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

Summary

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.

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.

Introduction

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 transcriptomic analysis to progress the identification of new therapeutic targets.

There are multiple models currently available to study wound healing, and each have their strengths and weaknesses. In vivo animal models, such as the db/db mouse² or streptozotocin-induced diabetic rats³ are widely used; however, wounds in rodent models heal via contraction, which is very different to the mechanism employed in the human body, and have limited success in translation to clinical trials^4,5. The benefits of using human tissue are well recognized⁴, but are complicated by the ethics of inflicting experimental wounds on individuals who are already known to sustain an impaired wound healing response. Consequently, studies on human subjects with diabetes is more commonly directed towards the inflammatory response rather than experimenting on excised tissue⁶. Organ-on-a-chip⁷ and artificial skin models⁸ are also available. These have the benefit of being able to analyze human cell contributions but give little indication of inter-patient variability. Thus, clinically relevant models to study the progress of wound healing in vulnerable patient populations could accelerate mechanistic understanding and drug discovery in this area.

The ex vivo wounding protocol described below is adapted from Stojadinovic and Tomic-Canic, 2013⁹. It is suitable for examining transcriptional data from controlled wounding of human tissue samples ex vivo and can be applied to clinical samples from patients with poor wound healing (e.g., type 2 diabetes, elderly individuals), in order to advance knowledge on how wound healing is impacted in these conditions and potentially how it can be restored.

Protocol

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 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…

Representative Results

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…

Discussion

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…

Materials

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