Tushar Ramesh1#, Lindsay S Moore2#, Neel Patel3, Kiranya Tipirneni4, Jason M Warram2,5, Jillian R Richter4, Erika M Walsh2, Geoffrey P Aaron2, Anthony B Morlandt6, Brian B Hughley2 and Eben L Rosenthal7*
1University of Alabama School of Medicine, Birmingham, AL, USA
2Department of Otolaryngology, University of Alabama at Birmingham, Birmingham, AL, USA
3Department of Psychiatry, University of Alabama at Birmingham, Birmingham, AL, USA
4Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA
5Department of Radiology, University of Alabama at Birmingham, Birmingham, AL, USA
6Department of Oral and Maxillofacial Surgery, University of Alabama at Birmingham, Birmingham, AL, USA
7Department of Otolaryngology, Stanford University, Stanford, CA, USA
#These authors contributed equally
Received: 13 April, 2017; Accepted: 19 April, 2017; Published: 21 April, 2017
Eben L Rosenthal, Professor of Otolaryngology and Radiology, Ann & John Doerr Medical Director, Stanford Cancer Center, Stanford, CA 94305, USA, Tel: (205) 934-9713; E-mail:
Ramesh T, Moore LS, Patel N, Tipirneni K, Warram JM, et al. (2017) Effects of Neoadjuvant Chemotherapy and Radiotherapy on Flap Perfusion in a Novel Mouse Model Using Standard Clinical Assessment and Near-Infrared Fluorescence Angiography. Arch Otolaryngol Rhinol 3(2): 038-042. DOI: 10.17352/2455-1759.000042
© 2017 Ramesh T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Reconstructive surgical procedure; Surgical flaps; Head and neck neoplasms; Radiation; Antineoplastic agents
Purpose: Minimizing surgical morbidity after local flap reconstruction is important in the management of cutaneous defects. Controversy exists in current literature regarding the effects of radiation and chemotherapy on flap perfusion. Neoadjuvant treatments can damage the microvasculature of the surgical bed through fibrosis, endothelial cell damage, and reduced cell proliferation, which collectively increase the likelihood of postoperative flap failure. The aim of this study is to examine the effects of neoadjuvant radiation and chemotherapy on skin flap perfusion.
Methods: Animals were divided into three groups: control (no treatment, n=4), radiation group (36-Gy administered to dorsal skin, n=4), and chemotherapy group (2 mg/kg IP cisplatin, n=4). Treatments were performed 15 days prior to random-pattern dorsal flap surgery, with a flap length-to-width ratio of 4:1 (4x1cm2). Flap perfusion was assessed via laser-assisted (Luna, Novadaq) indocyanine green dye angiography and standard clinical assessment.
Results: Fluorescence imaging was used to quantify flap perfusion as a fraction of healthy skin perfusion. Chemotherapy group flaps had poorer distal end perfusion than radiation or control group flaps (56% vs. 69% and 71%, respectively) on post-operative day (POD) 1. Clinical assessment of flap perfusion by experienced surgeons on POD2 found chemotherapy group flaps to be least viable. By POD5, 100% of chemotherapy group flaps had experienced complete flap loss as measured by clinical evaluation and perfusion imaging.
Conclusion: Flaps receiving neoadjuvant chemotherapy performed worse than those receiving local radiotherapy or no treatment. We demonstrate the detrimental effect of neoadjuvant chemotherapy on flap viability in this preclinical murine model.
The clinical management of locally advanced cancers of the head and neck pose a significant therapeutic challenge . Despite advances in the field, including the routine use of microvascular free tissue transfers in the reconstruction of defects, flap failure persists as a troublesome complication 2. The underlying factors that influence the development of flap failure are varied and complex, including comorbid systemic diseases, positive surgical margins, lymph node metastases, and prior radiotherapy and/or chemotherapy [1-3].
Radiation and chemotherapy are often administered neoadjuvantly, or prior to surgical intervention, to gauge the degree of tumor response to treatment [3,4] as sufficient downstaging through neoadjuvant treatments may allow an inoperable tumor to become a feasible candidate for surgical treatment . Additionally, therapy-induced tumor regression may also allow for a reduction in wound size and the sparing of critical neighboring structures . There is concern, however, that neoadjuvant therapies may lead to acute vascular injury, which may, in turn, increase the incidence of flap failure [5,6]. This may be due to capillary flow aberrations  and angiogenic alterations , predisposing flaps to poor perfusion and subsequently to failure.
Clinically, poor perfusion in a flap often signals the need for operative flap revision or salvage surgery. The current standard-of-care relies on qualitative markers of poor perfusion to guide surgeons in the decision-making process. This approach is subjective, and thus may be impacted by the surgeon’s level of experience and familiarity with the patient population, among other factors. Recent advances in near-infrared imaging technology have made it possible to assess perfusion in a quantitative manner. The LUNA fluorescence angiography system employs an indocyanine green (ICG) dye to allow for intraoperative and post-operative visualization of perfusion . Several groups have shown that perfusion assessments utilizing LUNA fluorescence angiography are reliable and accurate [9,10].
Random-pattern flaps with length-to-width ratios of 2:1 or less are reliably perfused throughout their length . Flaps with ratios greater than 2:1 have been shown to exhibit ischemia in distal regions due to decreased proximity to the pedicled end [12,13]. Studies conducted in rats bearing over-dimensioned 4:1 random-pattern flaps demonstrated ischemia in distal halves of flaps that, over time, led to flap necrosis in those regions [12,13]. In this study we create over-dimensioned 4:1 random-pattern local flaps in a murine model, anticipating a loss of perfusion in the distal halves of flaps. Our intention was to investigate the effects of neoadjuvant chemotherapy and radiotherapy on flap perfusion, which were evaluated by near-infrared imaging technology and standard clinical assessment.
Twelve female athymic nude mice were used in this study. Animals were caged in groups of 4 and fed a standard laboratory diet of food and water. Animal treatment and husbandry was in accordance with IACUC standards. Mice were divided into three treatment groups: control (no treatment, n=4), radiation group (36 Gy administered to dorsal skin, n=4), and chemotherapy group (2 mg/kg cisplatin, n=4). In the radiation group, mice received three 12 Gy treatments administered focally to dorsal skin flaps over a 6-day period. Cisplatin was administered intraperitoneally (IP). Mice were allowed a 15 day recovery period during which they were monitored daily for possible systemic side effects. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards (approved IACUC protocols) of the institution or practice at which the studies were conducted.
An X-Ray irradiator (XRAD320, Precision X-Ray, Inc. N. Bradford, CT, USA) designed for use with cell monolayers and small animals provided radiation therapy in this study. The XRAD320 was calibrated to administer a tube voltage of 320 kV and a tube current of 12.5 mA to generate high-energy electrons at a mean dose rate of 1.169 Gy/min to a total dose of 36 Gy. The Isovolt Titan (General Electric, Fairfield, CT, USA) was utilized to monitor exposure time, x-ray production voltage, and current; the Unidos dosimeter (PTW, Freiburg, Germany) recorded total radiation exposure as well as dose rate. A 20 mm thick aluminum collimator was used to effectively eliminate damaging low-energy x-ray exposure. For the procedure, anesthesia was induced using inhaled isoflurane or 2.5 ug/kg ketamine/xylazine. Animals were placed on either side of a shielded, radiation-safe box in the lateral decubitus position (Figure 1A). Target skin was exposed through a slit, allowing for focal delivery of radiation. The entire length of the flap was exposed to radiation treatment. Nose cones on either end ensured oxygen and isoflurane delivery for the duration of radiation exposure. A dosimeter was placed underneath the exposed skin to measure X-Ray dose. The dosimeter and radiation-safe box were secured to one another and to the base of the XRAD320 to insure homogeneity in radiation exposure among different treatment groups (Figure 1B). A three-eighths inch lead sheet was placed atop the radiation-safe box to minimize radiation exposure to the body of the mice (Figure 1).