Immunohistochemical analysis of collagen content and types in the rectus abdominis muscle of cadavers of different ages

PURPOSE: To assess the collagen content and types in the rectus abdominis muscle of cadavers of different ages. METHODS: Forty fresh adult male cadavers, at room temperature, were obtained from the Institute of Legal Medicine of Franca and dissected within 24 hours of death. The cadavers were divided into two groups: Group A (n=20), 18 to 30 years of age, and Group B (n=20), 31 to 60 years of age. Bilateral incisions were made in the middle portion of anterior rectus sheath 3 cm superiorly and 2 cm inferiorly to the umbilicus and four fragments of the rectus abdominis muscle were dissected. The samples were fixed in 10% buffered formalin and sent for immunohistochemical analysis to determine collagen content and types. RESULTS: Immunohistochemical results revealed higher amounts of type I and type III collagen in Group A. However, no difference in the amount of type IV collagen was found between the groups. CONCLUSION: The amount of type I and type III collagen was higher in group A.


Introduction
The most common defects of the abdominal wall are incisional hernias, tumor resections, trauma, congenital defects, and deformities resulting from the transposition of the rectus abdominis muscle, especially in breast reconstruction 1 .The incidence of these defects is high, with more than 11% of patients who undergo median laparotomy developing incisional hernias 2 .Up to 27% of patients who undergo breast reconstruction with a rectus abdominis muscle flap may develop donor-site bulging 3 .Incisional hernia formation may be related to local or systemic changes, which may compromise the healing process 4,5 .Although most incisional hernias are asymptomatic, they are associated with pain, incarceration, or strangulation in about 30% of the cases.Additionally, incisional hernia is the most common indication for reoperation after laparotomy 6 .
Collagen is an important component of fasciae and muscles that provides resistance to these structures 7 .At present, 19 types of collagen have been described 8 , with the types I, II and III being the most studied ones.Collagens type I, III, IV and V are found in striated muscles and only type I and III are found in fasciae 7 .Type I collagen is the most common and accounts for 90% of the total collagen in mammals.It is synthesized by fibroblasts, odontoblast, and osteoblasts and is usually organized into thick bundles, which confer resistance to structures.Type III collagen is composed of three alpha-1 chains and forms shorter and thinner fibers.It is synthesized by fibroblasts and reticular cells, and generally found associated with type I collagen in different ratios.Type III collagen is prevalent in tissues that require some degree of elasticity, such as the skin, muscles, fasciae, and ligaments.Type V collagen regulates the diameter of collagen fibers 9 .Fachinelli 10 reported that the amount of total collagen was 18.05% lower in the aponeurosis of cadavers with anterior abdominal wall hernias than in that of cadavers without hernia, and that the amounts of type I and type III collagen were, respectively, 20.5% and 7.3% smaller in cadavers with hernia.
Thus, variations in the amount of collagen and collagen type I/III ratio in the aponeurosis may be associated with abdominal wall hernias.However, no studies were found in the literature assessing collagen content in the rectus abdominis muscle.Therefore, the aim of this study was to assess the collagen content and types in the rectus abdominis muscle of cadavers of different ages.

Methods
The study was approved by the Research Ethics Committee of UNIFESP (process no.1492/08) and authorized by the Institute of Legal Medicine of Franca (SP, Brazil).Forty fresh adult male cadavers, at room temperature, were dissected within 24 hours of death.The exclusion criteria were: cadavers under 18 years of age, stored at low temperatures, with previous laparotomy, abdominal wall hernias, or abdominal trauma.
The height, weight, xyphoid-pubis distance, and distance between the iliac crests were measured and the body mass index was calculated.
The cadaver was placed in supine position and a xiphoid-to-pubis incision passing around both sides of the umbilicus was made with a scalpel through the skin and superficial fascia until the linea alba was exposed.The supra-aponeurotic dissection was limited superiorly by the coastal margins, bilaterally by the linea semilunaris, and inferiorly by the iliac crests and inguinal ligaments.
The midline between the rectus abdominis muscles was marked with crystal violet.Two sampling points were marked: the first, 3 cm above the umbilicus (supraumbilical level), and the second, 2 cm below the umbilicus (infraumbilical level).A 1.5-cm longitudinal incision was made in the anterior rectus sheath and two 1-cm 2 fragments were dissected from the central portion of each rectus abdominis muscle, one at the supraumbilical level and another from the infraumbilical level, for a total of 4 fragments per cadaver.
The fragments were named as following: ur, upper right; ul, upper left; lr, lower right; and ll lower left.The fragments were coded and then fixed in 10% buffered formalin, embedded in paraffin, and cut into 3-µm sections.Immunohistochemical analysis was performed using rabbit antibody (Novotec, France) diluted in the sequence 1:50, 1:100, 1:50, and 1:100 to detect type I, II, III, and IV collagen; human stomach/colon was used as a positive control and omission of the primary antibody, as a negative control.
Image acquisition and digitization was performed using a 3.2 megapixel digital camera (Olympus Q-Color3 Imaging System, USA) with 200 dpi resolution coupled to an optical microscope (Olympus).Images were acquired at x100 magnification from10 randomly chosen fields.
A scoring system was standardized for the immunohistochemical analysis of collagen content and types present in the histological sections (Table 1).

Results
Immunohistochemical analysis showed that the rectus abdominis muscle contains collagen fibers with different diameters, polygonal sections, and one or more peripheral nuclei.
Immunohistochemical results revealed significantly larger amounts of type I and III collagen in Group A than in Group B, absence of type II collagen in both groups, and similar amounts of type IV collagen in both groups (Figures 1 and 2).

Discussion
Because of the high incidence of abdominal wall hernias, a better understanding of the anatomy, physiology, and histology of the structures involved in this condition is needed 10 .
The skeletal striated muscle tissue is composed by two basic types of fibers with different metabolic, morphologic and contractile characteristics 11 .The relative amount and distribution of collagen fibers in the different muscles depends on the species, race, sex, age, muscle group, and on the individual itself.They also depend on the level of physical activity, lack of use, nutrition status, denervation, and chronic physiological stress.In this field of research, morphological and histochemical methods of analysis have been useful in the study of muscle anatomy and physiology 12 .
Although a large number of collagen types have been identified, the exact role of their structural functions in the different tissues of the body, especially in muscles, is still not well established.However, it is known that collagen in striated muscles has a structural function, connecting muscle fibers to ensure that these fibers are properly aligned.The tensile strength of collagen results from the unique structure of its fibers, fibrils, and molecules.More specifically, the tensile strength results from inter-and intramolecular crossed connections, orientation, density, and frictional forces between fibers, and physical and chemical interactions with other structural components of the extracellular matrix 13 .
The evaluation of the subtypes of collagen fibers in muscle biopsies has been essential for the diagnosis of different pathological and experimental conditions, such as hypertrophy caused by repetitive strain and sustained work, muscle atrophy due to lack of use or denervation, and longitudinal fiber splitting 11 .
There are few reports in the literature on the effects of ageing on human muscles, probably because of the difficulties associated with the performance of serial studies in humans, which may be one of the reasons why most of the studies use animal models [13][14][15][16] .Because it is difficult to obtain muscle biopsy samples from healthy individuals, we opted to use muscle fragments from cadavers, collected at the same site locations (3 cm above and 2 cm below the umbilicus) used in previous studies 15 .At these locations, the rectus abdominis muscle is completely surrounded by the muscle sheath.This is important to ensure sample homogeneity, since the amount of collagen may vary over muscle areas not covered by aponeurosis 1,[15][16][17][18][19] .
Male gender was used as an inclusion criterion, so that the observations were not affected by the number of pregnancies and possible diastasis recti abdominis, which is more common in women.The cadavers were divided into two groups: the Group A consisted of younger adults (18 to 30 years of age), and Group B consisted of older adults (31 to 60 years of age).The maximum age was set at 60 years so that possibly only changes in the functional equilibrium were present in the sample, thus avoiding the effect of senile changes common in older individuals 20 .
Studies in patients and cadavers assessing the amount of collagen in the fascia transversalis and anterior sheath of the rectus abdominis muscle have reported a link between quantitative changes in collagen content and hernia formation, but these studies have not compared different age groups 3,21 .Wolwacz Júnior et al. 3 did not observe degeneration of collagen fibers in the fascia transversalis associated with senility in patients with hernia.However, in that study, the percent area of collagen in samples of patients with hernia was half of that observed in samples of patients without hernia (controls).In another study, no difference in the concentration of collagen was found between patients with hernias and cadavers without hernias 22 .Immunohistochemical analysis of type I and type III collagen in the skin of 18 patients with (n=9) and without (n=9) inguinal hernia has shown quantitative and qualitative significant differences in collagen fibers and collagen type I/III ratio between groups 23 .Other investigators have reported no significant difference in the amount of collagen in the fascia transversalis of patients with hernia compared with cadavers without hernia, but significantly larger amounts of type III collagen were found in patients with Nyhus type IIIa hernias compared with patients with other types of inguinal hernia 22 .Fachinelli 10 observed that the amount of total collagen was 18.5% smaller in patients with anterior abdominal wall hernia compared to cadavers without hernia, and that the amounts of type I and type III collagen were, respectively, 20.5% and 7.3% smaller in patients with hernia.
None of the studies found in the literature compared the type and content of collagen in the rectus abdominis muscle of cadavers of different ages.Immunochemical results revealed that there was a significant difference in the amounts of type I and type III collagen between age groups.The results suggest that the muscle strength of the abdominal wall decreases with age, and that there is an increased risk of abdominal hernia formation associated with the fragility of the anatomic structure caused by the reduction in the amount of total collagen.

Conclusion
The amount of type I and type III collagen, determined by immunochemical analysis, was larger in the cadavers aged between 18 and 30 years compared with cadavers aged between 31 and 60 years.

TABLE 1 -
Qualitative evaluation of collagen by immunohistochemical analysis.