Experimental model of cutaneous radiation injury in rabbits

1 - ORIGINAL ARTICLE

MODELS, BIOLOGICAL

Experimental model of cutaneous radiation injury in rabbits¹

Rafael Panisi de Campos Meirelles^I; Bernardo Hochman^II; Americo Helene Junior^III; Rute Lellis^IV; Murillo Francisco Pires Fraga^V; Lydia Masako Ferreira^VI

^IFellow Master degree, Postgraduate Program of Translational Surgery, Federal University of Sao Paulo/Paulista School of Medicine (UNIFESP/EPM), Sao Paulo-SP, Brazil. Resident at Faculty of Medical Sciences of Santa Casa of Sao Paulo. Conception, design, intellectual and scientific content of the study, acquisition of data, surgical procedures, manuscript writing

^IIPhD, Associate Professor, Division of Plastic Surgery, Department of Surgery, UNIFESP, Sao Paulo-SP, Brazil. Conception of the study, manuscript writing, critical revision

^IIIPhD, Associate Professor, Division of Plastic Surgery, Department of Surgery, Faculty of Medical Sciences of Santa Casa of Sao Paulo, Brazil. Manuscript writing, critical revision

^IVMD, Affiliate Professor, Division of Pathology, Faculty of Medical Sciences of Santa Casa of Sao Paulo, Brazil. Surgical procedures and pathological analysis

^VPhD, Affiliate Professor, Division of Plastic Surgery, Department of Surgery, Faculty of Medical Sciences of Santa Casa of Sao Paulo, Brazil. Surgical procedures, manuscript writing, critical revision

^VIPhD, Full Professor, Division of Plastic Surgery, Department of Surgery, UNIFESP, Sao Paulo-SP, Brazil. Coordinator Medicine III, CAPES, Researcher CNPq. Manuscript writing, critical revision

^{Correspondence}

ABSTRACT

PURPOSE: To describe an experimental model of cutaneous radiation injury in rabbits.

METHODS: On this study eight six-month-old New Zealand male rabbits, with an average weight of 2.5kg were used. They were distributed in four groups (n=2 per group). The control group did not receive radiotherapy and the others received one radiotherapy session of 2000, 3000 and 4500 cGy, respectively. Photographic analysis and histopathological evaluation of the irradiated areas were carried out.

RESULTS: After 30 days, the animals from the control group had all their hair grown. In spite of that, the animals from group 2000 cGy had a 60-day alopecia and from group 3000 cGy, a 90-day alopecia. After the 30th day, the 3000cGy group demonstrated 90-day cutaneous radiation injuries, graded 3 and 4. One of the animals from group 4500 cGy died on the 7th day with visceral necrosis. The other from the same group had total skin necrosis. A progressive reduction of glands and blood vessels count and an increase on collagen deposition was observed.

CONCLUSION: The proposed experimental model is reproductable. This study suggests that the dosage 4500cGy is excessive and the 3000 cGy is the most effective for this experimental model of cutaneous radiation injury in rabbits.

Key words: Radiation Injuries. Radiotherapy Dosage. Models, Animal. Rabbits.

Introduction

Around 60% of cancer patients receive radiotherapy during their treatment^1,2. The most common complication of radiotherapy is the cutaneous radiation injury. The lack of experimental models of cutaneous radiation injury is one of the research challenges³. There are some experimental models described for small animals (murine, rats and mice)³. In spite of that, there are no reproductable models in literature for medium size animals.

The rabbits, medium size animals, because of their immunological similarity to humans are the most frequently used for cutaneous tests such as the Draize test⁴. Their antibody development is considered resistant to radiation, differently than rats, mice and murines that aren't capable of presenting a similar immune response⁵.

A experimental model is a materialization of a reality and it is as similar as possible to the desired study^6-8. Therefore, the development of an efficacy treatment for radiodermitis requires a reproductable experimental model with animals immunologically similar to humans, such as rabbits.

Methods

All the procedures followed the protocol approved at the Ethical Committee: UNIFESP/EPM-0264/12.

Eight six-month-old New Zealand male rabbits, with an average weight of 2.5kg, were used. They were distributed in four groups (n=2 per group). The control group did not receive radiotherapy and the others received one radiotherapy session of 2000, 3000 and 4500 cGy.

The animals were anesthetized with Ketamine intramuscular (gluteal region) and the dose was 0.5 ml/kg.

The dorsal skin was epilated (10x10 cm) with an OSTER A5 hair removal - blade size 40-10mm. The borders of epilation were standardized (from the occipital region, a 10x10cm square was drawn, having the vertebral column as the mid line).

The dorsal skin was elongated and fixed between two wood sticks. The sticks were fixed with an elastic string, giving five laps on each side (Figure 1).

The radiotherapy was performed in pairs of animals. After anesthesia, with the elongated dorsal skin, they were positioned on lateral decubitus and fixed together with three nylon 4.0 sutures, with a 10cm distance between them. They were then fixed to the table with adhesive tape (Figure 2).

The radiation machine used was ALCYON II for Telecobaltotheraphy. The distance applied was 70cm and the Bolus used was 0.5cm. The radiation field was 5x10cm (width x length) and the dosages were 2000cGY, 3000cGY and 4500cGy. The procedures were performed by the same researcher, assisted by a veterinarian and a radiotherapist at Arnaldo's Institute at Faculty of Medical Sciences of Santa Casa of Sao Paulo.

Serial photography of the dorsal skin surface was performed to evaluate for macroscopic evidence of radiation injury on days 15, 30, 60 and 90 post radiotherapy. The images were analyzed and classified by the same blinded radiotherapist.

The radiodermitis is classified based on skin appearance^9-12. Cox et al.¹³ published the Acute Radiation Morbidity Scoring Criteria to classify the effects of radiotherapy, grading 0 (no reaction), 1 (light eritema, dry desquamation, epilation), 2 (moderate eritema, exudative dermatitis and moderate edema), 3 (exudative dermatitis, intense edema) and 4 (ulceration, necrosis).

The camera used was a NIKON D90 on macro and automatic feature, with a 30cm distance from the animal.

The dorsal skin was divided in four quadrants and skin biopsy specimens were taken from the zone of irradiation on day 15, 30, 60 and 90 after external beam radiation therapy and fixed in 10% formalin ( n=2 per group). Specimens were paraffin-embedded and sectioned for hematoxylin and eosin staining. The histopathological analysis was performed by the same blinded pathologist. Fibrosis was assessed by picrosirius red stain (collagen content). The 'point-counting' technique proposed by Gundersen et al.¹⁴ was used to quantify the number of blood vessels, sweat glands, sebaceous glands, hair follicles and collagen using a reticulum of 100 points and 50 lines. Five random and non-coinciding fields were examined, totalling 500 points per slide at a magnification of x200. Points that were not incident upon solid areas were not considered. The values were expressed in percentage of positive points, in the total of points in solid areas, according to the following formula: P points= (Pi x100)/P parenchyma; where P points is the corrected percentage of marked points, Pi is the number of points that are incident upon the positivity of the items measured and P parenchyma is the total number of points that are incident upon the solid area.

Results

After 30 days of radiotherapy, the control group animals had all their hair grown. In spite of that, the animals from group 2000 cGy had a 60-day alopecia and from group 3000 cGy, a 90-day alopecia. The irradiated area from group 3000cGy demonstrated radiodermitis graded 3 (exudative dermatitis, edema) and 4 (ulceration - Figure 3) at 60 day and at 30 day, respectively. One of the animals from group 4500 cGy died on the 7th day after radiotherapy, because of visceral necrosis. The other animal from the same group had total skin necrosis after 30 days of radiotherapy and was sacrificed.

The epilated area was classified based on the Oncology Radiotherapy Group Scale (grade 0-4: Figure 4)¹⁵.

Rabbits exposed to radiation doses greater than 2000 cGy developed progressive skin thickening. The skin was thickened and inelastic with areas of non-healing ulceration (Figure 3). The histopathological analysis demonstrated thickening of the dermis, with exuberant and progressive collagen deposition over time (Figure 5).

A progressive reduction of glands and blood vessels count and an increase on collagen deposition was observed (Table 1).

Discussion

Although new techniques and sources of radiotherapy have been developed, the cutaneous tolerance is frequently a limiting factor during the radiotherapy treatment³. The skin exposition to ionized radiation causes acute and chronic effects. During the acute phase, the inflammatory cells, such as macrophages, are triggered and eritema, exudative dermatitis and ulceration may appear. The chronic phase is known as a proliferative phase, with an increase of myofibroblasts. The combination of perivascular fibrosis is due to myofibroblasts activation and its effects to the endotelial cells may cause hypovascularization and chronic fibrosis^16-20. This fibrosis is characterized by excessive collagen deposition, with stromal encasement of blood vessels, loss of hair follicles, and dermal thickening^21,22. The progressive collagen deposition, skin thickening and alopecia were features also found in our study and mimic a human radiation injury.

The dosages used on this study were similar to the murine model described by Thanik et al.³ On their study, from the 25 treated animals, three in the highest dose groups (≥ 4500 cGy) had inadequate lead shielding, resulting in tail exposure with subsequent necrosis and infection, and died within the first week. On our study, both animals from group 4500 cGy did not have a desired outcome.

The alopecia was noticed on groups 2000 cGy and 3000 cGy. The alopecia noticed after radiotherapy is caused by the damage to follicular cells associated with fibrosis²³. After radiotherapy, the tissue may become thick, inelastic, hyperchromic and hairless. Effects are thought to be dose, technique, and location dependent²⁴. Our results demonstrate this dose dependency since the alopecia was more persistent on group 3000cGy.

This model accurately reproduces the changes seen in humans subjects from radiotherapy-induced injury. It was possible to observe and classify the irradiated area by photographic analysis. This classification was not previously described for an experimental study. In spite of that, it could be used, since the dosages were comparable to dose rates to human treatment protocols and the macroscopic changes were similar²⁵.

The establishment of a reliable rabbit model to mimic the radiation-injury and study its pathogenesis and potential treatment is essential. Although, studies on radiation injury in rabbits have been described previously, there is no established rabbit model for creating a reproducible cutaneous injury pattern. Using a rabbit model offers many advantages in the study of radiation - induced cutaneous injury. It is ease handling, low cost and the rabbit, has an adequate donor site of fatty tissue, the dorsum, differently than mice²⁶.

Conclusion

The proposed experimental model is reproductable. This study suggests that the dosage 4500cGy is excessive and the 3000 cGy is the most effective for this experimental model of cutaneous radiation injury in rabbits. Therefore, a study with a greater sample is required to determine the most satisfactory dosage for this model.

Received: July 11, 2013

Review: Sept 10, 2013

Accepted: Oct 14, 2013

Conflict of interest: none

Financial source: none

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1

Research performed at Laboratory Animal Facility, Faculty of Medical Sciences of Santa Casa of Sao Paulo and Plastic Surgery Division, Federal University of Sao Paulo/Paulista School of Medicine (UNIFESP/EPM), Brazil. Part of Master degree thesis, Postgraduate Program in Translational Surgery. Tutor: Lydia Masako Ferreira.

Publication Dates

• Publication in this collection
  05 Dec 2013
• Date of issue
  Nov 2013

History

• Received
  11 July 2013
• Accepted
  14 Oct 2013
• Reviewed
  10 Sept 2013