human muscles contain red and white muscle fibers, arranged in a typical, combined mosaic pattern, resembling that of a chessboard. Depending on anatomical localization and function, the proportions of type I and type II muscle fibers vary, but type II fibers are nevertheless predominant with 60-65%, compared to 35-40% for type I fibers 9 .
Type II muscle fibers are darker in color, while type I fibers are of a paler color 10,9 . In the earliest studies investigating the difference between type I and II muscle fibers between genders 10 , the conclusion was drawn that some of the fibers are larger in men. Type II muscle fibers are usually larger in men compared to type I fibers, in contrast to women in whom type I fibers have an equal or greater diameter.
In 1970, in the study by Brooke and Kaiser 10 , who investigated biceps muscles, the conclusions were drawn, that are still valid in general, about the gender difference in skeletal muscles. The investigation of the vastus externus muscle by Lexell et al 11 in 1988 and Kobayashi 12 in 1991 did not demonstrate any significant differences in the diameter of muscle fibers between type I and type II fibers in men and women. As for the biceps brachii, a much higher percentage of type II fibers is present in men, while in women the percentages of both types are about equal. In contrast to that, the prevalence of type I and II fibers of the vastus externus muscle is approximately the same in both genders 13 .
It is thought that the established difference in the size of muscle fibers between men and women is determined by the fact that men are relatively higher and heavier than women, with a larger muscle mass and are more physically active. Androgenous hormones also play a role in the size of muscle fibers 14,15 . The difference between muscle fibers in men and women depends on the examined muscles as well 16 .
The effects of physical exercise and training on the muscle system have been investigated in numerous studies 17,13 . The results of these studies are in general contradictory, but some general principles do exist. It is evident that physical exercise and training of any kind increase the diameter of muscle fibers. In essence anaerobic activities lead to hypertrophy of type II muscle fibers, which is frequently seen in sprinters. In long distance runners, in whom aerobic metabolism is more significant, type I muscle fibers are usually larger.
Most authors agree that power training, for instance weightlifting, produces a significant hypertrophy of type II fibers, and lesser (if any) hypertrophy of type I muscle fibers 18 . It is well known that sprinters in general have a greater number of type II muscle fibers compared to sedentary controls, and long-distance runners have a greater number of type I fibers compared to untrained individuals. 6 Many authors believe that these two groups of runners have a genetically determined composition of muscles as to muscle fiber types, and that muscle fiber type conversion is negligible, if it occurs at all.
In the process of ageing, starting from the sixth decade and after 70 years of age, skeletal muscles structurally and functionally change, so that after 75 years of age, the power of muscles is reduced by 30-50%. The reason behind this reduction is the reduction of muscle fiber diameter. At 75 years of age, the diameter is reduced by 80% compared to the age of 25 years. Since the power of contraction is not linearly associated with muscle fiber diameter but it is proportional to the surface of muscle fiber transversal section, a diameter which is 80% of the normal diameter produces a 60% loss of muscle strength (contraction force).
Due to reduced elasticity, flexibility and joint diseases of different intensity, older individuals are less active, which is associated with the loss of muscle volume and contraction force. This is supported by the fact that ageing individuals have a selective atrophy of type II muscle fibers 19 . The effects of poorer nutrition in the elderly have not been sufficiently studied, although it is well known that cachexia is associated with atrophy of type II muscle fibers 20 .

Aim of the study
Based on the above information, it is possible that there is an association between ageing and loss of muscle mass in both genders. In view of the possible relationship between the process of ageing and muscle fiber changes in that process in both genders, our aim in this study was as follows:  To quantify in both genders during ageing the changes in size and shape of muscle fibers in the human psoas muscle, measuring the area, perimeter and Feret's diameter.

Material and methods
The study took place at the Institute of Anatomy, Institute of Histology and Embryology, Institute of Pathological Anatomy and Institute of Forensic Medicine, which represent the teaching and scientific bases of the Faculty of Medicine, University of Niš.

Material
The study material consisted of tissue samples of the right iliopsoas muscle taken The cadavers were divided into three age groups: first (I), with cases aged 30-49 years (n=10); second (II), with cases aged 50-69 years (n=10); and third (III), with cases aged 70 years and above (n=10).

Methods
The samples of the right psoas major sized 5x5.5 mm were taken utilizing the incision perpendicular to the muscle at the level of the mid-distance between the upper border of the twelfth thoracic vertebra (T12) and lower border of the fifth lumbar vertebra (L5).

Histological analysis
Histological analysis, as well as the identification of possible changes of the muscle fibers of the right iliopsoas muscle during ageing was based on light microscopy-based 8 assessment of their properties. The tissue of the psoas muscle was fixed in 10% buffered formalin during the next 24 hours. The obtained paraffin molds of the psoas major were used to obtain up to 5 µm thick tissue sections. We used classical hematoxylin-eosin (H&E) and PAS methods to identify the basic structures of the psoas muscle. The stained histological sections were then analyzed using light microscopy under 4x, 10x and 40x magnification. Digital images of the analyzed sections were obtained using a 1.3 megapixel digital camera.

Immunohistochemical analysis
Using immunohistochemical analysis, we established the presence of cells with positive reaction to applied immunohistochemical markers, by way of ultravision LP-HRP polymer (Cat. No. TL-125HL) detection technique for muscle fibers type II using a monoclonal antibody against myosin (anti-Myosin, Skeletal Muscle, Clone, MYSNO2, Ready to Use, Termo Scientific Lab Vision, Ca, 1:320).

Morphometric analysis of muscle fibers
Morphometric analysis of muscle fibers of the psoas major muscle was performed in 10 randomly selected visual fields per each analyzed case (270 visual fields in total for 27 analyzed cases). Stereological analysis of type I and type II muscle fibers in the analyzed visual fields was performed by measuring their volume density using a multipurpose test system, M168. Astereological analysis of type I and type II muscle fibers was performed by measuring their area (AMI and AMII), perimeter (BMI and BMII) and Feret's diameter (DFMI and DFMII) of the profile of transversally sectioned fibers.

Statistical analyses
Values are presented as mean values and SD. Independent samples t tests were used to test statistical differences between two samples. The normality of the distribution was validated by the Kolmogorov-Smirnov test. Data were analyzed using SPSS 16.0 (Statistical package for the social sciences, version 16.0, SPSS Inc, Chicago, IL, USA).

Morphological analysis
Morphological analysis involved histological analysis of transversal sections of the psoas muscle tissue stained with H&E and analysis of transversal sections of the psoas 9 muscle tissue stained using the immunohistochemical ultravision LP-HRP polymer detection technique.

Histological analysis
Classical fascicular structure of skeletal muscles was seen in transversal sections of m. psoas, stained with H&E, in all age groups. Connective tissue sheaths within the muscle had usual organization and contents; in more abundant connective tissue of the perimysium there were nerve elements, arterioles and venules, while more tender connective tissue of the endomysium contained capillaries and rare cells with slender projections enveloping individual muscle cells.

Immunohistochemical analysis
Identification of type II muscle fibers in transversal sections of the psoas muscle was performed using the anti-MYSN02 antibody, and immunopositivity was seen as a brown, fine-grained reaction of the sarcoplasm.
In the first age group, MYSN02-immunopositive type II muscle fibers were found in groups, rarely as individual fibers, and between them there were less numerous type I muscle fibers which did not show positive reaction. In transversal sections of the muscle, type I and II fibers had an irregular, polygonal shape, with sharper angles seen with type II fibers. Both muscle fiber types were of a similar diameter and with typically elongated or oval nuclei.
In the second age group, MYSN02-immunopositive type II muscle fibers were approximately equally prevalent as non-stained type I fibers. Type II muscle fibers were mostly polygonal, with sharp angles, in contrast to type I fibers which were more oval. In some transversal sections of the muscle, individual type II muscle fibers had a characteristic lamellar arrangement of myofibrils in the sarcoplasm, while in most type II muscle fibers immunopositivity manifested as a fine-grained reaction in the sarcoplasm.
In the third age group, MYSN02-immunopositive type II muscle fibers showed polymorpism of shape and thicknessthe cells were oval and with a smaller diameter compared to the non-stained type I muscle fibers. Immunopositive muscle fibers did not demonstrate regular distribution in the form of isolated groups, such as in younger age groups; instead, they were irregularly distributed between the non-stained type I fibers.
In all studied age groups, a smaller number of MYSN02-immunopositive type II muscle fibers was seen in female cases, compared to male ones (Fig. 01).

Morphometric analysis
The Kolmogorov-Smirnov tests showed that data were normally distributed. The average age of female cases was statistically significantly higher compared to male cases (T=2; SS=25; p=0.01) (Graph 1), which had a significant influence on the interpretation of observed gender-related differences when morphometric parameters of the psoas muscle fibers were concerned.

Insert Graph 1 here ˃˃˃
The average values of morphometric parameters of type I and type II muscle fibers in male and female cases are shown in Table 1.

Insert Table 1 here ˃˃˃
The results of t-test showed that there is no statistically significant difference (p>0.05) between the genders regarding given parameters (average area, perimeter and Feret's diameter of type I and II muscle fibers). The values in the male group were slightly higher than in female group (Graph 2) and it would be acceptable to claim that there is a tendency for higher values in males, but future studies are nedeed to confirm that.
Finally, average volume density of type II muscle fibers was statistically significantly greater in male cases (T=2.34; SS=25; p=0.028) (Graph 3). Nevertheless, since female cases were statistically significantly older than male cases, we could not decisively tell whether the observed difference was the consequence of ageing or genderrelated differences.

Discussion
The results of our study represent a morphological and morphometric analysis of changes affecting the fibers of the psoas muscle during ageing in cases of different ages and of both genders.
Morphological analysis of type I and type II muscle fibers of the psoas muscle demonstrated that in more advanced ages there occured changes affecting muscle fibers, especially type II ones. These changes were quantified in morphometric analysis and after that statistically analyzed, which provided an insight into their prevalence in cases of different ages, monitoring of their dynamics and analysis of their interrelatedness during the process of ageing.
Musculus psoas major, the object of our study, is a lower extremity muscle. Its origin is complex and involves lateral parts of the trunk and corresponding intervertebral discs from the twelfth thoracic to the fifth lumbar vertebra. Distally, the muscle joins the iliacus muscle forming the iliopsoas muscle, which attaches with its terminal tendon to the trochanter minor of the femur. The psoas major muscle has a flexion role, and is involved, together with adjacent muscles, in external rotation and adduction of the hip 21 . The psoas muscle therefore has an important dynamic and active postural function and belongs to lower extremity muscles which are important for everyday activities, such as walking, climbing the stairs, getting up from a chair, etc. Detection of age-related changes affecting the psoas muscle is thus of vital importance in the preservation of mobility and prevention of disability.
Arbanas et al. 21 (2009) have immunohistochemically and morphometrically analyzed the samples of the psoas muscle from 15 men aged 18 to 35 years in order to study the composition of muscle fibers. The age of this group of cases partly matches the age of the first group of our cases. The authors reported that the psoas muscle was mainly composed of type II muscle fibers (60%)fast-twitching, glycolytic and undergoing fatigue more quickly compared to type I fibers (40%)slow-twitching, oxidative and able to resist fatigue for longer periods of time. Similar to our own findings, they established that type I muscle fibers were characterized by a significantly greater transversal section area compared to type II fibers. Based on the composition of the psoas major muscle, Arbanas et al. 21 (2009) concluded that this muscle had complex dynamic and postural 12 functions. The results of our morphometric analysis revealed slightly greater volume density values of type I muscle fibers compared to type II fibers of the psoas muscle. This could indicate a possible predominance of type I fibers in our study. However, volume density is a stereological parameter the value of which is influenced by the number and area of the analyzed structure (in this case, a corresponding muscle fiber type). The fact that type I fibers have a significantly greater area of the transversal section compared to type II fibers and that the prevalence of type I fibers was approximately the same as the prevalence of type II fibers can account for a slightly higher value of their volume density compared to type II muscle fibers in our first age group. Our results therefore demonstrate a similar prevalence of type I and type II muscle fibers in our first (youngest) age group, which agrees with the results obtained by Arbanas et al. 21 (2009).
Histological changes in the skeletal muscles in older individuals are reflected in the reduction of muscle mass with simultaneous increase of fatty and connective tissue, as seen in our study as well. The size of type II muscle fibers is reduced, while the size of type I fibers remains unaffected. The reduction in size of muscle fibers with advancing age can be attributed to the loss of myosin heavy chains 22,20 . Moreover, the accumulation of "ringlike" and "torn" muscle fibers can be seen, then the accumulation of lipofuscin and nonmyelinated rod-like structures, as well as the reduction of number of blood vessels. Neuromuscular damage involves the increase of size of motor unit and reduction of number of motor neurons in the anterior horns of spinal cord gray matter. Furthermore, the process of ageing is associated with a reduced production of new muscle fibers, as the consequence of reduced activity of myosatellite cells. At the cellular level, muscle alterations associated with ageing involve proliferation of the sarcoplasmatic reticulum and T-tubular system and disorganization of sarcomeres, myofilaments and Z-lines 23 .
A significantly greater loss of type II muscle fibers compared to type I fibers can be explained by changes in the neuromuscular system associated with ageing, as well as the reduction of number and function of myosatellite cells as myogenic stem-cells which may differentiate into new muscle fibers 24,25 .
In our study, the obtained values of astereological parameters of type I and type II muscle fibers were greater in male cases compared to female ones, but these differences were not statistically significant. In addition, the average volume density of type I muscle 13 fibers was greater in female cases, but again the difference did not reach statistical significance. In contrast, the average volume density of type II muscle fibers was statistically significantly greater in male cases. In the literature, there is information about different distribution of type I and type II muscle fibers in different genders 10 . It was emphasized than in men both muscle mass and muscle fiber size was greater than in women. Individual muscle fibers are larger in men than in women. Type II muscle fibers are usually larger in men, but type I muscle fibers are of equal or greater diameter in women. It was thought that the gender difference was the consequence of greater height and weight of men compared to women, with greater muscle mass and more physically active 26,27 . The impact of male sex hormones cannot be neglected either. Androgenous hormones have an impact on muscle fiber size in men; it is well known that testosterone therapy leads to muscle hypertrophy 15 . Bennington 16 (1984) believed that some of the observed gender-related differences depended on the muscle which was analyzed. For instance, the biceps brachii muscle in men contains a markedly higher percentage of type II muscle fibers, while in women this muscle contains similar percentages of these two muscle fiber types. In our study, we also observed certain differences in some of the muscle fiber parameters between male and female cases. However, with the exception of volume density of type II muscle fibers, other differences in most of the parameters were not statistically significant. Since female cases were statistically significantly older than male cases, we could not decide with certainty whether the observed difference in the content of type II muscle fibers was the natural consequence of ageing or if it was gender related.

Conclusion
Based on the above elaborations of other authors' and our own study, the following conclusions may be drawn:  The loss of type II muscle fibers is associated with a continual (and of similar intensity) atrophy of type I and type II muscle fibers in both men and women.
The loss of type II muscle fibers, as well as the atrophy of type I and type II muscle fibers, demonstrate similar dynamics in both genders during ageing.
 The values of astereological parameters (area, perimeter and Feret's diameter) of type I and type II muscle fibers are higher in male cases, but without any statistical significance.
 The average volume density of type I muscle fibers is greater in female cases, but without any statistical significance.