A comparison of lidocaine, ropivacaine and dexamethasone toxicity on bovine tenocytes in culture.

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic. Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.

The use of 18F-fluoride and 18F-FDG PET scans to assess fracture healing in a rat femur model.

PURPOSE: Currently available diagnostic techniques can be unreliable in the diagnosis of delayed fracture healing in certain clinical situations, which can lead to increased complication rates and costs to the health care system. This study sought to determine the utility of positron emission tomography (PET) scanning with (18)F-fluoride ion, which localizes in regions of high osteoblastic activity, and (18)F-fluorodeoxyglucose (FDG), an indicator of cellular glucose metabolism, in assessing bone healing in a rat femur fracture model. METHODS: Fractures were created in the femurs of immunocompetent rats. Animals in group I had a fracture produced via a manual three-point bending technique. Group II animals underwent a femoral osteotomy with placement of a 2-mm silastic spacer at the fracture site. Fracture healing was assessed with plain radiographs, (18)F-fluoride, and (18)F-FDG PET scans at 1, 2, 3, and 4-week time points after surgery. Femoral specimens were harvested for histologic analysis and manual testing of torsional and bending strength 4 weeks after surgery. RESULTS: All fractures in group I revealed abundant callus formation and bone healing, while none of the nonunion femurs were healed via assessment with manual palpation, radiographic, and histologic evaluation at the 4-week time point. (18)F-fluoride PET images of group I femurs at successive 1-week intervals revealed progressively increased signal uptake at the union site during fracture repair. In contrast, minimal tracer uptake was seen at the fracture sites in group II at all time points after surgery. Data analysis revealed statistically significant differences in mean signal intensity between groups I and II at each weekly interval. No significant differences between the two groups were seen using (18)F-FDG PET imaging at any time point. CONCLUSION: This study suggests that (18)F-fluoride PET imaging, which is an indicator of osteoblastic activity in vivo, can identify fracture nonunions at an early time point and may have a role in the assessment of longitudinal fracture healing. PET scans using (18)F-FDG were not helpful in differentiating metabolic activity between successful and delayed bone healing.

Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats.

The objective of the present study was to assess the ability of bone marrow cells expressing BMP-2 created via lentiviral gene transfer to heal a critical sized femoral defect in a rat model. Femoral defects in Lewis rats were implanted with 5x10(6) rat bone marrow stromal cells (RBMSC) transduced with a lentiviral vector containing either the BMP-2 gene (Group I), the enhanced green fluorescent protein (LV-GFP) gene (Group IV), or RBMSC alone (Group V). We also included femoral defects that were treated with BMP-2-producing RBMSC transduced with lentivirus, 8 weeks after infection (Group III), and a group with 1x10(6) RBMSC transduced with a lentiviral vector with the BMP-2 gene (Group II). All defects (10/10) treated in Group I healed at 8 weeks compared with none of the femora in the control groups (Groups IV and V). In Group II, only one out of 10 femora healed. In Group III, 5 out of 10 femora healed. Significantly higher amounts of in vitro BMP-2 protein production were detected in Groups I, II, and III when compared to that of the control groups (p<0.05). Histomorphometric analysis revealed significantly greater total bone volume in defects in Group I and III when compared to control specimens (p<0.003). Biomechanical testing revealed no significant differences in the healed defects in Groups I and III when compared to intact, nonoperated femora with respect to peak torque and torque to failure. Our results indicate that BMP-2-producing RBMSC created through lentiviral gene transfer have the capability of inducing long-term protein production in vitro and producing substantial new bone formation in vivo.

Ex vivo antisense oligonucleotides to proliferating cell nuclear antigen and Cdc2 kinase inhibit graft coronary artery disease.

BACKGROUND: The long-term success of cardiac transplantation is limited by graft coronary artery disease (GCAD). Antisense oligonucleotides (ASs) to proliferating cell nuclear antigen (PCNA) and Cdc2 kinase (Cdc2 k) can arrest cell cycle progression and inhibit neointimal hyperplasia. Transforming growth factor-ss(1) (TGF-ss(1)) has been implicated in vascular smooth muscle cell (VSMC) activation. The role of TGF-ss(1) in GCAD remains unclear. We hypothesized that ASs to PCNA and Cdc2 k would inhibit VSMC proliferation and GCAD. METHODS AND RESULTS: In vitro VSMC proliferation was determined after pretreatment with AS solution or medium alone followed by angiotensin II stimulation. PVG-to-ACI rat heterotopic cardiac transplantation procedures were performed after ex vivo pressure-mediated transfection of ASs to PCNA and Cdc2k or saline alone. At postoperative days 30, 60, and 90, allografts were assessed for GCAD, percent neointimal macrophages and VSMCs, and TGF-ss(1) activity. AS pretreatment significantly attenuated VSMC proliferation. At postoperative day 90, percent affected arteries, percent occlusion, and intima-media ratio demonstrated severe GCAD in saline-treated allografts, whereas these parameters were significantly lower in AS-treated allografts. Percent neointimal macrophages and VSMCs was reduced in AS-treated allografts. TGF-ss(1) activity was increased in saline compared with AS-treated allografts and nontransplanted heart controls. CONCLUSIONS: ASs to PCNA and Cdc2 k inhibit VSMC proliferation in vitro and reduce GCAD, percent neointimal VSMCs and macrophages, and TGF-ss(1) activity in vivo. TGF-ss(1) may play a "response to injury" role in the development of GCAD. The prevention of GCAD via AS inhibition of cell cycle regulatory genes before reperfusion may offer a useful clinical alternative to current therapeutic strategies.

Optimization of ex vivo pressure mediated delivery of antisense oligodeoxynucleotides to ICAM-1 reduces reperfusion injury in rat cardiac allografts.

BACKGROUND: Our purpose was to optimize hyperbaric pressure as a vector for ex vivo transfection of antisense oligodeoxynucleotides (AS-ODN) to intercellular adhesion molecule-1 to limit reperfusion injury (RI) in cardiac allografts. We investigated the effects of increased pressure, incubation time, and AS-ODN concentrations on transfection efficiency and toxicity. METHODS AND RESULTS: PVG (RT1c) donor hearts were heterotopically transplanted to ACI (RT1a) recipients. Donor hearts were harvested and the various groups were treated at: (1) different pressure (1-9 atm) for 45 min with 80 micromol/liter AS-ODN; (2) different incubation times (15 min to 6 hr) at 5 atm with 80 micromol/liter AS-ODN; 3) different AS-ODN concentrations (80-240 micromol/liter) at 5 atm for 45 min. Hearts were procured 24 or 72 hr after transplantation. Transfection efficiency was determined with fluorescein-labeled AS-ODN. The degree of RI was determined with biochemical and histological analysis. Increasing pressure from ambient (1 atm) pressure to pressures as high as 9 atm leads to a increase in transfection efficiency from 1.7+/-.5 to 62+/-3.9% and a reduction in RI. Increased incubation time up to 45 min increased transfection efficiency and reduced RI, but longer incubation times induced significant toxicity to the allograft. Increased AS-ODN concentrations improved transfection and reduced RI. CONCLUSIONS: Hyperbaric pressure is a safe and effective vector for the ex vivo delivery of AS-ICAM-1-ODN to rodent cardiac allografts and results in a reduction in reperfusion injury.

Nuclear factor-kappaB transcription factor decoy treatment inhibits graft coronary artery disease after cardiac transplantation in rodents.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1. We hypothesized the use of ex vivo pressure-mediated delivery of transcription factor decoys (TFD) to NF-kappaB binding sites would decrease expression of adhesion molecules, and decrease reperfusion injury, acute rejection, and graft coronary artery disease (GCAD) in rat cardiac allografts. METHODS: Heterotopic heart transplants were performed on donor hearts treated with saline, 10 mg/kg LPS, 160 micromol/L NF-kappaB TFD, or 160 micromol/L scrambled sequence (NF-SC) TFD for 45 min at 78 psi (6 atm). Transfection efficiency was determined with FITC-labeled TFD. Reverse transcription-PCR and immunohistochemistry was used to analyze adhesion molecule mRNA and protein expression, respectively. Apoptosis was measured with DNA fragmentation analysis. Reperfusion injury was assessed with cardiac edema, neutrophil infiltration, and histology. Acute rejection was determined by daily palpation. Allografts were assessed at POD 90 for the development of GCAD by computer-assisted image analysis to determine intimal:medial ratio and myointimal proliferation. RESULTS: Hyperbaric pressure was an effective method of NF-kappaB TFD delivery (P<0.001 vs. controls). NF-kappaB TFD treatment led to decreased mRNA and protein expression of adhesion molecules. Treatment with NF-kappaB TFD led to a significant decrease in all reperfusion injury parameters compared to saline and NF-SC controls (P<0.01 vs. controls). Higher levels of apoptosis were seen in allografts treated with NF-kappaB TFD compared to control allografts. NF-kappaB TFD treatment prolonged allograft survival over saline and NF-SC controls (P<0.05). Myointimal proliferation and intimal:medial ratios in NF-kappaB TFD-treated allografts were significantly decreased compared to saline and NF-SC treatment (P<0.00001). CONCLUSIONS: Ex vivo pressure-mediated delivery of NF-kappaB TFD is an effective method to block adhesion molcule expression and reperfusion injury in the immediate posttransplant period. Further, NF-kappaB TFD treatment prolongs allograft survival and decreases GCAD.

Sulfasalazine inhibits reperfusion injury and prolongs allograft survival in rat cardiac transplants.

INTRODUCTION: Reperfusion injury is an inflammatory cell-mediated response that causes tissue damage immediately following transplantation, and has been implicated in the development of acute and chronic rejection. NF-kappaB is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1 following reperfusion. We hypothesized that treatment with sulfasalazine, a potent inhibitor of NF-kappaB, would decrease adhesion molecule expression, decrease reperfusion injury, and prolong allograft survival in rat cardiac transplants. METHODS: Heterotopic rat heart transplants were performed. Donor allografts were treated with saline, sulfasalazine (SSA), or lipopolysaccharide (LPS), a potent inducer of NF-kappaB activity. Reperfusion injury was assessed with cardiac edema (percent wet weight), neutrophil infiltration (MPO activity), and histologic damage (contraction band necrosis). Immunohistochemistry was performed to analyze protein expression. Acute rejection was determined by daily palpation. RESULTS: Treatment with a single 100 mg/kg intraperitoneal injection of sulfasalazine decreased reperfusion injury compared to saline controls (MPO activity, saline: 2.1+/-0.3, SSA: 1.2+/-0.31, P < 0.005; % wet weight, saline 77.6+/-1.1%; SSA 75.8+/-1.0%, P < 0.005; contraction band necrosis, saline: 13.1+/-2.5%, SSA: 6.1+/-3.4%, P < 0.001). LPS administration increased all parameters of reperfusion injury. Treatment with sulfasalazine prior to LPS also decreased reperfusion injury compared to LPS and saline groups. Sulfasalazine treatment decreased ICAM-1 and VCAM-1 protein expression. Administration of 500 mg/kg sulfasalazine increased graft survival to 15.4+/-1.8 days compared to saline (6.8+/-1.4 days, P < 0.005). CONCLUSION: Treatment with sulfasalazine is an effective method to decrease reperfusion injury and prolong allograft survival in a rat cardiac transplantation model.

Pressure delivery of AS-ICAM-1 ODN with LFA-1 mAb reduces reperfusion injury in cardiac allografts.

BACKGROUND: The goal of this study is to determine the effects of ex vivo hyperbaric pressure administration of AS-ICAM-1 ODN and systemic anti-LFA-1 mAb treatment on reperfusion injury in the rat cardiac allograft model. METHODS: A PVG to ACI functional heterotopic rat heart model was used. Donor hearts were treated with either saline or AS-ICAM-1 ODN and 5 atm of hyperbaric pressure for 45 minutes. Anti-LFA-1 mAb was administered systemically prior to reperfusion of the allograft. Allografts were procured 24 hours after transplantation for assessment of reperfusion injury or 72 hours to determine ICAM-1 protein expression. RESULTS: Ex vivo administration of AS-ICAM-1 ODN led to decreases in percentage wet weight (77.1+/-0.83% vs 78.7+/-1.0%, p < 0.05), myeloperoxidase activity (3.14+/-0.72 vs 4.07+/-0.59, p < 0.05), contraction band necrosis (6.4+/-6.47% vs 21.1+/-7.43%, p < 0.01), and ICAM-1 protein expression determined by immunohistochemistry compared to saline controls. Treatment with anti-LFA-1 mAb resulted in decreases in wet weight ratio (76.7+/-0.63%, p < 0.05 vs saline), myeloperoxidase activity (3.58+/-0.39, p < 0.05 vs saline) and contraction band necrosis (11.8+/-3.56%, p < 0.05 vs saline). Combination of pressure administration of AS-ICAM-1 ODN and anti-LFA-1 mAb decreased wet weight ratios (77.1+/-0.93%, p < 0.05 vs saline), myeloperoxidase activity (2.88+/-0.44, p < 0.01 vs saline), and contraction band necrosis (6.75+/-5.67%, p < 0.05 vs saline). CONCLUSIONS: Ex vivo pressure mediated delivery of AS-ICAM-1 ODN decreases ICAM-1 protein expression, reduces reperfusion injury in rodent cardiac allografts, and is more effective than anti-LFA-1 mAb treatment alone.