Microvascular Anastomoses for Cerebral Ischemia

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The results of blood flow before the closure of the skin on the day of the surgery were 0. One week later, the results were as follows: 0.

No significant differences in blood flow were detected between the groups. As shown in Fig 4 , the flap survival area in control group was 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.


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The different skin layers are numbered: epidermis 1 , dermis 2 and adipose tissue 3. Significant difference was found for angiogenesis-related genes: angiopoietin-2, Fgf2 and Vegfa. Ischemia is inevitable in all microsurgical free flaps performed in reconstructive procedures [ 40 ].

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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.


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  • 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. 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.

    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. 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.

    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.

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    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 ]. Simultaneously, the viable tissue counteracted the necrotic response retaining pro-regenerative M2 cells to promote tissue regeneration. As shown in the results section, any significant difference was observed when these genes were compared between groups.

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    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. 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. 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. 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.

    Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Ischemia reperfusion injury is associated with tissue damage and inflammation, and is one of the main factors causing flap failure in reconstructive microsurgery. Data Availability: All relevant data are within the manuscript. 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.

    Download: PPT. Flap model The experimental study was based on a free inguinal cutaneous flap [ 47 ].

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    Vascular patency assessment using transit-time ultrasound technology Transit-time ultrasound flowmeter and microvascular probes Transonic Systems Inc. Macroscopic measurement of the flap survival A macroscopic follow-up of the flaps was carried out evaluating the changes. Sample harvesting On day 7, animals were anesthetized for tissue sample harvesting. Methodological gene selection The selection of genes was performed according to Gene Ontology classification [ 49 , 50 ].

    Table 1. Results Vascular patency assessment using transit-time ultrasound technology All 28 microsurgical anastomoses were patent 1 week after the surgery day. Fig 4. 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. Fig 6. Quantitative expression of ischemia-reperfusion-related genes.

    Discussion Ischemia is inevitable in all microsurgical free flaps performed in reconstructive procedures [ 40 ]. References 1. Bennett N, Choudhary S Why climb a ladder when you can take the elevator? Plastic and reconstructive surgery Plastic and reconstructive surgery ā€” Microsurgery ā€” Plastic and reconstructive surgery eā€”e. You could not be signed in. Sign In Forgot password?

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    View all jobs. Fein and O. This article is only available in the PDF format. Download the PDF to view the article, as well as its associated figures and tables. Surgical treatment of vascular disease of the limbs, heart, and other body parts is well known, but until recently, few investigators dealt with operations to relieve ischemia due to cerebrovascular disease. Surgical techniques for cerebral revascularization are presented in this book. The basic anatomic, physiologic, and hemodynamic changes in stroke are discussed, followed by clinical and laboratory evaluation of direct and collateral circulation in the ischemic brain and basic laboratory studies in animals.

    There is information on the indications for operation, patient selection, management of risk factors, and a variety of operations. Most emphasis is placed on the most popular procedure, anastomosis of the superficial temporal artery to a branch of the middle cerebral artery. The follow-up of such operations is still brief the first human case dates only from , but there seems to be benefit for properly selected patients.

    Particular attention is paid to evaluation of changes in. Salazar JL. Microvascular Anastomoses for Cerebral Ischemia.