Research Progress Of Alzheimer’s Disease Syndromes Combined With Animal Models
Feb 11, 2025
Abstract Alzheimer's disease (AD) is a common neurodegenerative disease. Traditional Chinese medicine is effective in treating AD. The combination of disease and syndrome with animal model is the basis and premise of related research. In this paper, studies on non-transgenic AD animal models and AD disease and syndrome combined animal models are summarized. It was found that, there are eight common non-transgenic AD animal models, including aging type (natural aging type, rapid aging type, induced aging type) and injection-induced damage type (Aβ injection animal model, Tau damage type, cholinergic system damage model, neuroinflammation model, and aluminum poisoning induction model). AD syndromes combined with animal models include kidney deficiency/kidney deficiency/kidney deficiency pulp empty, phlegm turbidity blocking orifice, blood stasis blocking collaterals, phlegm stasis interlocking, liver depression and phlegm fluid stagnation heat. In this paper, the advantages and disadvantages of each model replication method are reviewed, in order to provide reference and support for the future study of AD disease and syndrome combined with animal models.
Keywords Alzheimer's disease; animal model; combination of disease and syndrome; research progress
Alzheimer's disease (AD) is a neurodegenerative disease closely related to aging, characterized by progressive cognitive and memory impairment[1]. The incidence of AD increases with age. AD is the most common cause of dementia, accounting for 60% to 70% of dementia cases[2]. There are nearly 15.07 million patients with dementia in my country, of which approximately 9.83 million are AD patients[3]. The disease has an insidious onset and is characterized by continuous progression, which seriously affects the patient's quality of life and imposes a huge economic burden on the patient's family and society[4].
Animal experiments are an important driving force for the development of medicine and a bridge between basic and clinical medicine. Establishing a correct animal model is an indispensable experimental basis and prerequisite for conducting animal experiments. Currently, the commonly used AD animal models are roughly divided into four types: "aging type, injection-induced type, transgenic type, and combined type" [5].
NEW HERBAL CISTANCHE SUPPLEMENTS FOR ANTI-Alzheimer's disease (AD
The disease-syndrome combined animal model is a derivative product developed under the guidance of the combination of the ideas of traditional Chinese medicine differentiation and modern medical theories. The characteristics of the model are that it replicates the same or similar factors as human diseases and syndromes in animals, while simulating information related to "disease" and "syndrome". It is more convincing than animal models of simple "disease" or simple "syndrome"[6]. In order to further explore the mechanism of action of traditional Chinese medicine in the treatment of AD and evaluate the efficacy of traditional Chinese medicine, establishing a standardized disease-syndrome combined animal model is an important means to promote the modernization of traditional Chinese medicine and is of great significance [7]. Therefore, this article systematically reviews the preparation methods of common non-transgenic AD animal models and their respective advantages and disadvantages. On this basis, it summarizes the existing AD disease-syndrome combined animal models, and summarizes their construction methods, evaluation models, etc., in order to provide a reference for the research on disease-syndrome combined animal models and provide an animal model basis for future TCM research on AD (Figure 1).
Figure 1 General of AD non-transgenic animal model and disease-syndrome combined animal model
Table1 Summary of characteristics of non-transgenic AD animal models
1.1 Animal models of aging AD
AD is a neurodegenerative disease closely related to aging. It is of great significance to construct an animal model that is similar to aging and consistent with the clinical manifestations of elderly patients to further study aging. Currently, the common aging AD animal models include three types: "natural aging type, rapid aging type and induced aging type".
1.1.1 Natural aging type
AD is common in people over 65 years old[21]. Natural aging models generally use mice and rats. The age of aged mice is 12 to 20 months, the age of early-aging rats is 21 to 26 months[22], and the age of late-aging rats is 30 to 32 months[23]. The natural aging model shows a decline in learning and memory abilities, accompanied by atrophy of brain neurons, which is consistent with the clinical manifestations of elderly patients[5]. Compared with non-human primates, rodent experiments have a shorter experimental cycle, are relatively economical, and save time and effort[8]. The disadvantage is that naturally aging animal models cannot simulate all the manifestations of AD and are prone to death during the experiment. For example, Lu Xia et al. [9] found high molecular weight Tau protein in the gray matter, white matter and spinal cord of rats over 24 months old. They also found that the content of medium molecular weight Tau in the gray matter and white matter of these rats was significantly higher than that in the adult group, which may be a compensatory response to the aging process. Gao Lin et al. [10] found that the memory ability of elderly rats (over 12 months old) was impaired, and a large amount of Aβ1-42 was deposited in the hippocampus, accompanied by shrinkage or loss of neurons and infiltration of glial cells.
1.1.2 Rapid aging type
The senescence accelerated mouse (SAM) is derived from inbreeding of AKR/J mice and is divided into nine substrains. Among them, SAMP8 is a widely recognized aging model. Its brain tissue contains a large amount of deposited amyloid β-protein (Aβ), indicating that the SAMP8 model has characteristics similar to the main pathological changes of AD. Therefore, it is a relatively mature model for studying AD[24] and is often used in the study of learning and memory deficits and aging-related mechanisms[25-26]. SAMP8 mice showed signs of aging such as hair loss at 4 months of age. At 5 months of age, a decrease in the number of neurons, abnormal neurotransmitter metabolism, and nerve cell atrophy were observed in the mice. At the same time, changes such as decreased learning and memory ability and cognitive impairment occurred. At 8 months of age, changes in dendrites, synapses, and neurons appeared, which were consistent with the clinical characteristics and neuropathological changes of AD patients. However, its short life cycle, high price, and complex mechanisms involved make it unsuitable for long-term research and single-site studies.
Research on the mechanism[27-28]. Zan Shujie et al. [11] found that SAMP8 mice showed signs of impaired spatial learning ability and short-term memory at 4 months of age, and the expression of Aβ1-42 in the hippocampus was significantly increased. Duan Liqi et al. [29] established a rapid aging mouse SAMP8 model and found that administration of Xixin Decoction improved their spatial learning and memory abilities and reduced the generation and deposition of Aβ1-42 in the hippocampus and colon tissues.
1.1.3 Induced aging
The induced senescence type is an AD animal model in which metabolic disorders are induced by subcutaneous or intraperitoneal injection of D-galactose (D-gal) to induce aging. This modeling method has the advantages of low experimental cost, stable experimental results, simple modeling method, short modeling time, and high repeatability, and has been widely used by scholars at home and abroad[30]. For example, YU et al. [31] used a D-gal-induced aging rat model and found that the escape latency of the model rats was prolonged and the time they stayed in the target quadrant was reduced after Morris water maze testing. In addition to the above advantages, the use of D-gal to construct an aging AD model still has the disadvantages of a long modeling cycle, the animals are very likely to develop other diseases or die during modeling, and there is no AD-related characteristic pathology such as Aβ deposition in the brain tissue[32]. Therefore, it is often used to establish a composite model and to construct an AD animal model together with other induction methods. For example, Qu Yan et al. [12] established an AD model by combining subcutaneous injection of D-gal with injection of Aβ1-42 oligomers into the hippocampus. The results showed that the model mice showed anxiety-like behaviors and impairment of spatial learning memory and episodic memory. In addition, there are also composite modeling methods such as D-gal combined with Aβ oligomers, D-gal combined with aluminum chloride (AlCl3), and D-gal combined with sodium nitrite [33], which can overcome the limitations of a single model that cannot simulate all the pathological manifestations of AD.
1.2 Injection-induced injury AD animal model
The etiology and pathogenesis of AD are relatively complex. Based on different pathogenesis, several hypotheses have been proposed, including the Aβ hypothesis, the abnormal phosphorylation of Tau protein hypothesis, the cholinergic hypothesis, the inflammatory damage hypothesis, and the aluminum poisoning hypothesis [34]. On the basis of fully understanding the etiology and pathogenesis of AD, establishing an AD animal model that is closely related to the disease hypothesis is of great significance to the treatment evaluation and cutting-edge research of AD.
1.2.1 Aβ injection animal model
The pathogenesis of AD is complex and involves multiple pathogenic factors, among which the Aβ hypothesis occupies a dominant position among current hypotheses[35]. The deposition of Aβ can trigger a cascade reaction, promote the structural degeneration of neuronal networks, induce synaptic damage and neuronal loss, and cause irreversible neuronal atrophy and neurodegeneration in the brain, which is a key factor causing AD[36]. The Aβ-induced AD animal model mainly involves injecting Aβ fragments into specific brain regions of animals to induce Aβ deposition. Commonly injected brain regions include the hippocampus and lateral ventricles. For example, Zhang Jingfan et al. [13] replicated the AD rat model by injecting 5 μL of Aβ1-42 solution (10 μg) into the bilateral hippocampus of the rats. After replication, the Morris water maze test results showed that the escape latency of the model rats was significantly prolonged compared with the blank group, and the number of times the platform was crossed and the time spent in the platform quadrant were significantly reduced;
At the same time, hematoxylin-eosin (HE) staining revealed that the number of neurons in the replicated model rats was reduced, and some of the cell nuclei showed shrinkage; a large number of Aβ-positive cells were also seen in the CA1 region of the hippocampus. The above research results suggest that injection of Aβ1-42 can cause Aβ deposition, damage to hippocampal neurons, and impaired learning and memory abilities in rats. The Aβ-induced model is an acute injury that can quickly produce an AD pathological model. Repeated injections are required to achieve stability. In addition, improper injection can easily lead to Aβ accumulation at the injection site, resulting in excessive local concentrations and local damage.
1.2.2 Tau protein damage
Tau protein, a hyperphosphorylated tubulin, plays an important role in AD neuronal apoptosis. The abnormal phosphorylation hypothesis of Tau protein holds that overphosphorylated Tau protein loses its ability to bind to microtubules and loses its original function. Overphosphorylated Tau protein aggregates in cells in the form of double helical filaments, straight filaments, and tangled skeletons to form intracellular neurofibrillary tangles (NFTs), which eventually lead to neuronal degeneration and death, thus forming AD[37]. Intracerebral injection of Tau protein can produce NFTs-like structures and accelerate the formation of related pathological characteristics in AD model mice[38-39]. The advantages and disadvantages of injecting Tau protein to replicate the AD model are the same as those of injecting Aβ. In addition, intracerebral injection of phosphatidic acid inhibitors such as okadaic acid is currently widely used, which can also lead to hyperphosphorylation of Tau protein. Hyperphosphorylated Tau protein is present in NFTs within neurons, which is an important pathological feature of AD[40]. This modeling method has the characteristics of simple operation, low cost, and short modeling cycle[41], but the model lacks Aβ amyloid lesions.
For example, ÇAKıR et al. [14] injected okadaic acid into the bilateral ventricles of rats to replicate the AD model. The results showed that the levels of caspase-3, phosphorylated-tau-(ser396), Aβ, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the cerebral cortex and hippocampal CA3 regions of the model rats were increased. The Morris water maze experiment showed that the injection of okadaic acid would impair the spatial memory ability of rats.
1.2.3 Cholinergic system damage model
The cholinergic system is responsible for regulating the body's learning and memory abilities, and the occurrence and development of AD is closely related to abnormalities in this system. Commonly used chemical agents that damage the cholinergic system include colchicine and scopolamine. This model lacks the typical pathological characteristics of AD, and some chemical agents that damage the cholinergic system have reversible effects, which is inconsistent with the irreversible neurological damage of AD[42]. Colchicine is an alkaloid derived from the seeds of the plant Colchicum. Intracerebroventricular injection of colchicine can cause progressive memory impairment and neurodegeneration in rats, which is characteristic of a sporadic model of AD [43]. LAKSHMISHA et al. [15] used colchicine to induce AD rat model to evaluate the effects of resveratrol and resveratrol combined with donepezil on the numerical expression of microglia and astrocytes in the frontal lobe and hippocampus of model rats. The results showed that the number of microglia in AD model rats was significantly increased. SIL et al.[16] found that intracerebral injection of colchicine can induce cerebral
Horse neuronal degeneration and neuroinflammatory response, and the increase in neuronal degeneration is consistent with the increase in neuroinflammatory response in this area, suggesting that this model rat may serve as a sporadic model of AD.
The cholinergic hypothesis holds that cholinergic deficiency caused by the loss of cholinergic cells and the reduction of acetylcholine (Ach) is an important cause of early AD[44-45]. Scopolamine is an M cholinergic receptor antagonist that can cross the blood-brain barrier to reach the nerve center and bind to the M receptors in the central nervous system, thereby blocking the binding of the receptors to Ach, inhibiting the function of the central nervous system, causing cholinergic disorders, and ultimately leading to manifestations such as learning and memory dysfunction [46-47]. Scopolamine is one of the most widely used methods for establishing AD models. It can inhibit learning and memory in the short term, but its effect is non-selective and cannot accurately identify the receptor type in a specific brain region. It also lacks characteristic pathological changes of AD, such as Tau protein hyperphosphorylation and Aβ deposition[48]. YERRAGURAVAGARI et al. [17] used scopolamine to replicate an AD rat model with learning and memory impairment and found that the learning and memory abilities of the replicated model rats were significantly reduced, the cholinesterase levels were significantly increased, and the brain-derived neurotrophic factor (BDNF) was decreased. In addition to the two chemical agents that damage the cholinergic system mentioned above, a few researchers have used the immunotoxin 192-IgG-saporin to establish an AD model of cholinergic system damage[49].
1.2.4 Neuroinflammation Model
Currently, more and more studies have shown that neuroinflammation also plays a crucial role in the occurrence and development of other degenerative diseases such as AD[2,50]. The neuroinflammation model of AD is mainly based on the cell model. Microglia and astrocytes are important mediators of neuroinflammation. The activation and proliferation of these two cells occur before the formation of Aβ and NFTs[51]. The AD neuroinflammation model is mainly induced by lipopolysaccharide (LPS) and streptozocin (STZ). LPS is a classic inflammatory agent and a component of the outer cell wall of Gram-negative bacteria [52-53]. If LPS enters the human body or brain, it causes inflammation and acts as an endotoxin, thereby promoting amyloid pathology, Tau pathology, and microglial activation, leading to neurodegeneration in AD [54].
XIE et al. [18] established a neuroinflammation model by intraperitoneal injection of LPS. The results showed that the microglia in the brain tissue of the model rats were activated, and the levels of IL-1β, TNF-α, and IL-6 in the brain tissue were increased, indicating that the neuroinflammation model was successfully replicated. Spontaneous activity, novel object recognition, and Morris water maze experiments showed that the model rats had impaired learning and memory abilities.
STZ is a nitrosourea derivative. Low-dose injection of STZ can disrupt the homeostasis of insulin signaling in the brain, leading to energy metabolism disorders, Aβ deposition in the brain, Tau hyperphosphorylation[55], and neuroinflammation[56], which are pathological changes similar to sporadic AD. It can also cause impaired learning and memory abilities and cognitive impairment[57]. The AD model induced by intracerebroventricular injection of STZ has been shown to increase the levels of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 in the brain[58] and upregulate nuclear factor-κB (NF-κB)[59]. However, this modeling method also has shortcomings such as unstable modeling success rate and high mortality rate during modeling[60]. GÁLL et al. [19]
In 2015, AD-like symptoms were induced in Wistar rats by intracerebroventricular injection of STZ. The results of open field, novel object recognition, and radial arm maze tests showed that the model rats showed cognitive behavioral decline and a significant increase in the level of astrocytes in the hippocampus.
1.2.5 Aluminum poisoning induction model
The "aluminum poisoning hypothesis" holds that aluminum is neurotoxic and can damage neurons after entering the brain, while also reducing the function of the cholinergic nervous system and learning and memory abilities [61]. In addition, aluminum can polymerize with Aβ in the brain, thereby producing a large number of oxygen free radicals, promoting lipid peroxidation, leading to membrane damage of hippocampal neurons, and causing cognitive dysfunction[62]. Aluminum poisoning models are usually established by intracranial or subcutaneous injection of AlCl3 in rats. There are also a few studies that simulate aluminum poisoning models by oral administration of AlCl3. This model is simple to make, low-cost, has a high success rate and a low mortality rate. It is also of great value in identifying early diagnostic markers for AD and further formulating strategies to prevent and treat AD. However, the aluminum poisoning model can only show a single AD pathological feature and usually needs to be combined with other modeling methods to accelerate the formation of AD pathology. For example, Cheng Houzhi et al. [20] fed rats with AlCl3 for 3 consecutive months. The results of the Morris water maze navigation experiment showed that the model rats had poor movement trajectory and deviated from the platform, indicating that the chronic aluminum poisoning animal model has a toxic effect on the body and can cause symptoms such as decreased learning and memory function and cognitive dysfunction.
