Skin Flap Models for Assessment of Angiogenesis

Abstract

Skin flaps are surgically elevated areas of skin and subcutaneous tissue transferred on their own vascular pedicle from a donor site to a recipient site that is devoid of skin. Skin flaps have been used in clinical reconstructive surgery for over 100 years and remain common procedures to this day. This chapter will describe numerous experimental animal skin flap models that have been developed over the last 40 years, and the various techniques applied to these models to increase their survival including vascular delay, ischemic preconditioning and flap prefabrication. Most of these techniques are now known to significantly increase the blood vessel numbers and blood flow to experimental skin flaps. Skin flap models and applied techniques illustrate various mechanisms by which angiogenesis can be stimulated and manipulated. Additionally, the manipulation of large vascular pedicles associated with flap prefabrication has led to a new and developing area in intrinsic vascularisation of tissue engineered chambers where new tissues and organs can be grown. Basic experimental flap models, techniques to enhance angiogenesis in these flaps and intrinsic vascularisation of tissue engineering chambers will be described in this chapter.

Geraldine M. Mitchell, Zerina Lokmic, and Shiba Sinha contributed equally to this chapter.

Appendices

Appendix

Methods for Rat AVL Construct and Mouse Flow Through Pedicle Construct and Skin Flap Tissue Processing, Visualization and Morphometric Assessment of Blood Vessels

2.1 Rat AVL Construct or Mouse Flow Through Pedicle Construct Weight Measurements

• 1. Place an empty 50 ml container on a four decimal balance (Mettler AE 260, DeltaRange^®, Mettler-Toledo, Switzerland) and record the initial weight (W _initial ).

• 2. Blot each construct (rat or mouse) carefully on Whatman filter paper then place the construct in the container on the balance and record the resulting weight (W _final ).

• 3. Determine the weight of the AVL construct or mouse chamber construct (W _construct ) by subtracting the initial weight from the final weight recorded: W _construct   = W _final   − W _initial .

2.2 Rat AVL Construct or Mouse Flow Through Pedicle Construct Volume Measurements

AVL or mouse construct volume is determined by a method described by Scherle in 1970 [152] in which the water displacement due to construct volume is determined by weighing in saline. To weigh the newly formed AVL construct

• 1. Tie off the AVL construct artery and vein or mouse chamber construct artery and vein at the point of entry into the chamber (approximately) with a long piece of 6.0 silk thread at harvest.

• 2. Fill a 50 ml plastic container with 40 ml of isotonic sterile saline (0.9% NaCl) solution, place on a balance and record the initial weight (W ₁ ).

• 3. Blot the AVL construct or mouse chamber construct on Whatman paper to remove excess fluids from the construct.

• 4. Attach the AVL or mouse flow through pedicle construct to a silk thread and lower the specimen suspended on the thread into the saline solution so that it is beneath the surface of the liquid but not touching the bottom or the sides of the container.

• 5. Record the resulting weight (W ₂ ).

• 6. To calculate the weight of the fluid displaced by the organ (W _O ), use the following formula: W _O   = W ₂ -W ₁

• 7. To determine the construct volume (V ₀ ) divide the weight of the fluid displaced (W ₀ ) by the AVL construct or mouse construct by the specific gravity of isotonic saline (g = 1.0048): V ₀   = W ₀ /g. As the specific gravity of saline is rounded off to g = 1.0, then: V ₀   ≈ W ₀   = W ₂   − W ₁ .

2.3 Slicing the AVL Construct Prior to Tissue Processing

Once weight and volume measurements are complete, the AVL construct should be sliced into 1 mm thick slices. However, if the construct is fragile, usually in the early stages of development (0–14 day construct), it should be fixed for 2 h in the desired fixative (10% neutral buffered formol saline or 4% paraformaldehyde) before attempting to slice. This will ensure that the construct structure does not disintegrate prematurely and therefore affect accurate histomorphometry.

The direction of slicing should be from the top of the construct, adjacent to the chamber lid, to the bottom of the construct, adjacent to the chamber base and from the femoral vessels point of entry into the construct towards the edge of the construct using a razor blade. Slice thickness should be as close as possible to 1–2 mm. This will ensure that cross sections of the AVL are obtained in these slices. After slicing, fixation should take place immediately for approximately 2 days prior to paraffin embedding. If paraffin embedding is delayed tissue should be stored in phosphate buffered saline (PBS) prior to processing. [If immediate fixation prior to slicing is not undertaken, some tissue slices can be removed and placed in liquid nitrogen for subsequent extraction of messenger RNA and total protein prior to fixation of the remaining slices].

2.4 Slicing of Skin Flaps

For histology and assessment of angiogenesis skin flaps should be either pinned out flat and fixed [or chemically cleared of all tissues and prepared as a whole mount – see [100]]. Skin flaps that are fixed must be sliced after fixation, at about 4–6 h after the fixative is applied. Flaps should then be cut ‘vertically” using a razor blade or scalpel from the epidermis to the fascia, at a slice thickness of 2–4 mm. The skin flap slices are returned to fixative for a minimum of 24 h, and then stored in phosphate buffered saline (PBS) overnight prior to histological processing (see below).

2.5 Tissue processing and sectioning

(applies to the AVL construct, mouse flow through pedicle construct and skin flap slices)

The following protocols have been determined by our group to provide optimal conditions that permit clear and consistent tissue staining and successful immunohistochemistry with a strong signal to noise ratio.

• 1. Post fixation, tissue slices are placed in a tissue processing cassette and stabilized in the cassette with biopsy pads.

• 2. The processing of tissue into paraffin blocks is done using a tissue embedding processor (HyperCenter XP, Shandon^®, Shandon Scientific Ltd, England), where the tissue is dehydrated through graded serial changes of alcohol and embedded into paraffin wax as follows:

  • i. 70% Ethanol – 20 min

  • ii. 90% Ethanol – 20 min

  • iii. 95% Ethanol – 20 min

  • iv. 100% Ethanol – 20 min

  • v. 100% Ethanol – 30 min

  • vi. 100% Ethanol −60 min

  • vii. Histolene – 20 min

  • viii. Histolene – 20 min

  • ix. Histolene – 30 min

  • x. Paraffin wax – 20 min under vacuum

  • xi. paraffin wax – 60 min under vacuum

• 3. Following the last step in wax, the tissue is transferred to an embedding stage and embedded into tissue moulds containing melted paraffin wax. Rat chamber slices are placed cut surface down on the base of the mould, all the slices from one construct in one mould, or if large, several slices from a construct are placed in one mould. Skin flap slices are also placed cut surface down on the base of the mould, again several slices may fit into one mould. (Mouse chamber constructs can be left whole, or cut into two halves at this point, either by a single cross section or longitudinal section cut in the mid line, and the two halves embedded cut surface down on the base of the embedding mould).

• 4. The moulds are then cooled down to 5°C to allow paraffin wax solidification.

• 5. Section paraffin blocks into 3–5 μm thick serial sections using a microtome (Reichert-Jung, Grale Scientific Australia), float on 3′ (Aminopropyl) triethoxy-silane (AAS) (Sigma^®, Sigma-Aldrich, St Louis, USA) coated slides for histology or poly-L-lysine coated slides (Polysine™, Menzel-Glaser^®, Germany) for immunohistochemistry and dry overnight in a 37°C oven.

2.6 Identification of Blood Vessels in Rat and Mouse Tissue Sections

To identify endothelial lined blood vessels in rat tissue, a two-step indirect immunoenzymatic method [153] is used to detect B. Simplicifolia lectin by a streptavidin biotin-based immunohistochemical protocol described below. Negative and positive control tissues were included in all immunohistochemical assessments. The negative control consisted of omitting B. Simplicifolia lectin from the reaction. The positive control tissues are rat skin tissues which label with this lectin on blood vessel endothelium. There is no absolutely definitive endothelial marker for rat tissue.

[It should be noted that a number of endothelial labels could be used, none of which are without some problems. The B. Simplicifolia lectin method described here is reliable and labels endothelial cells clearly – however it also labels macrophages and the observer needs also to be able to clearly distinguish small blood vessels from macrophages on morphological grounds. Another antibody which works very well in most rat tissues and labels endothelial cells very clearly is Factor VIII. However in the AVL chamber construct it will also label the fibrin matrix which persists for at least the first 2 weeks. This factor makes blood vessel identification very difficult and it is nearly impossible for the inexperienced to know what is true blood vessel labeling and what is not. We therefore have not included the Factor VIII protocol here. For mouse tissues we have generally used a CD31 antibody. This is a very clear label of endothelial cells, it does not label any inflammatory cells, but it is not specific to vascular endothelial cells as it also labels lymphatic endothelial cells.]

2.6.1 Lectin Iummunolabelling Protocol for Rat Tissue Blood Vessels

• 1. Dewax 3 μm thick serial tissue sections in histolene, graded ethanol (starting at 100% to 50%) and bring to water.

• 2. Wash sections in PBS, circle each tissue section with a wax pen. Apply Proteinase K for 5–8 min at room temperature, then wash the sections in three changes of PBS.

• 3. To block endogenous peroxidise and reduce the background that could be associated with the use of chromogen diaminobenzidine (DAB), blot the sections gently with tissues and treat the sections in 10% hydrogen peroxide (H₂O₂) in 50% methanol for 5 min. Wash sections in three changes of PBS.

• 4. Apply biotinylated B. Simplicifolia lectin solution (dilution 1:100) for 30 min at room temperature. Incubate the slides in a moist chamber to prevent drying out of the lectin solution. Wash sections in tris buffered saline (TBS) buffer.

• 5. Apply Streptavidin-HRP for 30 min at room temperature, also in a moist chamber, then wash sections in three changes of 0.05% Tween/TBS buffer.

• 6. To detect the bound lectin-streptavidin complex, use a chromogen such as DAB for 1–5 min. The sections are then washed in tap water, counterstained in Myer’s haematoxylin, dehydrated and cover-slipped with DPX mountant.

2.6.2 CD 31 Immunolabelling Protocol for Mouse Tissue Blood Vessels

• 1. For CD31 immunolabelling of mouse tissues a rat anti-mouse CD31 antibody is used (BD Pharmingen, San Jose, CA).

• 2. Paraffin sections are dewaxed and hydrated and have a TBS wash, followed by a peroxidase quench (3% H₂O₂ in 50%methanol/distilled water for 5 min) and another TBS wash.

• 3. Sections are digested with DAKO proteinase K that is warmed to room temperature (antigen retrieval) for 8 min, washed in TBS, and nonspecific binding blocked using DAKO protein block for 30 min. Blot excess fluid.

• 4. The primary antibody is applied at 1:100 in DAKO antibody diluent for 1 h, followed by a TBS wash.

• 5. The secondary antibody (DAKO rabbit anti-rat biotinylated immunoglobulin) is then applied at 1:300 for 30 min followed by a TBS wash.

• 6. An avidin-biotin-horseradish peroxidase (HRP) detection system (ABC Elite; Vector Laboratories, Burlingame, CA) is then used followed by a TBS wash.

• 7. Finally diaminobenzidine (DAB) color development for 5 min, is followed by a distilled water wash, and counterstaining with haematoxylin for 1 min.

• 8. Dehydrate sections in increasing concentrations of alcohol, clear and coverslip with DPX mountant.

2.7 Morphometric Analysis of Labelled Blood Vessels

To morphometrically assess the vessel development in the rat AVL model, mouse flow through pedicle model or skin flaps, it is necessary to define targets of interest and reference compartments. In our laboratory, to estimate the proportion and volume of newly developed tissue components, the morphometric technique described by Howard and Reed [154] is used. When assessing rat AVL constructs or mouse flow-through pedicle constructs, the targets of interest include new blood vessels, matrix and new connective tissue and the AVL or flow through pedicle itself. In addition to the target, a reference compartment is also defined, namely a compartment to which a target can be compared to. In AVL constructs, two reference compartments were defined. The first compartment consists of the entire AVL construct comprised of AVL, fibrin matrix, new blood vessels and connective tissue. All points counted in each compartment are added together to give the total reference compartment count. However, to determine the vascularisation of the newly developed connective tissue alone, the second reference compartment is defined as the newly synthesized connective tissue which consists of combined counts for blood vessels and the newly synthesized connective tissue. A number of software tools are available for automated and semi-automated quantification of blood vessels and tissue components. In our laboratory, the Computer Assisted Stereological Toolbox (CAST) system is used (CAST System, Olympus, Denmark).

Method: To count the targets of interest, select a random 3–5 μm tissue section which contains 4–6 slices of AVL construct (1–2 sections of mouse construct) or 2–3 sections of skin flap tissue. Only complete tissue sections should be counted (Note: each of these slices should be approximately 500 to 1,000 μm apart) and labelled with B. Simplicifolia lectin (rat tissue) or CD 31 (mouse tissue).

• 1. Place a section on the automatic stage and outline the area of interest (reference compartment- usually the entire tissue section, or only the new connective tissue area in rat AVL constructs). At this point is it necessary to determine the percentage of area to be counted so that a representation of all specimens can be achieved. This is done through trial and error and it is best to undertake a small pilot study to determine the percentage area suitable for counting and the number of points suitable for counting. In our case, depending on the model, this percentage value is between 5% and 20% of total area. The software package them randomly and uniformly selects the fields where the counts are to be completed. (Note at least 200 total points per chamber construct should be counted. We generally count between 300 and 800 per chamber construct).

• 2. To determine vascular volume density, the number of points falling on the blood vessels is divided by the number of points falling on the total reference compartment [51, 141]. The percent vascular volume is then obtained by multiplying that number by 100%. Furthermore, since AVL tissue volume (and mouse chamber construct volume) is available, the percent vascular volume can be multiplied by the organ displacement volume to obtain absolute (total) vascular volume within a construct. This parameter is of particular value when determining the effect of a treatment on the construct development. The final result is represented as a function of time or as the outcome of treatment effect.