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Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in
Rachael Williams1, Irene Castellano-Pelicena2, Aaiad H A Al-Rikabi3
1Centre for Skin Sciences, University of Bradford.
Abstract:
The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.
Insights
This study introduces a novel method to analyze gene expression in skin cells from patients with Type 2 diabetes mellitus (T2DM), using discarded surgical tissue to understand impaired wound healing and prevent amputations.
Area of Science:
- Biomedical Science
- Dermatology
- Molecular Biology
Background:
- Global prevalence of Type 2 diabetes mellitus (T2DM) is increasing, leading to severe complications like impaired wound healing.
- T2DM-related poor wound healing can result in non-healing sores, foot ulcers, and limb amputation.
- Existing research models do not fully replicate the complex wound healing process in T2DM patients.
Purpose of the Study:
- To develop and describe a novel ex vivo technique for studying gene expression in T2DM wound healing.
- To utilize surplus surgical skin tissue for analyzing cellular mechanisms of impaired wound healing in T2DM.
Main Methods:
- Collected donated skin samples from amputation or cosmetic surgery.
- Wounded skin ex vivo, cultured at an air-liquid interface, and fixed at various time points.
- Isolated specific cell types (keratinocytes, fibroblasts, vasculature) via laser capture microdissection for gene expression analysis (sequencing, microarray, qPCR).
Main Results:
- Identified inherent differences in gene expression between poorly healing and intact skin in diabetic and non-diabetic patients.
- The protocol successfully analyzed expression profiles of key cells involved in wound healing.
- This method uses tissue ordinarily discarded, overcoming ethical concerns of patient biopsies.
Conclusions:
- This technique provides a valuable tool for understanding the molecular basis of T2DM-associated chronic wounds.
- Insights gained can contribute to developing new therapeutic strategies for improving wound healing in T2DM patients.
- Enhanced understanding may help reduce the incidence of lower limb loss in T2DM.
