http://orcid.org/0000-0002-7150-9758
Figures
Abstract
Ischemia reperfusion injury is associated with tissue damage and inflammation, and is one of the main factors causing flap failure in reconstructive microsurgery. Although ischemia-reperfusion (I/R) injury is a well-studied aspect of flap survival, its biological mechanisms remain to be elucidated. To better understand the biological processes of ischemia reperfusion injury, and to develop further therapeutic strategies, the main objective of this study was to identify the gene expression pattern and histological changes in an I/R injury animal model. Fourteen rats (n = 7/group) were randomly divided into control or ischemia-reperfusion group (8 hours of ischemia). Microsurgical anastomoses were objectively assessed using transit-time-ultrasound technology. Seven days after surgery, flap survival was evaluated and tissue samples were harvested for anatomopathological and gene-expression analyses.The I/R injury reduced the survival of free flaps and histological analyses revealed a subcutaneous edema together with an inflammatory infiltrate. Interestingly, the Arginase 1 expression level as well as the ratio of Arginase 1/Nitric oxide synthase 2 showed a significant increase in the I/R group. In summary, here we describe a well-characterized I/R animal model that may serve to evaluate therapeutic agents under reproducible and controlled conditions. Moreover, this model could be especially useful for the evaluation of arginase inhibitors and different compounds of potential interest in reconstructive microsurgery.
Citation: Ballestín A, Casado JG, Abellán E, Vela FJ, Álvarez V, Usón A, et al. (2018) Ischemia-reperfusion injury in a rat microvascular skin free flap model: A histological, genetic, and blood flow study. PLoS ONE 13(12): e0209624. https://doi.org/10.1371/journal.pone.0209624
Editor: Lars-Peter Kamolz, Medical University Graz, AUSTRIA
Received: October 1, 2018; Accepted: December 7, 2018; Published: December 27, 2018
Copyright: © 2018 Ballestín et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: This work was supported in part by ISCIII projects (PI16/02164, CD17/00021, PI18/00911 and CP17/00021) (FMS-M, JGC), by a project from Junta de Extremadura (IB16168) and co-funded by the European Union (ERDF/ESF) (JGC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Microsurgical free-tissue transfer has become a common practice in reconstructive surgery to provide an efficient approach to restore the form and function after complex tissue defects due to trauma injuries or after oncological resections. The loss of skin, muscle and even bone especially after avulsion or amputation injuries or after tumor resections in the head, neck, trunk or extremities, represent reconstructive challenges for surgeons. Microsurgical reconstructions with composite or single free-tissue transfers are ideal options to cover these three-dimensional defects [1,2]. Well-perfused tissues are required to fill the defect, providing functional and aesthetic outcomes.
Although literature from previous studies shows that the final success rate of microsurgical free flap transfer is 90–95% [3,4,5,6,7,8,9], up to 25% of transferred flaps need revision because of complications [10,11,12,13]. These postoperative complications can be due to surgical problems (i.e., technical errors, prolonged tissue ischemia or venous congestion) [14,15], or the pathological condition of the patient (i.e., diabetes, hypertension or radiated patient) [16,17,18].
One of the most studied biological processes affecting flap survival is ischemia-reperfusion injury [3,4,19], which may lead to complete flap failure. In fact, it is important to note that, even partial flap necrosis leads to suboptimal aesthetic and functional results [20].
During last two decades, several research studies attempted attenuation of ischemia-reperfusion injury using different compounds and molecules [21,22,23,24,25,26]. Most of the studies have shown positive or promising results in preclinical settings [21,22,27,28] and some clinical trials are being conducted in this field (clinicaltrials.gov identifiers NCT01905501 and NCT03535623).
Any free flap transfer includes a period of ischemia followed by reperfusion. The ischemic interval lasts from completion of flap harvesting till the end of the microvascular anastomoses. When the ischemic period exceeds the tolerance of the tissue, an apoptotic/necrotic process is initiated. Additionally, a wide range of pathophysiological events occurs within the tissue after reperfusion [6,19,23]. Reactive oxygen species have been shown to trigger reperfusion injury and lead to recruitment of proinflammatory neutrophils in ischemic tissues [20].
The incidence of complications after flap transfer necessitates better understanding of the pathophysiological processes of ischemia reperfusion injury, which are associated with different biological/physiological processes such as metabolic dysfunction, cell death, hypoxia, inflammation and apoptosis or necrosis [29]. Gene expression analysis of a translational I/R model is crucial for evaluation of these biological processes and potential targets for therapeutic interventions.
Animal models are of major importance in understanding the pathophysiology of various biological processes in humans at the cellular and molecular levels [30,31]. Appropriate animal models are essential to predict the value and effect of novel therapeutic approaches and to assess the efficacy of new drugs [32]. With this background, the main objective of this study was to describe a clinically relevant model appropriate to conduct preclinical studies in the field of ischemia-reperfusion injury in reconstructive microsurgery.
Aiming at this, we firstly optimized the microsurgical and anatomical approach to harvest the free flap model. Secondly, the patency of the microvascular anastomoses was assessed using transit-time ultrasound technology. Third, post-mortem analyses were carried out to evaluate the histological changes. Finally the gene expression analysis was performed for a set of genes involved in inflammation, oxidative stress, angiogenesis and apoptosis.
The analysis of inflammatory-related genes was focused on Th1/Th2 cytokines (Interleukin 1 beta, Interleukin 6, Interleukin 10, Tumor Necrosis Factor) as well as on M1/M2-related mRNA expression (Arginase 1, Nitric Oxide Synthase 2), which has been found to be closely involved with ischemia reperfusion [33] and ischemic heart failure [34]. In the case of angiogenesis, this analysis was limited to the quantification of angiopoietin-2 (widely described in myocardial infarction and cerebral ischemia) [35,36], Fibroblast growth factor 2 and Vascular endothelial growth factor A (both related to hindlimb ischemia models) [37,38]. For oxidative-stress related genes, the analysis was focused on Superoxide dismutase 1 and Hypoxia-inducible factor 1, alpha subunit. In the case of Hypoxia-inducible factor 1, alpha, it has been shown to be differentially expressed in the context of microsurgical free muscle tissue transfer [39,40] and Superoxide dismutase 1 has been evaluated in an animal model for flap survival evaluation [41]. Finally, the analysis of apoptosis-related genes included the analysis of Caspase 3, Caspase 9, B-cell CLL/lymphoma 2. These proteins have a key role in the regulation of apoptosis [42,43], being frequently used to evaluate ischemia induction in animal models [44,45,46].
In summary, here we describe a well-characterized I/R animal model for the evaluation of therapeutic agents under reproducible and controlled conditions. This model could be especially interesting in reconstructive microsurgery and preclinical studies.
Materials and methods
Experimental design
Adult male Wistar rats (n = 14), weighing 290–350 g, were used in this study. The experimental procedures were approved by the ethical committee of animal experimentation of the Jesús Usón Minimally Invasive Surgery Center and were in accordance with the welfare standards of the regional government which are based on European legislation.
Previous to surgery, the rats were acclimatized in cages at 22–25°C with food and water ad libitum. All surgical procedures were performed under general inhalation anesthesia using sevoflurane. Enrofloxacin (7.5 mg/kg/day during 5 days), a broad spectrum antibiotic, and Meloxicam (1 mg/kg/day during 5 days), an anti-inflammatory and analgesic drug, were injected subcutaneously in all the animals after completion of the procedures. All animals were individually housed after surgery and postoperative protectors (patent number P201400272, Spanish Patent and Trademark Office) were placed on them to impede self-mutilation and injuries.
We used a caudal superficial epigastric skin free flap (3 × 6 cm) in our study. The surgical procedures were performed in 14 animals, randomly divided into two groups. The raised flaps in the animals of the control group (n = 7) did not suffer any ischemic insult (the vascular pedicle was not cut). The raised flaps in the ischemia-reperfusion group (n = 7) were subjected to ischemia for 8 hours prior to revascularization (detailed in the flap model section).
Blood flow of the flap pedicle was measured using a transit-time ultrasound flowmeter and microvascular probes (Transonic Systems Inc., Ithaca, NY) prior to repositioning of the skin to its original site using 4/0 silk sutures following a simple interrupted pattern. Microsurgical arterial and venous anastomoses were performed using 10/0 nylon sutures in the animals of the ischemia-reperfusion group (I/R Group). After the surgery, pictures were taken for planimetric analysis.
Seven days post-surgery the rats were anesthetized again. Macroscopic pictures were taken to assess the survival area of the flaps, then the flaps were raised again, the blood flow was evaluated, and the flaps were divided for histopathological and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis. Then, the animals were euthanized by rapid intracardiac injection of KCl (2 meq/kg, KCl 2M), under general inhalation anesthesia, according to the ethical committee recommendations. A schematic representation of the experimental design is shown in Fig 1.
Flap model
The experimental study was based on a free inguinal cutaneous flap [47]. A 3 x 6-cm skin flap was raised, being perfused by the left superficial caudal epigastric vessels. After dissection of the vascular pedicle, ligatures with 8/0 nylon sutures were performed on the proximal caudal femoral vessels, on the lateral circumflex femoral vessels and on the saphenous artery and vein [48] (Fig 2).
A) Ligatures of lateral circumflex femoral artery and vein. B) Ligatures of proximal caudal femoral artery and vein. C) Ligatures of saphenous artery and vein. Dotted lines represent the microsurgical anastomoses sites.
After completing the dissection, the flaps of the control group were relocated free of ischemia in their original site. The ischemia was induced in I/R group by cutting the artery and vein of the axial pattern flaps. Heparinized saline solution was used to perfuse the flap and thus remove stagnant blood from the microcirculation. Then, microsurgical anastomoses were performed (10/0 nylon suture) and removal of the clamps was done after the 8h of ischemia period.
Vascular patency assessment using transit-time ultrasound technology
Transit-time ultrasound flowmeter and microvascular probes (Transonic Systems Inc., Ithaca, N.Y.) were used to verify blood flow patency intraoperatively after the end-to-end anastomoses performed in the animals of the I/R group and also to verify again the blood flow one week after the procedure. Blood flow was also measured in the control group the day of the surgery and at the end of the study.
Macroscopic measurement of the flap survival
A macroscopic follow-up of the flaps was carried out evaluating the changes. Seven days after surgery, the surviving flap areas were measured by image analysis using the ImageJ software (program designed for scientific multidimensional images). The areas of survival and necrosis were measured in cm2 and the percentage of viable area was calculated as: (cm2 of viable area/ cm2 of total flap area) x 100.
Sample harvesting
On day 7, animals were anesthetized for tissue sample harvesting. All flaps were divided in 6 quadrants of 2 cm x 1.5 cm (Fig 3). Tissue samples from quadrants Q1 were fixed with 4% paraformaldehyde and were paraffin-embedded. Tissues were sliced at 4 μm for histological analysis. Tissue samples from quadrants Q4 were cryopreserved at -80°C till tested for RT-qPCR analysis. Greater tissue damage was found in the distal area of flaps. We focused our analyses on Q1 and Q4, which are the more distal quadrants to the flap vascular pedicle.
The quadrants Q1 were excised and fixed in 4% paraformaldehyde for further histological analysis. The quadrants Q4 were cryopreserved at -80°C for gene expression analysis.
Histological analysis
Paraffin-embedded samples were processed for hematoxylin/eosin staining and for Masson’s Trichrome staining. All preparations were visualized, scored and evaluated under light microscopy. A blinded histopathological evaluation of all samples was done.
The severity of inflammation for each sample was scored according to the presence of neutrophils, eosinophils, lymphocytes, plasma cells, macrophages, mastocytes and giant cells as follows: (1) Mild/without formation of aggregates, (2) Moderate/occasional aggregates, (3) Intense/packed. The severity of tissue damage was scored for necrosis, edema, hemorrhage and thrombosis as follows: (0) absent, (1) mild, (2) moderate, (3) intense. Fibrosis and vascular proliferation were assessed from the Masson’s Trichrome slides and scored as follows: (1) mild, (2) moderate and (3) intense.
Methodological gene selection
The selection of genes was performed according to Gene Ontology classification [49,50]. The first selection criteria was based on the rat (Rattus norvegicus) genes that were involved in the following biological processes with proved experimental evidence: response to ischemia (GO:0002931), regulation of inflammatory response (GO:0050729), regulation of angiogenesis (GO:0045766), regulation of necrotic cell death (GO:0010940), regulation of execution phase of apoptosis (GO:1900117), regulation response to oxidative stress (GO:0036473, GO:1902883, GO:1902884) and cornification (GO:0070268). A total of 165 genes were obtained. Then, we investigated these genes that regulate the same biological processes in humans (with proved experimental evidence), which lead to selection of 55 genes. From those, we selected Arginase, iNOS, TNF-α, IL-1β, IL6 and IL10 for monitoring the inflammatory response. In order to analyze the regulation of angiogenesis, we quantified VEGF, FGF and angiopoietin-2. Finally, HiF, SOD1, Bcl-2, Cas3 and Cas9 were used to assess the necrosis/apoptosis and oxidative stress processes. Gusb and 18S ribosomal RNA were used as housekeeping genes. Gene symbol, gene name, gene ID, Rat Genome Database (RGD) reference and ThemoFisher Assay ID Genes IDs are listed in Table 1.
RNA isolation, reverse transcription and real time polymerase chain reaction
Tissues were disrupted and homogenized in 1 ml of TRI-Reagent (Sigma, St. Louis, MO, USA). Then, chloroform was added and samples were incubated for 5–10 min at room temperature. After centrifugation of 15 min at 12,000 g, the aqueous phase was mixed with 500 μl of isopropanol and incubated at -80°C for 20 min to precipitate the RNA. Consecutive centrifugations and ethanol washings were performed. Finally, the pellets were resuspended in RNase-free 0.1% DEPC water (Invitrogen).
The cDNA was synthesized using iScript Reverse Transcription Supermix (BioRad, Hercules, CA, USA) from 1 μg of RNA in reverse transcription reaction for 20 min at 46°C, after a priming period of 5 min at 25°C and followed by RT inactivation at 95°C for 1 min. For qPCR amplification, commercial gene expression assay kits were used (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to manufacturer recommendations. The reaction was performed using TaqMan probes in a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc.) and the products were quantified by fluorescent method using 2-ΔCt expression with Gusb as housekeeping gene.
Statistical analysis
Statistical analyses were performed using SPSS 21 software (IBM Corp., Armonk, NY). All data are expressed as the mean ± standard deviations. Shapiro–Wilk test was used to determine normality of each variable. For parametric variables, differences between means of two groups were analyzed by a Student’s t-test and Levene’s test was used to evaluate the homogeneity of variance. For non-parametric variables, Mann-Whitney’s U test was used to determine differences between groups. In all analyses, p-values <0.05 were considered to indicate statistically significant differences between groups.
Results
Vascular patency assessment using transit-time ultrasound technology
All 28 microsurgical anastomoses were patent 1 week after the surgery day. The results of blood flow before the closure of the skin on the day of the surgery were 0.69 ± 0.26 mL/min for arteries and 0.54 ± 0.17 mL/min for veins. One week later, the results were as follows: 0.88 ± 0.45 mL/min for arteries and 0.83 ± 0.45 mL/min for veins. No significant differences in blood flow were detected between the groups.
Macroscopic measurement of the flap survival
Flap survival area in the control group was significantly higher than that in flaps that suffered 8 h of ischemic insult (p < 0.01). As shown in Fig 4, the flap survival area in control group was 84.11 ± 2.21%. In contrast, the flap survival area in the I/R group was 41.82 ± 15.99%.
Histological analysis
The histology of the distal flap area (Q1 quadrant from Fig 3) exhibited full-thickness skin necrosis, structural damage, subcutaneous edema and a large number of infiltrated inflammatory cells in all those flaps that suffered 8 hours of ischemia. In contrast, control skin flaps showed much less inflammatory cell infiltration (Fig 5).
At day 7 post-surgery, the skin flaps of euthanized animals were fixed in 4% paraformaldehyde, paraffin-embedded, and stained for hematoxylin-eosin (H&E) and Masson's trichrome (MT). Horizontal bars represent 100 μm. The different skin layers are numbered: epidermis (1), dermis (2) and adipose tissue (3).
The histology scores showed that the level of infiltration of neutrophils in the control group animals was significantly lower than that in the I/R group (p < 0.05). Also, a significant increase in necrosis severity was found in the I/R group when compared with that in the control group (p < 0.05) (Table 2).
Differences were statistically analyzed using Student’s t-test for variables with a parametric distribution and Mann-Whitney’s U-test for non-parametric variables. *p-values <0.05 were considered to indicate statistical significance.
Gene expression analysis
Expression of genes related with inflammatory response, angiogenesis, necrosis/apoptosis and oxidative stress was quantified in the harvested tissues (Q4 quadrant from Fig 3). No significant differences were found between groups when we compared expression of the following inflammation-related genes: IL1b, IL6, IL10, Tnf and iNOS. Significant difference was found for angiogenesis-related genes: angiopoietin-2, Fgf2 and Vegfa. Similarly, expression of oxidative stress-related genes (SOD1, HiF1a) and apoptosis/necrosis-related genes (Cas3, Cas9, Bcl2) did not show significant differences. Interestingly, our results demonstrated that Arginase 1 as well as the Arginase 1/Nitric Oxide Synthase 2 ratio showed a significant increase in expression in the I/R group (p<0.01) (Fig 6).
At day 7 post-surgery, total RNA from the skin flaps was isolated and qRT-PCR products were quantified by the 2-ΔCt method. Graphs represent the mean ± SD of independently performed experiments. The results were organized by groups of genes related with the following biological processes: A) Inflammatory response, B) Oxidative stress, C) Angiogenesis, and D) Necrosis/Apoptosis. Data were statistically analyzed using Student’s t-test for variables with a parametric distribution and Mann-Whitney’s U Test for non-parametric variables. Horizontal bars represent statistically significant differences (**p<0.01).
Discussion
Ischemia is inevitable in all microsurgical free flaps performed in reconstructive procedures [40]. The ischemia period is closely related with an inflammatory response, which is accelerated and augmented when the ischemic tissue is reperfused [4]. The process is dependent on a complex interaction between a variety of inflammatory mediators [51].
In this work, we have tried to improve the characterization of an animal model to study ischemia-reperfusion injury in the field of reconstructive surgery. The use of our refined preclinical model will hopefully instill greater confidence in clinicians when deciding which compounds are worthy of further investigation in preclinical or human clinical trials. The characterization of this microsurgical model for preclinical research is based on five different aspects: anatomy, ischemia-reperfusion injury induction, blood flow assessment, histology and gene expression.
In our ischemia/reperfusion injury animal model, after the anatomical dissections, vascular ligatures and anastomoses, hindlimb blood flow was not compromised in any animal. Furthermore, we did not observe any resulting pain or limp. In agreement with Kochi et al [48], our research model left intact three collateral routes through intramuscular networks in the quadriceps femoris muscle, biceps femoris muscle and the medial thigh muscles including the medial hamstring muscles and adductor muscles.
In our study, the surgical procedure was performed by microsurgical anastomoses to induce I/R injury. Although it is a time-consuming procedure that requires fine microsurgical skills, flaps undergoing anastomoses are more resistant to ischemia than those undergoing clamping [52]. Moreover, our surgical methodology is more translatable to a clinical scenario in free flap surgery.
During the surgical procedures, we have measured the blood flow using transit-time ultrasound technology, which is a novel and objective method of evaluation of microsurgical anastomoses [53,54]. Blood flow measurement is of crucial importance in microsurgery [55], as part of the tissue damage due to ischemia is responsible for slowing down blood flow and causing thrombosis [56]. The widely used manual patency test demonstrates whether there is blood flow through anastomoses sites, but it does not demonstrate the quality of the blood flow [57]. Transit-time flow measurement allows a quantitative and objective assessment of vascular function and helps to prevent failure of anastomoses [54,58,59]. Our research model ensured in all animals a good microvascular perfusion after 8 hours of ischemia (no statistical differences were observed). This test objectively demonstrated that our macroscopic, histological and genetic results were neither influenced by blood perfusion differences nor by the microsurgical technique, which is is essential to delineate valuable conclusions from our ischemia/reperfusion study.
The anatomopathological analysis of the free flaps at day 7 revealed infiltration of leukocytes, structural damage and edema in all those flaps that suffered 8 hours of ischemia. Control skin flaps had lower inflammatory cell infiltration than in the animals of I/R group (p < 0.05). Furthermore, a significant increase in necrosis severity was found in I/R group when compared with that in the control group (p < 0.05). These results are in agreement with those of previous studies in the area of ischemia/reperfusion injury [20,47,60].
Once we identified the histological changes, we next aimed to characterize the gene expression pattern of tissue samples from the control and I/R groups. The main goals of this analysis were to obtain an overview of the pathophysiology of ischemia reperfusion injury and to identify molecular biomarkers for preclinical studies and drug testing.
Interestingly, our analysis revealed the absence of significant changes in Th1/Th2 cytokine levels, which may indicate that most probably that other Th1/Th2 cytokines (i.e., IFN-γ, IL-2 or IL-4) may have differences but they were initially excluded from this study. Regarding to M1/M2–related mRNAs, the expression of Arginase 1 as well as the ratio Arginase 1/Nitric Oxide Synthase 2 showed a significant increase in the I/R group. Arginase 1 is released by M2 macrophages to metabolize L-arginine into ornithine and urea, and its expression is widely used as a classic M2 marker [61]. The significant increase of Arginase 1 expression may reflect a macrophage polarization towards and anti-inflammatory phenotype, however, it is interesting to note that the anatomopathological analysis demonstrated a decrease in the number of infiltrated macrophages in the I/R group. Considering these two changes (decrease in the number of infiltrating macrophages and increase of Arginase-1 expression), here we hypothesize that, in our experimental model, the I/R injury triggered the migration of M1 macrophages towards apoptotic and necrotic areas. Simultaneously, the viable tissue counteracted the necrotic response retaining pro-regenerative M2 cells to promote tissue regeneration. Additionally, the analysis of Arginase-1 expression in this I/R model could also be considered a useful biomarker for proof of concept studies and for the development of efficient therapies, but this aspect will be further discussed.
Apart from inflammatory-related genes, this study was also focused on three biological process associated with I/R injury: angiogenesis, oxidative stress and apoptosis. As shown in the results section, any significant difference was observed when these genes were compared between groups. The absence of significant differences among groups could reflect the maintenance of tissue homeostasis during ischemic insult in this I/R model. However, taking into account our macroscopic and histopathological results in this animal model, we should also consider the possibility that there are myriad genes that may have a key role in angiogenesis, oxidative stress or apoptosis and were not included in the selected panel of genes in this study.
Finally, it is important to highlight the relevance of the differential expression of Arginase 1 in the I/R free flap model. It is well known that the induction and activation of arginase is a common event that occurs in the initial stage of ischemic events [62,63].
The hypoxia and ischemic conditions shift arginine to be metabolized to ornithine and urea. This shift has been previously described in ischemic heart disease [63], renal ischemia-reperfusion injury [64] as well as in neurovascular injury after retinal I/R [65].
Given that arginase and iNOS is tightly regulated by direct protein modification or by induction of enzyme expression, our study was also focused on the simultaneous quantification of iNOS and their ratio.
Conclusion
Our results demonstrated an imbalance of arginase/iNOS ratio (with a predominant arginase activity), that could be interpreted as a biomarker to identify the initial phase of I/R injury in the tissue. It is important to note the limitations of this study lies in the fact that there are numerous genes that were not included in the qPCR analysis. In this sense, we should not exclude the importance of other biomarkers such as miRNAs or siRNAs with key role in angiogenesis, oxidative stress or apoptosis.
Additionally, it is important to point out the importance of this animal model to evaluate different therapies to counteract ischemia-reperfusion injury in skin free flaps. In particular, considering that arginase inhibition has been hypothesized as a promising therapeutic strategy for the treatment of I/R injury [66,67], this animal model could be especially useful for preclinical studies focused on the evaluation of arginase inhibitors such as NO scavengers or NOS inhibitors for a clinically safe ischemia time in free flap surgery.
Acknowledgments
The research project was performed at the Jesus Usón Minimally Invasive Surgery Center (CCMIJU) which is part of the ICTS “Nanbiosis.” The study was performed with the participation of the following Nanbiosis Units: U21-Experimental operating rooms, U22-Animal housing and U14-Cell Therapy. Special thanks to the medical illustrators Carmen Alía, Nuria González and María Pérez.
References
- 1. Bennett N, Choudhary S (2000) Why climb a ladder when you can take the elevator? Plastic and reconstructive surgery 105: 2266. pmid:10839429
- 2. Gottlieb LJ, Krieger LM (1994) From the reconstructive ladder to the reconstructive elevator. Plastic and reconstructive surgery 93: 1503–1504. pmid:7661898
- 3. Siemionow M, Arslan E (2004) Ischemia/reperfusion injury: a review in relation to free tissue transfers. Microsurgery 24: 468–475. pmid:15378577
- 4. Wang WZ, Baynosa RC, Zamboni WA (2011) Update on ischemia-reperfusion injury for the plastic surgeon: 2011. Plastic and reconstructive surgery 128: 685e–692e. pmid:22094770
- 5. Carroll WR, Esclamado RM (2000) Ischemia/reperfusion injury in microvascular surgery. Head & neck 22: 700–713.
- 6. Chang KP, Lai CS (2012) Micro-RNA profiling as biomarkers in flap ischemia-reperfusion injury. Microsurgery 32: 642–648. pmid:23097335
- 7. Shin MS, Angel MF, Im MJ, Manson PN (1994) Effects of 21-aminosteroid U74389F on skin-flap survival after secondary ischemia. Plastic and reconstructive surgery 94: 661–666. pmid:7938289
- 8. Yoshida WB, Campos EB (2005) Ischemia and reperfusion in skin flaps: effects of mannitol and vitamin C in reducing necrosis area in a rat experimental model. Acta cirurgica brasileira 20: 358–363. pmid:16186959
- 9. Harder Y, Amon M, Laschke MW, Schramm R, Rucker M, et al. (2008) An old dream revitalised: preconditioning strategies to protect surgical flaps from critical ischaemia and ischaemia-reperfusion injury. Journal of plastic, reconstructive & aesthetic surgery: JPRAS 61: 503–511.
- 10.
Bizeau A, Guelfucci B, Giovanni A, Gras R, Casanova D, et al. (2002) [15 years experience with microvascular free tissue transfert for repair of head and neck cancer defects]. Annales d'oto-laryngologie et de chirurgie cervico faciale: bulletin de la Societe d'oto-laryngologie des hopitaux de Paris 119: 31–38.
- 11. Bui DT, Cordeiro PG, Hu QY, Disa JJ, Pusic A, et al. (2007) Free flap reexploration: indications, treatment, and outcomes in 1193 free flaps. Plastic and reconstructive surgery 119: 2092–2100. pmid:17519706
- 12. Vega S, Smartt JM Jr., Jiang S, Selber JC, Brooks CJ, et al. (2008) 500 Consecutive patients with free TRAM flap breast reconstruction: a single surgeon's experience. Plastic and reconstructive surgery 122: 329–339. pmid:18626347
- 13. Chiu YH, Chang DH, Perng CK (2017) Vascular Complications and Free Flap Salvage in Head and Neck Reconstructive Surgery: Analysis of 150 Cases of Reexploration. Annals of plastic surgery 78: S83–S88. pmid:28166137
- 14. Koul AR, Patil RK, Nahar S (2013) Unfavourable results in free tissue transfer. Indian journal of plastic surgery: official publication of the Association of Plastic Surgeons of India 46: 247–255.
- 15. Yu P, Chang DW, Miller MJ, Reece G, Robb GL (2009) Analysis of 49 cases of flap compromise in 1310 free flaps for head and neck reconstruction. Head & neck 31: 45–51.
- 16. Shestak KC, Jones NF (1991) Microsurgical free-tissue transfer in the elderly patient. Plastic and reconstructive surgery 88: 259–263. pmid:1852818
- 17. Jones NF, Jarrahy R, Song JI, Kaufman MR, Markowitz B (2007) Postoperative medical complications—not microsurgical complications—negatively influence the morbidity, mortality, and true costs after microsurgical reconstruction for head and neck cancer. Plastic and reconstructive surgery 119: 2053–2060. pmid:17519700
- 18. Lo SL, Yen YH, Lee PJ, Liu CC, Pu CM (2017) Factors Influencing Postoperative Complications in Reconstructive Microsurgery for Head and Neck Cancer. Journal of oral and maxillofacial surgery: official journal of the American Association of Oral and Maxillofacial Surgeons 75: 867–873.
- 19. van den Heuvel MG, Buurman WA, Bast A, van der Hulst RR (2009) Review: Ischaemia-reperfusion injury in flap surgery. Journal of plastic, reconstructive & aesthetic surgery: JPRAS 62: 721–726.
- 20. Han HH, Lim YM, Park SW, Lee SJ, Rhie JW, et al. (2015) Improved skin flap survival in venous ischemia-reperfusion injury with the use of adipose-derived stem cells. Microsurgery 35: 645–652. pmid:26510716
- 21. Fries CA, Villamaria CY, Spencer JR, Rasmussen TE, Davis MR (2017) C1 esterase inhibitor ameliorates ischemia reperfusion injury in a swine musculocutaneous flap model. Microsurgery 37: 142–147. pmid:27088544
- 22. Liu YQ, Liu YF, Ma XM, Xiao YD, Wang YB, et al. (2015) Hydrogen-rich saline attenuates skin ischemia/reperfusion induced apoptosis via regulating Bax/Bcl-2 ratio and ASK-1/JNK pathway. Journal of plastic, reconstructive & aesthetic surgery: JPRAS 68: e147–156.
- 23. Cetin C, Kose AA, Aral E, Colak O, Ercel C, et al. (2001) Protective effect of fucoidin (a neutrophil rolling inhibitor) on ischemia reperfusion injury: experimental study in rat epigastric island flaps. Annals of plastic surgery 47: 540–546. pmid:11716267
- 24. Wang WZ (2009) Investigation of reperfusion injury and ischemic preconditioning in microsurgery. Microsurgery 29: 72–79. pmid:18946882
- 25. Fowler JD, Li X, Cooley BC (1999) Brief ex vivo perfusion with heparinized and/or citrated whole blood enhances tolerance of free muscle flaps to prolonged ischemia. Microsurgery 19: 135–140. pmid:10231122
- 26. Hong JP, Kwon H, Chung YK, Jung SH (2003) The effect of hyperbaric oxygen on ischemia-reperfusion injury: an experimental study in a rat musculocutaneous flap. Annals of plastic surgery 51: 478–487. pmid:14595184
- 27. Liu YY, Chiang CH, Hung SC, Chian CF, Tsai CL, et al. (2017) Hypoxia-preconditioned mesenchymal stem cells ameliorate ischemia/reperfusion-induced lung injury. PloS one 12: e0187637. pmid:29117205
- 28. Xiao YD, Liu YQ, Li JL, Ma XM, Wang YB, et al. (2015) Hyperbaric oxygen preconditioning inhibits skin flap apoptosis in a rat ischemia-reperfusion model. The Journal of surgical research 199: 732–739. pmid:26216750
- 29. Wu MY, Yiang GT, Liao WT, Tsai AP, Cheng YL, et al. (2018) Current Mechanistic Concepts in Ischemia and Reperfusion Injury. Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology 46: 1650–1667.
- 30.
Barre-Sinoussi F, Montagutelli X (2015) Animal models are essential to biological research: issues and perspectives. Future science OA 1: FSO63.
- 31. Ericsson AC, Crim MJ, Franklin CL (2013) A brief history of animal modeling. Missouri medicine 110: 201–205. pmid:23829102
- 32. Bonjour JP, Ammann P, Rizzoli R (1999) Importance of preclinical studies in the development of drugs for treatment of osteoporosis: a review related to the 1998 WHO guidelines. Osteoporosis international: a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA 9: 379–393.
- 33. Rao J, Lu L, Zhai Y (2014) T cells in organ ischemia reperfusion injury. Current opinion in organ transplantation 19: 115–120. pmid:24576906
- 34. Bansal SS, Ismahil MA, Goel M, Patel B, Hamid T, et al. (2017) Activated T Lymphocytes are Essential Drivers of Pathological Remodeling in Ischemic Heart Failure. Circulation Heart failure 10: e003688. pmid:28242779
- 35. Chen S, Guo L, Cui M, Sun L, Mi L (2012) Dynamic changes in serum angiopoietin-1, angiopoietin-2, and angiopoietin-2/angiopoietin-1 ratio in acute myocardial infarction patients treated with primary percutaneous coronary intervention. Biomarkers: biochemical indicators of exposure, response, and susceptibility to chemicals 17: 441–446.
- 36. Ma XL, Liu KD, Li FC, Jiang XM, Jiang L, et al. (2013) Human mesenchymal stem cells increases expression of alpha-tubulin and angiopoietin 1 and 2 in focal cerebral ischemia and reperfusion. Current neurovascular research 10: 103–111. pmid:23469950
- 37. Li J, Wei Y, Liu K, Yuan C, Tang Y, et al. (2010) Synergistic effects of FGF-2 and PDGF-BB on angiogenesis and muscle regeneration in rabbit hindlimb ischemia model. Microvascular research 80: 10–17.
- 38. Jazwa A, Florczyk U, Grochot-Przeczek A, Krist B, Loboda A, et al. (2016) Limb ischemia and vessel regeneration: Is there a role for VEGF? Vascular pharmacology 86: 18–30. pmid:27620809
- 39. Dragu A, Schnurer S, Surmann-Schmitt C, Unglaub F, Kneser U, et al. (2011) Expression of HIF-1alpha in ischemia and reperfusion in human microsurgical free muscle tissue transfer. Plastic and reconstructive surgery 127: 2293–2300. pmid:21617463
- 40. Schmidt Y, Bannasch H, Eisenhardt SU (2012) Ischemia-reperfusion injury leads to significant tissue damage in free flap surgery. Plastic and reconstructive surgery 129: 174e–175e;author reply 175e-176e. pmid:22186547
- 41. Schein O, Westreich M, Shalom A (2013) Effect of intradermal human recombinant copper-zinc superoxide dismutase on random pattern flaps in rats. Head & neck 35: 1265–1268.
- 42. Kale J, Osterlund EJ, Andrews DW (2018) BCL-2 family proteins: changing partners in the dance towards death. Cell death and differentiation 25: 65–80. pmid:29149100
- 43. Fogarty CE, Bergmann A (2017) Killers creating new life: caspases drive apoptosis-induced proliferation in tissue repair and disease. Cell death and differentiation 24: 1390–1400. pmid:28362431
- 44. Hernandez de GM, Garay FJ, Loureiro NE (2015) Neuroprotective action of valproic acid accompanied of the modification on the expression of Bcl-2 and activated caspase-3 in the brain of rats submitted to ischemia/reperfusion. Investigacion clinica 56: 377–388. pmid:29938967
- 45. Liu G, Wang T, Song J, Zhou Z (2013) Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats. Biomedical reports 1: 861–867. pmid:24649043
- 46. Li J, Han B, Ma X, Qi S (2010) The effects of propofol on hippocampal caspase-3 and Bcl-2 expression following forebrain ischemia-reperfusion in rats. Brain research 1356: 11–23. pmid:20707988
- 47. Ballestin A, Casado JG, Abellan E, Vela FJ, Alvarez V, et al. (2018) Adipose-Derived Stem Cells Ameliorates Ischemia-Reperfusion Injury in a Rat Skin Free Flap Model. Journal of reconstructive microsurgery.
- 48. Kochi T, Imai Y, Takeda A, Watanabe Y, Mori S, et al. (2013) Characterization of the Arterial Anatomy of the Murine Hindlimb: Functional Role in the Design and Understanding of Ischemia Models. PloS one 8.
- 49. Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, et al. (2004) The Gene Ontology (GO) database and informatics resource. Nucleic acids research 32: D258–261. pmid:14681407
- 50. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature genetics 25: 25–29. pmid:10802651
- 51. Frangogiannis NG, Smith CW, Entman ML (2002) The inflammatory response in myocardial infarction. Cardiovascular research 53: 31–47. pmid:11744011
- 52. Chafin B, Belmont MJ, Quraishi H, Clovis N, Wax MK (1999) Effect of clamp versus anastomotic-induced ischemia on critical ischemic time and survival of rat epigastric fasciocutaneous flap. Head & neck 21: 198–203.
- 53. Selber JC, Garvey PB, Clemens MW, Chang EI, Zhang H, et al. (2013) A prospective study of transit-time flow volume measurement for intraoperative evaluation and optimization of free flaps. Plastic and reconstructive surgery 131: 270–281. pmid:23076414
- 54. Shaughness G, Blackburn C, Ballestin A, Akelina Y, Ascherman JA (2017) Predicting Thrombosis Formation in 1-mm-Diameter Arterial Anastomoses with Transit-Time Ultrasound Technology. Plastic and reconstructive surgery 139: 1400–1405. pmid:28538566
- 55. Pafitanis G, Raveendran M, Myers S, Ghanem AM (2017) Flowmetry evolution in microvascular surgery: A systematic review. Journal of plastic, reconstructive & aesthetic surgery: JPRAS 70: 1242–1251.
- 56. Deheng C, Kailiang Z, Weidong W, Haiming J, Daoliang X, et al. (2016) Salidroside Promotes Random Skin Flap Survival in Rats by Enhancing Angiogenesis and Inhibiting Apoptosis. Journal of reconstructive microsurgery 32: 580–586. pmid:27276197
- 57. Krag C, Holck S (1981) The value of the patency test in microvascular anastomosis: Correlation between observed patency and size of intraluminal thrombus: An experimental study in rats. British journal of plastic surgery 34: 64–66. pmid:7459527
- 58. Di Giammarco G, Pano M, Cirmeni S, Pelini P, Vitolla G, et al. (2006) Predictive value of intraoperative transit-time flow measurement for short-term graft patency in coronary surgery. The Journal of thoracic and cardiovascular surgery 132: 468–474. pmid:16935097
- 59. Walpoth BH, Bosshard A, Genyk I, Kipfer B, Berdat PA, et al. (1998) Transit-time flow measurement for detection of early graft failure during myocardial revascularization. The Annals of thoracic surgery 66: 1097–1100. pmid:9769011
- 60. Fitzal F, Valentini D, Mittermayr R, Worseg A, Gasser IH, et al. (2001) Circulatory changes after prolonged ischemia in the epigastric flap. Journal of reconstructive microsurgery 17: 535–543. pmid:11598828
- 61. Roszer T (2015) Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms. Mediators of inflammation 2015: 816460. pmid:26089604
- 62. Hein TW, Zhang C, Wang W, Chang CI, Thengchaisri N, et al. (2003) Ischemia-reperfusion selectively impairs nitric oxide-mediated dilation in coronary arterioles: counteracting role of arginase. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 17: 2328–2330.
- 63. Schluter KD, Schulz R, Schreckenberg R (2015) Arginase induction and activation during ischemia and reperfusion and functional consequences for the heart. Frontiers in physiology 6: 65. pmid:25814956
- 64. Raup-Konsavage WM, Gao T, Cooper TK, Morris SM Jr., Reeves WB, et al. (2017) Arginase-2 mediates renal ischemia-reperfusion injury. American journal of physiology Renal physiology 313: F522–F534. pmid:28515179
- 65. Shosha E, Xu Z, Yokota H, Saul A, Rojas M, et al. (2016) Arginase 2 promotes neurovascular degeneration during ischemia/reperfusion injury. Cell death & disease 7: e2483.
- 66. Tratsiakovich Y, Yang J, Gonon AT, Sjoquist PO, Pernow J (2013) Arginase as a target for treatment of myocardial ischemia-reperfusion injury. European journal of pharmacology 720: 121–123. pmid:24183975
- 67. Pernow J, Jung C (2013) Arginase as a potential target in the treatment of cardiovascular disease: reversal of arginine steal? Cardiovascular research 98: 334–343. pmid:23417041