A New Technique for Development of Rabbit Ear Keloid Model

Abdul Mannan Babar1* and Abdul Hannan Nagi2

1Department of Surgery, Postgraduate Medical Institute, Ameeruddin Medical College, Lahore General Hospital, Lahore, Pakistan

2Department of Pathology, Experimental Research Laboratory, University of Health Sciences, Lahore, Pakistan

ABSTRACT

Keloids and hypertrophic scars are an exaggeration of fibroplasia in dermal repair. Keloids are found solely in humankind, and not in animals. Thus far no practicable animal model of Keloid has been made. Recently a prototype rabbit ear keloid model has been produced by the authors by giving Transforming Growth Factor Beta 1 injection, and then skin punch excision on rabbit ear. In the present follow-up study consisting of eight refind techniques, best Keloid was produced by the technique where Transforming Growth Factor Beta 1 injection was followed by ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium punch excision, leaving behind only dorsal skin.

Article Information

Received 03 August 2016

Revised 07 September 2016

Accepted 14 September 2016

Available online 11 January 2017

Authors’ Contributions

Both the authors have made equal contributions in designing the study, experimental work and writing the article.

Key words

Cicatrix, Hypertrophic, Keloid, Transforming Growth Factor beta 1.

* Corresponding author: ambabar@gmail.com

0030-9923/2016/0004-1161 $ 8.00/0

Copyright 2016 Zoological Society of Pakistan

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.1.sc8

Keloids and hypertrophic scars are an exaggeration of fibroplasia in dermal repair. Keloids are basically benign tumours of skin. Hypertrophic scars represent a related entity, but they do not spread to surrounding tissue as do keloids (Rabello et al., 2014). Keloids and hypertrophic scars form as a consequence of wounds. They may form months or years after initial insult. They vary in size, and minor wounds can produce major keloids (Carmassi et al., 2015). Precise cause of keloids and hypertrophic scars is unknown. Nevertheless several causes are considered as risk factors. They may be internal or external (Babar, 2015a, b).

Keloids occur solely in humankind, and not in animals. This is the major obstacle against systematic enquiry on these lesions, as primary studies on humankind are not practical, and not ethical. Research on keloids has typically been clinical, with inadequate objective evidence. Thus far no practicable animal model of keloid has been made. Recently a prototype rabbit ear keloid model has been produced by the authors by giving transforming growth factor beta 1 injection, and then skin punch excision on rabbit ear (Nagi and Babar, 2015; Babar and Nagi, 2016).

Many types of rabbit ear hypertrophic scar have been produced until now by 5 mm (Xiao and Xi, 2013) and 6 mm circular ventral skin excision (Morris et al., 1997; Li et al., 2001), 6 mm circular dorsal skin excision (Li et al, 2001), 7 mm circular ventral skin excision (Kryger et al., 2007; Wang et al., 2015), 10 mm circular ventral skin excision (Diao et al., 2013), 15 mm x 45 mm rectangular ventral and dorsal skin excision (Li et al, 2001), 20 mm x 50 mm rectangular ventral skin excision (Xiang et al., 2004), 7 mm and 10 mm circular ventral skin and perichondrium excision (Jia et al., 2011), 7 mm circular ventral skin and perichondrium excision and nicking of cartilage (Kloeters et al., 2007), 6 mm circular ventral skin excision along with excision of two main sensory nerves (Yagmur et al., 2011) and circular ventral skin excision and applying pathogen-associated molecular patterns (PAMPs) of dead Pseudomonas aeruginosa and damage-associated molecular patterns (DAMPs) of homogenized dermis sample (Qian et al., 2015). These all are induced models. The purpose of this study was to make an ideal rabbit ear keloid model.

Material and methods

This was a quasi-experimental study done at Experimental Research Laboratory. Its duration was twelve months. Sample comprised of eight New Zealand White rabbits. Sampling technique was simple convenience.

Anaesthesia to rabbits was given with mixture comprising Ketamine and Xylazine. Study zone was cleaned using normal saline, and ethyl alcohol swab prior to creation of wound.

Eight rabbits were each given injection of Transforming Growth Factor Beta 1 (TGFB1) (Santa Cruz Biotechnology, 10410 Finnell Street Dallas, Texas 75220, USA; Tel +1-8004 57-3801; email: scbt@scbt.com; URL: www.scbt.com) 100 pg in 0.1 ml water, prepared in insulin syringe.Then one of the following eight different surgical techniques of punch excision was used: (a) ventral skin excision, (b) dorsal skin excision, (c) ventral skin and ventral perichondrium excision, (d) dorsal skin and dorsal perichondrium excision, (e) ventral skin, ventral perichondrium, and cartilage excision, (f) dorsal skin, dorsal perichondrium, and cartilage excision, (g) ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium excision, and (h) dorsal skin, dorsal perichondrium, cartilage, and ventral perichondrium excision.

Skin punch excision was done using a 4 mm skin biopsy punch (Acu-Punch; Acuderm Inc., 5370 NW, 35 Terrace, Ft. Layderdale, FL 33309, USA; Tel +1-8003 27-0015; URL: www.acuderm.com). It is a pre-sterilized prepacked single-use skin punch biopsy gadget, which has a circular sharp edge acting as a knife. When pressure is applied at its back end, and circular movements are given, it cuts a disc of skin and underlying cartilage if required. Perichondrium is cut with a sharp pointed scissors. If there is bleeding, it is stopped with cotton gauze pressure for a short time.

Wounds were cleaned with sterilized gauze and dried with hot air dryer. Dressing was done with hydrocolloid sheet. Usual post-operative care was given. Standard wound care was given. Hydrocolloid sheet was permitted to drop off on its own. Results were recorded at twenty eight days.

Results and discussion

Rabbits were inspected at twenty eight days. Out of eight rabbits, best Keloid was created by technique (g) where injection of TGFB1 was given, followed by ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium punch excision, leaving behind only dorsal skin. Gross height of this keloid was 1.355 mm. Scar elevation index (SEI) was calculated to be 3.45 (Table I).

Keloid was a clinically substantial. Its consistency was hard. It was dome-shaped, and colour was red. It had smooth surface, and there were no hair on it. Even after follow-up of one year, keloid was still present (Fig. 1A).

On gross examination, keloid specimen was hard, and was palpated as distinct nodule. Its colour was red, and surface smooth. On dissection, there was a gritty sensation. Colour of cut surface was pale. Microscopically, keloid depicted homogeneous, brilliantly eosinophilic, haphazardly dispersed, glassy, hyalinized collagen. Fibroblasts were scarce, and lied parallel to collagen (Fig. 1B).

In this study, the authors used eight different techniques to find out ideal method for production of rabbit ear keloid model. Out of eight techniques, best keloid was produced by the technique where TGFB1 injection 1 was followed by ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium punch excision, leaving behind only dorsal skin.

TGFB1 is a strong chemotactic factor for fibroblasts and excites them to build main extracellular matrix constituents like collagen. It is manufactured by macrophages, and also by extracellular matrix itself. It is formed in greater amounts in keloids and keloid fibroblasts react to lesser amounts of TGFB1 than non-keloid fibroblasts. Adding exogenous TGFB1 results in stimulation of fibroblasts, which enhances secretion of collagen, and ultimately production of keloids.

Table I.- Size of keloids produced by different techniques.

Technique Code | Tissues excised with 4 mm Skin Punch, after injection of 100 pg TGF Beta 1 | Gross Keloid Height (mm) | Ranking No
a | Ventral skin | 0.512 | 8
b | Dorsal skin | 0.550 | 7
c | Ventral skin and ventral perichondrium | 0.939 | 6
d | Dorsal skin and dorsal perichondrium | 0.954 | 4
e | Ventral skin, ventral perichondrium, and cartilage | 1.049 | 3
f | Dorsal skin, dorsal perichondrium, and cartilage | 1.253 | 2
g | Ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium | 1.355 | 1 (Best)
h | Dorsal skin, dorsal perichondrium, cartilage, and ventral perichondrium | 0.918 | 5

Table II.- Comparison of techniques of production of rabbit ear keloid models.

Author | Babar and Nagi, (2016) | Morris et al. (1997) | Li et al. (2001) | Kloeters et al. (2007) | Kryger et al. (2007) | Jia et al. (2011) | Diao et al. (2013) | Xiao and Xi (2013) | Wang et al. (2015)
Parameter
Clinical
Duration | 365+ days | 288 days | 262 days | NR | NR | NR | NR | 180 days | NR
Gross
Scar elevation index (SEI) | 3.45 | 2.72 | NR | 1.70 | 1.67 | 2.10 | 2.07 | 1.73 | 2.93
Texture | Hard | Palp able | NR | NR | NR | Protr usive | Palp able | NR | NR
Microscopic
Hyalinized collagen | Abundant | Absent | NR | Absent | Absent | NR | Absent | Absent | Absent
Fibro asts | Scant | NR | Increa sed | NR | Decre ased | NR | NR | NR | NR

NR, Not reported.

Our technique of production of rabbit ear keloid model is superior to previous techniques, as keloids produced by us had maximum scar elevation index, hard texture, long duration, abundant hyalinized collagen, and scant fibroblasts. Hyalinized collagen, which is pathognomonic, characteristic and hallmark of keloid was not reported by any of the previous authors. Morris et al, whose technique has been considered as gold standard so far, reported: ‘No hyalinized collagen such as seen in keloids was present.’ Comparison with previous keloid production techniques is given in Table II.

Conclusion

Best rabbit ear Keloid model can be produced by giving TGFB1 injection, followed by ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium excision

Acknowledgment

This study was funded by Higher Education Commission, Govt. of Pakistan, Islamabad, vide Project No. 20-1427/R&D/09.

Conflict of interest statement

We declare that we have no conflict of interest.

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