Astrocytes Ovariectomy Combined Injection Of D-galactose In Rat Composite Model Of Ad Pathological Changes And Dysfunction

Objective:1.To develop a new multiplicity AD model by long-term intraperitoneal administration of D-gal in OVX rats to further address the synergistic effects of estrogen deprivation and oxidative stress on the development and progression of AD.2.To investigate whether astrocytes are involved in the pathogenesis of this AD model.Methods:Three-month-old female Sprague-Dawley rats were randomly and equally divided into five groups:the intact control group(C),the OVX and D-gal injected group(O+D),the OVX-only group(O),the sham operated and D-gal injected group(D)and the OVX,D-gal and 17-βestradiol injected group(O+D+E).After six weeks,several behavioral tests as well as ChAT immunohistochemistry of cholinergic neuron in basal forebrain and Aβimmunohistochemistry in hippocampus, observation of electron microscope,ultrastructural analysis of lipofuscin deposition and the density of synapses have been performed to evaluate the success of this AD model.To investigate the involvement of astrocytes in the pathogenesis of this AD model,We detected the expression of GFAP by immunohistochemistry and western-blotting, levels of GSH,ultrastructural changes of the astrocytes,with special attention on astrocytes surrounding the axonal terminals and brain microvessels as well as the permeability of blood-brain barrier by intravascular injection of HRP in the hippocampus in ovariectomized rats injected with D-gal for 2 and 6 weeks respectively.Results:1.Compared with C groups,behavior and pathology of the rats was prominently impaired in the D,O,O+D and O+D+E groups, especially the O+D groups,versus with which the learning impairment was attenuated significantly in the O+D+E animals.The more serious damages of O+D groups as follows:(1)The mean number of square entries was increased in the open field testing;The training frequency required to attain the learning criterion was markedly increased in the Y-maze;The number of errors was highest and the step-down latency decreased dramatically in step-down type passive avoidance testing.(2) The decreased number of cholinergic neuron suffered atrophy,indicated by the small soma and deficiency of the processes.(3)Many Aβimmunoreactive neurons which absent in the other four groups,were easily detected in the hippocampus mainly located at the pyramidal layer of CA1,subiculum and granular cell layer of the dentate gyrus.(4) Ultrastructural changes:Nuclear degeneration with condensed chromatin aggregated in the rim of the nucleus with a disrupted and incomplete nuclear membrane.Lipofuscin accumulated in the cytoplasm some of which contained granulovacuolar bodies.NFT appeared in the cytoplasm or dendrites of neurons.Some mitochondria showed disrupted cristae and a swollen,twisted or vacuolar appearance.Synapse degeneration was frequently observed in the CA1 region of the hippocampus,the obvious increased lipofuscin and decreased synapses.2.(1)The 6w O+D rats required longer times to reach the platform than control and 2w experimental rats.The control and 2w O+D rats spent the majority of their time swimming in the quadrant where the platform was located,while the 6w O+D animals tended to spend their time in all quadrants.No statistical differences were observed between the control and 2w O+D groups.(2)6w O+D rats showed a prominent loss of cholinergic terminals and AChE activity in the hippocampus,compared to the control and 2w O+D groups.No significant differences were found between the control and 2w O+D groups.(3)2w and 6w O+D groups showed significant increase in the protein levels of GFAP,compared to the control.No statistical significances were observed between the two O+D groups.(4)The 6w O+D group exhibited significant decrease of GSH levels,compared to the control and 2w groups.A slight increase in GSH levels was observed in the 2w O+D group,but the difference was not significant,relative to the control.(5)Activated astrocytes were widespread in the all regions of the hippocampus in 2w and 6w O+D rats, relative to the controls.These activated glial cells exhibited hypertrophy, with very thick,highly ramified and intensely immunostained branches, compared to resting form with long,slender processes in the controls. Furthermore,many GFAP-positive astrocytes in the 6w O+D rats underwent degeneration,characterized by apparent breakdown of cell bodies.(6)Eelectron microscopy showed that some astrocytes were degeneration in the hippocampus of 6w O+D rats,identified by reduction or devoid of glycogen granules and glial intermediate fibers,and swollen or aberrant appearance of mitochondria.Especially,in the aggregated area of synapse,extremely swollen astrocytic process with watery appearance often engulfed the degenerated synaptic structures.Swollen perivascluar astrocytic endfeet surrounding the brain capillary were frequently observed in both 2w and 6w O+D rats.Especially,in the 6w O+D rats,irregular-shaped microvessels were lined by distorted or vacuolized endothelium.The above pathological changes were not evident in the controls.(7)Perivascular areas of HRP leakages were observed in some regions in the hippocampus or the cerebral cortex of O+D rats,the marker showed an intravascular localization in the controls.Conclusions:1.These results strongly suggest that estrogen deprivation and oxidative stress behave synergistically to enhance the development and progression of AD.2.OVX combined with D-gal injection may serve as an ideal AD rodent model capable of mimicking pathological,neurochemical and behavioral alterations in AD.3. Biochemical and pathological alterations of astrocytes may partially contribute to exacerbating neuronal deficits in course of this AD model.