| Objective To explore the role and mechanisms of physiological dose of17-β-estrodiol(E2) replacement therapy on vascular dementia caused by cerebral chronic hypoperfusionby detecting the pathological changes of vascular-neuron following bilateral commoncarotid artery occlusion (BCCAO), which might provide a new treatment strategy forvascular dementia caused by chronic hypoperfusion.Methods1Animal model of BCCAO: SPF adult SD female rats (body weight280~300g) were used through the study. BCCAO was carried out one week afterbilaterally ovariectomized. Experimental animals were randomly divided into earlygroups: sham3d, BCCAO (6h,1d,3d,7d), BCCAO3d+placebo, BCCAO3d+E2andchronic groups: sham3m, BCCAO3m, BCCAO3m+placebo, BCCAO3m+E2-treatmentfor3m continuously.2Evans Blue (EB) and fluorescein sodium (NaF) dye extravasationand the expression of tight junction protein ZO-1, Occludin were measured to evaluatethe extent of BBB disruption at different time point following BCCAO.3Immunoglobulin G (IgG) leakage into the parenchyma was detected to assessed thedamage of microvessel at different time point.4Vascular morphology and densityindicated in cortex and hippocampal were investigated to illustrate its pathologicalchanges following BCCAO.5Electron microscope (EM) were used to detect the changesof neurovascular unit (NVU) ultrastructure changes in different groups.6Western blotand confocal were used to detect the expression and the location of VEGF and MAP2following BCCAO and E2-treatment groups.Results1Compared to sham group, EB and NaF exudation significantly increasedfrom1d after BCCAO and peaked at3d and then markedly decreased at7d but still muchhigher than sham group (P<0.05).2Western Blot results showed both ZO-1andOccludin levels at BCCAO3d or3m were significantly decreased compared to sham3dor3m respectively (P<0.05).3Both in cortex and CA1region, a large number ofimpaired vascular was surrounded by a halo of IgG immunoreactivity in BCCAO(3d or3m) groups compared to sham(3d or3m). Moreover, weaker staining of IgG aroundmicrovascular was observed in BCCAO3m group than that in BCCAO3d in CA1region.There was no significant IgG leakage in E2(3d or3m) groups.4The positive structuresof RECA-1was significantly increased in BCCAO groups (3d,3m) compared to shamgroups (3d,3m) in both cortex and CA1region in bin50~500(P<0.05). However, E2treatment show a remarkable decrease in bin50~500(P<0.05). In bin2001~infinity, thequantity of microvessels dramatically decreased in BCCAO groups (3d,3m) compared to sham (3d,3m)(P<0.05). And E2treatment can increase microvessels density in bin2001~infinity (P<0.05).5Both in BCCAO3d and3m, severe angioedema, dilated bloodwall with basement membrane degradation and damaged endothelial cells were observedin microvessel, and hippocampal neurons were severe impaired evidenced by nuclearmembrane distortion, hyperchromasia and disintegrated in mitochondria. Additionally,astrocytes were observed severe damage appearing swollen with disintegrated cristae inmitochondria both in BCCAO3d and3m. In striking contrast, E2-treatment preservedultrastructure of NVU following BCCAO.6Western blot analysis showed VEGFexpression significantly decreased at BCCAO3d and3m groups compared to sham3d or3m respectively, while E2-treatment markedly inversed the changes (P<0.05). MAP2expression markedly attenuated at BCCAO3d,7d,3m compared with sham3d or3m.Continuously E2-treatment could enhance the levels of VEGF and MAP2. Also,immunostaining results showed that MAP2staining significantly decreased in BCCAO3d,7d and3m groups. E2treatment could increase the expression of MAP2. Intriguingly,VEGF immunostraining occurred different distribution following BCCAO, displayingthat VEGF significantly increased mainly localized in hippocampal neurons at BCCAO6h and1d, however in BCCAO3d,7d and even3m VEGF predominantly co-localizedwith astrocyte that closely consistented with astrocyte excessive activation response toBCCAO insult. The immunohistochemistry investigations perfectly mirror the westernblot results.Conclusion1Down-regulation of tight junction proteins such as ZO-1, occludinginduces the structural and functional damage of NVU in the early stage of BCCAO.2Continously physiological dose of E2replacement therapy protects BBB integrity, NVUultrastruture from chronic hypoperfusion insult via up-regulating, at least in part, theexpressions of VEGF and MAP2. |
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