Establish Of Aβ-AD Animal Model And NGF Treatment

ObjectiveAlzheimer disease ( AD) is a high risk central nervous system degenerative disease. Deposit of β amyloid protein ( Aβ) is central point in the pathogenesis of AD. Decreasing of Choline Acetyl Transferase (ChAT) activity is considered to be the key biochemical feature. AD is closely related with apoptosis, and nerve growth factor (NGF) is the most important nutritional factor of the central choline system, NGF has obvious anti -apoptic effects.In this study, we build rat model of AD, observe the effects of intra — ventricular Aβ on the behavior, ChAT, to establish an ideal AD animal model. Observe apoptosis and expression of NGF, study the mechanism of AD. Give NGF through intra -ventricular injection, observe its behavior, ChAT change, apoptosis situation. Observe treating effects of NGF to AD, discuss the mechanism of treatment.Methods1. establish of AD model; behavior observe and determination of ChAT activity.24 rats is divided into 4 groups: control group, 7 days after Aβ injection group, 14 days after Aβ injection group, 28 days after Aβ infection group, each group 6 rats. The rats is anesthesed with abdominal babitorates, and given Aβ1 -42 into right lateral ventricle, 7μg each time, continues for 3 days. The rats were killed 7, 14 and 28 days after Aβ injection. Before killed, the rats were preformed with Morris water maze test to observe behavior change. Rats werekilled by de - brain method, and the brains were put into liquid nitrogen.2. Morris water maze test.The maze is made up of circular wash tube and one platform under the water. The test is conducted 5 blocks in 3 days. Every test, the rats were put into water at east, west, south and north point at the wash tube, record the time that the rats find the platform under the water, that is the escape latency time. The test is performed before killed.3. ChAT activity determinationTake every rat' s hippocampus, basal ganglion, frontal lobe, parietal lobe to prepare 5% harmonized brain tissue. In 70 μl reaction volume contains: sample or blank control liquid 20μl, 1.14g/L acetyl co - A 10μl, 70 mmol/L cho-line 8μl, 1mmol/L neostigmine bromide 7μl, 3 mmol/L NaCl 7μl, 3. 7 x 104Bq/ml 14C - CoA 10μl, detect the cpm in liquid β - counter. Let the cpm of control group 100% , then calculate the relative ChAT activity.4. neuron apoptosis and NGF expression in AD brain.24 rats is divided into 4 groups; control group, 7 days after Aβ injection group, 14 days after Aβ injection group, 28 days after Aβ infection group, each group 6 rats. The rats is anesthesed with abdominal babitorates, and given Aβ1 -42 into right lateral ventricle, 7μg each time, continues for 3 days. The rats were killed 7, 14 and 28 days after Aβ injection, when killed, the rats were perfused with 100 ml poly formaldehyde, then the rat brain were keeped into formaldehyde at least for 72 hours, then perform TUNEL apoptosis and NGF im-munochemical stain.5. TUNEL apoptosis stain and analysisTake the brain sample in formalin, bed with wax, cut saggital slide that contain all the 4 brain areas, named hippocampus, basal ganglion, frontal lobe, parietal lobe. The slide was 7μm thick. Perform TUNEL stain according to directions in the stain kit. The nuclei of positive cell was stained brown - yellowish, calculate the percent of positive neuron, namely neuron apoptosis index (NAI).6. NGF immunochemical stain and image analysisThe wax tissue slides were undertaken NGF immunochemical stain accord-ing to the directions of the kit. Stain 2 slides every rat brain, under high power (400 x ) microscope, analysis the image by UIC's Metamorph Image System V6.4, get the average gray value of positive stained cells.7. NGF treatment of AD.Treatment groups were divided into early treat group and late treat group. The early treat group were given NGF 60ng intraventricularly once a week after 3 days of Aβ injection for 4 weeks. The late treat group begins after 3 weeks of Aβ injection, and the regimen was same with the early group. Observe the behavioral change, undertook water maze task, determine the ChAT activity of different brain area, undertook TUNEL apoptosis stain.Results1. Morris water maze results; at 28 days after Aβ injection, the escape latency is obviously prolonged compared with control group, suggest that the memory and learning ability is damaged.2. Effects of Aβ on ChAT activity. ChAT activity was decreased in hippocampus and lobe 2 weeks after Aβ injection, and was decreased generally at the 4th week, especially in hippocampus.3. Effects of Aβ on neuron apoptosis. After 2 weeks, the apoptotic neurons in basal Meynert nucleus and hippocampus were increased compared with the control group, especially at the Meynert nucleus, reaching 19.7%.4. Effects of Aβ on NGF expression. There were no changes at 7 days after Aβ injection. At 14 and 28 days after Aβ injection, NGF positive cells decreased in basal forebrain, and the average gray value is much higher compared with control group.5. Effects of NGF treatment on rats'behavior. The water maze test escape latency is obviously shortened in NGF treatment group compared with AD group, and had no difference with control group.6. Effects of NGF treatment on ChAT activity. ChAT activity is increased compared with untreated group. In early treat group, the activity retained to a-bout 80% of the control group, higher than that of the late treat group.