Hernia DOI 10.1007/s10029-014-1213-0
Collagen fibers in the rectus abdominis muscle of cadavers of different age E. N. C. Calvi • F. X. Nahas • M. V. Barbosa J. A. Calil • S. S. M. Ihara • Y. Juliano • L. M. Ferreira
Received: 20 July 2013 / Accepted: 3 January 2014 Ó Springer-Verlag France 2014
Abstract Purpose To assess collagen content and types in the rectus abdominis muscle of cadavers of different ages. Methods Forty fresh adult male cadavers within 24 h of death were obtained from an Institute of Legal Medicine and divided by age at death into Group 1 (mean age, 23.3 years; range, 18–30 years; n = 20) and Group 2 (mean age, 46.2 years; range, 31–60 years; n = 20). From each cadaver, samples of the rectus abdominis muscle measuring 1 cm2 were collected 3 cm superiorly and 2 cm inferiorly to the umbilicus. Histological sections were prepared and stained with picrosirius red and Masson’s trichrome stain for visualization of total collagen fibers, and immunohistochemical analysis was performed to distinguish types I, II, III, IV and V collagen. Results No significant differences in total collagen were found between groups by Masson’s trichrome staining. However, picrosirius red staining revealed a significantly greater amount and higher concentration of total collagen and types I and III collagen in Group 1 than in Group 2 (P \ 0.05). All but type II collagen were detected by immunohistochemistry in both groups. No significant E. N. C. Calvi F. X. Nahas (&) M. V. Barbosa J. A. Calil L. M. Ferreira Division of Plastic Surgery, Universidade Federal de Sa˜o Paulo (UNIFESP), Rua Napolea˜o de Barros 715, 4o. andar, Sa˜o Paulo, SP 04023-062, Brazil e-mail: [email protected]
S. S. M. Ihara Department of Pathology, Universidade Federal de Sa˜o Paulo (UNIFESP), Sa˜o Paulo, SP, Brazil Y. Juliano Department of Biostatistics, Universidade de Santo Amaro (UNISA), Sa˜o Paulo, SP, Brazil
difference in type IV collagen was found between groups. Type V collagen was detected by immunohistochemistry in both groups, but quantification was not possible due to background staining. Conclusion The amounts of types I and III collagen in the rectus abdominis muscle were significantly smaller in older subjects. Keywords Collagen Staining and labeling Histology Rectus abdominis
Introduction The abdominal wall and musculoaponeurotic structures provide protection, compression, and support for the abdominal contents. The integrity of these structures is of fundamental importance in the control of bodily functions, such as urination and defecation, as well as during labor and delivery, and forced breathing. They are also responsible for flexion and rotation of the trunk, and spine stability . Abdominal tissue excess and diastasis of the rectus abdominis muscles may lead to physical, social and psychological problems . The most common defects of the abdominal wall are inguinal and incisional hernias [3, 4]. It is estimated that 1 in 5 men and 1 in 17 women will develop hernia at some point in their lives. Besides the socio-economic impact of surgical interventions for hernia repair, recurrence rates of up to 30 % have been reported [5, 6]. Collagen is essential for maintaining the strength and structural integrity of tissues [5, 6]. Collagen is an important component of fasciae and muscles that confers resistance to these structures. At present, more than 18 types of
collagen have been described, and types I, II, and III collagen are the most studied. Types I, III, IV and V collagen are found in striated muscles, and only types I and III are present in the fasciae [7, 8]. Fachinelli and Trindade  found that the amounts of total collagen, and types I and III collagen in the fascia were respectively 18.05, 20.5, and 7.3 % smaller in patients with anterior abdominal-wall hernias compared with a cadaver control group without hernias. Thus, variations in collagen content in fasciae and muscles may be associated with the presence of defects or deformities of the abdominal wall. Collagen content in fasciae has been extensively studied and its correlation with incisional hernia has been established [6, 10, 11]. This raised the question of whether the collagen content in the muscle is also correlated with hernia formation. Although the collagen content in the muscle (1–2 %) is smaller than that of the fasciae, collagen is the main component of the connective tissue, which provides structural strength to the muscle. No studies were found that investigated the collagen content in the rectus abdominis of cadavers. Therefore, the aim of this study was to evaluate the amount and types of collagen fibers in the rectus abdominis muscle of cadavers at different ages at death.
Materials and methods This study was approved by the Institutional Research Ethics Committee and authorized by the Institute of Legal Medicine. Forty fresh adult male cadavers at room temperature (22–25 °C), obtained from the Institute of Legal Medicine, were dissected within 24 h of death. The exclusion criteria were female gender, age at death under 18 years or over 60 years, storage at low temperatures, previous laparotomy, abdominal wall hernias, or abdominal trauma. The cadavers were divided by age at death into Group 1 (mean age, 23.3 years; range, 18–30 years; n = 20) and Group 2 (mean age, 46.2 years; range, 31–60 years; n = 20). For each cadaver, the height, weight, xyphoidpubis distance, and distance between the iliac crests were measured and the body mass index was calculated. The two groups were then compared based on these characteristics and were found to be homogeneous.
costal margins, laterally by the linea semilunaris, and inferiorly by the iliac crests and inguinal ligaments. The medial edge of the rectus abdominis muscles was marked with crystal violet. Two sampling reference points were marked on the abdomen: the first, 3 cm above the umbilicus in the cranial direction (supraumbilical level), and the second, 2 cm below the umbilicus in the caudal direction (infraumbilical level). After performing an incision 1 cm lateral to the medial edges of the rectus abdominis muscle with a no. 10 scalpel blade, four 1-cm2 tissue segments were dissected per cadaver from the rectus abdominis muscle (two from the supraumbilical level and two from the infraumbilical level), as shown in Fig. 1. The center of each segment was marked (Fig. 1), according to the experimental model described by Calvi et al. . The segments were coded as: s (supraumbilical), i (infraumbilical), l (left), and r (right). Thus, segments dissected from the right or left side at the supraumbilical level were coded sr and sl, respectively, and those dissected from the right or left side at the infraumbilical level were coded ir and il, respectively. Histological procedures The specimens were fixed in 10 % phosphate-buffered formalin and sent to the laboratory of pathology for histological and immunohistochemical analyses of collagen fibers. The muscle segments were identified in ascending numerical order so that the observer remained blinded to the age of subjects during histological analysis. After fixation in 10 % phosphate-buffered formalin for 24 h, the specimens were dehydrated in increasing concentrations of ethanol, cleared in xylol, embedded in paraffin at 60 °C, cut into 4-lm thick sections with a rotary
Surgical procedure The cadaver was placed in supine position and a xiphoidto-pubis incision passing around both sides of the umbilicus was made with a scalpel (no. 10 blade) through the skin and superficial fascia until the linea alba was exposed. The supra-aponeurotic dissection was limited superiorly by the
Fig. 1 Location of sampling sites on the rectus abdominis muscle in cadavers
Hernia Table 1 Staining methods used for the identification of collagen fibers
Table 3 Standards for quantification of the different types of collagen by immunohistochemical analysis
Type I collagen, fibroblast, and other cells
Type I and III collagen
Table 2 Primary antibody and dilution used in immunohistochemical analysis for each type of collagen Staining
Type I collagen
Type II collagen
Type III collagen
Type IV collagen
Type V collagen
microtome (Leica, Germany), and specifically stained for collagen (Table 1). Sections for histological analysis were routinely stained with hematoxylin and eosin (H&E) for detection of histopathological changes, which were used as an inclusion/ exclusion criterion; samples with histopathological changes were excluded from the study. All segments were analyzed by immunohistochemistry for visualization and quantification of the different types of collagen fibers. Primary antibodies at different dilutions were used in the immunohistochemical analysis (Table 2); the secondary antibody was obtained from a LSAB kit (primary antibody and peroxidase-conjugated streptavidin; Dako Cytomation, UK). The quantification of total collagen from sections stained with picrosirius red and Masson’s trichrome, as well as the quantification of each type of collagen by immunohistochemistry were performed digitally. Micrographs were taken by a digital camera (Olympus Q-Color3; resolution 200 dpi) coupled to an optical microscope (Olympus BX-40; magnification 1009). Ten random fields of view were examined on each section. The magnification, light intensity, condenser distance, as well as parameters of the image capture software, such as the intensity, brightness and contrast of the captured images were standardized to ensure the uniformity of results. Sections stained with picrosirius red were examined by polarized light microscopy . The captured images were processed with the Corel PhotoPaint software (Corel Corporation, Mountain View, CA, USA) to generate color masks in order to separate stained areas. The mask images were then analyzed with the UTHSCSA ImageTool 3.0 software (University of Texas Health Science Center, San Antonio, TX, USA). For
the quantification of collagen, color images were converted to grayscale images and the area, content, concentration, and percentage of collagen were measured. A scoring system ranging from 0 (no collagen) to 3 (abundant collagen) was used to evaluate collagen content (Table 3). Statistical analysis Parametric and nonparametric tests were used for the analysis of the collected data, taking into account the nature of the variables. The Wilcoxon test was used for comparisons among samples (sr, sl, ir, and il samples) within groups of data collected from sections stained with picrosirius red and Masson’s trichrome. The Mann–Whitney test was used for comparisons between groups of data collected using the picrosirius red and Masson’s trichrome staining, and immunohistochemical analysis. All statistical tests were performed at a significance level of 0.05 (P \ 0.05). The GraphPad Prism software (GraphPad Software, San Diego, CA, USA) and Microsoft Office Excel software (Microsoft Corp., Redmond, WA, USA) were used for data analysis.
Results No significant differences in total collagen were found within and between groups for the four sampling sites by Masson’s trichrome staining (Fig. 2). Also, no significant difference in type I collagen was found between groups by Masson’s trichrome staining, which is not highly specific for type I collagen. Sections stained with H&E, picrosirius red, and Masson’s trichrome are shown in Fig. 3. Types I and III collagen were observed in sections stained with picrosirius red. It is possible to distinguish between type I and type III of collagen using picrosirius red, but their staining colors are very similar, which may lead to an incorrect identification. Thus, type I and III collagens were quantified together. Picrosirius red staining revealed a significantly greater amount and higher concentration of
Fig. 2 Total collagen in samples from Group 1 (age at death, 18–30 years) and Group 2 (age at death, 31–60 years) collected at the right (R) and left (L) supraumbilical level, and right (R) and left (L) infraumbilical level of the rectus abdominis muscle. Picrosirius
red and Masson’s trichrome staining. The results are expressed as the relative amount of total collagen in a field view of 0.66 lm2. Asterisks indicate statistical significance (P \ 0.05)
Fig. 3 Micrographs of sections of the rectus abdominis muscle stained with (a, b) hematoxylin-eosin; (c, d) Masson’s trichrome; (e) picrosirius red, under polarized light; and (f) picrosirius red,
without polarized light. (left column) micrographs from Group 1; and (right column) micrographs from Group 2. 9100
Fig. 4 Immunohistochemical results showing the content of types I, II, III, and IV collagen in samples of the rectus abdominis muscle for Group 1 and Group 2. The results are expressed as relative amounts of types I, III and IV collagen in a field view of 0.66 lm2. Asterisks indicate statistical significance (P \ 0.05)
total collagen and types I and III collagen in Group 1 than in Group 2 (P \ 0.05). Immunohistochemistry is a more specific technique and confirmed the histological results. The amounts of types I, III, and IV collagen detected by immunohistochemistry for each group are shown in Fig. 4.
Discussion Collagen is the most abundant extracellular fibrous protein in humans, accounting for about 25 % of the total protein in the body and 1–9 % of the lean muscle mass. Type I collagen is the most common form of collagen, representing 90 % of the total collagen in mammals. It is usually organized into thick bundles, which confer resistance to structures, and found in ligaments, tendons and fasciae. Type III collagen is synthesized by fibroblasts and reticular cells and forms shorter and thinner fibers. It is generally found associated with type I collagen in varying proportions, and is more abundant in tissues with some degree of elasticity, including the skin, muscles, fasciae, and ligaments. However, it has been difficult to determine the contribution of individual biological components of the extracellular matrix to the mechanical properties of muscles . The relative amount and distribution of the various types of collagen fibers in different muscles depend on the species, race, sex, age, muscle group, and on the individual itself. These characteristics are influenced by the level of physical activity, disuse, nutritional status, denervation, and chronic physiological stress. In this field of research, the use of morphological and histochemical methods of analysis has proved to be important for the study of muscle anatomy and physiology . In the present study, it was observed that the rectus abdominis muscle contains collagen fibers of different diameters, with polygonal cross-sections, and one or more
peripheral nuclei. Although various types of collagen have been identified, the exact role of their structural function in the different tissues of the body, particularly in muscles, is not fully understood. However, it is known that collagen provides a structural function in striated muscles by connecting muscle fibers and ensuring their proper alignment. The tensile strength of collagen results from the unique structure of its fibers, fibrils, and molecules. More specifically, the tensile strength depends on factors, such as intraand intermolecular cross-links, orientation and concentration of collagen fibers, frictional forces, and physical and chemical interactions of collagen with other structural components of the extracellular matrix . Senescence causes adverse changes in skeletal muscle, including reduced muscle function due to loss of mass, elasticity, and strength . Few studies have reported on the effects of aging in human muscles, probably due to the difficulties inherent in conducting serial studies in humans, which is why the vast majority of studies are performed in animal models. A problem that arises when human tissues are obtained from cadavers for histological studies is the time elapsed between death and tissue fixation due to changes in tissues after death. Therefore, a short time should have elapsed between death and fixation of tissue samples to minimize changes in the histoarchitecture of muscle tissues. For this reason, in the present study, tissue samples were collected and fixed within 12–24 h of death. During this period, enzyme activity remains unchanged and postmortem artifacts are absent or minimal [1, 7, 9, 10]. Only male cadavers were studied to avoid changes in the abdominal musculoaponeurotic system caused by previous pregnancies. This experimental model was based on that described by Calvi et al.  and the sampling locations (3 cm above and 2 cm below the umbilicus) were based on the experimental model of Nahas and Ferreira . In previous studies, the tensile strength of the aponeurotic system has been measured at these same levels [17–20]. The cadavers were divided by age at death into Group 1, which was composed by younger individuals, in the age range of 18–30 years, and Group 2 consisted of older individuals, in the age range of 31–60 years. Subjects younger than 18 years were not included in the study because muscles are still developing at this age, and those older than 60 years were also excluded because senile changes may occur in the muscles of older adults. In order to avoid bias, the muscle segments were identified in ascending numerical order so that the observer remained blinded to the age of the cadavers. In skeletal muscles, collagen is present in three fibrillar forms (types I, III and V collagen) and one non-fibrillar form (type IV collagen), with types I and III collagen being the most abundant. According to Fachinelli and Trindade
, type I collagen accounts for 90 % of the total collagen in the muscle. In the present study, similar amounts of types I and III collagen were detected by immunohistochemistry in the muscle samples from both age groups. Histological analysis was carried out to quantify the types of collagen present in the rectus abdominis muscle. H&E staining was used to evaluate the quality and suitability of the specimens. Both the picrosirius red and Masson’s trichrome staining were used to detect total collagen (types I and III collagen). However, picrosirius red staining is more specific for collagen because Masson’s trichrome also stains fibroblasts and other cells. These staining methods are the most widely used for quantification of collagen fibers, but the measurements are usually made at only three or four fields of view . In order to collect more consistent data, ten fields of view per section were chosen randomly and examined to determine the collagen content and types. Thus, 1,600 images were captured from histological sections for each staining method. Quantitative analysis was performed with a software package that allows the gradation of selected color images for the counting of histological features. Collagen content was determined as the ratio of collagen-positive pixel count to total pixel count in a field view of 0.66 lm2. The ImagePro Plus software has been used for this type of analysis, but it has limitations, such as complexity in operation and high cost, and requires increased analysis time because images are processed individually . The ImageTool 3.0 software was used in the study because it is free, has a simple interface, and is suitable for the analysis of biological samples. This software allows the quantification of the relative amounts of collagen fibers and their classification according to a single threshold defined by the user. There are several histochemical methods for detecting collagen in tissues. Picrosirius red and immunohistochemistry contributed significantly to the identification of the different types of collagen. On the other hand, staining by the Mallory, Masson, and Van Gieson trichrome methods, although widely used, is not as sensitive as by picrosirius red and immunohistochemistry [9, 11]. Picrosirius red for tissue staining is a solution of Sirius red F3B (0.1 %) in saturated aqueous picric acid. The molecules of collagen are rich in basic amino acids, which strongly react with acid stains such as picrosirius red. This stain has long molecules that bound parallel to the long axis of the collagen, promoting intensification of its natural birefringence . No significant differences were found in total collagen between groups as determined by Masson’s trichrome staining. However, picrosirius red, which is more specific for collagen, revealed that the amounts of type I and III collagen were significantly greater in Group 1 (younger adults) than those of Group 2 (older individuals). This result was also confirmed by immunohistochemistry.
Age-related changes in the distribution of collagen fibers in various organs other than the rectus abdominis muscle have been widely studied but with conflicting results. Most authors have reported that these changes are more associated with the state of aggregation of collagen molecules and tissue organization of collagen fibers than with quantitative changes in collagen content . Other studies [6, 21] have measured collagen content in patients and cadavers attempting to correlate the characteristics of individuals to pathologies. However, collagen content was not evaluated according to age group, as in the present study. Hernia repair is the second most common surgical procedure in our field . Anatomical factors alone are not sufficient to explain the occurrence of hernias. Meyer et al.  found quantitative and qualitative differences in collagen from the fascia transversalis between patients with hernia and cadavers without hernia, but no significant difference in total collagen was observed. The authors concluded that the weakening of the aponeurosis may be attributed to a higher proportion of type III collagen to type I collagen in patients with hernia . Bo´rquez Morales  investigated by immunohistochemistry the content of types I and III collagen in the skin of nine patients with primary inguinal hernia and nine patients without hernia. The authors reported qualitative and quantitative differences in collagen fibers and type I/III collagen ratio between groups. The findings suggest that there may be a relationship between decreased abdominal muscle strength in these age groups and increased predisposition to the development of abdominal hernias due to the fragility of the anatomical structure. Despite the findings of several studies, Haus et al.  contended that variations in the collagen content of tissues throughout life have no effect on the concentration of intramuscular collagen. The authors found that both endomysial collagen concentration and enzymatically mediated collagen cross-links are little affected by age. However, an increased number of non-enzymatically mediated collagen cross-links were observed in the muscles of older individuals . The various concentrations and associations of extracellular matrix components result in specific adaptations of particular organs, allowing the maintenance of homeostasis, cellular differentiation at every stage of life, and adaptation to different stimuli . Strong abdominal muscles (with high collagen content), as found in young individuals, prevent abdominal distension. The overstretching of the aponeurosis and subsequent hernia formation may result from weak muscles, leading to the distension of the abdominal wall. Previous studies have indicated that there is a correlation of collagen content in the fascia with occurrence of anterior abdominal wall hernia [11, 28–30]. However, fascia collagen content is not the only culprit in hernia formation, which is multifactorial. The decrease in muscle collagen
content may also contribute to hernia formation and may make difficult the repair of the abdominal wall by local tissues. Aging is probably not the only contributing factor for incisional hernia. Physical activity, diet and genetic factors may also play a role in the amount of collagen present in the muscle. However, a decrease in collagen content with age may cause a general distension of the muscle, resulting in weakening of the abdominal wall. This study is not an attempt to solve the problem of incisional hernia, but to be one more step toward this goal and serve as a basis for further studies. Conclusion The content of types I and III collagen in the rectus abdominis muscle was smaller in the older group. Conflict of interest EC declares no conflict of interest. FN declares no conflict of interest. MB declares no conflict of interest. JC declares no conflict of interest. SI declares no conflict of interest. YJ declares no conflict of interest. LF declares no conflict of interest. Ethical standards The study procedures comply with the current laws of the country where they were performed. The study was approved by the Institutional Research Ethics Committee and authorized by the Institute of Legal Medicine.
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