Role of autophagy and apoptosis in wound tissue of deep second-degree burn in rats

Abstract

Objectives: The pathogenesis of burn wound progression is poorly understood. Contributing factors include continuous loss of blood perfusion, excessive inflammation, and elevated apoptosis levels in wound tissue. Macroautophagy (here referred to simply as "autophagy") is associated with many chronic diseases. The authors hypothesized that autophagy is involved in burn wound progression in a rat model of deep second-degree burn.

Methods: Deep second-degree burns were modeled using a brass rod heated to 100°C applied for 6 seconds to the back skin of Wistar rats. Full-thickness biopsies were obtained from burned and nonburned controls at several times postburn. Western blotting and immunohistochemical (IHC) staining determined expression of the autophagy markers Light Chain 3 (LC3) and beclin-1. Apoptosis was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and laser Doppler flowmetry (LDF)-measured tissue perfusion. Myeloperoxidase (MPO) activity assay measured inflammation. Hematoxylin and eosin (H&E) and Masson's trichrome staining-determined pathology and wound depth.

Results: The LC3 and beclin-1 protein level in burn wounds decreased to one-fourth of normal levels (p<0.01) over 24 hours and then began to increase but still did not reach their normal level. TUNEL-positive cells in burn wounds were 3.7-fold (p<0.01) elevated over 48 hours and then decreased slightly, yet still remained higher than in normal skin. The burn wound progressed in depth over 72 hours. In addition, significant decrease in LDF values and upregulation of MPO activity were observed. Enhanced LC3-positive cells were observed in the deep dermal layer of burn wounds as shown by IHC staining.

Conclusions: A reduction in autophagy and blood flow and an increase in apoptosis and inflammation were observed in burn wounds early during the course of burn injury progression. This suggests that autophagy, complemented by apoptosis, play important roles in burn progression. Enhanced autophagy in the deep dermis may be a prosurvival mechanism against ischemia and inflammation after burn injury.

Figure 1

Western blot (WB) showing the expression of LC3 and beclin‐1 protein in wound and normal tissue at different time points postburn (A). Expression of LC3II/I and beclin‐1 in the burn wound tissue was lower than in normal skin (B, C). Data are presented as means ± SE (burn vs. normal control, *p < 0.05, **p < 0.01; n = 8 per group). LC3 = Light Chain 3.

Figure 2

TUNEL staining of normal skin (A) and burn wound tissue 24 (B), 48 (C), and 72 (C) hours postburn. Cells with shrunken brown stained nuclei were considered positive (black arrows). The TUNEL‐positive cells were mainly observed in the epithelium of hair follicles or blood vessels (A‐D). Quantitative analysis demonstrated that TUNEL‐positive cells in burn wounds were elevated about 3.7‐fold over 48 hours and then decreased slightly, yet remained higher than in normal skin (E; scale bars = 50 μm, burn vs. normal control, *p < 0.05, **p < 0.01; n = 8 per group). TUNEL = terminal‐deoxynucleoitidyl transferase mediated nick end labeling.

Figure 3

H&E and Masson staining of normal skin and burn wounds. Masson staining was a good indicator of burn wound depth. The red dye intensity reflects the tissue necrosis boundary.16 The red dye intensity of burn wounds at 48 and 72 hours postburn (C, D) was markedly higher than that at 6 (A) and 24 hours postburn (B), indicating progression of wound depth with time. Fewer residual cutaneous appendages and more severe collagen denaturation were seen at 48 and 72 hours (C, D) than at 6 or 24 hours (A, B). H&E staining of burn wounds at 6 (E) and 48 hours postburn (F) showing more profuse inflammatory infiltrates at 48 hours. (G, H) Masson and H&E staining of normal skin. (A‐D, G, H scale bars = 200 μm; E, F scale bars = 100 μm). H&E = hematoxylin and eosin.

Figure 4

LDF values and MPO activity in burn wounds and normal skin. (A) LDF values in the burn wounds were significantly decreased compared to the control group. LDF values in the burn wounds decreased to one‐fourth of normal level over 12 hours and then began to rise but remained below (p < 0.01) the baseline normal level. (B) MPO activities in burn wounds were significantly increased compared with the control. The levels of MPO activity in burn wounds were increased 5.3‐fold over 48 hours and then decreased but were still far higher (p < 0.01) than in normal tissue. Alterations in the LDF value were significant at all the time points postburn. Changes in the levels of MPO activity between burn and control groups were significant at the time points of 24, 48, and 72 hors postburn. The values described herein were mean ± SE (burn vs. control group, *p < 0.05, **p < 0.01, n = 8 per group). LDF = laser Doppler flowmetry; MPO = myeloperoxidase.

Figure 5

IHC staining of LC3 (black arrows, cells with brown cytoplasm) in the deep dermis of normal skin (A) and burn wounds (B‐D). (B) 24 hours postburn; (C) 48 hours postburn; (D) 72 hours postburn. At all time points after burn, the cells expressing LC3 were increased in the deep dermis of burn wound tissue compared to that of control. Scale bars = 50 μm. (E) Quantitative analysis of the immunohistochemical staining showed that the expression of LC3 in the deep dermis of burn wounds was significantly higher than that of the control at all time points (p < 0.01 at 24 and 72 hours; p < 0.05 at 48 hours postburn). The values herein were mean ± SE (burn vs. normal control, *p < 0.05, **p < 0.01, n = 8 per group). IHC = immunohistochemical; LC3 = Light Chain 3; LDF = laser Doppler flowmetry.