Name: the superficial inferior epigastric artery axial flap to study ischemic preconditioning effects in a rat model
Uploaded: 2023-01-27T14:45:00
Description: Watch this Scientific Journal Video about The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model at JoVE.com

This work was funded by Massachusetts General Hospital (W.G.A) and Shriners Children&#39;s Boston (B.U, K.U, C.L.C). Y.B and I.F.v.R are funded by Shriners Hospitals for Children (Proposal ID: #970280 and #857829 respectively).

The Massachusetts General Hospital Institutional Animal Care and Use Committee approved the experimental protocol (IACUC- protocol #2022N000099).The authors followed the ARRIVE (Animal Research: Reporting In Vivo Experiments) guideline checklist for this work. All animals received humane care following the National Institute of Health Guide for the Care and Use of Laboratory Animals. A total of 12 male Lewis rats (250-350 g, 8-10 weeks old) were used for all experiments.
1. Anim...

<ol>
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E., Jennings, R. B., Reimer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+with+ischemia:+a+delay+of+lethal+cell+injury+in+ischemic+myocardium.">Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium.</a> Circulation. 74 (5), 1124-1136 (1986).</li><li>Akcal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+ischemic+preconditioning+and+postconditioning+can+minimise+skin+flap+loss:+experimental+study.">Combination of ischemic preconditioning and postconditioning can minimise skin flap loss: experimental study.</a> Journal of Plastic Surgery and Hand Surgery. 50 (4), 233-238 (2016).</li><li>Ulker, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+ischemic+preconditioning+increase+flap+survival+by+ADORA2B+receptor+activation.">Does ischemic preconditioning increase flap survival by ADORA2B receptor activation.</a> Clinical Hemorheology and Microcirculation. 75 (2), 151-162 (2020).</li><li>Min, S. -. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+groin+flap+model+redesigned+for+evaluating+treatment+effects+on+ischemia-reperfusion+injury.">The rat groin flap model redesigned for evaluating treatment effects on ischemia-reperfusion injury.</a> Journal of Surgical Research. 222, 160-166 (2018).</li><li>M&#252;cke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green+videoangiography-assisted+prediction+of+flap+necrosis+in+the+rat+epigastric+flap+using+the+flow&#174;+800+tool.">Indocyanine green videoangiography-assisted prediction of flap necrosis in the rat epigastric flap using the flow&#174; 800 tool.</a> Microsurgery. 37 (3), 235-242 (2017).</li><li>Wang, D., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green+angiography+for+continuously+monitoring+blood+flow+changes+and+predicting+perfusion+of+deep+inferior+epigastric+perforator+flap+in+rats.">Indocyanine green angiography for continuously monitoring blood flow changes and predicting perfusion of deep inferior epigastric perforator flap in rats.</a> Journal of Investigative Surgery. 34 (4), 393-400 (2021).</li><li>Berkane, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+secure+pedicled+flaps+using+perioperative+indocyanine+green+angiography:+a+prospective+study+about+10+cases.">How to secure pedicled flaps using perioperative indocyanine green angiography: a prospective study about 10 cases.</a> World Journal of Surgery and Surgical Research. 4 (1), 1319 (2021).</li><li>Alstrup, T., Christensen, B. O., Damsgaard, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICG+angiography+in+immediate+and+delayed+autologous+breast+reconstructions:+peroperative+evaluation+and+postoperative+outcomes.">ICG angiography in immediate and delayed autologous breast reconstructions: peroperative evaluation and postoperative outcomes.</a> Journal of Plastic Surgery and Hand Surgery. 52 (5), 307-311 (2018).</li><li>K&#252;ntscher, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+by+brief+extremity+ischemia+before+flap+ischemia+in+a+rat+model.">Ischemic preconditioning by brief extremity ischemia before flap ischemia in a rat model.</a> Plastic and Reconstructive Surgery. 109 (7), 2398-2404 (2002).</li><li>Liu, R. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+analysis+of+indocyanine+green+fluorescence+angiography+for+prevention+of+anastomotic+leakage+in+colorectal+surgery.">Cost analysis of indocyanine green fluorescence angiography for prevention of anastomotic leakage in colorectal surgery.</a> Surgical Endoscopy. 36 (12), 9281-9287 (2022).</li><li>Cheng, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Devices+for+ischemic+preconditioning+of+the+pedicled+groin+flap.">Devices for ischemic preconditioning of the pedicled groin flap.</a> The Journal of Trauma. 48 (3), 552-557 (2000).</li><li>Xiao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+innovative+and+economical+device+for+ischemic+preconditioning+of+the+forehead+flap+prior+to+pedicle+division:+a+comparative+study.">An innovative and economical device for ischemic preconditioning of the forehead flap prior to pedicle division: a comparative study.</a> Journal of Reconstructive Microsurgery. 38 (9), 703-710 (2022).</li><li>K&#252;ntscher, M. V., Hartmann, B., Germann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+ischemic+preconditioning+of+flaps:+a+review.">Remote ischemic preconditioning of flaps: a review.</a> Microsurgery. 25 (4), 346-352 (2005).</li></ol>

This article describes a reproducible fasciocutaneous flap model harvested in rats, allowing I.P. evaluation. This step-by-step surgical protocol gives research groups a reliable model to test different I.P. regimens. By preventing any vascularization other than the pedicle, this model allows for studying the flap's neovascularization from the wound bed and margin. This study performed the ligation on POD5, as previous studies have observed the autonomization of this flap in rats on POD5-711,...

Plastic and reconstructive surgery is constantly in evolution. The development of muscle, fasciocutaneous, and perforator flaps has made it possible to offer better-quality reconstructions while reducing morbidity. Combining this improved anatomical knowledge with enhanced technical skills, reconstructive surgeons can perform free flap transfers when defects are not close to any local solution. However, while perforator flap surgery is currently the most advanced technique in reconstructive surgery, the literature reports a 5% failure rate in free flap transfers1,2,3, and up to 20% for pedicled flap reconstruction4,5,6. Partial to total flap failure occurs when the flap&#39;s pedicle is compromised, therefore it is essential to continuously search for improvements to the current techniques. One of the methods to improve flap survival is to promote its neovascularization on the wound bed, thus allowing perfusion by a source other than the pedicle. Ischemic preconditioning (I.P.) has been initially described in a heart model7, demonstrating that an organ exposed to controlled ischemia survives to a higher degree after losing its primary blood supply by undergoing ischemia-induced neovascularization. Several authors have studied this cornerstone principle to optimize flap survival in preclinical and clinical models8,9,10.
The advantage of this technique over other methods to improve flap survival is its ease of implementation, consisting of clamp/declamp tests of the blood source. In the rat model, previous authors used the superficial inferior epigastric artery (SIEA) flap to study I.P. by clamping the main pedicle11,12,13. Nonetheless, several technical issues can be encountered with this model, and the literature lacks well-described protocols.
Therefore, this work aims to provide researchers with a detailed description of a rat SIEA flap procurement technique with an extended dissection of the femoral vessels to allow I.P. studies on an axial fasciocutaneous flap model. This model retains the integrity of the epigastric vessels and instead manipulates the femoral vessels, which are more resilient. We share our experience and tools to improve the study of this phenomenon and increase the replicability of this procedure.

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			<td>Lone Star elastic stays</td>
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			<td>Lone star Self-retaining retractor&#160;</td>
			<td>Cooper Surgical</td>
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			<td>Metronidazole tablets USP</td>
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			<td>Micro spring handle scissors&#160;</td>
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			<td>Microsurgical needle holder</td>
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			<td>Needle holder</td>
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			<td>Nosecone for Anesthesia&#160;</td>
			<td>World Precision Instruments</td>
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			<td>Pixel analysis software</td>
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			<td>Ragnell Scissors&#160;</td>
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			<td>Small animal ear tag</td>
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			<td>Style 1005-1</td>
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			<td>Peco Services Ltd</td>
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			<td>Dynarex Corporation</td>
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			<td>Sterile water for injection and irrigation&#160;</td>
			<td>Hospira</td>
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			<td>Surgical scrub &#8211; BD ChloraPrep Hi-Lite Orange 3 mL applicator with Sterile Solution</td>
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			<td>Webcol Alcohol prep pads&#160;</td>
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the superficial inferior epigastric artery axial flap to study ischemic preconditioning effects in a rat model

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

All flaps were viable on POD5, showing good vascularization by the SIEA alone. Figure 5 shows the flap before and after IV fluorescein injection, showing a complete vascularization.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64980/64980fig05.jpg" /> 
Figure 5: Immediate intravenous fluorescein angiography (POD0). This assessment shows the flap&#39;s vascu...

Watch this Scientific Journal Video about The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model at JoVE.com

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model

This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing ...

This surgical protocol provides a reliable model to study ischemic preconditioning regimen in fasciocutaneous flaps to improve the outcomes of these surgeries. This protocol is easy to learn, reproducible and reliable. The main difference with other protocols is the use of femoral vessels to provoke the ischemic preconditioning, avoiding harming small vessels.It can eventually lead to optimizing an ischemic preconditioning protocol for free and pedicled flap reconstruction to improve flap survivability and reduce the critical period of the flap vulnerability. This method can contribute to the field of reconstructive surgery and lends itself well to the study of ischemia reperfusion injuries. To begin, place the properly sedated animal in a supine position.Shave the abdomen from below the inguinal crease to above the level of the xiphoid process. Apply a hair removal cream on the shaved area and wait for five minutes before cleaning it. Using a sterile skin pen and a ruler, first mark the midline of the animal's abdomen and then mark the inguinal crease.Before making the incision, indicate the position of the desired flap on the animal's body by drawing an oval or a rectangle, having a vertical length of up to six centimeters and a horizontal width of about three centimeters extending cranially from the inguinal crease. Now draw five or six equidistant marks perpendicular to the flap limits. Then using a pair of Ragnell scissors, make a longitudinal incision of three to four centimeters on the inguinal crease.Be sure to pull skin upward to avoid damaging vessels. Expose and identify the femoral and epigastric vessels using a 4 jewelers microsurgical forceps by opening and closing the forceps to separate the fascia and to gain access to the vessels beneath the inguinal fat pad. Use the inguinal incision to start the flap incision using the Ragnell scissors.Pay attention to undermining the full thickness of the skin and the connective tissue over the abdominal muscle. As the tip of the flap is initially freed from the surrounding skin, continue the flap procurement by undermining from the distal to the proximal part using the Ragnell scissor tips to separate the flap from the muscle while cauterizing any perforator vessels and dermal plexus vessels around the flap. Once the flap is entirely harvested, aim to encapsulate the entirety of both branches of the superficial inferior epigastric artery or SIEA with the flap by gently lifting the flap upward to visualize the vessels.Next, separate the fat pads on the inferior aspect on both the medial and lateral sides of the flap. Use the bipolar cautery to cauterize the fat pads close to the border of the incision carefully without harming the superficial inferior epigastric pedicle. Before proceeding for the femoral vessel preparation after completion of the flap harvesting, inject the animal with a single dose of 17.5 international units of sodium heparin via the penile vein.To allow a better view of the vessels, place the animal within a lone star self-retaining retractor. Now working under a surgical microscope having a 40X magnification, use a couple of 4 microsurgical forceps to dissect the femoral vessels both proximally and distally until the emergence of the superficial epigastric vessels. On the distal femoral vessels, clean the fascia and gently free the nerve from the artery and vein.Using an 8-0 nylon suture, ligate the distal femoral vessels by circumventing the microsurgical needle holder under the artery and vein, clasping the suture and tying off these vessels right after the emergence of the epigastric vessels, leaving a one millimeter distance after the pedicle origin. On the proximal femoral vessels, repeat the same process of cleaning the connective tissue. However, separate the artery and vein from each other to allow efficient clamping.To induce intermittent ischemia, place microsurgical clamps separately on each proximal femoral artery and vein. When the ischemic injuries are completed, suture the flap to its original position lining the marks drawn preoperatively. Suture the flap using a running suture with 3-0 nylon at the inguinal crease medially, around the flap, and ending at the inguinal crease laterally.To verify the blood supply to the flap, inject 0.25 milliliters of 10%sterile fluorescein sodium into the the penile vein using the same technique and tools described for the previous injection. After three minutes, shine a long wave UV lamp at 366 nanometers to reveal the fluorescent areas corresponding to the perfused areas. After closure and verification, spread crushed metronidazole along the sutures to prevent auto-mutilation and spray liquid bandage on the same area.To remove the flap from the epigastric blood supply at the end of the ischemic preconditioning period, cauterize inferior to the fat pad along the inferior border of the flap. Immediate intravenous fluorescein angiography on postoperative day zero or POD0 showed complete vascularization of the flap by the SIEA alone. The tissue, including the whole flap paddle, was well perfused.The angiography of the negative control on POD5 and POD10 showed full viability of the flap paddle. All animals in this group where no ligation was performed on the feeding vessels remained healthy. For the positive control group, the absence of green fluorescence immediately after ligation showed no perfusion of the flap, proving the absence of neovascularization.This was confirmed on POD10 with observed necrosis of 85%of the skin paddle. Interestingly, the distal tip was viable and neovascularized. Statistical analysis of the flap surface viability on POD10 showed 99.5%survivability by the negative control group whereas the positive control group showed 11.25%survivability.An example experimental group meant to study ischemic preconditioning showed 13.67%survivability demonstrating that the particular ischemic preconditioning protocol was inefficient in this model. The two major steps are flap harvesting and vessel dissection under the microscope. The most important piece of advice is to have good-quality micro instruments.The next goal is to try different ischemic preconditioning protocols by varying the ischemia durations and rhythms to select the regimen allowing the best flap survivability following early flow disruption. This technique by providing a good model for ischemia fusion injuries can lead to new findings on ischemic preconditioning applied to modern reconstructive surgery and reconstructive transplant.

Research

JoVE Journal

Immunology and Infection

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

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An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

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Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

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In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

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Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

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Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

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Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

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Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

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Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...