Establishment of a Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus via Brain Stereotactic Technology

doi:10.12300/j.issn.1674-5817.2025.030

Abstract

Abstract:

Objective To establish a rat model of Alzheimer's disease (AD) expressing human triple mutant amyloid precursor protein (APP) in the hippocampus, and to provide a model for the study of disease mechanisms and drug development. Methods Twenty-four 12-week-old SPF-grade female SD rats were randomly divided into a blank control group, a virus control group and an experimental group, with eight rats in each group; among them, the experimental group received a stereotaxic injection of adeno-associated virus (AAV) carrying the human triple mutant APP and NanoLuc luciferase genes into the hippocampus. In vivo imaging was used to observe viral expression in the brains of rats in each group, the novel object recognition test was used to assess the recognition memory of the rats in each group, real-time fluorescent quantitative PCR was used to detect the expression level of the APP gene, HE staining was used to examine the brain histopathology, Nissl staining was used to assess the hippocampal lesions, and immunohistochemistry was used to detect the deposition of amyloid β-protein (Aβ). Results In vivo imaging showed that reporter fluorescence was detected in the brains of rats in both experimental and virus control groups. Fluorescence quantitative PCR showed that the expression level of the APP gene was significantly increased in the brains of rats in the experimental group (P<0.01). Novel object recognition test revealed that the recognition memory of rats in the experimental group was significantly reduced compared with that of the blank control group (P<0.01). Six months after recombinant AAV virus infection, HE staining and Nissl staining of brain tissues showed that the number of neurons and Nissl bodies in the CA1 region of the hippocampus in the experimental group was reduced and disorganized; immuno-histochemistry testing of the CA1 region of the hippocampus and the pyramidal cell layer of the experimental group revealed prominent brown deposits, indicating Aβ protein deposition. Conclusion The rat model successfully established by stereotaxic injection and AAV-mediated delivery of human triple mutant APP gene exhibits typical AD features, providing a valuable animal model for studying AD pathology and developing drug therapies targeting Aβ protein deposition.

Key words: Alzheimer's disease, Amyloid precursor protein, human triple mutant gene, Brain stereotaxis, Rat model

CLC Number:

R-332

XIAO Linlin,YANG Yixuan,LI Shanshan,et al. Establishment of a Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus via Brain Stereotactic Technology[J]. Laboratory Animal and Comparative Medicine, 2025, 45(3): 269-278. DOI: 10.12300/j.issn.1674-5817.2025.030.

Figures/Tables 5

Figure 1 Schematic diagram of recombinant adeno-associated viral vector structureNote：A， structure of the empty viral plasmid； B， structure of the plasmid carrying the human triple mutant APP gene， APPsla-L-A represents the human triple mutant APP gene， Nluc represents the NanoLuc luciferase gene， and SYN promoter is the neuron-specific promoter.

Figure 2 In vivo imaging of rats two weeks and six months after virus injectionNote：BC， VC， Exp are blank control group， virus control group， and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively. The luciferase reporter activity was observed 10 min after intraperitoneal injection of the luciferase substrate Furimazine into rats in each group. 2 weeks later， fluorescent luminescence could be detected in the head area of the experimental group and the virus control group， and 6 months later， the fluorescence intensity of both the experimental group and the virus control group was weakened， but it could be detected； the fluorescence was not detected in the blank control group.

Figure 3 Novel object recognition test to assess recognition memory of ratsNote：A shows rats exploring two identical objects； B is a new object replaced； C is the result of the new object recognition experiment using distance as a statistical index； D is the result of the new object recognition experiment using time as a statistical index. RI：Recognition index.BC， VC， Exp are blank control group， virus control group and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively， 8 rats per group （n=8）； **P<0.01， compared with blank control group； #P<0.05， compared with virus control group.

Figure 4 APP gene expression and sequencing results in rat hippocampusNote： A is the result of real-time fluorescence quantitative PCR. BC， VC， Exp are blank control group， virus control group and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively. 4 rats in each group （n=4）； Compared with blank control group， **P<0.01； Compared with virus control group， ##P<0.01. B is the result of real-time fluorescence quantitative PCR product sequencing comparison， and the yellow highlighted part is the indicated mutation site.

Figure 5 HE staining （A）， Nissl staining （B）， and immunohistochemical staining （C） results of rat hippocampal regionNote： BC， VC， Exp are blank control group， virus control group and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively. The areas indicated by black arrows are crumpled necrotic cells. The areas indicated by red arrows are cells with sparse Nissl bodies and light staining. The area indicated by blue arrow is the area of amyloid β-protein deposition.

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