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BACKGROUND: The purpose of this study was to evaluate learning curves for an existing microsurgical training model. We compared efficiency and amount of training needed to achieve proficiency between novice microsurgeons without operative experience versus those who had completed a surgical internship. METHODS: Ten novice microsurgeons anastomosed a silastic tube model. Time to perform each anastomosis, luminal diameter, and number of errors were recorded. RESULTS: First year residents improved up to a brief plateau at 10 repetitions, followed by continued improvement. Second year residents improved up to a plateau at 10 repetitions with no further improvement thereafter. There was no significant difference in luminal area or errors between groups. CONCLUSION: Residents with no operative experience can benefit from early exposure to microsurgical training. These interns continue to improve with additional repetitions while second year residents achieve proficiency with fewer repetitions.
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Anastomosis Quirúrgica/educación , Competencia Clínica/normas , Microcirugia/educación , Entrenamiento Simulado , Técnicas de Sutura/normas , Anastomosis Quirúrgica/normas , Evaluación Educacional , Humanos , Internado y Residencia , Curva de Aprendizaje , Microcirugia/normasRESUMEN
PURPOSE: Hyperbaric oxygen (HBO2) therapy is used to improve the survival of compromised flaps. Compromised flaps are complications encountered postsurgically, or in traumatic degloving or avulsion injuries. Failed flaps lead to persistence of the defect, requirement of another donor site, and psychosocial sequelae. Although evidence of the benefit of HBO2 therapy is significant, there is no consensus on the optimal treatment regimen. The purpose of this study is to examine whether twice-daily treatments (BID HBO2) provide additional benefit compared to daily treatments (QD HBO2) in a rat compromised random flap model. METHODS: A rat random flap model was used with subjects divided into three groups: 1) control group; 2) QD HBO2; and 3) BID HBO2, where HBO2 was performed with 100% oxygen at 2.5 atmospheres absolute/ATA (253 kPa) for 90 minutes. After 10 days, areas of flap necrosis were measured and biopsies were taken for histologic analysis. Statistical analysis was performed using ANOVA and paired t-tests. A P-value ⟨0.05 was considered significant. RESULT: Both treatment groups had significantly increased mean flap survival compared to controls (P⟨0.05). There was no significant difference in flap survival between the QD and BID groups. Capillary proliferation in the QD group was increased compared with controls. CONCLUSION: Both QD and BID HBO2 protocols can significantly decrease random flap necrosis. However, the results of this study suggest there is no additional benefit gained with BID treatments. Clinical studies are warranted to confirm these findings and assist in formalization of protocols for the use of HBO2in treating compromised random flaps.
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Supervivencia de Injerto , Oxigenoterapia Hiperbárica/métodos , Análisis de Varianza , Animales , Capilares/anatomía & histología , Masculino , Necrosis/patología , Ratas , Ratas Sprague-Dawley , Colgajos Quirúrgicos/irrigación sanguínea , Colgajos Quirúrgicos/patología , Factores de TiempoRESUMEN
BACKGROUND: The purpose for the present study was to determine which anesthetic method, local anesthesia versus tumescent, is superior for liposuction in terms of adipose-derived stem cell (ASC) survival in lipoaspirate; which component, lidocaine versus lidocaine with epinephrine, in anesthetic solutions could affect ASC survival; and which mechanism, necrosis versus apoptosis, is involved in lidocaine-induced ASC death. METHODS: Human lipoaspirates were harvested using standard liposuction technique. Individuals scheduled for liposuction on bilateral body areas gave consent and were included in the study. On one area, liposuction was conducted under local anesthesia with lidocaine/epinephrine. On the contralateral area, liposuction was accomplished with tumescent wetting solution containing lidocaine/epinephrine. Lipoaspirates were processed for the isolation of stromal vascular fraction (SVF). ASC survival was determined by the number of adherent ASCs after 24 h of SVF culture. Lidocaine dose-response (with or without epinephrine) on cultured ASCs was examined. Lidocaine-induced ASC apoptosis and necrosis was determined by Annexin V-FITC/Propidium Iodide (PI) assay and analyzed by flow cytometry. RESULTS: All of the participants were female adults. The average age was 45 ± 4.0 years (±SEM) and the average BMI was 28 ± 1.0 (±SEM). Lipoaspirate samples (n = 14) treated by local anesthesia (n = 7/group) or tumescent anesthesia (n = 7/group) were investigated. Liposuction sites were located in the hip or thigh. The average number of adherent ASCs was 1,057 ± 146 k in the local anesthesia group, which was significantly lower than the 1,571 ± 111 k found in the tumescent group (P = 0.01). ASC survival was significantly lower in the lidocaine group and in a dose-dependent manner as compared to the correspondent PBS controls (P < 0.05 or P < 0.01). ASC survival was significantly lower in both the lidocaine and lidocaine with epinephrine groups when compared to PBS controls. Annexin/PI assay showed that ASC apoptosis (but not necrosis) in the lidocaine group was significantly higher than that in the corresponding PBS control (P = 0.026). CONCLUSIONS: Tumescent anesthesia is the superior method for liposuction with respect to ASC preservation compared to local anesthesia. Lidocaine could cause significant ASC apoptosis.
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Anestésicos Locales/administración & dosificación , Apoptosis/fisiología , Lidocaína/administración & dosificación , Adulto , Anestesia Local , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lipectomía , Persona de Mediana EdadRESUMEN
Acellular dermal matrix products are popular in various aspects of surgical reconstruction including hernia repairs and breast reconstructions. The goal of this study was to determine quantitative collagen weights of AlloMax (C. R. Bard, Inc. [Davol], Warwick, RI) and of contralateral dermis for composition comparison. A rehydrated sample of AlloMax was subcutaneously implanted on the dorsum of 18 male Wistar rats. Rats were randomly assigned to groups on the basis of in vivo implant time: 1, 3, and 6 weeks. At the end of the implant time, the AlloMax was removed and a section of contralateral dermis was excised as a control. Hydroxyproline, rat Collagen I and Collagen III, and neoangiogenesis were determined in the sections. The results are reported as mean ± standard error of the mean. Analysis of variance was used to evaluate the between-group differences. A p value of 0.05 or less was considered significant. Hydroxyproline was significantly increased in the 6-week AlloMax implant (26.19 ± 1.05 vs. 15.03 ± 3.29). Collagen I and Collagen III were significantly increased following 3 weeks in vivo (612.5% ± 98.0 vs. 312.9% ± 82.7, p < 0.05 Collagen I). Neoangiogenesis was significantly increased at 3 and 6 weeks in vivo (2.3 ± 0.3 and 1.9 ± 0.3). Acellular AlloMax was rapidly incorporated into the rat dorsum. The measurement parameters were greater than or equivalent to contralateral dermis in this study.
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Dermis Acelular/metabolismo , Colágeno/metabolismo , Trasplante de Piel , Cicatrización de Heridas/fisiología , Animales , Dermis/metabolismo , Hidroxiprolina/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: Hyperbaric oxygen (HBO) mitigates ischemia-reperfusion (IR) injury via a nitric oxide mechanism that is nitric oxide synthase (NOS) dependent. The purpose of this study was to investigate this NOS-dependent mechanism by examining isoform-specific, tissue-specific, and time-specific upregulation of NOS mRNA, protein, and enzymatic activity. METHODS: We raised a gracilis flap in Wistar rats that were separated into early and late phases. Treatment groups included nonischemic control, IR, HBO-treated ischemia-reperfusion (IR-HBO), and nonischemic HBO control. We harvested tissue-specific samples from gracilis, rectus femoris, aorta, and pulmonary tissues and processed them by reverse transcription polymerase chain reaction and Western blot to determine upregulation of isoform-specific NOS mRNA and protein. We also harvested tissue for NOS activity to investigate upregulation of enzymatic activity. Data are presented as mean ± standard error of the mean with statistics performed by analysis of variance. P ≤ 0.05 was considered significant. RESULTS: There was no increase in NOS mRNA in the early phase. In the late phase, there was a significant increase in endothelial-derived NOS (eNOS) mRNA in IR-HBO compared with IR in gracilis muscle (79.4 ± 22.3 versus 36.1 ± 4.5; P < 0.05) and pulmonary tissues (91.0 ± 31.2 versus 30.2 ± 3.1; P < 0.01). There was a significant increase in the late-phase eNOS pulmonary protein IR-HBO group compared with IR (235.5 ± 46.8 versus 125.2 ± 14.7; P < 0.05). Early-phase NOS activity was significantly increased in IR-HBO compared with IR in pulmonary tissue only (0.049 ± 0.009 versus 0.023 ± 0.003; P < 0.05). CONCLUSIONS: The NOS-dependent effects of HBO on IR injury may result from a systemic effect involving an early increase in eNOS enzymatic activity followed by a late-phase increase in eNOS protein expression within the pulmonary tissues.
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Oxigenoterapia Hiperbárica , Óxido Nítrico Sintasa/metabolismo , Daño por Reperfusión/prevención & control , Animales , Aorta Abdominal/enzimología , Pulmón/enzimología , Masculino , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/enzimología , Ratas , Ratas Wistar , Daño por Reperfusión/enzimologíaRESUMEN
BACKGROUND: Autologous fat grafting has gained popularity, particularly with the discovery of adipose-derived stem cells (ADSC). The possibility of freezing lipoaspirates (LA) for later use has intriguing clinical potential. However, the effect of LA cryopreservation on ADSC is unclear. OBJECTIVES: The authors explore the effect of LA cryopreservation on ADSC viability. METHODS: Human LA (n = 8) were harvested using a standard technique. Lipoaspirate samples were either processed immediately as fresh LA (A) or stored at -20°C and then at -80°C for 30 days with (B) or without (C) freezing medium. Stromal vascular fraction (SVF) was separated from adipocytes and either cultured to obtain purified ADSC or processed for the isolation of 3 distinct ADSC subpopulations (CD90(+)/CD45(-), CD105(+)/CD45(-), and CD34(+)/CD31(-)). Apoptosis and necrosis were determined by an annexin V/propidium iodide assay and quantified by flow cytometry. The capability of ADSC for long-term proliferation and differentiation was also examined. RESULTS: There were no significant differences in the apoptosis and necrosis of adipocytes, SVF, or ADSC between groups A and B. However, cell viability in SVF and ADSC was significantly compromised in group C as compared with group B (P < .01) due to higher ADSC apoptosis but not necrosis. The viable ADSC isolated from fresh or frozen LA were cultured for more than 20 passages and demonstrated similar patterns and speed of proliferation with strong capability to differentiate, evidenced by cell doubling time and positive staining with Oil Red O (Sigma-Aldrich, St Louis, Missouri) and alkaline phosphatase. CONCLUSIONS: Lipoaspirates cryopreservation had a significant impact on ADSC apoptosis but not on ADSC necrosis, proliferation, or differentiations. Freezing medium provides significant protection against ADSC apoptosis.
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Adipocitos/citología , Criopreservación , Células Madre/citología , Adulto , Apoptosis , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citometría de Flujo , Humanos , Necrosis , Temperatura , Factores de TiempoRESUMEN
INTRODUCTION: Medical training is increasingly focused on patient safety, limiting the ability to practice technical skills in the operative arena. Alternative methods of training residents must be designed and implemented. METHODS: Three expert microsurgeons were solicited to develop two drills to help residents acquire the basic subset of skills in microsurgery. The first drill was performance of five consecutive simple interrupted sutures on a rubber glove. Expert proficiency was considered a drill time of two standard deviations from expert mean. The drill was performed up to 10 times until completion of the task at expert proficiency. The second drill was performance of an anastomosis on silastic tubing. Residents performed the drill sequentially until performing two consecutive drills at expert proficiency. RESULTS: Eight residents with no microsurgical experience volunteered. Six of the eight residents were able to perform the rubber glove drill at expert proficiency within 10 attempts, with an average of 5.3. All of the residents were able to perform two consecutive silastic tubing drills at expert proficiency within nine attempts, with an average of 5.4. CONCLUSION: Residents were able to acquire a basic subset of microsurgical skills within a reasonable time period using these drills.
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Competencia Clínica , Educación de Postgrado en Medicina , Evaluación Educacional , Internado y Residencia , Microcirugia/educación , Análisis de Varianza , Anastomosis Quirúrgica/normas , Humanos , Técnicas de Sutura/normasRESUMEN
Ischemia/reperfusion (I/R) injury in the skeletal muscle is inevitable in many vascular and musculoskeletal traumas, diseases, free tissue transfers, and during time-consuming reconstructive and transplantation surgeries. Although skeletal muscle has a higher tolerance to ischemia than other organs, prolonged ischemia can still result in significant complications, including muscle necrosis and apoptosis. One of the major goals in the treatment of ischemia is early restoration of blood flow (reperfusion) to the area at risk. However, reperfusion has led to a new pathophysiologic condition called "reperfusion injury," a phenomenon which actually provokes a distinct degree of tissue injury. The purpose of this review is to examine the current state of understanding of I/R injury as well as to highlight recent developments on I/R interventions including our own experience in this particular field. We expect, as our acquired experience and the increased knowledge of underlying mechanisms of I/R injury, more effective interventions aimed to reduce I/R injury will be developed to interfere with or modulate this particular pathophysiologic processes.
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Músculo Esquelético/lesiones , Daño por Reperfusión/etiología , Daño por Reperfusión/terapia , Heridas y Lesiones/complicaciones , Heridas y Lesiones/terapia , Animales , Humanos , Daño por Reperfusión/fisiopatología , Heridas y Lesiones/fisiopatologíaRESUMEN
BACKGROUND: Our recent studies have shown that ischemia/reperfusion (I/R) produces significant necrosis and apoptosis in the cells of skeletal muscle. Our previous studies also demonstrated that melatonin provides significant protection against superoxide generation, endothelial dysfunction, and cell death in the skeletal muscle after I/R. Mitochondria are essential for cell survival, because of their roles as ATP producers as well as regulators of cell death. However, the efficacy of melatonin on I/R-induced mitochondrial dysfunction in the skeletal muscle in vivo has not been demonstrated in the literature. MATERIALS AND METHODS: Vascular pedicle isolated rat gracilis muscle model was used. After 4 h of ischemia followed by 24 h of reperfusion, gracilis muscle was harvested, and mitochondrial as well as cytosolic fractions were isolated. Mitochondrial dysfunction was determined by the alteration of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c. Three groups were designed; sham I/R, I/R-V (I/R with vehicle), and I/R-Mel (I/R with melatonin). Melatonin or vehicle was given intravenously 10 min prior to reperfusion and 10 min after reperfusion. RESULTS: We found that the capability of uptake of fluorescent JC-1 dye in skeletal muscle cells was substantially improved in I/R-Mel group compared with I/R-V group. Melatonin significantly inhibited the outflow of cytochrome c from mitochondria to cytoplasm, which was demonstrated in the I/R-V group. CONCLUSIONS: Melatonin significantly attenuates I/R-induced mitochondrial dysfunction, such as the depolarization of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c, from the mitochondria.
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Melatonina/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Animales , Antioxidantes/farmacología , Bencimidazoles/farmacocinética , Carbocianinas/farmacocinética , Citocromos c/metabolismo , Colorantes Fluorescentes/farmacocinética , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Superóxidos/metabolismoRESUMEN
BACKGROUND: Fat grafting is a growing field within plastic surgery. Adipose-derived stem cells (ASCs) and stromal vascular fracture (SVF) may have a role in fat graft survival. Our group previously demonstrated a detrimental effect on ASC survival by the lidocaine used in tumescent solution. Sodium bicarbonate (SB) buffers the acidity of lidocaine. The purpose of this study was to determine whether SB buffering is a practical method to reduce ASC and SVF apoptosis and necrosis seen with common lidocaine-containing tumescent solution. METHODS: Human patients undergoing bilateral liposuction for any indication were included in this study. An internally controlled, split-body design was utilized. Tumescent liposuction on one side of the body was conducted with tumescent containing lidocaine. On the opposite side, liposuction was conducted by adding SB to the tumescent. Tumescent solution and lipoaspirate pH were measured. Lipoaspirate from each side was processed for SVF isolation and ASC culture. The number of viable ASCs was counted and SVF apoptosis/necrosis was examined. RESULTS: The pH of the SB-buffered tumescent was significantly higher than that of the standard tumescent, an effect also seen in the lipoaspirate. Adipose-derived stem cell survival in the SB-buffered lipoaspirate was approximately 53% higher. However, there was no significant difference in SVF apoptosis and necrosis between the groups. CONCLUSIONS: The acidic standard tumescent solution commonly used in liposuction diminishes ASC viability from lipoaspirates. Sodium bicarbonate buffering tumescent solution can enhance ASC viability, but does not affect SVF apoptosis and necrosis. We recommend buffering tumescent with SB to potentially improve fat graft take. Our findings advocate for further research investigating mechanisms and optimal harvest techniques that maximize SVF/ASC survival and the clinical effect on overall fat graft viability.
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BACKGROUND: Hyperbaric oxygen (HBO) inhibits ischemia reperfusion (IR) -induced neutrophil adhesion to endothelium through an unknown mechanism. This study evaluates the effect of HBO on IR-stimulated neutrophil adhesion and polarization of expressed CD18 adhesion molecules using a novel in vitro adhesion assay and confocal microscopy. MATERIALS AND METHODS: Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps on coverslips pretreated with ICAM. Percent adherence to ICAM and CD18 polarization was evaluated in the following five groups: (1) Nonischemic control, n = 15; (2) 4 h ischemia (IR, n = 15); (3) 4 h ischemia with HBO treatment (100% oxygen at 2.5 atmospheres absolute (IR + HBO, n = 15)); (4) 4 h ischemia with 100% oxygen at room temperature and pressure (RTP) (IR + normobaric hyperoxia, n = 5); and (5) 4 h ischemia with 8% oxygen at 2.5 atmospheres absolute (IR + hyperbaric normoxia, n = 5). Direct HBO treatment of neutrophils was also evaluated. RESULTS: Neutrophils exposed to IR plasma showed a significant increase in percent adherent (0.8 +/- 0.1% versus 16.7 +/- 2.2%, P < 0.05) and polarized cells (6.2 +/- 1.7% versus 43.9 +/- 12.2%, P < 0.05) compared to controls. Hyperbaric oxygen significantly reduced the adhesion and polarization to 1.6 +/- 0.3 and 4.1 +/- 2.5%, respectively (P = < 0.05). Normobaric hyperoxia and hyperbaric normoxia did not affect neutrophil adherence or CD18 polarization following IR. Direct HBO treatment of neutrophils did not change the percent of polarized cells in IR. CONCLUSIONS: Hyperbaric oxygen inhibits IR-induced neutrophil adhesion by blocking CD18 surface polarization and requires plasma exposure to HBO. Treatment with oxygen or pressure alone is not effective.
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Antígenos CD18/metabolismo , Oxigenoterapia Hiperbárica , Neutrófilos/metabolismo , Daño por Reperfusión/prevención & control , Animales , Adhesión Celular , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismoRESUMEN
BACKGROUND: Ischemia-reperfusion (IR) injury is seen in many settings such as free flap salvage and limb replantation/revascularization. The consequences-partial/total flap loss, functional muscle loss, or amputation-can be devastating. Of the treatment options available for IR injury, hyperbaric oxygen (HBO) is the most beneficial. HBO inhibits neutrophil-endothelial adhesion through interference of CD18 neutrophil polarization in IR, a process mediated by nitric oxide. The purposes of this study were to examine the involvement of vascular endothelial growth factor (VEGF) in the beneficial HBO effect on CD18 polarization and neutrophil adhesion and investigate the effect of plasmin on VEGF expression in skeletal muscle following IR injury. METHODS: A rat gracilis muscle model of IR injury was used to evaluate the effect of VEGF in IR, with and without HBO, on neutrophil CD18 polarization and adhesion in vivo and ex vivo. Furthermore, we investigated the effects that plasmin has on VEGF expression in gracilis muscle and pulmonary tissue by blocking its activation with alpha-2-antiplasmin. RESULTS: HBO treatment following IR injury significantly decreased neutrophil polarization and adhesion ex vivo compared with the IR group. Anti-VEGF reversed the beneficial HBO effect after IR with polarization and adhesion. In vivo adhesion was also increased by anti-VEGF. HBO treatment of IR significantly increased the VEGF protein in both gracilis and pulmonary vasculature. Alpha-2-antiplasmin significantly reversed the HBO-induced increase of VEGF in gracilis muscle. CONCLUSIONS: These results suggest that HBO inhibits CD18 polarization and neutrophil adhesion in IR injury through a VEGF-mediated pathway involving the extracellular matrix plasminogen system.
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Both adipose-derived stem cells (ASCs) and stromal vascular fraction (SVF) have been demonstrated to have regenerative properties with therapeutic potential for numerous diseases through local or topical applications. However, it is unclear whether ASC or SVF can be delivered systemically through an intra-arterial infusion. The purpose of this study was to examine the microcirculatory response in vivo on local intraarterial infusion of autogenic ASCs or SVF in a vascular pedicle isolated rat cremaster microcirculation model. MATERIALS AND METHODS: Fat tissue was surgically harvested from the flanks of male Sprague-Dawley rats (n = 12) and processed for SVF isolation. Some SVF samples were cultured for 24 hours for ASC purification. The autogenic SVF (1 × 105) cells (n = 6) or purified ASC (1 × 105) cells (n = 6) cells were infused into the microcirculation of cremaster muscle at a speed of 0.05 mL/min through the cannulation of femoral artery. As this is a vascular pedicle isolated preparation, the infused SVF or ASC cells went nowhere but the cremaster muscle. The video image of the microcirculation was monitored in real time during infusion. RESULTS: Arteriole diameter was measured as A1 (100-160 µm), A2 (40-80 µm), and A3/A4 (10-30 µm). Capillary perfusion was quantified in 18 capillary fields of each muscle. There was a significant increase in the diameter of terminal arterioles (P = 0.049) and the capillary density (P = 0.02) after ASC intraarterial infusion. However, a significant cell aggregation, embolisms, and arterial obstruction were observed in the microcirculation in every case during SVF infusion. CONCLUSIONS: Intraarterial infusion is an appropriate route for the delivery of autogenic ASCs, but not of SVF. SVF-induced microembolisms were the reason for narrowing or blocking the lumen of terminal arterioles, resulting in no flow in the corresponding capillaries.
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BACKGROUND: Our previous study demonstrated that lidocaine has a negative impact on adipose-derived stem cell (ASC) survival. Currently for large-volume liposuction, patients often undergo general anesthesia; therefore, lidocaine subcutaneous anesthesia is nonessential. We hypothesized that removing lidocaine from tumescent might improve stromal vascular fraction (SVF) and ASC survival from the standard tumescent with lidocaine. Ropivacaine is also a commonly used local anesthetic. The effect of ropivacaine on ASC survival was examined. METHODS: Adults who underwent liposuction on bilateral body areas were included (n = 10). Under general anesthesia, liposuction on 1 area was conducted under standard tumescent with lidocaine. On the contralateral side, liposuction was conducted under the modified tumescent without lidocaine. Five milliliters of lipoaspirate were processed for the isolation of SVF. The adherent ASCs were counted after 24 hours of SVF culture. Apoptosis and necrosis of SVF cells were examined by Annexin/propidium iodide staining and analyzed by flow cytometry. RESULTS: Average percentage of live SVF cells was 68.0% ± 4.0% (28.5% ± 3.8% of apoptosis and 3.4% ± 1.0% of necrosis) in lidocaine group compared with 86.7% ± 3.7% (11.5% ± 3.1% of apoptosis and 1.8% ± 0.7% of necrosis) in no-lidocaine group (P = 0.002). Average number of viable ASC was also significantly lower (367,000 ± 107) in lidocaine group compared with that (500,000 ± 152) in no-lidocaine group (P = 0.04). No significant difference was found between lidocaine and ropivacaine on ASC cytotoxicity. CONCLUSIONS: Removing lidocaine from tumescent significantly reduced SVF and ASC apoptosis in the lipoaspirate. We recommend tumescent liposuction without lidocaine, particularly if patient's lipoaspirate will be used for fat grafting.
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BACKGROUND: In the present study, the authors hypothesized that adipose-derived stem cells in cell culture may secrete multiple cytokines in the supernatant, which might have a significant impact in vivo on the reperfusion-induced microcirculatory alterations and endothelial dysfunction. METHODS: Fat tissue was surgically harvested from rat flanks and processed for adipose-derived stem cell isolation; cells (1 × 10(6)) were subcultured for 3, 6, 9, and 12 days without passage. The postcultivated medium was harvested with medium change every 3 days. After centrifugation, the supernatant was collected and stored at -20°C. Supernatant collected on day 9 was analyzed for eight oxidative stress cytokines by an enzyme-linked immunosorbent assay strip. The effect of the supernatant on the reperfusion-induced microcirculatory alterations was examined in the vascular pedicle of isolated rat cremaster muscles subjected to 4 hours of ischemia followed by 2 hours of reperfusion. RESULTS: Enzyme-linked immunosorbent assay results demonstrated that adipose-derived stem cells produced several highly expressed cytokines in the supernatant. The average concentration of interleukin-6, in particular, was 5-fold higher compared with control. The reperfusion-induced vasospasm, arteriole stagnation, and the capillary no-reflow that often appear in the early phase of reperfusion were eliminated by adipose-derived stem cell supernatant. CONCLUSIONS: Adipose-derived stem cells in cell culture display cytokine secretory properties that enable the cells to act through paracrine signaling. The supernatant even without cells could be used as a paracrine agent to interfere with the reperfusion-induced microcirculatory alterations and endothelial dysfunction.
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Adipocitos/metabolismo , Citocinas/metabolismo , Microcirculación , Daño por Reperfusión/fisiopatología , Daño por Reperfusión/terapia , Células Madre/metabolismo , Animales , Células Cultivadas , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/complicacionesRESUMEN
Previously, we have demonstrated a late phase protection of ischemic preconditioning in the microcirculation of cremaster muscle. This microvascular protection was blocked by a non-specific NOS inhibitor. The purpose of present study was to evaluate endothelial function in the terminal arteriole of cremaster muscle after 24-h of ischemic preconditioning followed by 4-h warm ischemia and to evaluate eNOS and iNOS gene and protein expression at 24 h after ischemic preconditioning in the cremaster muscle. A vascular pedicle isolated cremaster muscle in male SD rats underwent 45-min of ischemic preconditioning and 24 h later, 4-h of warm ischemia followed by reperfusion. Endothelial-dependent and -independent vasodilatation was evaluated on day 2 after 4-h ischemia and 60-min of reperfusion. Cremaster muscles were harvested at 24 h after ischemic preconditioning for measuring of eNOS and iNOS gene expression by reverse transcriptase polymerase chain reaction (RT-PCR) and protein expression by western blotting analysis. We found that IPC significantly attenuated endothelial dysfunction induced by 4-h warm ischemia and reperfusion. The expression of eNOS and iNOS mRNA shown a 229% and 135% increase respectively in IPC treated cremaster muscles as compared to normal cremaster muscles (P<0.05). The expression of eNOS and iNOS protein exhibited a 133% and 148% increase respectively in IPC treated cremaster muscles as compared to normal cremaster muscles (P<0.05). There was no statistically significant difference between normal cremaster muscle and sham IPC treated cremaster muscle. The results suggest that IPC preventing vascular endothelial dysfunction from ischemia/reperfusion injury may be due to the enhanced NOS expression. These results combined with the results from our previous studies suggest that IPC-induced microvascular protection in the skeletal muscle may act through a NOS-dependent mechanism.
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Células Endoteliales/fisiología , Regulación Enzimológica de la Expresión Génica , Precondicionamiento Isquémico , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa/genética , Acetilcolina/farmacología , Animales , Masculino , Óxido Nítrico Sintasa/fisiología , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Acral myxoinflammatory fibroplastic sarcoma is an extremely rare soft-tissue sarcoma. It typically presents as an inflammatory mass in the distal extremities of adult patients. The authors present a review of the available literature as well as a discussion on the surgical management of a patient with acral myxoinflammatory fibroplastic sarcoma who originally requested conservative management but ultimately required a two-digit ray amputation after local recurrence.
Le sarcome fibroblastique myxoinflammatoire acral est un sarcome des tissus mous d'une extrême rareté. En général, il prend la forme d'une masse inflammatoire des extrémités distales chez des patients adultes. Les auteurs présentent une analyse des publications et un exposé sur la prise en charge chirurgicale d'un patient atteint d'un sarcome fibroblastique myxoinflammatoire acral qui a commencé par demander une prise en charge prudente, mais a finalement apté pour l'amputation de deux orteils après une récurrence locale.
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BACKGROUND: Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. METHODS: Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90/CD45, CD105/CD45, and CD34/CD31) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. RESULTS: The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90/CD45, 7.5 ± 1.0 percent expressed CD105/CD45, and 26.4 ± 3.8 percent expressed CD34/CD31. CD34/CD31 adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105/CD45 adipose-derived stem cells (p < 0.05). CONCLUSIONS: Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.
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Adipocitos Blancos/patología , Células Madre Adultas/patología , Apoptosis , Lipectomía , Grasa Subcutánea/patología , Adipocitos Blancos/citología , Adulto , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Centrifugación , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Necrosis , Osteoblastos/citología , Fenotipo , Grasa Subcutánea/cirugíaRESUMEN
Ischemia-reperfusion injury occurs when tissue is reperfused following a prolonged period of ischemia. It is a subject of interest to plastic surgeons involved in replantation, free tissue transfer, and composite tissue allotransplantation, as it can have a significant impact on the overall success of these procedures. The purpose of this article is to review the recent progress in the investigation of ischemia-reperfusion injury in skeletal muscle and skin and to highlight the potential clinical implications of therapeutic interventions aimed at reducing ischemia-reperfusion injury.
Asunto(s)
Colgajos Tisulares Libres/irrigación sanguínea , Microcirugia , Músculo Esquelético/irrigación sanguínea , Daño por Reperfusión/fisiopatología , Reimplantación , Piel/irrigación sanguínea , Trasplantes , Animales , Apoptosis/fisiología , Humanos , Concentración de Iones de Hidrógeno , Precondicionamiento Isquémico/métodos , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Mitocondrias Musculares/fisiología , Membranas Mitocondriales/fisiología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/terapia , Supervivencia Tisular , Trasplante HomólogoRESUMEN
BACKGROUND: Recently, nitrite has been rediscovered as a physiologically relevant storage reservoir of nitric oxide in blood and it can readily be converted to nitric oxide under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of nitrite on reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle. METHODS: The authors used a vascular pedicle isolated rat cremaster model that underwent 4 hours of warm ischemia followed by 2 hours or 17 hours of reperfusion. At 5 minutes before reperfusion, normal saline, sodium nitrite (0.20 µM/minute/kg), or nitrite mixed with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (potassium salt) (0.2 mg/minute/kg) was infused into the microcirculation of ischemic cremaster by means of intraarterial infusion. Ischemia-reperfusion-induced microcirculatory alterations were measured after 2 hours of reperfusion. Microvasculature of the cremaster muscle including the vascular pedicle was harvested to determine the mitochondrial dysfunction. The blood concentration of methemoglobin was also measured to determine the toxicity of nitrite. RESULTS: The authors found that nitrite significantly attenuated ischemia-reperfusion-induced vasoconstriction, arteriole stagnation, and capillary no-reflow in the early phase of reperfusion and the depolarization of mitochondrial membrane potential and cytochrome c release in the late phase of reperfusion. Nitrite-induced protection was significantly blocked by a nitric oxide scavenger (potassium salt). The methemoglobin results showed that the doses of nitrite we used in the present study were safe. CONCLUSION: The supplementation of a low dose of nitrite, directly into the microcirculation of ischemic muscle through local intraarterial infusion, significantly attenuated ischemia-reperfusion-induced microcirculatory alterations in vivo and mitochondrial dysfunction in vitro in the microvasculature of skeletal muscle.