在大鼠切除创面愈合模型中进行伤口愈合的形态学评估

Lígia Reis de Moura Estevão; Puebla Cassini-Vieira; Ana Greice Borba Leite; Apolônia Agnes Vilar de Carvalho Bulhões; Lucíola da Silva Barcelos; Joaquim Evêncio-Neto

doi:10.21769/BioProtoc.3285

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Peer-reviewed

Morphological Evaluation of Wound Healing Events in the Excisional Wound Healing Model in Rats

在大鼠切除创面愈合模型中进行伤口愈合的形态学评估

Lígia Reis de Moura Estevão email

Puebla Cassini-Vieira

Ana Greice Borba Leite

Apolônia Agnes Vilar de Carvalho Bulhões

Lucíola da Silva Barcelos

JE Joaquim Evêncio-Neto

发布: 2019年07月05日第9卷第13期 DOI: 10.21769/BioProtoc.3285 浏览次数: 11631

评审: Gal HaimovichChaitali BasoleAnonymous reviewer(s)

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Abstract

Skin wound healing is a complex process involving different events such as blood coagulation, inflammation, new blood vessels formation, and extracellular matrix deposition. These events can be observed by using histology techniques. However, the lack of the standardization of such parameters impacts on the reproducibility of results. Here, we describe a protocol to perform macroscopic and microscopic analyses of the events that occur during skin wound healing using the experimental model of excisional wounds in rats.

Keywords: Rat excisional wound (大鼠切除创面)

Histological score (组织学评分)

Angiogenesis (血管生成)

Inflammation (炎症)

Collagen deposition (胶原沉积)

Epithelialization (上皮形成)

Background

Skin wounds can be induced by extrinsic factors such as surgical incision, accidental injury or trauma, or by intrinsic factors such as those produced by infection and chronic ulcers. The skin wound healing is a complex process involving cellular, molecular and humoral components to tissue reestablishment after injury. In healthy adult mammals, it involves an early inflammatory phase, characterized by infiltration of leucocytes, with subsequent formation of granulation tissue (new tissue formed), epithelization, and matrix remodeling (Balbino et al., 2005; Estevão et al., 2017). An accurate assessment of wound healing events is critical to reproducible data, with histological studies being the gold standard in this area of knowledge. Here, we describe a protocol using an excisional wound healing model in rats to demonstrate the wound creation, wound closure evaluation and qualitative analysis of microscopic events (inflammation, extracellular matrix deposition, angiogenesis, and epithelialization) in skin wounds.

Materials and Reagents

Glass slides (Micro Slides, VWR, catalog number: 48311-703)
Coverslip slides (IMEB, catalog number: CG1-2450)
Wistar rats (Rattus norvergicus albinus), 250-300 g, and 90-100 days old
Paraffin (Fisher Scientific, catalog number: AC416770020)
Ketamine hydrochloride (Sigma, catalog number: K113)
Xylazine hydrochloride (Sigma, catalog number: K4138)
Formaldehyde (Fisher Scientific, catalog number: SF100-4)
Ethanol (Fisher Scientific, catalog number: BP2818100)
Xylene (Fisher Scientific, catalog number: X3S-4)
Xylene-based mounting medium (Source Mount, catalog number: 9277722)
Hematoxylin (Merck, catalog number: 517-28-2)
Potassium alum (Merck, catalog number: 7784-24-9)
Mercury oxide (Merck, catalog number: 21908-53-2)
Eosin (Merck, catalog number: 15086-94-9)
Phloxine B (Merck, catalog number: 18472-87-2)
Acetic Acid (Merck, catalog number: 64-19-7)
Sodium phosphate dibasic (Merck, catalog number: 7558-79-4)
Sodium phosphate monobasic (Merck, catalog number: 7558-80-7)
Phosphotungstic acid hydrate (Merck, catalog number: 12501-23-4)
Chromotrope 2R (Merck, catalog number: 4197-07-4)
Fast green FCF (Merck, catalog number 2353-45-9)
Harris Hematoxylin (see Recipes)
Eosin (stock solution) (see Recipes)
Eosin phloxine (see Recipes)
10% buffered formaldehyde (see Recipes)
Gomori (see Recipes)

Equipment

Digital caliper (Digimess, King Tools, catalog number: 502.150BL)
Hair removal machine (Wahl and Toshico)
Tweezers 14 cm (Golgan, Fibra cirúrgica, catalog number: 003439)
Straight Metzenbaum Scissors (WPI, catalog number: 501747)
8 mm diameter circular biopsy punch (Kruuse, catalog number: 273693)
10 mm diameter circular biopsy punch (Integra Miltex, catalog number: 12-460-406)
LEICA^® manual microtome (LEICA, model: M2125RT)
Trinocular Microscope with HD camera (LEICA, model: DM500)
Histological Bath (Lupetec, model: BH2015)

Procedure

English

中文翻译

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Estevão, L. R. D. M., Cassini-Vieira, P., Leite, A. G. B., Bulhões, A. A. V. D. C., Barcelos, L. D. S. and Evêncio-Neto, J. (2019). Morphological Evaluation of Wound Healing Events in the Excisional Wound Healing Model in Rats. Bio-protocol 9(13): e3285. DOI: 10.21769/BioProtoc.3285.