A Reliable Experimental Model of Fat Necrosis Occurring in Pedicled Flaps Using the fa/fa Zucker Rat

Abdel-nasser M. Khallaf, MD*

Paul D. Smith, MD†

Wyatt G. Payne, MD†

Theodore J. Strickland, MD†

Francis Ko, BS†

M. C. Robson, MD†

*Department of Plastic Surgery, Al-Azhar University, College of Medicine, Naser City, Cairo, Egypt

†The Institute of Tissue Regeneration, Repair and Rehabilitation, Department of Veterans Affairs Medical Center, Bay Pines, Florida, and The Department of Surgery, University of South Florida, Tampa, Florida

KEY WORDS: Fat necrosis, animal model, pedicled flap, TRAM flap, Zucker rat

Abstract

Introduction: Fat necrosis remains an unresolved problem facing the plastic surgeon. Attempts to find preventative or therapeutic measures to deal with fat necrosis have been hindered by lack of a reliable experimental animal model. The creation of a transverse rectus abdominis musculocutaneous (TRAM) flap on a fa/fa Zucker genetically obese rat is described as a model to mimic human fat necrosis.

Materials and Methods: TRAM flaps were surgically raised in 10 fa/fa Zucker rats. Postoperative monitoring for color changes and palpable firmness at 24 hours, 48 hours, and 7 days were noted. Histologic stains were performed on flap fat specimens at 7 days to determine extent of fat necrosis.

Results: All flaps survived. Consistent amounts of firmness, minimal amounts of histologic fat necrosis and inflammation were noted.

Conclusions: This model consistently produces clinical and histologic changes seen in the clinical scenario. Because of the reliability and reproducibility of this model, it can be used to evaluate techniques such as medical stimulation of angiogenesis, control of inflammation, or surgical augmentation of the vascular supply to prevent or treat clinical fat necrosis.

INTRODUCTION

Fat necrosis is an unresolved problem facing the plastic surgeon. It can occur anywhere there is adipose tissue. Although it can occur spontaneously in unmanipulated body fat,1 it is more common after trauma or surgical procedures. A certain degree of fat necrosis occurs in virtually all cases of free fat graft autotransplantation.2 In pedicled flaps such as the transverse rectus abdominis musculocutaneous (TRAM) flap commonly used for breast reconstruction, fat necrosis occurs in 12% to 35% of cases.3,4

With specialized mammographic techniques of the flap, this figure has been reported as high as 40%.5

Fat necrosis has been defined as the formation of a small firm area (or areas) of scar tissue caused by ischemic necrosis of subcutaneous fat in the absence of necrosis of the overlying skin.3 However, it has also been stated that hyperemia of adipose tissue promotes depletion of fat cells and that reduction in volume of fat grafts is in part ascribable to hypervascularization during the healing process.6 Attempts to study this apparent contradiction or to develop possible therapies for the prevention or treatment of fat necrosis is difficult in the clinical setting, but no good reliable experimental animal model of fat necrosis mimicking the clinical situation exists. A pedicled fat flap of the inguinal and hypogastric fat pad isolated on a vascular pedicle consisting of the femoral and superficial inferior epigastric arteries and veins has been used to study fat necrosis.7 This model lacks overlying skin and does not correlate to the clinical scenario. A rat rectus abdominis musculocutaneous flap was reported based on either the superior or inferior epigastric vessels.8 When a single artery was included in the pedicle and the flap placed outside of the abdominal skin, this model serves well to study partial skin loss of the flap and its prevention or treatment.9 The rat has little subcutaneous fat and, therefore, this model does not provide a mechanism to study fat necrosis.

Two genetic rodent models of obesity, the leptin receptor-negative fa/fa Zucker rat and the leptin-deficient ob/ob mouse have excessive adipose tissue mass and could serve as models to study fat necosis.10 We have used one of these rodents, the fa/fa Zucker rat, to develop a reliable experimental model of fat necrosis occurring in pedicled flaps.

MATERIALS AND METHODS

Genetically obese leptin receptor-negative fa/fa Zucker rats were purchased commercially (Harlan Sprague-Dawley, Inc., Indianapolis, IN). The rats weighed 250 to 350 g, were 7 to 8 weeks in age, and of both genders. The rats were housed on a 12-h light/dark cycle and given food and water ad libitum. All experimental protocols involving the fa/fa Zucker rats were approved by The Animal Care Use Committee of the Department of Veterans Affairs Medical Center, Bay Pines, Florida.

All operations on the rats were performed under intraperitoneal sodium pentobarbital anesthesia, 35 mg/kg body weight, using aseptic surgical techniques. Once the animals were anesthetized, the abdomens were shaved and the skin prepared with povidone-iodine and 70% alcohol. The transverse rectus abdominis musculocutaneous flaps were designed and elevated as described by Dunn et al.8 and the flap placed in an overlay position modified from the position suggested by Clugston et al.9 Specifically, a hexagonally shaped template 5 cm by 2.5 cm at its greatest dimensions was used to outline the proposed flap centered on the umbilical dimple (Fig. 1). The tips of the template typically reached the midaxillary line bilaterally. The flap was raised as a unipedicle superiorly based flap attempting to capture the maximal number of periumbilical musculoskeletal perforating vessels. Flap elevation included part of the rectus abdominis muscle and anterior rectus sheath encompassing the flap’s vascular pedicle. The remainder of the muscle and the posterior rectus sheath was approximated with absorbable sutures. The flap was overlayed superficial to the approximated fascial layer and the skin was closed with a single layer of continuous nylon sutures insetting the flap to its original template shape and dimensions.

Postoperatively, the flaps were monitored for color changes and palpable firmness at 24 hours, 48 hours, and 7 days. On postoperative day 7, the animals were killed and the fat harvested from the flaps, fixed, sectioned, and stained with hematoxylin and eosin (H&E). The histologic sections were evaluated by a blinded pathologist to determine the degree of fat necrosis and flap viability. A standard measuring grid was used in all assessments. The representative biopsies were graded on a six-point scale for inflammation and a six-point scale for fat necrosis. The inflammation scale ranked the specimens from 0 (no inflammation) to 5 (severe multicellular inflammatory changes in greater than 75% of the specimen). Similarly, fat necrosis was ranked 0 to 5 with 0 being normal healthy fat and 5 being severe necrosis and liquefaction in over 75% of the specimen (Table 1). Examples of the biopsies are shown in Figure 2 of the various sections evaluated for each flap, a mode number was chosen for the inflammation scale and the fat necrosis scale.

RESULTS

Transverse rectus abdominis musculocutaneous flaps were created on 10 fa/fa Zucker rats. All 10 of the flaps survived. Eight of the flaps showed no color changes during the period of observation and two flaps showed cyanosis of the skin paddle in the area lateral to the contralateral rectus muscle border. All 10 flaps were firm to palpation throughout at the three observation time points (Table 2). One rat (No. 4) had the contralateral tip become dark and quite hard on day 7.

Histologically, a degree of fat necrosis occurred in all 10 flaps. The mode ranking for both the inflammation scale and the fat necrosis scale was remarkably consistent for the 10 rats (Table 3). Evaluating the 10 flaps as a group, the mode inflammation rank was 3 and the mode fat necrosis rank was 4.

DISCUSSION

Fat necrosis remains a difficult problem in plastic surgery. Its occurrence appears to be unpredictable and its location within the subcutaneous adipose tissue random. When occurring in a reconstructive flap, it can range from a relatively minor spontaneously resolving complication to a major problem resulting in infection and liquefaction which requires surgical drainage.3 Attempts to prevent or treat fat necrosis has been difficult because of a lack of a reproducible animal model that mimics the human condition. The ability to systematically and objectively measure fat necrosis in an animal model is the first step in determining causes and solutions to this problem.

The availability of two genetically obese rodent models with abundant subcutaneous fat presented an opportunity to develop a reliable model for human fat necrosis. The ob/ob mouse is too small to raise a significant musculocutaneous flap. However, the larger fa/fa Zucker rat combined with the recently reported rodent transverse rectus abdominis musculocutaneous flap has resulted in a dependable model mimicking the clinical scenario. The Zucker rat model concentrates on fatty tissue with a lower metabolic demand and more precarious blood supply. These factors in combination allow for a greater susceptibility to ischemia and fat necrosis development.

The consistency of both the inflammatory changes and the fat necrosis in this model provides an opportunity to study possible preventative or therapeutic measures for fat necrosis. It could be used to surgically augment or deprive vascular supply to the flap. It could also be used to evaluate the response to various medications. Compounds could be tested to increase local blood supply, stimulate angiogenesis, or decrease inflammation. Results from such experimental manipulations of this model could then be tested in the human to attempt to alleviate problems associated with the complication of fat necrosis.

REFERENCES

1. Kiryu H, Rikihisa W, Furue M. Encapsulated fat necrosis: A clinicopathological study of 8 cases and a literature review. J Cutan Pathol 27:19–23, 2000.

2. Billings E, May JW. Historical review and present status of free fat graft autotransplantation in plastic and reconstructive surgery. Plast Reconst Surg 83:368–381, 1989.

3. Kroll SS, Gherardini G, Martin JE, et al. Fat necrosis in free and pedicled TRAM flaps. Plast Reconstr Surg 102:1502–1507, 1998.

4. Jewell RP, Whitney TM. TRAM fat necrosis in a young surgeon’s practice: is it experience, technique, or blood flow? Ann Plast Surg 42:424–427, 1999.

5. Eidelman Y, Liebling RW, Buchbinder S, et al. Mammography in the evaluation of masses in breasts reconstructed with TRAM flaps. Ann Plast Surg 41:229–233, 1998.

6. Smahel J. Adipose tissue in plastic surgery. Ann Plast Surg 16:444–453, 1986.

7. Goldberg I, Knight KR, Mahoney JE, O’Brien BM. A biochemical and histological comparison of vascularized and free fat grafts in the rabbit. Ann Plast Surg 30:334–340, 1993.

8. Dunn RM, Huff W, Mancoll J. The rat rectus abdominis myocutaneous flap: A true myocutaneous flap model. Ann Plast Surg 31:352–357, 1993.

9. Clugston PA, Perry LC, Fisher J, Maxwell GP. A rat transverse abdominis musculocutaneous flap model: Effects of pharmacological manipulation. Ann Plast Surg 34:154–161, 1995.

10. Alvarez B, Carbo N, Lopez-Sorano J, et al. Effects of interleukin-15 (IL-15) on adipose tissue mass in rodent obesity models: Evidence for direct IL-15 action on adipose tissue. Biochim Biophys Acta 1570:33–37, 2002.

Figure 1. Design of transverse rectus abdominis musculocutaneous flap on a genetically obese fa/fa Zucker rat.

Figure 2. Examples of histological sections of flap adipose tissue seven days postoperatively (40¥ magnification). A) normal fat of subcutaneous adipose tissue prior to raising of flap (0 on inflammation scale, 0 on fat necrosis scale); B) biopsy demonstrating minimal inflammatory infiltrate consisting of acute and chronic inflammatory cells and minimal fat necrosis (1 on inflammation scale, 1 on fat necrosis scale); C) section demonstrating 25–50% infiltration by inflammatory cells and marked (50–75%) fat necrosis (3 on inflammation scale, 4 on fat necrosis scale); and D) section demonstrating >75% multicellular inflammatory changes and necrosis of all fat cells (5 on inflammation scale, 5 on fat necrosis scale)

Table 1. Scales for Inflammation and Fat Necrosis in Flap Adipose Tissue

Inflammation Fat Necrosis

0 no inflammation 0 normal adipose tissue

1 minimal inflammation 1 minimal fat necrosis

2 inflammation < 25% of section 2 fat necrosis < 25% of section

3 inflammation > 25% to < 50% of section 3 fat necrosis > 25% to < 50% of section

4 inflammation > 50% to < 75% of section 4 fat necrosis > 50% to < 75% of section

5 inflammation > 75% of section 5 fat necrosis > 75% of section

Table 2. Clinical Observations on TRAM Flaps

Table 3.

Rat Observation

No. Time Color Softness

1 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

2 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

3 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

4 24 h Cyanosis tip Firm

48 h Cyanosis tip Firm

7 d Dark tip Hard

5 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

6 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

7 24 h Cyanosis tip Firm

48 h Cyanosis tip Firm

7 d Cyanosis tip Firm

8 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

9 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

10 24 h No changes Firm

48 h No changes Firm

7 d No changes Firm

Rat Inflammation Scale Fat Necrosis Scale

No. Rank (mode) Rank (mode)

1 3 4

2 3 4

3 2 4

4 4 4

5 3 4

6 3 4

7 4 5

8 3 4

9 5 5

10 3 4

group mode 3 4