Histology of the rectus abdominis muscle in rats subjected to cranial and caudal devascularization

PURPOSE: To investigate the microscopic changes in the rectus abdominis muscle in rats subjected to five delay procedure. METHODS: 30 male holtzmann rats, weighting between 250 and 350 grams, were used. The animals were divided into five groups (n=6): A – cranial section of the right muscle; B – caudal section of the right muscle; C – craniocaudal section of the right muscle; D – cranial section reflecting the right muscle in the craniocaudal direction; E – caudal section reflecting the right muscle in the caudocranial direction. On the seventh day after surgery, a resection of the cranial and caudal fragments of the right and left muscles, respectively, was performed for microscopic analysis. Histological alterations were quantified and the right and left (control) muscle fragments compared. Fisher’s exact test was used for statistical purposes with a significance level of 5%. RESULTS: The comparison between right and left muscles showed statistically significant differences in group A – inflammatory infiltrate in the cranial fragment (p=0.015); in group C – inflammatory infiltrate (p=0.000) and necrosis (p=0.015) in the caudal fragment; and in group E – edema in the caudal fragment (p=0.000). No significant alterations were noted in groups B and D. CONCLUSIONS: Irrigation exclusively through the perforating muscle vessels is inappropriate; irrigation exclusively through the cranial pedicle causes milder, insignificant histological alterations, when compared with irrigation exclusively through the caudal pedicle; irrigation exclusively through the caudal pedicle causes more pronounced, but still insignificant, histological alterations when compared with other forms of devascularization.


Introduction
The Transversal Rectus Abdominis Myocutaneous flap (TRAM) has been considered the standard autogenous tissue for breast reconstruction ever since its description by Hartrampf et.al. 1 , despite reported complications among at-risk patients such as smokers, the obese and diabetics [1][2][3] .Among 300 patients in good health who were subjected to breast reconstruction employing this flap, however, complications were rare, with the most frequent one being partial or total loss of the flap, which occurred in 6.2% of the patients.This complication occurs due to tension in the vascular pedicle, hematomas and technical errors.Among the patients that presented complications, 32% had previously been subjected to radiotherapy and 26% were smokers [3][4][5] .
Considering the importance of the rectus abdominis muscle in breast reconstruction, this study was conducted on rats with the goal of assessing microscopic alterations in this muscle after the use of five devascularization techniques.

Methods
This was a prospective study of 30 males, Holtzmann, whose weight varied between 250g and 350g.The project was approved by the Animal Experimentation Ethics Committee, Protocol No. 106/2006 (CETEA/UFMG).In all stages of the experiment, the ethical precepts that guide animal experimentation were observed [6][7][8] .
The 30 animals were randomly distributed into five groups of six.In each group of animals, a different type of devascularization of the right rectus abdominis muscle was performed as described below: • Group A -section of the cranial epigastric vessels • Group B -section of the caudal epigastric vessels • Group C -section of the cranial and caudal epigastric vessels • Group D -section of the cranial and perforating epigastric vessels, maintaining irrigation through the caudal epigastric vessels • Group E -section of the caudal and perforating epigastric vessels, maintaining irrigation through the cranial epigastric vessels Anesthesia and preparation for the surgical procedure All the animals were subjected to intraperitoneal anesthesia using ketamine chloridrate (Ketalar ® ), at a dosage of 50mg/kg, together with 2-(2.6xilidino)-5.6dihidro-4H-1.3-tiazine(Rompun ® ), at a dosage of 1.0 ml/kg, administered through a plastic insulin syringe (U-100) connected to 13x8 needles.Next, trichotomy of the abdomen, antisepsis with povidone-iodine detergent and the placement of sterile drapes were performed.

Surgical procedure
The surgical procedure was performed in accordance with the specific group of animals: Transversal section of the aponeurosis and muscle at the cranial threshold, cauterization of the cranial epigastric vessels.Medial and lateral sections of the anterior aponeurosis, 3.5 cm wide, and the release of the right muscle from its posterior aponeurosis, in the cranio-caudal direction.Suture of the aponeurosis identical to that of the skin (Figures 2 and 3).
• Group E -incision and exposition identical to group D. Transversal section of the aponeurosis and muscle at the caudal threshold, cauterization of the caudal epigastric vessels.
Median and lateral section of the anterior aponeurosis, 3.5 cm wide, and release of the right muscle from its posterior aponeurosis in the caudocranial direction (Figures 4 and 5).The animals were euthanized while still under anesthesia through the sectioning of the abdominal aorta.

Anatomopathological exam
The muscle fragments were processed in blocks of paraffin and the histological cuts were colored with hematoxylin and eosin (HE), and analyzed through optic microscopy.The following alterations were investigated in the five groups of animals by the same pathologist: inflammatory infiltrate, muscle fiber necrosis, vascular alterations, foreign body type reactions and edema (Figure 6), quantitatively classified as: • N (Normal) -no alterations present; • + -alterations of up to 33%; • ++ -alterations of between 34 and 66%; • +++ -alterations of between 67 and 100%.

Variables investigated and statistical analysis
The dependent variable was represented by the devascularized right rectus abdominis muscle and the independent variable by the surgical devascularization techniques.For each group of animals, the microscopic alterations in the cranial and caudal fragments of the right muscle (devascularized) were compared with the left muscle (control) through Fisher's exact test with a significance level of 5%.

Results
The comparison of the histological alterations between the left and right rectus abdominis muscles showed significant results in the following groups (Table 1): • Inflammatory infiltrate in the cranial segment of group A (p = 0.015) and in the caudal segment of group C (p = 0.000); • Necrosis in the caudal segment of group C (p = 0.015); • Edema in the caudal segment of group E (p = 0.000).In groups B and D, there were no significant differences between the left and right muscles in terms of the investigated histological alterations.An increase in necrosis and inflammatory infiltrate found in the animals in groups D and E when compared with the other groups was also noted, however it was not statistically significant (Figure 7).

Discussion
Flaps are broadly employed in the reconstruction of defects caused by surgery, trauma, burns, etc.This type of reconstruction may, however, result in complications, the most common of which is partial or total necrosis of the flaps.This has stimulated research aimed at reducing the risks of said complication.
Miller et al. 9 investigated the supply of blood to the TRAM through angiographies of the internal mammary artery and the observation of the vascular anatomy of patients that had been subjected to breast reconstruction, demonstrating the applicability and safety of this flap.
Ishii et al. 10 and collaborators proposed the bipediculated transversal flap of the rectus abdominis muscle for high risk patients, aiming at increasing local blood supply.Results were satisfactory, showing a reduced number of complications, but were not confirmed by other authors [11][12][13] .
Wagner et al. 14  The rat has proven an excellent study model for TRAM research 16 , having been used by several authors.Özgentas et al. 17 In the present study, the simultaneous interruption of blood flow in the cranial and caudal epigastric vessels caused necrosis in the distal muscle fragment among the animals in Group C. This proved the inadequacy of irrigation exclusively through the perforatory vessels.Despite the dominance of the cranial epigastric vascular pedicle in rats having been defined in the literature 17,18 , no significant histological alterations were found between the animals in Group D and their respective controls.This probably was due to the smaller size of the group sampled.
An increase in necrosis and inflammatory infiltrate was found among the animals in groups D and E, but this was not statistically significant (Figure 7).
Considering that TRAM is one of the most widely employed techniques in breast reconstruction, studies show that complications persist.It is therefore necessary to proceed with further studies, especially concerning blood irrigation of the muscles, in order to improve the technique.
Transversal incision of the skin and superficial fascia, one centimeter wide, in the cranial threshold of the right rectus abdominis muscle.Section of the anterior aponeurosis and of the muscle at this point, and cauterization of the cranial epigastric vessels with a bipolar scalpel.Suture of the anterior aponeurosis with two simple stitches and continuous intradermic suture of the skin, both with mononylon 6-0 thread.• Group B -Incision and suture identical to Group A in the caudal threshold of the right rectus abdominis muscle and cauterization of the caudal epigastric vessels.• Group C -Incision and suture identical to groups A and B, in the cranial and caudal thresholds of the right rectus abdominis muscle and cauterization of the cranial and caudal epigastric vessels (Figure 1).• Group D -Median xifopubic incision exposing the anterior aponeurosis of the right rectus abdominis muscle.

FIGURE 1 -
FIGURE 1 -Group C -anterior abdominal wall of rat displaying both cranial and caudal devascularization of the right rectus abdominis muscle.

FIGURE 2 -
FIGURE 2 -Group D -anterior abdominal wall of rat displaying cranial devascularization of the right rectus abdominis muscle, reflected in the craniocaudal direction.

FIGURE 3 -
FIGURE 3 -Group D -anterior abdominal wall of rat displaying cranial devascularization of the right rectus abdominis muscle, sutured in its bed after the muscle was reflected in the craniocaudal direction.

FIGURE 4 -
FIGURE4 -Group E-anterior abdominal wall of rat displaying caudal devascularization of the right rectus abdominis muscle, reflected in the caudocranial direction.

FIGURE 5 -
FIGURE5 -Group E-anterior abdominal wall of rat displaying caudal devascularization of the right rectus abdominis muscle, sutured in its bed after the muscle was reflected in the caudocranial direction.
A, B, C, D and E in terms of the percentage with some alteration in the variables investigated.* p value with statistical significance

FIGURE 7 -
FIGURE 7 -Frequency and intensity of muscle fiber necrosis in groups A, B, C, D and E.
reported their experiences with 341 unilateral breast reconstructions.They recognized increased tissue necessity, larger flap extension and microvascular dysfunction as conditions which justify the use of the bivascular flap.The univascular flap, they stated, should be reserved for patients with no risk factors.Boyd et al. 15 identified, in humans, a dominance of the role played by the caudal epigastric vascular pedicle in the rectus abdominis muscle, along with the presence of numerous perforating muscular vessels acting in the local collateral circulation.
,18 demonstrated, through microangiography, the physiological dominance of the cranial epigastric vessels in relation to the caudal epigastric vessels.The study of the rectus abdominis muscle for the purpose of reconstructing defects in the corporal surface are part of a line of research initiated by Lopes Filho and Lázaro da Silva 19 .These authors adopted the term Retramcur (Transversal Rectus Abdominis Myocutaneous flap) and performed various types of blood flow dissections aimed at bilateral cranial autonomization of the skin, superficial fascia and rectus abdominis muscle.The group of animals in which only the deep superior epigastric vessels were sectioned presented a smaller area of necrosis in the flap 19 .Based on knowledge of the vascular anatomy of the anterior abdominal wall of the rat, various devascularization models for the rectus abdominis muscle were produced in an attempt to clarify, from a histological perspective, the possible repercussions resulting from the modification of its normal vascular physiology.