RESUMO
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.
Assuntos
Anestésicos Locais/administração & dosagem , Apoptose/fisiologia , Lidocaína/administração & dosagem , Adulto , Anestesia Local , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Lipectomia , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Derme Acelular/metabolismo , Colágeno/metabolismo , Transplante de Pele , Cicatrização/fisiologia , Animais , Derme/metabolismo , Hidroxiprolina/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
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.
Assuntos
Oxigenoterapia Hiperbárica , Óxido Nítrico Sintase/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Aorta Abdominal/enzimologia , Pulmão/enzimologia , Masculino , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologiaRESUMO
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.
Assuntos
Adipócitos/citologia , Criopreservação , Células-Tronco/citologia , Adulto , Apoptose , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Necrose , Temperatura , Fatores de TempoRESUMO
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.
Assuntos
Competência Clínica , Educação de Pós-Graduação em Medicina , Avaliação Educacional , Internato e Residência , Microcirurgia/educação , Análise de Variância , Anastomose Cirúrgica/normas , Humanos , Técnicas de Sutura/normasRESUMO
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.
Assuntos
Melatonina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Antioxidantes/farmacologia , Benzimidazóis/farmacocinética , Carbocianinas/farmacocinética , Citocromos c/metabolismo , Corantes Fluorescentes/farmacocinética , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Superóxidos/metabolismoRESUMO
Although most cleft palates can be closed with conventional mucoperiosteal flap techniques, the occasional wide cleft or difficult fistula has few options for an early 1-stage reconstruction. Distraction osteogenesis (DO) has the potential to close the palate with both hard and soft tissues as well as mitigating the possibility of future oronasal fistula. A right unilateral 5-mm cleft was surgically created in 15 New Zealand white rabbits. In group 1 (N = 5), no further repair was performed (NR); in group 2 (N = 5), mucoperiosteal flaps were used to close the cleft for a soft-tissue-only repair (STR); in group 3 (N = 5), a unilateral osteotomy in the palate on the noncleft side allowed distraction of the palatal shelf across the cleft until closed (DO). Clinical examination, Micro-computed tomography bone density, direct cephalometry, and histology were evaluated at 8 weeks after the completion of distraction. Bone mineral density (BMD; mg/mL) data were obtained from micro-computed tomography scans of both the cleft and noncleft sides for each rabbit, and a ratio was obtained [(BMDc/BMDnc) x100]; NR = 1.38, STR = 44.27, DO = 88.36, P = 0.007. Facial measurements revealed no growth disturbances as a result of DO. Histologic evaluation revealed increased organization of new bone in DO group compared with NR and STR. Clinically, DO group rabbits did not show any increase in feeding disturbances, infection, or wound healing. The success of membranous facial bone distraction has been applied to a new model for palatal repair with the potential to ameliorate the problems associated with soft-tissue-only repair.
Assuntos
Fissura Palatina/cirurgia , Osteogênese por Distração/métodos , Palato/cirurgia , Animais , Densidade Óssea/fisiologia , Remodelação Óssea/fisiologia , Cefalometria , Fissura Palatina/patologia , Arco Dental/patologia , Modelos Animais de Doenças , Masculino , Maxila/patologia , Desenvolvimento Maxilofacial/fisiologia , Mucosa Bucal/cirurgia , Osteoblastos/patologia , Osteotomia/métodos , Palato/patologia , Palato Duro/patologia , Palato Mole/cirurgia , Coelhos , Distribuição Aleatória , Retalhos Cirúrgicos , Microtomografia por Raio-XRESUMO
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.
Assuntos
Antígenos CD18/metabolismo , Oxigenoterapia Hiperbárica , Neutrófilos/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Adesão Celular , Masculino , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Adipócitos/metabolismo , Citocinas/metabolismo , Microcirculação , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/terapia , Células-Tronco/metabolismo , Animais , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicaçõesRESUMO
Nitric oxide has been reported to be beneficial in preserving muscle viability following ischemia-reperfusion injury. The purpose of this study was to evaluate the influence of nitric oxide via L-arginine on leukocyte adhesion following ischemia-reperfusion injury. Intravital videomicroscopy of rat gracilis muscle was used to quantify changes in leukocyte adherence. The gracilis muscle was raised on its vascular pedicle in 48 male Wistar rats. The animals were assigned to one of five groups: (1) nonischemic control; (2) ischemia-reperfusion; (3) ischemia-reperfusion and L-arginine; (4) ischemia-reperfusion and Nomega-nitro-L-arginine methyl ester (L-NAME); and (5) ischemia-reperfusion, L-NAME, and L-arginine. All groups that included ischemia-reperfusion were subjected to 4 hours of global ischemia followed by 2 hours of reperfusion. L-Arginine (10 mg/kg) and L-NAME (10 mg/kg) were infused into the contralateral femoral vein beginning 5 minutes before reperfusion, for a total of 30 minutes. The number of adherent leukocytes was counted at baseline and at 5, 15, 30, 60, and 120 minutes after reperfusion (reported as mean change from baseline, +/- SEM). Groups were compared by repeated-measures analysis of variance (five groups, five times). P < or =0.05 was accepted as significant. L-Arginine significantly reduced leukocyte adherence to venular endothelium during reperfusion when compared with the ischemia-reperfusion group (1.39 +/- 0.92 versus 12.78 +/- 1.43 at 2 hours, p < 0.05). Administration of L-NAME with L-arginine showed no significant difference in adherent leukocytes when compared with the ischemia-reperfusion group (10.28 +/- 2.03 at 2 hours). The nitric oxide substrate L-arginine appears to reduce the deleterious neutrophil-endothelial adhesion associated with ischemia-reperfusion injury. L-NAME (nitric oxide synthesis inhibitor) given concomitantly with L-arginine reversed the beneficial effect of L-arginine alone, indicating that L-arginine may be acting via a nitric oxide synthase pathway. These results suggest an important role for nitric oxide in decreasing the neutrophil-endothelial interaction associated with ischemia-reperfusion injury.
Assuntos
Arginina/farmacologia , Leucócitos/fisiologia , Músculo Esquelético/irrigação sanguínea , Traumatismo por Reperfusão/fisiopatologia , Animais , Adesão Celular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Membro Posterior , Leucócitos/efeitos dos fármacos , Masculino , Microcirculação , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/fisiologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Retalhos Cirúrgicos/irrigação sanguínea , Vênulas/patologiaRESUMO
Clinical reports of full-thickness skin necrosis have raised concern about the thermal and dermal ischemic effects of ultrasound-assisted liposuction. The purpose of this study was to evaluate skin perfusion in patients treated with ultrasound-assisted liposuction or suction-assisted liposuction. Patients (n = 75) were studied prospectively in the perioperative period surrounding their suction-assisted liposuction (31 patients) or ultrasound-assisted liposuction (64 patients). The laser Doppler flowmeter was used to monitor skin perfusion in the treated regions preoperatively, intraoperatively, and postoperatively at a series of time intervals. The effects of the anesthetic, wetting solution, and type of liposuction (suction-assisted liposuction or ultrasound-assisted liposuction) on skin perfusion were measured. Anesthetic induction significantly increased measured skin perfusion. Wetting solution infusion significantly decreased skin perfusion (-57.4 percent +/- 2.0) by 15 minutes postinfusion. Skin perfusion in the ultrasound-assisted liposuction group was significantly greater than that of the suction-assisted liposuction patients at 1 hour, 1 day, and 1 week postoperatively; however, by 2 to 5 weeks, no difference in skin perfusion was noted and skin perfusion had returned to preoperative levels in both groups. Although skin perfusion in the suction-assisted liposuction group was significantly lower than in the ultrasound-assisted liposuction group in the early postoperative period, no differences in skin perfusion between the groups were noted beyond 1 week postoperatively, suggesting that neither technique impairs perfusion.
Assuntos
Lipectomia/métodos , Pele/irrigação sanguínea , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
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.
Assuntos
Adipócitos Brancos/patologia , Células-Tronco Adultas/patologia , Apoptose , Lipectomia , Gordura Subcutânea/patologia , Adipócitos Brancos/citologia , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Centrifugação , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Necrose , Osteoblastos/citologia , Fenótipo , Gordura Subcutânea/cirurgiaRESUMO
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.
Assuntos
Isquemia/tratamento farmacológico , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/irrigação sanguínea , Nitritos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Análise de Variância , Animais , Citocromos c/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Potenciais da Membrana , Metemoglobina/efeitos dos fármacos , Metemoglobina/metabolismo , Microcirculação/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de Referência , Traumatismo por Reperfusão/tratamento farmacológicoAssuntos
Adipócitos , Criopreservação/métodos , Lipectomia/métodos , Células-Tronco , Sobrevivência Celular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Controle de Qualidade , Valores de Referência , Refrigeração/métodos , Estudos de Amostragem , Fatores de Tempo , Coleta de Tecidos e Órgãos/métodosRESUMO
BACKGROUND: Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis. MATERIALS AND METHODS: Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined. RESULTS: The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis. CONCLUSIONS: DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.
Assuntos
Apoptose/fisiologia , Ductos Biliares Intra-Hepáticos/citologia , Metamorfose Biológica/fisiologia , Petromyzon/fisiologia , Animais , Antitireóideos/farmacologia , Apoptose/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/fisiologia , Atresia Biliar/patologia , Fragmentação do DNA , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Larva/efeitos dos fármacos , Larva/fisiologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/fisiologia , Fígado/anatomia & histologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Metamorfose Biológica/efeitos dos fármacos , Compostos de Potássio/farmacologiaRESUMO
BACKGROUND: Hyperbaric oxygen decreases ischemia-reperfusion-induced neutrophil/intercellular adhesion molecule-1 adhesion by blocking CD18 polarization. The purpose of this study was to evaluate whether this hyperbaric oxygen effect is nitric oxide dependent and to determine whether nitric oxide synthase is required. METHODS: A gracilis muscle flap was raised in nine groups of male Wistar rats. Global ischemic injury was induced by clamping the gracilis muscle pedicle artery and vein for 4 hours. The hyperbaric oxygen treatment consisted of 100% oxygen at 2.5 atm absolute during the last 90 minutes of ischemia. Groups were repeated with and without various nitric oxide synthase inhibitors and carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), a nitric oxide scavenger. Normal neutrophils were exposed to activated plasma on intercellular adhesion molecule-1-coated coverslips (percentage adherent) and labeled with fluorescein isothiocyanate/antirat-CD11b for confocal microscopy (percentage polarized). The percentage of adherent and polarized cells was reported as mean + or - SEM. Statistical analysis was by analysis of variance. A value of p < or = 0.05 was considered significant. RESULTS: C-PTIO-treated ischemia-reperfusion/hyperbaric oxygen plasma showed a significant increase in the percentage polarization of CD18 compared with ischemia-reperfusion/hyperbaric oxygen-untreated plasma from 4.1 + or - 2.5 percent to 33.7 + or - 7.7 percent (p < or = 0.05). The nitric oxide scavenger C-PTIO also increased the percentage of adherent cells from 1.6 + or - 0.4 percent to 20.3 + or - 5.9 percent (p < or = 0.05). Administration of N-nitro-L-arginine methyl ester and other nitric oxide synthase inhibitors before hyperbaric oxygen treatment restored neutrophil adhesion and CD18 polarization to ischemia-reperfusion control values, significantly greater than ischemia-reperfusion/hyperbaric oxygen alone. CONCLUSION: These results suggest that the hyperbaric oxygen reduction of ischemia-reperfusion-induced neutrophil polarization of CD18 and adherence to intercellular adhesion molecule-1 is mediated through a nitric oxide mechanism that requires nitric oxide synthase.
Assuntos
Antígenos CD18/metabolismo , Oxigenoterapia Hiperbárica/métodos , Neutrófilos/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Análise de Variância , Animais , Antígenos CD18/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Microscopia de Polarização , Músculo Esquelético/irrigação sanguínea , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Probabilidade , Distribuição Aleatória , Ratos , Ratos Wistar , Traumatismo por Reperfusão/terapiaRESUMO
BACKGROUND: The purpose of the present study was to investigate ischemia-reperfusion-induced apoptosis and necrosis in endothelial cells isolated from skeletal muscle. METHODS: A vascular pedicle isolated rat gracilis muscle model was used. After surgical preparation, clamps were applied to the vascular pedicle to create 4 hours of ischemia and released for reperfusion (ischemia-reperfusion group, n = 9). Clamping was omitted in sham ischemia-reperfusion rats (sham ischemia-reperfusion group, n = 9). The muscle samples were harvested after 20 hours of reperfusion for the process of cell isolation. One hundred thousand cells from each sample were stained by monoclonal anti-CD146-fluorescein (a principal marker for mature endothelial cells), Annexin V-PE, or 7-aminoactinomycin D to detect and quantify apoptotic and necrotic cells. Twenty thousand cells from each sample were scanned and analyzed by flow cytometry. RESULTS: The average +/- SEM of CD146-fluorescein-positive cells was 20.0 +/- 2.9 percent, suggesting that these cells might be endothelial cells from the muscle microvasculature. In the population of gated CD146-fluorescein-positive cells, the average percentage of apoptotic cells (stained by Annexin V-PE) was 15.9 +/- 2.2 percent in the sham ischemia-reperfusion group and 33.5 +/- 5.3 percent in the ischemia-reperfusion group (p < 0.01), the average percentage of necrotic/apoptotic cells (stained by both 7-aminoactinomycin D and Annexin V-PE) was 17.8 +/- 4.1 percent in the sham ischemia-reperfusion group and 39.2 +/- 3.1 percent in the ischemia-reperfusion group (p < 0.01). CONCLUSIONS: Given the results of the present study, the authors hypothesize that the endothelial cells lining microscopic blood vessels are among the major contributors to ischemia-reperfusion-induced cell apoptosis and necrosis detected from rat skeletal muscle.
Assuntos
Células Endoteliais/fisiologia , Isquemia/fisiopatologia , Músculo Esquelético/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Apoptose , Modelos Animais de Doenças , Células Endoteliais/patologia , Isquemia/patologia , Masculino , Microcirculação , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Necrose , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologiaRESUMO
Soft tissue coverage for avascular wounds is necessary in reconstructive surgery. Several authors have demonstrated successful treatment of problem wounds using artificial dermis. This study evaluates in an animal model the potential for neovascularization of artificial dermis in devascularized and avascular wound beds. Forty rats were assigned to four groups: (1) control, full-thickness skin graft was replaced on the vascular wound bed; (2) Integra, Integra placed over the full-thickness wound; (3) fascia, the spinotrapezius fascia exposed and Integra placed over the wound; and (4) fascia/Parafilm, a Parafilm layer placed under the raised fascia with Integra over the fascia. Laser Doppler readings were taken at baseline over the intact skin and then over the created wound beds. Biopsies of the full-thickness skin graft and the neodermis were obtained on postoperative day 14 and histologically evaluated for neovascularization. The laser Doppler readings confirmed the nature of the surgically created, poorly vascularized and avascular wound beds. Subsequent biopsies of the artificial dermis in these wound beds, however, demonstrated active neovascularization. This study demonstrates that Integra artificial dermis can serve as an effective dermal substitute in avascular wounds. Lateral ingrowth of capillaries into the dermal substitute may explain the successful integration of this artificial dermis.
Assuntos
Sulfatos de Condroitina , Colágeno , Neovascularização Fisiológica , Pele Artificial , Cicatrização/fisiologia , Animais , Fluxometria por Laser-Doppler , Masculino , Modelos Animais , Ratos , Ratos Wistar , Ultrassonografia DopplerRESUMO
Necrosis was considered to be the solo mechanism for ischemia/reperfusion (I/R)-induced cell death. Recent evidence from I/R models of the heart, liver, kidney, and brain indicates that apoptosis is a major contributor to I/R-induced cell death. However, evidence of I/R-induced apoptosis in skeletal muscle is sparse and divided. The purpose for the present study was to investigate I/R-induced necrosis and apoptosis in the cells isolated from rat skeletal muscle. A rat gracilis muscle model was used. After surgical preparation, clamps were applied on the vascular pedicle to create 4 h of ischemia and released for 24 h of reperfusion (I/R, n = 10). Clamping was omitted in sham I/R rats (sham I/R, n = 10). The muscle samples were harvested after 24 h of reperfusion for the process of cell isolation. Cells were stained by Propidium Iodide (PI) or Annexin V-FITC or both. Twenty thousand cells from each muscle sample were scanned and analyzed by flow cytometry. The average percentage of live cells was 45 +/- 2% in the I/R group versus 65 +/- 3% in the sham I/R group (p < 0.01). The average percentage of necrotic cells was 18 +/- 1% in I/R versus 12 +/- 1% in sham I/R (p < 0.01). The average percentage of apoptotic cells was 40 +/- 3% in I/R versus 27 +/- 3% in sham I/R (p < 0.01). Our results clearly demonstrated that I/R not only causes necrosis, but also accelerates apoptosis in the cells isolated from rat skeletal muscle.