Standardized Pre-clinical Surgical Animal Model Protocol to Investigate the Cellular and Molecular Mechanisms of Ischemic Flap Healing

Background Some of the most complex surgical interventions to treat trauma and cancer include the use of locoregional pedicled and free autologous tissue transfer flaps. While the techniques used for these reconstructive surgery procedures have improved over time, flap complications and even failure remain a significant clinical challenge. Animal models are useful in studying the pathophysiology of ischemic flaps, but when repeatability is a primary focus of a study, conventional in-vivo designs, where one randomized subset of animals serves as a treatment group while a second subset serves as a control, are at a disadvantage instigated by greater subject-to-subject variability. Our goal was to provide a step-by-step methodological protocol for creating an alternative standardized, more economical, and transferable pre-clinical animal research model of excisional full-thickness wound healing following a simulated autologous tissue transfer which includes the primary ischemia, reperfusion, and secondary ischemia events with the latter mimicking flap salvage procedure. Results Unlike in the most frequently used classical unilateral McFarlane’s caudally based dorsal random pattern skin flap model, in the herein described bilateral epigastric fasciocutaneous advancement flap (BEFAF) model, one flap heals under normal and a contralateral flap—under perturbed conditions or both flaps heal under conditions that vary by one within-subjects factor. We discuss the advantages and limitations of the proposed experimental approach and, as a part of model validation, provide the examples of its use in laboratory rat (Rattus norvegicus) axial pattern flap healing studies. Conclusions This technically challenging but feasible reconstructive surgery model eliminates inter-subject variability, while concomitantly minimizing the number of animals needed to achieve adequate statistical power. BEFAFs may be used to investigate the spatiotemporal cellular and molecular responses to complex tissue injury, interventions simulating clinically relevant flap complications (e.g., vascular thrombosis) as well as prophylactic, therapeutic or surgical treatment (e.g., flap delay) strategies in the presence or absence of confounding risk factors (e.g., substance abuse, irradiation, diabetes) or favorable wound-healing promoting activities (e.g., exercise). Detailed visual instructions in BEFAF protocol may serve as an aid for teaching medical or academic researchers basic vascular microsurgery techniques that focus on precision, tremor management and magnification. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12575-023-00227-w.

Using prechilled porcelain mortar and pestle, frozen tissue is crushed into a fine powder.Using prechilled spatula (LN2 is poured in the Styrofoam container and the spatulas are placed in the cooling cryogenic rack) the powder is scooped and poured in 10 ml Potter-Elvehjem borosilicate glass tissue grinder containing 1 -2 ml of WCL and kept in a glass beaker with ice.Powder is homogenized with 10-20 strokes using overhead stirrer set to 2000 rpm.The lysate is transferred into 1.5 ml or 2 ml Eppendorf tube, which is then placed in the rubber bucket on ice.Other samples are processed as necessary.At the end, the lysates are centrifuged at 10,000×g for 10 min at 4°C to remove insoluble material.The supernatants are transferred into a new set of tubes that are either stored at -80°C or immediately used to prepare the samples for SDS-PAGE or LDS-PAGE followed by a traditional or Multistrip Western Blotting as described previously [1].

Isolation of tissue-derived mRNA
For the isolation/purification of large and small RNA from flap biopsies, we use commercial phenolfree Animal Tissue RNA Purification Kit (Norgen, ON, Canada, #25700) supplemented with RNAse-Free DNAse I kit (Norgen, #25710).We follow the manufacturer's recommendations but introduce minor adjustments.Briefly, ~150 mg of each frozen skin sample is crushed into fine powder under liquid nitrogen using mortar and pestle.The powder is collected into prechilled 10 mL size Potter Elvehjem tissue grinder containing 1 ml of ice-cold RL lysis buffer.The tissue is then homogenized using motorized overhead stirrer attached to the PTFE pestle, which is continuously wiped with RNAse AWAY reagent surface decontaminant (Thermo Fisher Scientific, #7003) before and after processing of each sample.The homogenate is then diluted with 850 µL of nuclease-free molecular biology grade water, transferred into 2 mL DNA low-binding Eppendorf tubes and treated with 75 µL RNAse-free 20 mg/ml proteinase K aqueous solution at 55°C dry-heat bath for 30 min.Proteinase K aids in the removal of the various proteins present in fiber-rich tissues including collagen, contractile proteins, and connective tissues.The digested sample is then centrifuged at 14,000×g for 2 min to filter out insoluble debris.To reduce viscosity, the supernatant of each sample is passed through the individual disposable QIAshredder spin-columns (Qiagen, #79656).The filtrate is mixed with 100% ethanol at 1:1 vol/vol ratio and subjected to on column RNA binding, on-column DNAse treatment, column wash and RNA elution steps.The nucleic acid (NC) concentration, purity ratio and quality are measured by NanoDrop spectrophotometer and Qubit 4 fluorometer in triplicate (Qubit RNA IQ Assay, Thermo Fisher Scientific).
The NanoDrop requires just 1 µL of sample and by simply measuring at 230, 260 and 280 nm, one can obtain the total amount of NC present (260 nm), any protein contamination (280 nm) and any phenol or other solvent contaminants (230 nm) present in the sample.The Qubit RNA XR (extended range) and HS (high-sensitivity) assay dyes that selectively bind only to intact RNA can also be used to quantitate RNA.NC concentrations are measured using the fluorescence signal of the sample and a calibration curve that is generated from standard samples of known concentration and fit to appropriate regression models.Alternatively, RNA integrity is measured by Agilent 2100 bioanalyzer (Agilent Technologies Inc, PaloAlto, CA).
2. Refrigerated tabletop centrifuge -for sample pre-clearance and isolation of large-size EVs.
3. Ultracentrifuge with applicable rotor -for isolation of medium and small-size EVs.
13. Sterile 100x20 mm tissue culture dishes -for cutting the tissue into fine pieces.
16. Tube revolver/rotator with speed adjustment and rotisseries/paddles fit for 15 ml or 50 ml tubes (e.g., Thermo Scientific, #88881001) -used at 25 rpm, 37°C and 5% CO2 to facilitate tissue digestion.17. 100 mM RNAse-free EDTA solution (used at 1 mM final concentration) -to stop digestive reaction.Due to sample processing variability, it is recommended to isolate EVs using at least 3 technical replicate (TR) samples in parallel.Tissues are washed from blood in PBS, weighted, cut into small ~5 mm pieces, and placed into 15 ml Protein LoBind tubes containing 10 ml of enzymatic digestion solution with/without DNAse I. Tissues are incubated for 2 h in tube revolver/rotator at 25 rpm, 37°C and 5% CO2.To stop enzymatic reaction, 100 μL of 100 mM EDTA is added to reach 1 mM final concentration.
Each digestate is passed through 70 µm cell strainer, centrifuged at 400×g for 10 min at 4°C, and thereafter passed through 40 µm cell strainer, followed by centrifugation at 800×g for 10 min at 4°C and addition of TEIB to a final 40 mL volume and further centrifugation at 2,000×g for 30 min at 4°C.Removal of larger particles by vacuum-assisted filtration using 0.45 μm cellulose acetate filters is optional but strongly recommended.After this step, the filtrates may be stored at 4°C for a week or processed immediately.Freezing is not recommended, especially if EVs are to be analyzed for the metabolic processes.For the isolation of larger size EVs (microvesicles, MV), 34 ml of solution is centrifuged at 14,000×g for 1 h at 4°C.          panel.Place the origin point of the Ruler tool on the precision scale (upper panel) and drag it while holding "Shift" button as a straight line for 10 mm (middle panel).After stopping, enter the value "10" and "mm" in the input tabs "Logical Length" and "Logical Units", respectively.Click OK.

Figure S2 .
Figure S2.A variation of BEFAF model using a quadruplicate biopsy design (left panel), biopsy excision (middle panel) and the temporary protection of wound bed after a partial excision of the tissue (right panel).

Figure S4 .
Figure S4.Principles of simple-interrupted suturing.A. Pass a needle through the inner tissue to avoid piercing the skin.B. Throw a couple of loops onto the tip of needle holding scissors and grab an end of the suture to pass it through these loops.C. Close the knot in the direction shown.D. Make two additional loops on the scissors.E-F.Grab the end of a suture and close the knot in the direction shown.Cut the sutures, leaving short ends at the base of the knot.

Figure S5 .
Figure S5.Temporary sutures placement order (upper left panel) and directions (upper right panel) for the left-side BEFAF with real-time suturing order examples (bottom panel) covering suture placement at points 1-6 (A), 7-8 (B), 9-12 (C) and remaining 13-18 (D).Multiple suture points help to achieve even distribution of flap tension.Horizontal lines at the end of BEFAF indicate future biopsy excision areas.

Figure S6 .
Figure S6.Visual directions on how to pass a needle through the corners of left (left and middle panel) and right (right panel) base of the BEFAF.The suturing directions (represented by black arrows) for the remaining corner of the right flap (not shown) are identical to that indicated in the left image.

Figure S7 .
Figure S7.A photograph of male SD rat who's left BEFAF did not recover from the 4 h of PI after 2 h of REP1 due to the development of spontaneous venous thrombosis, which was confirmed by close inspection of the pedicle (left panel) before any attempt to salvage a flap.

Figure S8 .
Figure S8.Principles of simple continuous running suturing at the end of the survival surgery.

Figure S9 .
Figure S9.Animal recovering after major survival surgery in the cage supplied by water, regular chow diet, Nutra-Gel Diet™ diet and bacon softies.Once the anesthesia wears off, the animal resumes regular activities.Despite the placement of protective collar, leaner female rats may start picking up on sutures (inlet image).

Figure S10 .
Figure S10.Preparation for digital planimetric analysis using commercial "Adobe Photoshop" software.A. Upper panel.Open a digital photo of one or both flaps placed next to a precision ruler and activate "Measurement Log" panel, which is under "Window" menu.Bottom panel.Set a custom measurement scale in millimeters.Open "Image" menu, "Analysis" panel and "Set Measurement Scale" subpanel.Then click on "Custom".B. A digital Ruler tool should automatically appear along with an opened custom "Measurement Scale"

Figure S11 .
Figure S11.Digital planimetric analysis using commercial "Adobe Photoshop" software.A. Estimation of total flap area.Using "Quick Selection" tool located in the left toolbar (upper left panel) and a fine "60px" diameter brush (upper middle panel), select the total area of the flap (upper right panels).Delete the unwanted selected regions by holding "Alt" button.Flap margins are shown by bright green lines.Once all flap margins are included in the selection, click on the "Record Measurements" button in the "Measurement Log" window.Once the values appear in the window (bottom panel), proceed to the calculation of either necrotic or viable flap area.B. Gross estimation of necrotic flap area.Deselect previous selection of the flap by clicking on "Deselect" panel under "Select" window (left panel).Using "Quick Selection" tool and a fine "30px" diameter brush, select visibly necrotic flap areas (right panel).Delete the unwanted selected regions by holding "Alt" button.Click on the "Record Measurements" button in the "Measurement Log" window.Repeat the same steps for another flap.C. Export of data.Recorded measurements can be exported as .txtfile (upper panel) to the chosen destination and then opened in MS Office Excel program as a datasheet (bottom panel).Unwanted data fields may be hidden.The percentage of flap survival (FS%) is calculated as the total flap area minus the necrotic flap area divided by the total flap area (in square millimeters) multiplied by 100.

Figure S12 .
Figure S12.Examples of statistical simulated flap-study related data analysis.Upper left panel.Example1: paired T-test of left and right fap survival of multiple subjects (N = 8).Upper right panel.Example 2: comparison of flap conditions independent of animal gender by RM-ANOVA (N = 8).Bottom left panel.Example 3: comparison of gender-dependent flap response/signals by 2-way RM-ANOVA (N = 5 per each group).Bottom right panel.Example 4: comparison of gender-dependent flap response/signals by 3-way ANOVA (N = 7 per each group).

Figure S13 .Figure S14 . 9 (
Figure S13.Non-acceptable (upper panel) and acceptable (bottom panel) quality of raw unprocessed micrographs of Masson's Trichrome-stained histological sections of rat's BEFAF.The subpar quality image is too blurry, while the proper one has an adequate focus and contrast.Both micrographs suffer from a minor vignetting artefact mostly pronounced on the lower right side of the image.

Figure S15 .
Figure S15.Expected size distribution and concentration of MV-2.5K-SN-F (dashed line) and EXO-10K-SN-F (solid line) particles derived from 2000 mg of unperturbed control condition abdominal rat skin.Nanoparticle tracking analysis was performed at fixed camera level = 14, detection threshold = 3 and syringe pump speed = 35 settings over 7 repeated measurements with 60 s video capture time in 100-fold or 3200-fold diluted primary analyte (PA) samples using PBS, respectively.