Age-related changes of brain iron load changes in the frontal cortex in APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) as a neurodegenerative brain disorder is a devastating pathology leading to disastrous cognitive impairments and dementia, associated with major social and economic costs to society. Iron can catalyze damaging free radical reactions. With age, iron accumulates in brain frontal cortex regions and may contribute to the risk of AD. In this communication, we investigated the age-related brain iron load changes in the frontal cortex of 6- and 12-month-old C57BL/6J (C57) and APPswe/PS1ΔE9 (APP/PS1) double transgenic mouse by using graphite furnace atomic absorption spectrometry (GFAAS) and Perls' reaction. In the present study, we also evaluated the age-related changes of DMT1 and FPN1 by using Western blot and qPCR. We found that compared with 6-month-old APP/PS1 mice and the 12-month-old C57 mice, the 12-month-old APP/PS1 mice had increased iron load in the frontal cortex. The levels of DMT1 were significantly increased and the FPN1 were significantly reduced in the frontal cortex of the 12-month-old APP/PS1 mice than that in the 6-month-old APP/PS1 mice and 12-month-old C57 mice. We conclude that in AD damage occurs in conjunction with iron accumulation, and the brain iron load associated with loss control of the brain iron metabolism related protein DMT1 and FPN1 expressions.

Effects of angiotensin II and its receptor blockers on migration and endothelin-1 expression of rat vascular adventitial fibroblast subpopulations / 药学学报

The study is to investigate the effect of angiotensin II (Ang II) and its receptor blockers on migration and endothelin-1 (ET-1) expression of rat vascular adventitial fibroblast subpopulations. Vascular adventitial fibroblasts were individually expanded by using cloning rings, and the effects of Ang II on the migration of adventitial fibroblast subpopulations were evaluated by Transwell. Fluorescence quantitative-PCR detected the expression of preproET-1 mRNA induced by Ang II, and its receptor antagonists losartan and PD-123319. The concentration of ET-1 was determined by ELISA. It showed that spindle shaped and epithelioid shaped cells were isolated by using cloning rings, named as spindle cells and round cells. RT-PCR showed that fibroblast subpopulations did not have leukocytes, endothelial cells and smooth muscle cells, namely pure cell lines. Compared with respective control cells, two subpopulations had transferring ability. Ang II significantly improved round cells migration in a concentration-dependent manner, and had no obvious influence on spindle cells migration. Ang II (1 x 10(-8) - 1 x 10(-6) mol x L(-1)) significantly increased the expression of preproET-1 mRNA in round cells (P < 0.01), and had no significant effect on the expression of preproET-1 mRNA in spindle cells. Losartan blocked the expression of preproET-1 mRNA induced by Ang II in round cells, and had no significant effect on the expression of preproET-1 mRNA in spindle cells. The effects of Ang II and ET-1 receptor inhibitors on the release of ET-1 were similar to the expression of preproET-1 mRNA. The results indicate that there are two cell subpopulations: round cells and spindle cells in rat vascular adventitial fibroblasts. Ang II significantly improved cells migration, and increased the expression of ET-1 in round cell subpopulation. It suggested that there may be different migratory mechanisms in two cell subpopulations, and the two subpopulations may play a different role in vascular remodeling and reparative process.

Construction and expression of adeno-associated virus vector harboring human hypoxia inducible factor-1α gene / 第二军医大学学报

Objective: To construct a recombinant adeno-associated virus (AAV) vector harboring human hypoxia inducible factor-1α (HIF-1α) gene and to express it in primarily cultured neurons in the hippocampus. Methods: Human HIF-1α gene was inserted into AAV vector plasmid pSNAV 2.0 to generate a recombinant plasmid pSNAV-HIF-1α, then pSNAV-HIF-1α was transfected into BHK-21 cells and selected by G418. The construction of rAAV-HIF-1α was achieved by infecting G418-resistant BHK-21 cells with recombinant herpes simplex virus. The recombinant rAAV-HIF-1α was confirmed by polymerase chain reaction(PCR). The titer of rAAV-HIF-1α was detected by dot-blot with digoxin labelled cytomegalovirus probe. The purity of rAAV-HIF-1α was detected by SDS-PAGE. The expression of HIF-1α protein was determined by Western blot. Results: PCR demonstrated that human HIF-1α gene was successfully inserted into rAAV-HIF-1α. The Virus titer was 1 × 1012/ml and the virus purity exceeded 98%. Western nblot analysis demonstrated that rAAV-HIF1α infected hippocampal neurons expressed HIF-1α protein. Conclusion: rAAV-HIF-1α has been successfully constructed and it is capable of expressing HIF-1α in cultured hippocampal neurons.

Effect of intracerebroventricular injection of rAAV-HIF-1α on hippocampal neuronal apoptosis in a rat model of Alzheimer disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of intracerebroventricular injection of rAAV-HIF-1α on hippocampal neuronal apoptosis in a rat model of Alzheimer disease (AD).</p><p><b>METHODS</b>Thirty-two male SD rats (250-300 g) were randomized into 4 groups (n=8), including the normal control group without any treatment, AD model group with right intracerebroventricular injection of 2 µl Aβ25-35 (10 mg/m1), sham-operated group with right intracerebroventricular injection of 2 µl normal saline, and AD+ rAAV-HIF-1α group with right intracerebroventricular injection of 10 µl rAAV-HIF-1a (1×10¹² v.g./m1) one week after Aβ25-35 injection. The rats were sacrificed to detect the expression of HIF-1α and apoptosis of hippocampal neurons 5 weeks after Aβ25-35 or saline injection.</p><p><b>RESULTS</b>Western blotting showed that the expression of HIF-1α was significantly higher in AD+rAAV-HIF-1α group (451.59±34.39) than in normal control group (229.05±41.28) and sham-operated group (216.29±37.08) (P<0.05) without significant difference between the latter two groups. The apoptotic ratio of the hippocampal neurons was significantly higher in AD model group ([19.49±2.59]%) than in normal control group ([5.41±0.75]%) and sham-operated group ([5.28±0.66]%) in (P<0.05), and intracerebroventricular injection of rAAV-HIF-1α resulted in a significant reduction of the apoptotic ratio in the AD rats ([12.07±2.06]%) (P<0.05).</p><p><b>CONCLUSION</b>Intracerebroventricular injection of rAAV-HIF-1α can inhibit hippocampal neuronal apoptosis in the rat model of AD.</p>