RESUMO
Objective To investigate the effects of electroacupuncture (EA) on affected and healthy limbs and on the regulation of insulin-like growth factor-1 (IGF-1) mRNA and proteins in the cerebral cortex early after focal ischemic.Methods A model of focal cerebral ischemia was established in 54 SD rats using the suture occlusion method.They were then randomly divided into an affected limb therapy group (ALTG,n =18),an unaffected limb therapy group (UALTG,n =18),and a control group (CG,n =18).Each group had a 7-day subgroup,a 14-day subgroup and a 21-day subgroup with 6 rats in each.Rats in the experimental groups received EA beginning 24h after the occlusion.Rats in each subgroup were sacrificed in a random order on the 7th,14th and 21st days and the ischemic cerebral cortexes were quickly dissected.The specimens were frozen in liquid nitrogen before being analysed for IGF-1 mRNA expression by RT-PCR and for IGF-1 protein by Western blotting.Results ①After occlusion,IGF-1 protein levels in the ischemic cortexes of the CG declined from the 7th through the 21st day.Rats in the ALTG had significantly higher levels compared with the CG at all time points.The UALTG had the highest values on the 14th day,but was lower than the ALTG and higher than the CG at the 21st day.②IGF-1 mRNA levels in the ischemic cortexes of the UALTG declined from the 7th through the 21st day.At day 7 the results of the UALTG were 6.8 times higher than the CG,and the ALTG was 3.0 times higher.At day 14 levels in the UALTG were significantly lower than those in the ALTG.At that point the results of the UALTG rats were 3.3 times higher than those of the CG and the ALTG was 5.7 times higher.On day 21 levels in both the UALTG and ALTG were significantly lower than in the CG.Conclusions EA intervention at an early stage of focal cerebral ischemia can improve the expression of IGF-1 mRNA and protein levels in the ischemic cortex.Treating the unaffected limb can evoke more IGF-1 mRNA expression earlier and with relatively longer duration,and generate relatively longer protein increases.EA administered to the unaffected limb was more effective in the early stage of stroke.
RESUMO
An automated procedure using monolithic-phase based on-line extraction is described for pharmaceutical component analysis in plasma by LC-MS/MS. In this approach, a short monolithic C(18) 4.6 mm x 10 mm cartridge is used for high flow extraction at 4 mL/min. Plasma samples were subjected to protein precipitation first with acetonitrile, and the supernatant was diluted and loaded onto a monolithic cartridge. Sample elution was accomplished with narrow-bore LC-MS/MS system. A method for determination of Amprenavir (APV) and Atazanavir (AZV) in human plasma was developed with this approach. After 0.1 mL of plasma was transferred into each well of a 96-well plate by a liquid handler, the rest of sample preparation time typically only takes about 20 min. A Phenomenex Luna C18(2) 2.0 mm x 150 mm analytical column was used for the separation at a flow rate of 0.3 mL/min. The run time for each sample was 4 min. The standard curve range was 2.77-1520 ng/mL for Atazanavir, and 4.50-2560 ng/mL for Amprenavir. The accuracy (%bias) at the lower limit of quantitation (LLOQ) for Atazanavir was 2.7% and the precision (%CV) at the LLOQ was 7.9%, while the accuracy at LLOQ for Amprenavir was -1.3% and the precision at LLOQ was 7.8%. The inter-day %bias and %CV of the quality control samples of Atazanavir were < or = 4.5% and < or = 6.5%, respectively. The inter-day %bias and %CV of the quality control samples of Amprenavir were < or = 1.1% and < or = 7.2%, respectively. Coefficients of determination, a measure of linearity, ranged from 0.993 to 0.999. Very low carry-over (0.006%) even after high standard sample was demonstrated in the monolithic-phase based method. Other characteristics of such method include high recovery and good tolerance to matrix effect, which was demonstrated by 12 lots of plasma. The back pressure of the monolithic extraction cartridge remained the same after 450 samples injected. The performance of the monolithic-phased on-line extraction method was compared with that done by an automated 96-well liquid-liquid extraction procedure, which was carried out using hexane:ethyl acetate as the extraction solvent. The results showed that similar precision and accuracy were achieved by both methods.
