The effect of exercise preconditioning on stroke outcome in ovariectomized mice with permanent middle cerebral artery occlusion.

Exercise preconditioning has been shown to be effective in improving behavioral and neuropathological indices after cerebral ischemia. We evaluated the effect of exercise preconditioning, 17ß-estradiol, and their combination on stroke outcome using an experimental model of stroke in ovariectomized (OVX) mice. OVX mice were randomly assigned to 4 groups as follows: control (stroke), exercise (exercise and stroke), estradiol (17ß-estradiol and stroke), and exercise+estradiol (exercise and 17ß-estradiol and stroke). Exercise preconditioning was performed on a treadmill 5 days/week, 40 min/day, at a speed of 18 m/min for 4 weeks. 17ß-estradiol was gavaged (40 µg/kg per day) for 4 weeks. Stroke was induced by permanent middle cerebral artery occlusion (pMCAO), and neurological deficits were evaluated 1, 2, and 7 days after stroke. Then, the serum concentrations of matrix metalloproteinase-9 (MMP-9) and interleukin-10 (IL-10) and infarct volumes were assessed. Exercise preconditioning and 17ß-estradiol induced a better outcome compared with the control ischemic mice, which was manifested by decrease in MMP-9, increase in IL-10, diminished infarct volume, and improved neurological deficits. Concomitant administration of 17ß-estradiol and exercise also significantly improved these parameters. Exercise preconditioning or administration of 17ß-estradiol alone or in combination before pMCAO induced significant neuroprotection in OVX mice.

Detection of unknown deletions in beta-globin gene cluster using relative quantitative PCR methods.

beta-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in beta-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real-time PCR assay for copy number analysis of beta-globin gene cluster. The copy number of target fragments (i.e. beta, delta or (G)gamma-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire beta-globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real-time PCR for analysis of copy number variations in beta-globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown beta-thalassemia deletions in routine diagnosis of beta-thalassemia mutations.