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Objective To evaluate the role of NOX2 in bupivacaine-induced production of reactive oxygen species (ROS) in nerve cells.Methods SH-SY5Y cells were seeded in culture plates and divided into 4 groups (n =11 each) using a random number table:small interfering RNA (siRNA) negative control group (group NC),siRNA negative control plus bupivacaine group (group NC +B),NOX2 siRNA group and NOX2 siRNA plus bupivacaine group (group NOX2 siRNA + B).In NC and NOX2 siRNA groups,the cells were transfected with negative siRNA and NOX2 siRNA,respectively,and then incubated in the culture medium for 24 h.In NC+B and NOX2 siRNA+B groups,cells were transfected with negative siRNA and NOX2 siRNA,respectively,new plates were used,the cells were incubated for 3 h with bupivacaine at the final concentration of 1.5 mmol/L,the culture medium was then replaced,and the cells were incubated until 24 h.The level of intracellular ROS was measured using the fluorogenic probe dihydroethidium,the cell apoptosis was determined by TUNEL,and the expression of activated caspase-3 and caspase-9 was detected using Western blot.Apoptosis rate was calculated.Results Compared with group NC,the level of ROS and apoptosis rate were significantly increased,and the expression of activated caspase-3 and caspase-9 was up-regulated in group NC+B (P＜ 0.05),the level of ROS was significantly increased,and the expression of activated caspase-3 and caspase-9 was up-regulated (P＜0.05),and no significant change was found in apoptosis rate in group NOX2 siRNA+B (P＞0.05),and no significant change was found in the level of ROS or apoptosis rate (P＞0.05),and the expression of activated caspase-3 and caspase-9 was significantly up-regulated in group NOX2 siRNA (P＜ 0.05).Compared with group NC+B,the level of ROS and apoptosis rate were significantly decreased,and the expression of activated caspase-3 and caspase-9 was down-regulated in group NOX2 siRNA+B (P＜0.05).Conclusion NOX2 is involved in the pathophysiological mechanism of bupivacaine-induced burst production of ROS in nerve cells.
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Objective To prepare the rat model of type 2 diabetes mellitus (T2DM), and to ob-serve the characteristics of peripheral neuropathy. Methods High fat and high sugar diets were fed for 8 weeks to induce insulin resistance and then low dose streptozotocin ( STZ) was injected intraperitoneally to induce type 2 diabetes mellitus models in Sprague Dawley rats. Blood glucose and serum insulin levels con-tinuous were monitored. Tactile allodynia in response to von Frey ( VF) filament stimulation of the plantar hind paws and paw withdrawal thermal latency ( PWTL) to plantar test were used as the criterion for diabetic neuropathy. Instruments AD was used to detect nerve conduction velocity ( NCV) of sciatic nerve in rat and the morphological and pathological changes of sciatic nerve were detected by electron microscope. Results The characteristics of T2DM rats by peripheral neuropathy in this method were that 50％ force withdrawal threshold and PWTL were measured. Both values of diabetic rats were decreased from the day of STZ injec-tion until 4 weeks after STZ injection, and then increased 8 weeks after STZ injection (50％ force withdraw-al threshold values, (11.8 ±0.8)g, (8.4 ±0.7)g and (16.2 ±1.4)g; PWTL (10.2 ±0.9)s, (8.3 ± 1. 2)s and (13. 2 ± 1. 0)s. These results indicated that tactile sensation changed from hypersensitive to hy-posensitive. Compared to the NC group, the sciatic nerve motor and sensory conduction velocity were signifi-cantly decreased at 4 and 8 weeks in DM group, respectively. Compared to DM group at 4 weeks, the sciat-ic nerve motor and sensory conduction velocities were further decreased in the DM group at 8 weeks. Con-clusively, sciatic nerve showed obvious demyelination and axonal collapse. Conclusions T2DM rat model was successfully induced by high fat and sugar diet combined with small dose of STZ injection. The rat mod-el has typical pathological change of peripheral nerve. It might provide a particularly advantageous tool for investigations of diabetes and its chronic complications.
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Objective To evaluate the effect of diabetic peripheral neuropathy on peripheral neurotoxicity induced by local anesthetics in rats.Methods Sixty healthy adult male SPF Sprague-Dawley rats,aged 6 weeks,weighing 150-180 g,were divided into either control group (n =18) or diabetic peripheral neuropathy group (n=42) using a random number table.The rats were fed a high-fat and high-sucrose diet for 8 weeks,and streptozotocin (STZ) 30 mg/kg was injected intraperitoneally to induce diabetes mellitus which was confirmed by blood glucose level≥ 16.7 mmol/L.The mechanical paw withdrawal threshold to yon Frey filament stimulation and thermal paw withdrawal threshold were measured.The decrease in reaction thresholds to thermal and mechanical stimuli (changing from sensitivity to insensitivity) was observed after STZ injection.At 4 weeks after STZ injection,the rats showing a marked hyperalgesia served as early diabetic group.At 8 weeks after STZ injection,the rats showing a marked insensitivity to pain served as late diabetic group.Experiments were carried out in early or late diabetic rats,and ordinary Sprague-Dawley rats of the same age were used as control group.Left sciatic nerve block was performed with 2％ lidocaine 0.2 ml.Before the sciatic nerve block and at 1 week after the sciatic nerve block,the nerve conduction velocity of the left sciatic nerve and F-wave minimal latency were measured,and the sciatic nerve block time was recorded.Results Compared with the baseline before block,the nerve conduction velocity was significantly decreased,and the F-wave minimal latency was prolonged in late diabetic rats (P＜0.05).Compared with control group,the sciatic nerve block time was significantly prolonged in late diabetic group (P＜0.05).Conclusion Diabetic peripheral neuropathy aggravates peripheral neurotoxicity induced by local anesthetics in rats.