Targeting Cx43 to Reduce the Severity of Pressure Ulcer Progression.

In the skin, repeated incidents of ischemia followed by reperfusion can result in the breakdown of the skin and the formation of a pressure ulcer. Here we gently applied paired magnets to the backs of mice to cause ischemia for 1.5 h and then removed them to allow reperfusion. The sterile inflammatory response generated within 4 h causes a stage 1 pressure ulcer with an elevation of the gap junction protein Cx43 in the epidermis. If this process is repeated the insult will result in a more severe stage 2 pressure ulcer with a breakdown of the epidermis 2-3 days later. After a single pinch, the elevation of Cx43 in the epidermis is associated with the inflammatory response with an increased number of neutrophils, HMGB1 (marker of necrosis) and RIP3 (responsible for necroptosis). Delivering Cx43 specific antisense oligonucleotides sub-dermally after a single insult, was able to significantly reduce the elevation of epidermal Cx43 protein expression and reduce the number of neutrophils and prevent the elevation of HMGB1 and RIP3. In a double pinch model, the Cx43 antisense treatment was able to reduce the level of inflammation, necroptosis, and the extent of tissue damage and progression to an open wound. This approach may be useful in reducing the progression of stage 1 pressure ulcers to stage 2.

Connexin43 in Post-Surgical Peritoneal Adhesion Formation.

OBJECTIVE: Post-surgical peritoneal adhesions are a serious problem for the quality of life and fertility. Yet there are no effective ways of preventing their occurrence. The gap junction protein Cx43 is known to be involved in fibrosis in several different organs and disease conditions often associated with inflammation. Here we examined the Cx43 dynamic expression in an ischemic button model of surgical adhesions. METHODS: Using the mouse ischemic button model, Cx43 antisense was delivered in Pluronic gel to attenuate Cx43 expression. The severity of button formation and immunofluorescence analysis of Cx43 and TGF-ß1 were performed. The concentration of tissue plasminogen activator via ELISA was also performed. RESULTS: As early as 6 h after button formation, the Cx43 levels were elevated in and around the button and some weak adhesions were formed. By 24 h Cx43 levels had increased further and adhesions were more defined. At 7 days the adhesions were much more robust, opaque, and vascularized, requiring blunt or sharp dissection to break them. Cx43 antisense attenuated its upregulation and, reduced the number and severity of adhesions that formed. CONCLUSION: Targeting Cx43 after surgical procedures may be a potential therapeutic strategy for preventing adhesion formation or at least reducing their severity.

Bio-Mimicking Acellular Wet Electrospun Scaffolds Promote Accelerated Integration and Re-Epithelialization of Full-Thickness Dermal Wounds.

Scaffolds can promote the healing of burns and chronic skin wounds but to date have suffered from issues with achieving full skin integration. Here, we characterise the wound response by both tissue integration and re-epithelialization to a scaffold using wet electrospinning to fabricate 3D fibrous structures. Two scaffold materials were investigated: poly(ε-caprolactone) (PCL) and PCL + 20% rat tail type 1 collagen (PCL/Coll). We assessed re-epithelisation, inflammatory responses, angiogenesis and the formation of new extracellular matrix (ECM) within the scaffolds in rat acute wounds. The 3D PCL/Coll scaffolds impeded wound re-epithelisation, inducing a thickening of wound-edge epidermis as opposed to a thin tongue of migratory keratinocytes as seen when 3D PCL scaffolds were implanted in the wounds. A significant inflammatory response was observed with 3D PCL/Coll scaffolds but not with 3D PCL scaffolds. Enhanced fibroblast migration and angiogenesis into 3D PCL scaffolds was observed with a significant deposition of new ECM. We observed that this deposition of new ECM within the scaffold was key to enabling re-epithelialization over the scaffold. Such scaffolds provide a biocompatible environment for cell integration to lay down new ECM and encourage re-epithelisation over the implanted scaffold.

Characterisation of an ischemia reperfusion model for the formation of a stage I pressure ulcer in mouse skin.

Pairs of magnets were applied to the loose skin on the backs of mice in order to cause ischemia for periods of 1.5, 2, 2.5 and 3 h followed by reperfusion. We found 1.5 h of ischemia resulted in the most reliable outcome of blanched skin but no redness or skin breakdown. Histological analysis at 4 h of reperfusion showed, in the centre of the insult, condensed nuclei in the epidermis and sebaceous glands with a build up of neutrophils in the blood vessels, and a reduction in the number of fibroblasts. At 24 h, spongiosis was seen in the epidermis and pockets of neutrophils began to accumulate under it, as well as being scatted through the dermis. In the centre of the insult there was a loss of sebaceous gland nuclei and fibroblasts. Four days after the insult, spongiosis was reduced in the epidermis at the edge of the insult but enhanced in the centre and in hair follicles. Leukocytes were seen throughout the central dermis. At 8 days, spongiosis and epidermal thickness had reduced and fibroblasts were reappearing. However, blood vessels still had leukocytes lining the lumen. The gap junction protein connexin 43 was significantly elevated in the epidermis at 4 h and 24 h reperfusion. Ischemia of 1.5 h generates a sterile inflammatory reaction causing the loss of some cell types but leaving the epidermis intact reminiscent of a stage I pressure ulcer.