A Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus using Brain Stereotactic Technology

doi:10.12300/j.issn.1674-5817.2025.030

Abstract

Abstract:

Objective To construct a rat model of Alzheimer's disease (AD) in the hippocampal brain region expressing human triple mutant amyloid precursor protein (APP), and to provide a model for mechanistic study and preclinical research. Methods Twenty-four SD rats were randomly divided into negative control, virus control and experimental groups. The experimental group was prepared by stereotactic injection of AAV carrying the human triple mutant APP genes and the NanoLuc luciferase gene. In vivo imaging was used to observe the expression of the virus, qPCR was used to examine the expression level of the mutant APP, the behavioral experiments were conducted to detect the cognitive and memory ability, the pathological changes in the hippocampus were detected by HE staining and Nissl staining, and the deposition of Aβ protein was detected by immunohistochemistry. Results The in vivo imaging results showed that the AAV was successfully expressed in the rat brain; The qPCR results showed that the expression level of APP genes was significantly increased in the rat of experimental group (p<0.01); The recognition of new objects test observed that the cognitive and memory abilities of the experimental group of rats were significantly decreased compared with the blank control group or the empty virus group (p<0.01); Six months after recombinant AAV virus infection, the HE staining and Nissl staining of brain tissue showed that the number of neuronal cells and Nissl bodies in hippocampal CA1 area of the experimental group was reduced and disorganized, the deposition of Aβ protein in CA1 region and conical cell layer of hippocampus of experimental rats was detected by immunohistochemistry. Conclusion The rat model successfully constructed by brain stereotaxic injections of AAV expressing human triple mutant APP genes can reflect typical AD characteristics, providing a rat model for the AD pathological and pharmacological research based on Aβ protein deposition.

Key words: Alzheimer's disease, Human triple mutant APP, Brain stereotaxis, Rat model

CLC Number:

Q95-33

XIAO Linlin,YANG Yixuan,LI Shanshan,et al. A Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus using Brain Stereotactic Technology[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.030.

Figures/Tables 7

Figure 1 Diagram of adeno-associated viral vectorsNote：A， Structure of the empty viral plasmid； B， Structure of the plasmid carrying the human triple mutant APP gene； APLsla-L-A in the vector diagram represents the human triple mutant APP genes， 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：Luciferase activity was observed 10 min after intraperitoneal injection of luciferase substrate Furimazine in each group of rats. A，In vivo imaging a rat in the blank control group； B， In vivo imaging a rat two weeks after injection of the empty virus； C，In vivo imaging a rat two weeks after injection of of AAV virus expressing the human triple mutant APP genes in the experimental group； D， In vivo imaging a rat survived for 6 months in the blank control group； E， In vivo imaging a rat six months after injection of the empty virus； F， In vivoimaging a rat six months after injection of the mutant APP genes in the experimental group.

Figure 3 Results of APP expression detection in rat hippocampusNote：A， qPCR results，compared with blank control group， **P<0.01； compared with empty virus group，##P<0.01； B， sequencing comparison results of qPCR products， yellow highlights are mutation sites.

Figure 4 Experimental results of new object recognitionNote：A， Exploratory behavior graphs of rats exploring two identical objects； B， Exploratory behavior graphs of rats exploring new and old objects； C， Results of new object recognition experiments using distance as a statistical index； D， Results of new object recognition experiments using time as a statistical index； NC， VC， and Exp are blank control group， empty virus group， and experimental group， respectively； compared with the blank control group，**P<0.01； compared with the airborne virus group， #P<0.05， n=8 .

Figure 5 HE staining results of rat hippocampal regionNote：NC， VC， Exp are the HE staining results of the blank control group， the empty virus group， and the experimental group， respectively. Scale bars are shown. The areas indicated by arrows are crumpled necrotic cells.

Figure 6 Staining results of Nystrom's body in the hippocampus of the ratNote：NC， VC， Exp are the Nissl staining results of the blank control group， the empty virus group， and the experimental group， respectively. Scale bars are shown. Areas indicated by arrows are cells with sparse nidus and light staining.

Figure 7 Immunohistochemical results of rat hippocampal regionNote：NC， VC， Exp are the immunization results of blank control group， empty virus group， and experimental group， respectively. Scale bars are shown. The area indicated by the arrow is the area of Aβ protein deposition.

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