Pathological Study On Cerebral Amyloid Angiopathy With Experimantal Alzheimer’s Disease In Rats

BackgroundAlzheimer’s disease (AD) is the most common cause of dementia, accounting for50%of cases, although there is growing awareness that AD is often mixed with other dementia causes. Dementia is a clinical term that refers to the development of progressive cognitive deterioration associated with an inability to perform normal activities of daily living. When dementia afflicts the young, there is usually a single pathologic process present on autopsy such as the neuritic plaques and neurofibrillary tangles, which are indicative of Alzheimer’s disease. AD is characterized by memory disorders, aphasia, disuse, agnosia, visual spatial ability damage, abstract thinking, and ability to calculate damage, personality and behavior change. Lack of specific evidence is responsible for Alzheimer’s disease. Now the precise mechanism of AD is unknown. There are many hypothesizes about what AD is. It including protein abnormally aggregated. Most consider that abnormal aggregated protein is the hallmarked of AD, such as the deposition of amyloid-β peptides and the hyperphosphorylated microtubule-associated protein tau. Some research provides that neurotransmitter plays an important role in the developing AD. And in addition,a rapidly growing body of evidence indicated oxidative stress, inflammation and genetic mutation are associated with increased AD morbidity.Recent studies revealed a significant comorbidity of Alzheimer’s disease and cerebrovascular disease suggesting that cerebrovascular dysregulation is an important feature of Alzheimer’s disease. A dysfunction of the vascular system may result in damage of the neurovascular unit, initiating a cascade of events. So far, lots of researchers present a hypothesis that vascular risk factors may play an important role in initiation of Alzheimer’s disease by damaging brain capillary endothelial cells and the neurovascular unit.A large number of epidemiological studies suggest that, including Rotterdam Study, The vascular hypothesis of AD is summarized and modified from different recent studies. Old age, atherosclerosis, stroke, hypertension, transient ischemic attacks, cardiac disease, apolipoprotein E (ApoE), elevated homocysteine levels, hyperlipidemia, metabolic syndrome and diabetes are risk factors for both vascular dementia and AD. Such risk factors can degrade cerebral hypoperfusion, which induce the decrease of cerebral metabolism and degrade basement membranes, endothelial cells,subsequently damaging the integrity of the BBB, and potentially leads to dementia. Small ischaemic lesions, which in isolation would not alter cognition, substantially aggravate the dementia.Neurons and astrocytes are in close proximity and are functionally coupled to smooth muscle cells and endothelial cells. Their interaction in the normal state and their coordinated response to injury has led to the concept that these cells constitute a functional unit, termed the neurovascular unit.Large cerebral arteries branch into smaller arteries and arterioles that run along the surface of the brain (pial arteries).These consist of an endothelial cell layer, a smooth muscle cell layer and an outer layer of leptomeningeal cells,termed adventitia, which is separated from the brain by the Virchow-Robin space. It is generally thought that the penetrating intracerebral vessels are separated from brain parenchyma by the surrounding perivascular spaces also known as Virchow-Robin spaces. It has been viewed for a long time that the Virchow-Robin space is the normal anatomy in the central nervous system. The relationship between the VRS and the peripheral lymphatics is well established. The patterns of drainage initially led to the concept that CSF serves a "lymphatic" function through its exchange with brain ISF along paravascular spaces. In the absence of conventional lymphatic vessel in the brain, VRS is widely distributed in the periphery of the arteries and veins in the central nervous system, except in the periphery of the capillaries. As the primary prelymphatic system in the brain, it shares the same anatomical structures with the cerebral circulation system.There is a significant body of evidence suggesting that Aβaccumulates in the wall of cerebral vessels and in the brains of AD individuals because of imbalances between its production and clearance from the brain. Aβ1-40and Aβ1-42are the two main form of Aβ. Aβ1-42is more likely to deposit within the brain, the main component of senile plaque. On the other hand, Aβ1-40deposits more around the vessel walls instead of the brain tissue. If any of the clearance pathways are disrupted, soluble Aβwill accumulate and promote the formation of toxic Aβoligomeric and aggregated species, which have devastating effects on the neurovascular unit. The soluble Aβ peptide becomes oligomerization and then the amyloid fibril. Under certain circumstances, the soluble AP peptide become mature through oligomerization and fibrillization, A mature Aβ1-42amyloid fiber is known to consist of a P-sheet structure. There is neurotoxicity in both β amyloid fiber and β oligomers. These neurotoxicity may be the main course of AD.Amyloid is deposited in the walls of arteries and capillaries as erebral amyloid angiopathy (CAA) in the brains of older individuals and of those with Alzheimer disease (AD). CAA may occur throughout arteries in the cerebral hemispheres and in the cerebellum. CAA predominantly affects small arteries and not the larger intracranial arteries which contain more smooth muscle cells; furthermore, CAA rarely, if ever, occurs in extracranial arteries. Aβenters perivascular spaces mainly at the capillary level and then flows along vessel walls into periarterial compartments. Aβaccumulates in pericapillary and periarterial drainage pathways causing CAA. CAA is very prominent and is associated with the blockage of lymphatic drainage in brain.Therefore, in these study, we prepare an Alzheimer’s disease rat models by Aβ fiber injection in bilateral hippocampus with a stereotactic frame. The cognitive function of different groups of rats was tested and pathologies were observed. In this way, we explore the concept that the accumulation of Aβ amyloid within the brain and the continuous pathologies in the walls of blood vessels, in order to find out the accumulation pattern of AP peptides.Methods:1、Aβ1-42was prepared as stock solution at a concentration of1μg/μL in sterile0.1M phosphate-buffered saline (PBS)(pH7.4) and aliquots were stored at-20℃.Aβ1-42solution was aggregated by incubation at37℃for7days before use.2、40Sprague Dawley rats as subjects were randomly separated into2groups:the experiment group and the control group. Each group was20rats. These two groups are separated into4sub-groups, each sub-group5rats. In brief, rats were anesthetized with intraperitoneal injection of chloral hydrate. Under light anesthesia, Alzheimer’s disease rat models were prepared by Aβ fiber injection in bilateral hippocampus with a stereotactic frame. The injection volume of Aβ fiber was delivered gradually. The control group was prepared by sterile PBS injection in bilateral hippocampus with a stereotactic frame. 3、The cognitive function of different groups of rats was tested with a Morris water maze. Each rat was allowed a120-second swim to find the platform and was left on the platform for15seconds. A probe test was performed after the last training session. The platform was removed and each rat was allowed a free120-second swim.4、Animals were anesthetized using an intraperitoneal injection and were perfused intracardially with0.9%NaCl followed by phosphate buffer0.01mol/L containing4%paraformaldehyde. Brains were removed and quickly frozen in liquid nitrogen. Frozen brains were stored at the Ultra Low Temperature Freezer.5、Frozen brains were mounted on a freezing microtome and serially cut into6μm coronal sections. For histology, sections were studied from each brain, taken from the anterior hippocampus level. Sections were stained with HE staining and Congo Red staining as well as double-labeling indirect immunofluorescence on β amyloid fiber and a-actin.6、All the experimental data are expressed by the mean±tandard deviation. Comparisons were performed using One-way ANOVA analysis or Two-way repeated measures analysis of variance, P<0.05was considered significant, using the statistical software SPSS13.0analysis.Results:1、The spatial reference memory was analyzed using a water-maze procedure. When rats started training at4and12weeks after peptide injection, acquisition profiles decreased with training. Animals tested at4and12weeks after beta-amyloid injection showed significant increases in latencies, compared with control rats (P<0.05), indicating a significant alteration of acquisition performance. Two-way repeated-measures analysis of variance showed a significant effect between experiment group and control group (P<0.001). Probe test analysis revealed that beta-amyloid treated rats at weeks4and12showed a significant decreased compared to control group (P<0.05)2、HE staining:Loss of granule cells in the hippocampus layers was measured in the brain in the experimental group. Beta-amyloid induced a significant increase in pyramidal cells in the hippocampus subfields from week1to week12. In the frontal cortex, beta-amyloid injection provoked more apoptosis, cellular degeneration and mortifies than rats after sterile PBS injection. Pathologies as described gradually increased as time went by. No significant modification was observed after sterile PBS injection.3、Congo Red staining:As a major pathology, beta amyloid deposit was analyzed in the rat brain structures. Our findings revealed that beta amyloid levels in the hippocampus were increased2weeks after beta amyloid treated rats, when compared to control rats. It is likely that beta amyloid deposit in the small arteries.as time went by, beta amyloid exhibited varying degrees of CAA. Beta amyloid injection induced complete infiltration in the small vessels in12week sub-group. By contrast, the sterile PBS injection induced no modification of CAA in each time-course sub-group.4、Immunofluorescence:Immunofluorescence revealed a prominent beta amyloid accumulation of dot-like aggregates mainly in hippocampal arterioles. Immunostainings of frozen brain sections from beta-amyloid injected rats with Ap and a-actin antibodies showed only little evidence of extravascular parenchymal amyloid deposition in1week and2week sub-group. However, extracellular vascular amyloid deposits were observed in4week sub-group. Vascular amyloid deposits were more pronounced observed in12week group, especially in arteriole in hippocampus. By contrast, no beta-amyloid aggregated in veins.Conclusions:1、Under light anesthesia, we succeeded in preparing the Alzheimer’s disease rat models by Aβ fiber injection in bilateral hippocampus with a stereotactic frame. After the evaluation tested with a Morris water maze, the cognitive function of different groups of rats showed a significant difference. The spatial reference memory significantly increased in latencies after Aβ fiber injection compared to after sterile PBS injection. HE staining showed Alzheimer-type pathology in AP fiber injection rats. Congo Red staining of Aβ fiber injection rats, Green birefringence was observed in the brain tissue. According to the results above, Aβ fiber injection in bilateral hippocampus with a stereotactic frame stimulated AD special pathological changes.2、Morris water maze test analysis revealed that Aβ fiber injection treated rats showed obvious damage in advanced cognitive functions. There were significant differences in the results of the escape latency and the times acrossing platform between the experiment and the control groups.3、Congo Red staining show that the Aβ fiber was deposited gradually into the small vessels in the brain, induced thickening, stenosis and occlusion in small vessels. As time went by, the influence of Aβ fiber deposition on small vessels became more prominent.4、The immunofluorescence staining show the dynamic course that the Aβ fiber migrated to the small vessels in experimental groups.5、Cerebral amyloid angiopathy is a major pathogenesis in Alzheimer’s disease...