Lidocaine-induced ASC apoptosis (tumescent vs. local anesthesia).

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.

The effect of hyperbaric oxygen on nitric oxide synthase activity and expression in ischemia-reperfusion injury.

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.

The effect of lipoaspirates cryopreservation on adipose-derived stem cells.

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.

Contributing variables to post mastectomy tissue expander infection.

Tissue expander breast reconstruction is a common post mastectomy breast procedure performed by plastic surgeons. The purpose of this study was to define the incidence of breast reconstruction prosthetic infection, relate patient characteristics with increased rate of infection, and analyze the influence of postoperative complications to expander/implant infection. A retrospective, single-institution chart review of 195 women with post mastectomy tissue expander/implant reconstructions performed from 2006 through 2008 was conducted. Total periprosthetic infection rate was calculated. Patient factors, operative technique, and noninfectious complications were analyzed and related to increased periprosthetic infection rate. A binary logistic regression model was fitted using periprosthetic infection as the dependent variable and 12 patient characteristics as independent variables, followed by a step-wise model for best fit with a limited number of independent variables. The overall periprosthetic infection rate per patient over the 2 year period was 5.1%. The incidence of periprosthetic infection per reconstructed breast was 3.2%. Odds ratio estimates indicated that the presence of cellulitis increased the odds of periprosthetic infection more than 200 times (p = <0.0001), and inpatient procedures increased the odds 16 times (p = 0.02). Other variables (i.e., age > 65, DM, flap necrosis, smoking, dehiscence, AlloDerm, etc) failed to reach statistical significance (p > 0.05). Postoperative breast cellulitis and inpatient status appear to be significant risk factors for increased periprosthetic infection. No significant increase in periprosthetic infection rate was noted with other variables in this model.

Melatonin attenuates I/R-induced mitochondrial dysfunction in skeletal muscle.

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.

Reperfusion-induced neutrophil CD18 polarization: effect of hyperbaric oxygen.

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.

Nocardial septic arthritis of the wrist diagnosed and treated by arthroscopy.

This is the first case of Nocardial septic arthritis of the wrist that has been reported without any cutaneous presentation. It was successfully diagnosed and treated with wrist arthroscopy. Arthroscopy is less traumatic and done through smaller incisions than the traditional open technique.

Elimination of reperfusion-induced microcirculatory alterations in vivo by adipose-derived stem cell supernatant without adipose-derived stem cells.

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.

NOS upregulation attenuates vascular endothelial dysfunction in the late phase of ischemic preconditioning in skeletal muscle.

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.

The effects of the nitric oxide donor SIN-1 on ischemia-reperfused cutaneous and myocutaneous flaps.

Ischemia-reperfusion injury causes tissue damage that leads to a decrease in bioavailability of nitric oxide. The authors hypothesized that an exogenous supply of nitric oxide will have beneficial effects on survival of skin and skeletal muscle subjected to ischemia-reperfusion injury. By using the nitric oxide donor SIN-1 (3-morpholino-sydnonimine) the effects of direct intraarterial infusion of an exogenous source of nitric oxide in reperfused flaps was studied. Bilateral island buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs, for a total of 32 flaps. Flaps were subjected to 6 hours of ischemia followed by 18 hours of reperfusion. Flaps on one side of each animal were randomized to be treated with the nitric oxide donor (treatment group). The contralateral side was treated with an equivalent volume of saline vehicle (infusion control) SIN-1, or saline was administered as a continuous direct intraarterial infusion at the onset of reperfusion and continued during the observation period. Outcomes measured were tissue neutrophil accumulation by using myeloperoxidase assay and tissue survival (intravenous fluorescein and nitroblue tetrazolium for skin and muscle, respectively). In both skin and myocutaneous flaps, SIN-1 treatment caused a significant improvement in survival and a decrease in neutrophil accumulation. Nitric oxide may play an important role in the pathophysiologic process of ischemia-induced reperfusion injury in skin and skeletal muscle. Nitric oxide donors may be a promising family of therapeutic agents for the prevention of ischemia-induced reperfusion injury in cutaneous and myocutaneous flaps.

Differing flow patterns between ischemically challenged flap skin and flap skeletal muscle: implications for salvage regimens.

In this study, the authors tested the hypothesis that there is a significant difference in spatial patterns of reflow in skin as opposed to skeletal muscle after an ischemic insult. The authors believe that this pathophysiologic difference between the two flap types has significant implications for flap salvage strategies. Bilateral buttock skin flaps (10 x 18 cm) and latissimus dorsi myocutaneous flaps (10 x 20 cm) were elevated in Landrace pigs (n = 7). Flaps on one side of the animal were randomly assigned to 6 hours of arterial occlusion, with the contralateral side acting as control. At 15 minutes, 1 hour, and 4 hours after reflow, radioactive microspheres (15 microm) were injected into the left ventricle. After 18 hours of reperfusion, skin and muscle viability were estimated by intravenous fluorescein and soaking in nitroblue tetrazolium, respectively. Flow rates in the skin with an ischemia-reperfusion injury were significantly reduced (30 to 53 percent), at all time intervals, compared with controls. The flow rate in the fluorescent skin with ischemia-reperfusion injury of the latissimus dorsi flaps (0.037 ml/min/g at 15 min) was greater than in that of the buttock flaps (0.018 ml/min/g). The muscle flaps with ischemia-reperfusion injury had significantly higher flow rates than control muscle flaps at all time intervals studied (at 1 hour, 0.32 ml/min/g compared with 0.16 ml/min/g, respectively). In flap skeletal muscle, an early hyperemic phase during reperfusion maintains a significant blood flow to all regions, including the area of the flap that is destined for necrosis. In flap skin, however, there is a marked decrease in flow rates. These differences have important implications for the intravascular delivery of therapeutic agents to the damaged portions of the flap. Transdermal drug delivery systems should be explored as an alternative to intravascular regimens for the salvage of flap skin with ischemia-reperfusion injury.

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps.

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.

Effect of liposuction on skin perfusion.

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.

Acral myxoinflammatory fibroblastic sarcoma: A case report and literature review.

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.

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

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.

Nitrite attenuates ischemia-reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.

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.

Hyperbaric oxygen inhibits ischemia-reperfusion-induced neutrophil CD18 polarization by a nitric oxide mechanism.

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.

Levels of evidence in research published in The Journal of Bone and Joint Surgery (American Volume) over the last thirty years.

BACKGROUND: The recent emphasis on evidence-based medicine has led to increasing levels of evidence being published in surgical journals. The purpose of the present study was to review the levels of evidence in reports published in The Journal of Bone and Joint Surgery (American Volume) over the last thirty years. METHODS: We reviewed all of the articles published in The Journal in the years 1975, 1985, 1995, and 2005. Cadaver studies, animal studies, basic-science studies, review articles, Instructional Course Lectures, and correspondence were excluded. Articles were scored according to The Journal's levels of evidence for a primary research question. RESULTS: A total of 1058 articles were reviewed. Of these, 134, 123, 120, and 174 articles met the inclusion criteria for the years 1975, 1985, 1995, and 2005, respectively, and were ranked according to level of evidence. The number of articles for each level of evidence rating was then expressed as a percentage of the total number of articles meeting the inclusion criteria for that year. There was a significant trend toward higher levels of evidence, with the combined percentage of Level-I, II, and III studies increasing from 17% to 52% (p < 0.01). The percentage of Level-I studies increased from 4% in 1975 to 21% in 2005. The average level of evidence rating improved from 3.72 to 2.90 during the study period. CONCLUSIONS: The level of evidence in The Journal has improved significantly over the last thirty years.