Ischemia-reperfusion injury in a rat microvascular skin free flap model: A histological, genetic, and blood flow study

Abstract

Ischemia reperfusion injury is associated with tissue damage and inflammation, and is one of the main factors causing flap failure in reconstructive microsurgery. Although ischemia-reperfusion (I/R) injury is a well-studied aspect of flap survival, its biological mechanisms remain to be elucidated. To better understand the biological processes of ischemia reperfusion injury, and to develop further therapeutic strategies, the main objective of this study was to identify the gene expression pattern and histological changes in an I/R injury animal model. Fourteen rats (n = 7/group) were randomly divided into control or ischemia-reperfusion group (8 hours of ischemia). Microsurgical anastomoses were objectively assessed using transit-time-ultrasound technology. Seven days after surgery, flap survival was evaluated and tissue samples were harvested for anatomopathological and gene-expression analyses.The I/R injury reduced the survival of free flaps and histological analyses revealed a subcutaneous edema together with an inflammatory infiltrate. Interestingly, the Arginase 1 expression level as well as the ratio of Arginase 1/Nitric oxide synthase 2 showed a significant increase in the I/R group. In summary, here we describe a well-characterized I/R animal model that may serve to evaluate therapeutic agents under reproducible and controlled conditions. Moreover, this model could be especially useful for the evaluation of arginase inhibitors and different compounds of potential interest in reconstructive microsurgery.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Experimental design.

Fig 2. Flap model.

A) Ligatures of lateral circumflex femoral artery and vein. B) Ligatures of proximal caudal femoral artery and vein. C) Ligatures of saphenous artery and vein. Dotted lines represent the microsurgical anastomoses sites.

Fig 3. Tissue sampling diagram.

The quadrants Q1 were excised and fixed in 4% paraformaldehyde for further histological analysis. The quadrants Q4 were cryopreserved at -80°C for gene expression analysis.

Fig 4. Comparison of skin flap survival areas between groups by Student’s t-test (**p

Fig 5. Representative histological images.

At day 7 post-surgery, the skin flaps of euthanized animals were fixed in 4% paraformaldehyde, paraffin-embedded, and stained for hematoxylin-eosin (H&E) and Masson's trichrome (MT). Horizontal bars represent 100 μm. The different skin layers are numbered: epidermis (1), dermis (2) and adipose tissue (3).

Fig 6. Quantitative expression of ischemia-reperfusion-related genes.

At day 7 post-surgery, total RNA from the skin flaps was isolated and qRT-PCR products were quantified by the 2-ΔCt method. Graphs represent the mean ± SD of independently performed experiments. The results were organized by groups of genes related with the following biological processes: A) Inflammatory response, B) Oxidative stress, C) Angiogenesis, and D) Necrosis/Apoptosis. Data were statistically analyzed using Student’s t-test for variables with a parametric distribution and Mann-Whitney’s U Test for non-parametric variables. Horizontal bars represent statistically significant differences (**p<0.01).