| BackgroundAlzheimer’s disease (Alzheimer disease, AD) is the most common in the elderly patients with degenerative disease of nervous system, is the most common type of senile dementia, is the main factors causing the people over 60 years old,have serious mental burden to society and family. Its clinical features are insidious onset, the gradual emergence of memory loss, cognitive dysfunction, abnormal behavior and social obstacles. The condition usually progressive, gradually lost the ability to live independently, with onset after 10～20 years due to complications and death. The pathological features of AD include:extracellular amyloid beta (Aβ) of senile plaques and neurofibrillary tangles in the cell formed by the aggregation of tau and abnormal protein aggregation formation (neurofrillary tangle, NFT) decrease of synapses and neuronal loss. The etiology of AD is still unknown, is generally believed that may be associated with genetic and environmental factors. AD there are two main types: hereditary and sporadic, the incidence of hereditary AD mainly and gene mutation, it has become clear that the APP gene, PS1 gene, PS2 gene, tau gene and UBQLN1 protein gene (ubiquilin-1). And emits the pathogenesis of AD is more complex, at present, is clear about mutation and APOE gene, and there are still many hypothesis, abnormalities such as protein (mainly AP and Tau protein), abnormal consumption, neurotransmitter insulin resistance (IR), oxidative stress, inflammation mechanism of hypothesis.In recent years, found that about 70%～100% of AD in patients with cerebral tissue amyloid degeneration of cerebral vascular disease (Cerebral amyloid angiopathy, CAA), CAA even before the formation of senile plaques,but its pathogenesis is still unclear,restricted the clinical diagnosis and treatment of AD. Study on the distribution and reduce the cognitive function of brain tissue in patients with AD CAA is confirmed to have the great correlation with cognitive impairment, therefore is also one of the main pathological feathers of AD.A large number of epidemiological survey, aging, arteriosclerosis, hypertension, diabetes, high cholesterol, homocysteine, high levels of homocysteine is not only the vascular dementia (vascular dementia, VaD) of risk factors,but also risk factor of AD, these risk factors can damage brain hemodynamics, the cerebral blood flow perfusion decreases,cause cerebral ischemia and hypoxia,affecting brain tissue metabolism, leading to endothelial cell dysfunction,neurovascular unit physiological function injury and a series of pathological changes. Because of the close correlation between AD characteristic neuropathological changes and cerebral hemodynamic changes and brain energy metabolism disorder and therefore, more and more scholars think, AD should be classified into vascular diseases. Therefore, at present, vascular mechanism of AD by more and more researchers referred to the primary position.At present, the exact mechanisms of AD occurrence of CAA are still not clear, but more and more researchers think that CAA is likely to be due to brain tissue metabolism produced AP by perivascular spaces clear disorder caused by. The perivascular spaces (Perivascular spaces, PVS)is also called Virchow-Robin spaces(VRS), is the normal anatomic structures in the central nervous system, is wrapped in small tissue space around the arteries and veins, VRS will be separated the blood vessels and surrounding brain tissue, VRS filled with interstitial fluid, instead of cerebrospinal fluid. The physiological significance of VRS as a main pathway of brain interstitial fluid discharged from the main solute removal, solution and macromolecules metabolic waste brain metabolism produce more, such as soluble Aβ, from the structure and function, it think the perivascular spaces is lymphatic channels of the central nervous system. Brain interstitial fluid by intracerebral arteries around gap into the subarachnoid space of large artery perivascular spaces, and epidural lymph tube into the brain outside the lymphatic system, is mainly to cervical lymph node,thus,the walls of blood vessels in the brain is the brain anatomical structure of circulation and brain lymph circulation share, lesions of the vessel wall is not only can affect cerebral blood circulation, but also lead to cerebral lymphatic circulatory disorder.Production of soluble Aβ in central nervous system under normal circumstances can clear the brain through receptor mediated pathways into the blood,scavenging enzymes,phagocytosis, VRS removal,which will not lead to deposition, when Aβ increase or remove barriers then abnormal aggregated insoluble fiber with neurotoxicity.Abnormal Aβ fiber accumulation cannot transendothelial migration, cerebral lymphatic circulation become the most main clearance pathways.Aβ easily deposition in the location in a drainage process, resulting in CAA,on the other hand, the formation of CAA can block VRS, leading to lymphatic circulatory disturbance, form a vicious cycle; therefore,lymph circulation disorders in the incidence of CAA may play an important role.Aβ1-40 and Aβ1-42 are two main type Aβ,and Aβ amyloid protein by soluble polypeptides into insoluble amyloid fibrils form is the key to its deposition in the brain. Metabolic activity in the brain to produce soluble Aβ polypeptide secreted into the extracellular to become soluble Aβ peptide intermediates, when accumulated to a certain extent spontaneously polymerize to form soluble oligomers, raw fiber and other forms, under certain conditions to the polypeptide chain between thepsheet folding form together to form insoluble amyloid fiber (AP fiber) form,extremely easy to amyloid plaque formation and deposition. The A｛1-40 are more easily deposited in the vessel,Aβ1-42 fiber is the main component of senile plaques, the study found that Aβ1-42 fiber is necessary for the formation of senile plaques and CAA, i.e., only when the Aβ1-42 first deposition can gather Aβdeposition of more on the vessel. Aβ1-42 fiber is the most toxic form of Aβin the brain tissue, Aβ1-42 fiber sedimentary formation of senile plaques and CAA is the main reason leading to the occurrence of AD.Cerebral amyloid angiopathy (CAA) is a kind of age-related disease characterized by Aβ deposition in leptomeningeal and cortical small artery wall. Brain tissue of patients with AD in the Aβ in perivascular spaces (VRS) drainage process easy to local deposition,the main sedimentary arterioles in cortex and spinal cord around the VRS and the adjacent structure (mainly the vascular wall and astrocyte glial limiting membrane), gradually formed in the local CAA deposition. The pathological changes of CAA mainly characterized as amyloid deposition in the brain parenchyma and cerebral vascular adventitia and membrane area without visible uniform structure.Under light microscope, the amyloid appears smooth muscle cell basement membrane in the adventitia and membrane, immunohistochemistry confirmed amyloid mainly for amyloid beta protein.Astrocytes are the main component of glial cells in the central nervous system, by other cell contact and connection,with nutrition and support neurons,modulate neu rotransmitter release,energy metabolism,signal transduction,ion buffering,flow contro 1 function of the central nervous system, while minor injuries,astrocytes immediate ac tivation and repair the damage,it is very important to maintain homeostasis of central nervous system. In addition, recent studies have shown that crack on small vascular b rain astrocytes surrounding between adjacent foot processes in regulating Aβdepositio n in the perivascular spaces are very important role. But less research has done in the process of formation of pathological changes of perivascular astrocytes on CA A.The role of astrocytes in CAA formation is still not clear,so it is very important to f orm the pathological changes of the astrocytes in the process of the discussion of AD, contribute to the pathogenesis of AD to ascertain from another angle.This study by stereotactic bilateral intrahippocampal injection of Aβ1-42 fiber, pathological changes of treble-labeling indirect immunofluorescence staining of dynamic observation of brain tissue CAA forming process of middle and small perivascular astrocytes,further understand the role of perivascular astrocytes on brain tissue Aβ deposition in blood vessels, with a new target to explore AD treatment and prevention.Methods1、Animals and groups:Healthy adult SPF male SD rats 40,weight 250 ± 10g, provided by experimental animal center of Southern Medical University. The rats were randomly divided into model group and control group, with 20 rats in each group,and each group was divided into 4 subgroups:1w、2w、4w、12,2 rats in 1w and 2w subgroup,8 rats in 4w and 12w subgroups.2、Aβ1-42 fiber preparation:Aβ1-42 powder dissolved in sterile 0.01mmol/L 1mL PBS solution (1g/L), with Aβ1-42 suspension was placed under the constant temperature of 37 ℃ water bath shaker,continuous oscillating water bath 7 days aging into Aβ1-42 fiber, used for the preparation of an AD animal model.3、The AD animal model preparation:Model group rats by stereotactic technique into rats with bilateral hippocampal injection has been prepared Aβ1-42 fiber, each side of 5ul, the rats in the control group using the same method to the rat bilateral intra hippocampal injection of equivalent sterilization PBS solution.4、Morris water maze test:4w and 12w subgroup rats in Model group and control group were to do Morris water maze test in the corresponding time points.5、Cerebral blood flow detection:Rats in 12w sub-group in model and control group were to detect the cerebral blood flow with a laser doppler instrument on the day of operation and after 1w,2w,3w,4w and 8w,12w respectively.6、Materials:Subgroups of rats were in aortic perfused and fixated in the corresponding time point, then take out the brain tissue specimens to keep in the-80 °C refrigerator after quick frozen in liquid nitrogen.7、Pathologic staining:Brain tissue in rats of each group were for continuous frozen sections, HE staining, Congo red and labeled Aβ, a-actin, GFAP, three indirect immunofluorescence staining.8、Statistical analysis:The Morris water maze test results are expressed by the mean ±standard deviation, escape latency of rats were analyse using repeated measures analysis of variance, explore the platform number with single factor analysis of variance.Laser doppler measurement of cerebral blood flow using independent sample t test.Statistical analysis was performed using SPSS 13.0 software, taking P＜0.05 significant difference.Results1、Morris water maze test:After 4 days training,the escape latency in each sub-group rats is gradually shortened.12w model rats showed significant increase in each day’s escape latencies,compared with 12w control rats (P<0.05), 4w model rats on the first day and the second day the escape latency were increased compared with 4w control rats (P＜0.05), but there was no differences on the third and forth day. Compared with the total escape latency,significant differences was evident between the two groups (P＜0.001). Significant decrease was shown between model groups and the control groups in the time of searching platform in Morris water maze test (P＜0.05)2、CBF:Model group rats of cerebral blood flow was gradually decreased over time, while the control group rats no obvious changes of cerebral blood flow, there was no differences on the time of 0、1w、2w (P>0.05), significant differences was evident between the two groups on the time of 3w、4w、8w、12w (P＜0.01).3、HE staining:Pathological changes in the brain of the model groups showed decreased of granule cells and pyramidal cells in the hippocampus layers, neuron degeneration, VRS dilation, small vessel wall degeneration, luminal stenosis. The pathological manifestations as described gradually increased as time went by. The control group, HE staining showed no obvious pathological changes.4、Congo red staining showed no amyloid deposition in 1w model group of rats brain tissues, in 2w model group rats cerebral parenchymal and vascular peripheral space were visible with a small amount of orange red deposits of amyloid, in 4w model group rats showed perivascular spaces of amyloid deposition was significantly increased than 2w group, the surrounding brain parenchyma note the amount of dispersion in the distribution of amyloid,visible perivascular interstitial tissue is loose,12w model group can be seen a large number of orange amyloid deposition in the vascular wall surrounding the whole,markedly dilated perivascular spaces, contributing to wall thickening, lumen narrow or occlusion. The control group rat brain tissues had no obvious amyloid deposition.5、Immunofluorescence:Immunofluorescence revealed no β-amyloid deposited in perivascular spaces in 1w model group,intact astrocytes surrounding vessels is visible; In 2w model group a small amount of granular Aβ deposition around hippocampal arterioles accompanying by surrounding astrocytes fuzzy structure and disorder.Immunostainings of frozen brain sections from 4w model group observed more perivascular amyloid deposits than 2w model group,astrocytes around vessel with amyloid deposition GFAP staining enhanced, aggregation and hyperplasia with fuzzy structure. Vascular amyloid deposits and astrocytic GFAP were more pronounced observed in 12w model group than 4w model group and also found amyloid deposits in venules besides in arteriole in hippocampus. The rats brain tissue in model groups did not see the classic amyloid plaque formation. By contrast, no AP deposited in vessels in control groups, astrocytes were less and intact.Conclusions1、We have succeeded in making the AD rat models by injection Aβ1-42 fiber in bilateral hippocampus with a stereotactic frame. The advanced cognitive function of different groups of rats was evaluated with a Morris water maze test.The results showed that escape latencies in model group rats were significant prolonged than the control group. HE and Congo Red staining showed dilated perivascular spaces, CAA like pathologic changes in brain tissue of rats in the model group. The granular amyloid was deposited in vessels wall and diluated VRS with surrounding astrocytes aggregation. The above experiment can confirm that Alzheimer’s disease animal model for the study of modeling make success, it can be used for further experimental research.2 、Water maze experiment results show that the ability of learning and memory of the rats in the model group decreased significantly compared with the control group, it showed that there was obvious damage of higher cognitive function of the rats in the model group.3、Laser doppler measurement results show that the model group rats of the cerebral blood flow gradually reduced, while the control group rats blood flow has no obvious change.4、Neuron degeneration, VRS diluation, vascular wall thickening and luminal stenosis was visible in the brain tissue of model rats.5、Congo red staining showed that the blood vessel of brain tissue in rats of model group in each subgroup and hippocampus in the surrounding space and the wall of small vessels were visible on the amyloid deposition,and with the passage of time, the blood vessel wall deposition of amyloid more,perivascular space expanding obviously.6、Three immunofluorescence Aβ, α-actin and GFAP labeled staining showed that brain tissue Aβα gradually migrated and deposited and formed CAA in the vascular wal, surrounding the small blood vessels,astrocytes increased. Aβ1-42 fiber found early deposited mainly in the arteriolar wall, later also vein wall were found on Aβ1-42 fiber deposition eventually lead to pathological changes of small vessel wall and astrocytes aggregation appear around small blood vessels of different degrees of stenosis or occlusion,leading to cerebral vascular pulsation weakened, form a vicious cycle,leading to the occurrence of CAA.7、This study illuminated that cerebral amyloid angiopathy and astrocytes proliferation and damage maybe the highlights pathological changes in the early process of Alzheimer’s disease. |
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