Tight junction disruption through activation of the PI3K/AKT pathways in the skin contributes to blister fluid formation after severe tibial plateau fracture

Abstract

Background: Acute compartment syndrome (ACS) is an orthopedic emergency that commonly occurs after severe tibial plateau fracture. Fracture blisters form on the skin, and it was found in our previous study that when blisters form, the compartment pressure significantly decreases. However, the potential mechanism underlying this pressure decrease has not yet been elucidated. Methods: To obtain a comprehensive understanding of the changes that occur after blister formation on the skin, the changes in tight junction expression in the skin after tibial plateau fracture were observed. Blister samples and normal skin were collected from patients with bicondylar tibial plateau fractures with or without blisters. The epidermis thickness was measured, and the difference in the levels of K1, K5, K10, and skin barrier proteins such as claudin 1, claudin 2, and occludin between the two groups was evaluated by immunochemistry analysis, immunofluorescence, Western blotting, and qPCR. Results: The skin was thinner and the levels of K1, K5, and K10 were significantly decreased in blistered skin. Furthermore, the PI3K/AKT pathway was found to be activated, and the tight junction expression was significantly decreased in blistered skin. This indicates that the paracellular pathway, which is essential for accelerating fluid accumulation in blisters and indirectly decreases compartment pressure, was activated. Conclusion: Changes in the tight junction expression after blister formation may underlie blister fluid formation and indirectly explain the decrease in compartment pressure under blistered skin after severe tibial plateau fracture.

Keywords: acute compartment syndrome; fracture blisters; paracellular pathway; tibial plateau fracture; tight junctions.

FIGURE 1

(A) Anterior-posterior X-rays of a Schatzker VI tibial plateau fracture. (B) Lateral view of this fracture. (C) Fracture appearance with a serous-filled blister observed on the skin around the fracture site (proximal tibia) after admission. (D) Blister became larger after 1 day, and the patients were not subjected to fasciotomy. When a blister was observed, the intracompartmental pressure decreased significantly.

FIGURE 2

(A) Anterior-posterior X-rays of another Schatzker VI tibial plateau fracture. (B) Lateral view of this fracture. (C) Fracture appearance with a blood-filled blister. The blood-filled blister was larger than the serous blister observed in Figure 1. (D) Blister became larger after 1 day, and a similar phenomenon in which the intracompartmental pressure decreased after the appearance of the blood-filled blister was observed.

FIGURE 3

(A) Normal skin was stained with HE, and the cornified layer in the epidermal layer was intact. (B) Blistered skin was stained with HE and observed by light microscopy. The epidermal layer was disrupted (blue arrow) and separated from the dermis, with a small portion of the stratum basale retained (dotted yellow line), which explained the quick recovery of the blistered skin (red arrow). (C) Comparison of skin thicknesses in the two groups. CG: control group; BG: blister group.

FIGURE 4

Immunohistochemical images of the two groups with antibodies against cytokeratins K1, K5, and K10. The epidermal layer was disrupted and separated from the dermis, while a small portion of the stratum basale was retained in the BG. The cytokeratins were distributed nonhomogeneously in the residual middle layer of the epidermis. The intensity scores for K1, K5, and K10 in the two groups were compared, and the expressions of K1, K5, and K10 were significantly decreased in the BG. K1: cytokeratin 1, K5: cytokeratin 5, K10: cytokeratin 10. (Bars = 200 μm, *: p < 0.05, **: p < 0.01, and ***: p < 0.001).

FIGURE 5

(A–C) Immunofluorescence analysis of normal and blistered skin. Images of immunofluorescence staining of the cytokeratins K1, K5, and K10 in the two groups. In accordance with the immunohistochemical results, the number of positively expressing cells was compared for K1, K5, and K10, and the positive expression rate was significantly decreased in the BG. (D) Representative Western blots of cytokeratins K1, K5, and K10 in control and blistered human skin. It was observed that the expression of the three cytokeratins was significantly decreased in the BG group. *: p < 0.05, **: p < 0.01, and ***: p < 0.001).

FIGURE 6

Immunohistochemical images of PI3K and AKT in normal and blistered skin. The number of cells expressing PI3K or AKT in the two groups was compared, and the expression of PI3K or AKT increased significantly in blister skin compared with normal skin. (Bars = 200 and 50 μm, *: p < 0.05, **: p < 0.01, and ***: p < 0.001).

FIGURE 7

Immunofluorescence images of occludin, claudin 1, and claudin 2 in normal and blistered skin. The nuclei were stained with DAPI (blue), and red fluorescence indicates the lineage markers. The OCLN (A), CLDN1 (B), and CLDN2 (C) levels in each group were compared, and it was illustrated that the expression of these TJ markers decreased significantly in blister skin compared with normal skin. OCLN: occludin, CLDN1: claudin 1, CLDN 2: claudin 2. (Bars = 200 and 50 μm, *: p < 0.05, **: p < 0.01, and ***: p < 0.001).

FIGURE 8

(A) Representative Western blots of the proteins OCLN, CLDN1, and CLDN2 in control and blistered human skin. (B) Representative Western blots of the proteins PI3K, AKT, and P-AKT in control and blistered human skin. (C) Expressions of the OCLN, CLDN1, and CLDN2 proteins were significantly decreased and those of PI3K, AKT, and pAKT were significantly increased in blistered skin compared with normal skin. OCLN: occludin, CLDN1: claudin 1, CLDN 2: claudin 2. (*: p < 0.05, **: p < 0.01, and ***: p < 0.001). (D) mRNA expression levels of occludin and claudins 1 and 2 were measured by qRT–PCR, and it was found that occludin, claudin 1, and claudin 2 expressions were decreased in blistered skin. In contrast, the mRNA expression levels of PI3K, p-PI3K, and AKT were significantly increased in blistered skin. The results are expressed as the mean ± SEM. ***: p < 0.001.