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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes

Published: January 13th, 2021

DOI:

10.3791/62091

1Centre for Skin Sciences, University of Bradford, 2The Charles Institute of Dermatology, School of Medicine, University College Dublin, 3University of Baghdad
* These authors contributed equally

This technique provides a guide to inflicting ex vivo wounds, performing laser capture microdissection and quantifying changes in gene expression related to poor wound healing processes in diabetes using clinically relevant human tissue.

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.

The incidence of type 2 diabetes is growing globally, driven by an obesity epidemic and physical inactivity. Poor wound healing is common in these patients and up to 25% of patients will develop a chronic non-healing wound1. The mechanisms underpinning this are complex and incompletely understood, limiting the discovery rate for new therapeutics. One of the contributing factors to this is the lack of a suitable model for studying wound healing in type 2 diabetes patients. Thus, the purpose of this method is to provide a physiologically relevant ex vivo model for examining wound healing in those at risk of chronic wounds, allowing for t....

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This protocol relies on the provision of human surgical tissue. Ethical approval and informed patient consent were obtained prior to experimentation, and the study conformed with the principles outlined in the Declaration of Helsinki.

1. Collection of tissue and ex vivo wounding

  1. Collect surgical tissue following limb amputation/surgery into a sterile container containing Dulbecco's Modified Eagle Medium (DMEM) with 5% penicillin-streptomycin-fungizone, 2 mM L-glutamine.......

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Following the protocol, a 48 h timepoint was chosen to generate representative results. The creation of the initial wound in surplus tissue from elective cosmetic surgery can be seen in Figure 2A where the excised wound is clearly visible. Haematoxylin and eosin staining confirms that this has generated a full thickness wound (Figure 2B). After 48 h, partial closure of the wound is visible under the light microscope (Figure 2C). His.......

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As the incidence of chronic disorders such as type 2 diabetes increases globally, the need for techniques that can facilitate pathophysiologically relevant studies becomes more urgent. The protocol described above provides a standardized method for examining transcriptomic data from ex vivo healing wounds utilizing human tissue.

This protocol is dependent on the provision of surplus clinical tissue for which ethical permission has been granted from the relevant authority, and from pat.......

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ICP was supported by the European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no.: 607886. RW was supported by Aveda, Hair Innovation & Technology, USA. RB, SS were supported by the Centre of Skin Sciences, University of Bradford.

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Name Company Catalog Number Comments
Arcturus RiboAmp PLUS kit ThermoFisher Scientific KIT0521 RNA amplification kit
Diffuser Caps 0.5mL MMI K10028161 Laser capture microdissection caps; 50 pack
Dulbecco’s Modified Eagle Medium (DMEM) Sigma-Aldrich D6046 With 1000 mg/L glucose, L-glutamine, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Foetal Bovine Serum Thermo Fisher Scientific 10270106 Cell culture supplement
H&E Staining Kit Plus MMI K10028305 Rnase-free haematoxylin and eosin staining kit
High capacity cDNA reverse transcription kit Applied Biosystems 4368814 Reverse transcription kit
L-glutamine Thermo Fisher Scientific 25030149 Cell culture supplement
MembraneSlides MMI K10028153 Laer capture microdissection slides; 5 per box
Netwell Mesh Insert Corning 3479 Cell culture insert
Penicillin-Streptomycin-Fungizone Thermo Fisher Scientific 15070-063 Cell culture supplement
15290-026
OCT Tissue-Tek Sakura 4583 Cryostat-compatible cutting medium
PBS Thermo Fisher Scientific 10209252 Five tablets per 100ml sterile water and then autoclaved for cell culture use
RNeasy Micro Kit Qiagen 74004 RNA extraction kit
RNase Away Sigma-Aldrich 83931 RNase spray
Sterile blades Scientific Laboratory Supplies INS4974 Tissue dissection implements
Support Slide MMI K10028159 Laser capture microdissection support slide, RNase-free
Surgical scissors Scientific Laboratory Supplies INS4860 Tissue dissection implements
Surgical forceps Scientific Laboratory Supplies INS2026 Tissue dissection implements
SYBR Green Supermix Applied Biosystems 4344463 Quantitative PCR mastermix

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