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Intraventricular injection of amyloid to establish a rat model of Alzheimer's disease
Intraventricular injection of amyloid to establish a rat model of Alzheimer's disease
Abstract Objective To observe the β-amyloid (A [beta]) intraventricular injection of rat model of Alzheimer's disease (AD) and on the behavior of rats, choline acetyltransferase (of ChAT) activity, apoptosis, nerve growth Factors such as factor (NGF) levels. Methods Aβ1242 injected into the lateral ventricle of rats at 1 week, 2 weeks, 4 weeks to observe escape latency in Morris water maze, measuring ChAT activity in the brain, in situ end labeling NGF apoptotic staining and immunohistochemical staining. Results The escape latency of Morris water maze was prolonged in the second week after Aβ1242 injection, which was more obvious at 4 weeks. After 2 weeks, the activity of ChAT in hippocampus and cortex decreased. After 4 weeks, the activity of ChAT in the brain decreased widely, with the hippocampus being the most significant. After 2 weeks, the apoptotic cells in the Meynert nuclear region of the basal forebrain increased more than other brain regions, and NGF positive cells were significantly reduced. Conclusion Intraventricular injection of Aβ can mimic changes in AD behavior, reduce ChAT activity in the brain, decrease NGF content in basal forebrain, and induce apoptosis of cholinergic neurons, which can be used as an AD study model.
[Key words] Alzheimer's disease β amyloid choline acetyltransferase nerve growth factor
[Chinese Library Classification Number] R749. 1 [Document Identification Code ] A
The central link in the pathogenesis of Alzheimer Disease (AD) is the deposition of β-amyloid protein (Aβ) in the brain [1]. The primary degeneration of various neurons, especially cholinergic neurons. , brain choline acetyltransferase (ChAT) activity decreased, is a landmark biochemical change of AD [2]. Neurodegenerative diseases such as AD are closely related to the occurrence of apoptosis [3], and nerve growth factor (NGF) is an important neurotrophic factor in the central cholinergic system, which has obvious anti-apoptotic effects [4] ].
The biggest obstacle to basic research and drug development in AD is the lack of an ideal animal model similar to changes in pathology, biochemistry, and behavior of AD. In this study, an AD rat model was established by intraventricular injection of Aβ, and the behavioral changes in rats, changes in ChAT activity in various parts of the brain, neuronal apoptosis, and changes in NGF levels were observed, providing an animal model for further AD treatment studies.
1 Materials and methods
111 Animal grouping and A β intraventricular injection of 48 male Wistar rats, weighing 260 ~ 300 g, were randomly divided into 4 groups: control group, 7 days after injection Αβ group, group 14 days, 28 days groups 12 only. After anesthesia, the rats were slowly injected into the right ventricle with a micro-syringe (018 mm after the bregma, 111 mm beside the midline, and 316 mm deep). The Aβ model group was injected with Aβ1242 (purchased from AnaSpec, USA), 7 μg each time for 3 consecutive days, and the control group was injected with normal saline for 7 μL for 3 consecutive days.
1 1 2 　 Morris water maze test Refer to the literature method [5] to record the escape latency required for the rat to find the platform. The experimental groups were tested on the 5th to 7th, 12th to 14th, and 26th to 28th day after the last drug injection, and the control group was tested at the same time.
113 group were drawn from injected Aβ last 1 7 post drug injection days, 14 days, 28 days slaughter drawn, based on the control group 28 days after the last drug injection times. Six fast decapitated brains were taken from each group and immediately frozen in liquid nitrogen for ChAT activity to be measured. Another 6% paraformaldehyde was perfused and fixed in 4% paraformaldehyde solution for more than 72 hours. TUNEL apoptosis staining and NGF immunohistochemical staining were performed.
1 1 4 　 ChAT activity detection and quantitative analysis of cryopreserved brain tissue in liquid nitrogen were prepared from hippocampus, basal ganglia, frontal lobe and parietal cortex to prepare 5% (V / W) brain tissue homogenate, according to Fonnum [6] radioimmunoassay, at 70 μL The total reaction volume contained 20 μL of sample solution or blank control solution, 1114 g/L acetyl-CoA (Sigma) 10 μL, 70 mmol/L choline (Sigma) 8 μL, 1 mmol/L bromide neostigmine (Sigma) 7 μL, 3 mmol/L sodium chloride 7 μL, 317 × 104 Bq/ mL 14C2 acetyl-CoA 10 μL, and the number of decays per minute (cpm) was measured on a liquid scintillation meter. The relative activity of ChAT in each part of each group was calculated by the number of decays in each part of the control group being 100%.
1 1 5 　 TUNEL Apoptosis Staining and Semi-quantitative Analysis Sagittal section sections of the basal ganglia, hippocampus, frontal lobe, and parietal lobe were selected and subjected to TUNEL apoptosis staining according to the TUNEL Apoptosis Staining Kit (Wuhan Boster). Two slices of each mouse were stained, and a total of 100 neurons were counted in each field under a high power field (400 ×). The positive percentage of apoptotic neurons, ie, the neuronal apoptosis index (NAI), was calculated.
1 1 6 　 NGF immunohistochemistry and image analysis taking the above-described paraffin sections by NGF immunohistochemistry kit (Wuhan Boster Corporation) staining operation instructions. Two sections were stained in each mouse brain, and five fields of view were taken under high power field (400 ×) for each stained piece. The average gray value of positive stained cells was obtained by UIC Image Analysis software from UIC of USA. ).
117 statistical analysis of measurement data as mean ± standard deviation (x ± s) represented among the groups ChAT activity, water maze test latency, of NGF positive neurons average gray value of t-test was used to compare neuronal apoptosis index Comparisons between groups were performed using the χ 2 test.
2 results
2 1 1 　 Morris water maze test results showed that the escape latency of each group was: control group (1211 ± 514) s; 7 days after Aβ injection (1411 ± 519) s, 14 days after Aβ injection (2116 ± 617) s, Aβ The group was (4510 ± 1013) s at 28 days after injection. The 7-day incubation period after Aβ injection was unchanged from the control group. The incubation period was prolonged 14 days after injection ( P < 0105), and the incubation period was significantly prolonged 28 days after injection ( P < 0101), suggesting that the learning and memory function was impaired.
2 1 2 　 The effect of Aβ intraventricular injection on ChAT activity The effect of Aβ intraventricular injection on ChAT activity in the brain is shown in Table 1.
Table 1 　 Effects on A β ChAT Activity (cpm)
Group of lobular basal ganglia
Control group 1208. 5 ±49. 8 3662. 3 ±118. 5 2584. 0 ±98. 8 1184. 2 ±53. 2
7 days after Aβ injection 1183. 8 ± 26.7 3559. 7 ± 120. 3 2513. 0 ±99. 8 1139. 3 ±43. 6
14 days after Aβ injection 1033. 4 ±47. 92) 3507. 5 ±126. 6 2234. 2 ±84. 52) 1088. 3 ±63. 01)
28 days after AB injection 950. 1 ±67. 52)3228. 5 ±153.
02) 1833. 0 ±57. 62) 941. 8 ±42. 42)
1) Compared with the control group, P < 0. 05 2) compared with the control group, P < 0. 01
After intraventricular injection of Aβ for 7 days, there was no significant decrease in ChAT activity in brain tissue at various sites.
After 14 days, the activity of ChAT in hippocampus and frontal lobe decreased significantly. After 28 days, the activity of ChAT in each brain area decreased significantly ( P < 0101), and the decrease in hippocampus was the most obvious, which decreased to 71% in the control group.
2 1 3 　 Effects on A β brain neuronal apoptosis TUNEL staining apoptotic nuclei were stained brown positive apoptotic cells, the control group and 7 days after injection of individual areas of the brain, see scattered apoptotic cells, 14 days after injection of Aβ visible A small number of apoptotic cells increased significantly after 28 days, and the NAI increased. The Meynert nucleus of the basal forebrain reached 1917%, and the hippocampus, frontal and parietal lobes were 617%, 512%, and 413%, respectively. The neuronal apoptosis in the Meynert nuclear region was obvious. More than hippocampus and cortex ( P < 0101). Meynet nuclear apoptosis staining is shown in Figure 1.
2 1 4 　 Effect of NGF on the expression level of A β in the brain of NGF immunohistochemical staining in the hippocampus, frontal, parietal, basal forebrain were found positive cells stained brown cytoplasm, no significant change in A [beta] 7 days after the injection, 14 to 28 days It can be seen that the NGF positive cells in the basal forebrain area are significantly reduced, the staining is shallow, and the expression levels of NGF in other brain regions are not significantly changed. NGF staining of the basal forebrain is shown in Figure 2. The average gray value of the positive stained cells was obtained using the UIC Image Metamorph Image System V416 image analysis software.
Table 2 　 NGF immunohistochemical staining cells mean gray value
Group of lobular basal ganglia
Control group 135. 48 ± 17.27 129. 67 ± 18.49 126. 87 ± 9. 56 138. 46 ± 13.54
After 7 days of Aβ injection, 129. 32 ± 14.47 134. 57 ± 10. 38 125. 48 ± 8. 21 139. 76 ± 9. 72
14 days after Aβ injection 131. 21 ± 17.82 149. 65 ± 9. 431) 129. 24 ± 12. 59 128. 45 ± 12. 43
28 days after Aβ injection 126. 34 ± 15.97
175. 54 ± 12.
942) 116. 75 ±11. 16 128. 27 ±10. 03
1) Compared with the control group, P < 0105 2) compared with the control group, P < 0101
3 discussion
Data accumulated over the past decade or so indicate that the cholinergic neurons of the forebrain basal ganglia, the hippocampus, and the pathways between them are important structural foundations for learning and memory function. Destroying these structures or aging can cause degradation of these regions. Lead to serious mental impairment [7]. ChAT is a biosynthetic enzyme of ACh, which is present in cholinergic neurons and is a marker for measuring the function of cholinergic neurons. It is also a successful biochemical marker for the success of AD animal models [2,8]. To date, most AD models (such as aging animal replacement models, cholinergic system damage models, transgenic mouse models) have mimicked the early development of memory impairment or pathological features in AD, limiting their application. In this experiment, acute intracerebroventricular injection of Aβ1242 was used in rats, and the performance of rat water maze test decreased gradually within 4 weeks, and AD appeared, which was consistent with the literature [9]. The activity of ChAT in the hippocampus, basal ganglia, and frontal and parietal neocortex was significantly decreased to 70% of the control group, indicating that the effect of Aβ intraventricular injection on ChAT activity in the brain is extensive and obvious. Aβ deposition is the central link and pathological features of AD. This model comprehensively simulates the pathological, behavioral and biochemical changes of AD. Therefore, intraventricular Aβ injection can be used as an ideal method for making AD models. The decline in ChAT activity actually reflects the decline or degeneration of cholinergic neurons. In recent years, there is increasing evidence that the main mode of cholinergic neuronal degeneration is apoptosis [3]. In this experiment, it was found that the activity of ChAT decreased, and the number of apoptotic neurons in the basal ganglia and hippocampus increased, and the basal forebrain region where cholinergic neurons aggregated was more obvious. Therefore, the decrease in ChAT activity may be due to Aβ. Caused by apoptosis of cholinergic neurons. The basal forebrain cholinergic neurons emit fibers that are projected into the hippocampus, marginal lobes, and cerebral cortex. NGF produced in the target cells of these sites is taken up by the axonal terminals of cholinergic neurons and transported to the cell body via axoplasmic reverse. The ganglion is the main trophic factor of the basal forebrain and septal cholinergic neurons. The level of NGF in the basal forebrain neurons decreased at 2 weeks after intraventricular injection of Aβ, which was most obvious at 4 weeks. It is speculated that in this model, NGF has weakened the nutrition and support of basal forebrain central cholinergic neurons, resulting in nerves. Meta-apoptosis causes a series of behavioral and biochemical changes in AD. If gene therapy can be used to supplement the basal forebrain NGF in time, it will be possible to treat AD symptoms and delay the progression of AD. At present, gene therapy AD is a hot topic at home and abroad, and it is our next research direction [10]. In summary, Aβ intraventricular injection caused AD-like behavioral changes in rats, decreased ChAT activity in the brain, decreased NGF content in the basal forebrain, and apoptosis of cholinergic neurons, which can be used as an animal model for AD studies.