Modeling method for inducing mouse cognitive impairment by METH and HIV-1 Tat proteins
By injecting HIV-1 Tat protein and METH in mice, combined with multiple behavioral tests, a mouse cognitive dysfunction model was established that synergistically induced by METH and HIV-1 Tat protein, solving the problem that the existing model cannot simulate both The problem of synergistic effects has achieved more accurate research and drug screening.
Patent Information
- Application Number
- CN202411517150.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2025-02-11
AI Technical Summary
Existing research models usually only consider the effects of METH or HIV-1 Tat protein on the nervous system alone, ignore the synergistic effects under the combined effect of the two. The steps to build an animal model are cumbersome, costly and poorly repeatable, and it is impossible to fully simulate the actual situation. Clinical situations lead to limited clinical relevance and predictive value of the study results.
By injecting HIV-1 Tat protein into the lateral ventricle of the brain stereotaxic mice and injecting METH solution intraperitoneal 7 consecutive days, a mouse cognitive dysfunction model was established that synergistically induced by METH and HIV-1 Tat protein, combined with open field experiments, New object recognition experiments, elevated cross maze experiments and water maze experiments evaluate cognitive function and anxiety-like behavior in mice.
This method can more comprehensively simulate the impact of the joint action of METH and HIV-1 Tat proteins on the nervous system in the human body, improve the clinical relevance and predictive value of the research results, simplify the steps of building animal models, reduce costs, and increase the The repetitiveness of the model is supported in large-scale research and drug screening.
Smart Images
Figure CN119405451A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of animal experimental model construction, and more specifically, to a modeling method for inducing cognitive dysfunction in mice by using METH and HIV-1Tat protein. Background Art
[0002] METH (methamphetamine) belongs to the amphetamine stimulant class. Abuse of METH can cause serious physical and psychological harm, increase the economic burden, and have adverse effects on public health and social development. It is widely recognized that METH can seriously damage the central nervous system and cause complex neurotoxicity. Short-term intake of METH can lead to euphoria, hyperactivity, and changes in the cardiovascular system, which are similar to other types of stimulants. However, long-term or large-scale use of METH can lead to METH use disorder, triggering a series of serious physiological and neurological problems, including weight loss, muscle atrophy, periodontal tissue necrosis, brain damage, and impaired attention, memory, and executive function. In fact, patients with METH use disorder are often accompanied by anxiety and cognitive impairment. There is a certain correlation between the two, and cognitive function is affected in many ways.
[0003] Due to the psychoactive effects and susceptibility to addiction after using METH, abusers can easily be infected and spread HIV by sharing syringes or engaging in high-risk sexual behavior after taking drugs. HIV-1 Tat protein is a key regulatory protein encoded by HIV-1 virus, which plays a vital role in the transcription and replication of the virus. Tat protein can cross the blood-brain barrier and enter the brain tissue, and act together with Tat secreted or released from infected cells in the brain to act on NMDAR, mGluR1, dopamine transporter, etc., to damage the nervous system. Tat protein has a close relationship with the blood-brain barrier. It can affect the permeability of the blood-brain barrier, induce inflammatory response, and recruit lymphocytes and monocytes in the blood into the brain tissue. In addition, Tat protein can also interact with neuronal receptors, induce oxidative stress response, interfere with the cellular calcium regulation system, and then cause damage to the nervous system. These effects may lead to diseases such as HIV-associated cognitive dysfunction (HAND) and HIV-associated dementia (HAD), and patients mainly show motor dysfunction, cognitive and behavioral impairment, etc.
[0004] A large number of studies have shown that METH abusers infected with HIV are more likely to develop HAND earlier or even more easily than HIV-infected people with no history of drug abuse, but the specific mechanism of action has not been clarified. Therefore, an animal model of cognitive dysfunction synergistically induced by METH and HIV-1 Tat protein is urgently needed to promote our further in-depth research, better understand its mechanism of action, and lay the foundation for the study of effective prevention and treatment strategies.
[0005] However, the existing research models have the following defects:
[0006] 1. Existing research models usually only consider the effects of METH or HIV-1 Tat protein on the nervous system alone, while ignoring the synergistic effect of the two together. This single-factor model cannot fully simulate the actual clinical situation, resulting in limited clinical relevance and predictive value of the research results.
[0007] 2. Existing technologies for constructing animal models are often cumbersome, costly, and have poor reproducibility, which limits the feasibility of large-scale research and drug screening.
[0008] 3. Existing animal models may lack sensitivity and specificity when evaluating cognitive dysfunction, resulting in the inability to accurately evaluate the effects of drug interventions or the accuracy of disease models.
[0009] 4. Existing animal models may lack sensitivity and specificity when evaluating cognitive dysfunction, resulting in the inability to accurately evaluate the effects of drug interventions or the accuracy of disease models. Summary of the invention
[0010] The purpose of the present invention is to provide a method for modeling cognitive dysfunction in mice induced by METH and HIV-1 Tat protein, and to more accurately study the mechanism of cognitive dysfunction by establishing a new experimental animal model to simulate the neurotoxicity under the joint action of METH and HIV-1 Tat protein, and to provide an effective experimental platform for drug screening and the development of treatment strategies. Through this method, it is possible to better understand how METH and HIV-1 Tat protein affect cognitive function and provide a scientific basis for the development of new treatment strategies.
[0011] The above technical purpose of the present invention is achieved through the following technical scheme: a method for modeling cognitive dysfunction in mice induced by METH and HIV-1 Tat protein, comprising: stereotaxic injection of 10 μg / kg HIV-1 Tat protein into the lateral ventricle of the brain of each mouse once, and then intraperitoneal injection of 2 mg / kg METH solution into each mouse for 7 consecutive days.
[0012] The present invention is further configured as follows: the mice are 6-8 week old C57BL / 6J male mice.
[0013] The present invention is further configured to include behavioral tests after modeling, wherein the behavioral tests include open field tests, new object recognition tests, elevated plus maze tests and water maze tests.
[0014] The present invention is further configured to include: collecting data from behavioral experiments and analyzing them using statistical software to evaluate the effects of METH and HIV-1 Tat proteins on the cognitive function of mice.
[0015] The present invention is further configured as follows: the open field test and the elevated plus maze test evaluate anxiety-like behavior of mice.
[0016] The present invention is further configured as follows: the water maze experiment and the novel object recognition experiment evaluate the cognitive function of mice.
[0017] By adopting the above technical scheme, the experimental animal model of the present invention models cognitive dysfunction induced by METH and HIV-1 Tat protein, and this method may have certain applicability to the study of other types of neurodegenerative diseases. The present invention simulates the synergistic effect of the joint action of METH and HIV-1 Tat protein on the nervous system by simultaneously considering the effects of METH and HIV-1 Tat protein on the nervous system, and more comprehensively simulates the actual clinical situation. This helps to improve the clinical relevance and predictive value of the research results, and the present invention provides a method for constructing an animal model with simplified steps and low cost, which improves the repeatability of the model, thereby making large-scale research and drug screening possible.
[0018] The present invention adopts multiple behavioral tests such as open field test, novel object recognition test, elevated plus maze test and water maze test when evaluating cognitive dysfunction. These tests can evaluate the cognitive function and anxiety-like behavior of mice more sensitively and specifically, so as to more accurately evaluate the effect of drug intervention or the accuracy of disease model. Through multiple behavioral tests including open field test, novel object recognition test, elevated plus maze test and water maze test, the present invention can comprehensively evaluate the cognitive function and anxiety-like behavior of mice, and provide richer data support for drug screening and the development of treatment strategies. The present invention also includes collecting data of behavioral experiments and analyzing them using statistical software, which helps to more objectively evaluate the effects of METH and HIV-1 Tat proteins on the cognitive function of mice.
[0019] In summary, the present invention has the following beneficial effects:
[0020] 1. The present invention establishes an experimental animal model of cognitive dysfunction induced by METH and HIV-1 Tat protein. This method can more comprehensively simulate the actual clinical situation. METH and HIV-1 Tat protein exist simultaneously in the human body and affect the nervous system. This model helps researchers to more accurately understand how these two substances act together on the nervous system, thereby causing cognitive dysfunction.
[0021] 2. The present invention provides a method for constructing an animal model with simplified steps and low cost. Through precise dosage and injection method, the model construction process is more standardized and repeatable, which not only improves the efficiency of the experiment, but also makes large-scale research and drug screening possible. In addition, by using a variety of behavioral tests, such as open field tests, new object recognition tests, elevated plus maze tests, and water maze tests, the cognitive function and anxiety-like behavior of mice can be comprehensively evaluated, providing richer data support for drug screening and the development of treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Schematic diagram of the modeling of cognitive dysfunction in mice synergistically induced by METH and HIV-1 Tat protein in the embodiment of the present invention;
[0023] Figure 2 Schematic diagram of METH and HIV-1 Tat protein synergistically inducing anxiety-like behavior in mice in an embodiment of the present invention;
[0024] in, Figure 2 A is a representative trajectory diagram in the open field experiment; Figure 2 B is the total distance traveled by the mouse in the open field test; Figure 2 C is the number of times the mouse entered the central area in the open field test; Figure 2 D is the time the mouse stayed in the center area in the open field test; Figure 2 E is a representative trajectory diagram in the elevated plus maze experiment; Figure 2 F is a graph showing the time mice spent in the open arm in the elevated plus maze test;
[0025] Figure 3 Schematic diagram of METH and HIV-1 Tat protein synergistically inducing cognitive dysfunction in mice in an embodiment of the present invention;
[0026] in, Figure 3 A is a representative trajectory diagram in the water maze experiment; Figure 3 B is the escape latency during the water maze experimental training; Figure 3 C is the percentage of time that mice spend exercising in the first quadrant in the water maze experiment; Figure 3 D is the movement speed of mice in the water maze experiment; Figure 3 E is the exploration time of the new object after 2 h of training in the new object recognition experiment; Figure 3 F is the number of explorations of new objects after 2 h of training in the new object recognition experiment; Figure 3 G is the exploration time of the new object after 24 h of training in the new object recognition experiment; Figure 3 H is the number of explorations of new objects after 24 hours of training in the new object recognition experiment. DETAILED DESCRIPTION
[0027] The present invention is further described in detail below by specific embodiments. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. In addition, those who do not indicate specific technical operation steps or conditions in the examples are all carried out according to the technology or conditions described in the literature in this area or according to the product instructions. Those who do not indicate the manufacturer of the reagents or instruments used are all conventional products that can be obtained through commercial purchase.
[0028] Selection and grouping of mice: C57BL / 6J wild-type male mice aged 6-8 weeks were selected and randomly divided into a control group, a METH-treated group, a HIV-1 Tat protein-treated group, and a METH+HIV-1 Tat protein-treated group.
[0029] Example 1: A method for modeling cognitive dysfunction in mice induced by METH and HIV-1 Tat protein, comprising:
[0030] Stereotaxic injection: After the mice were anesthetized, HIV-1 Tat protein was injected into the lateral ventricle of the mice using stereotaxic technology.
[0031] Dosage and time of administration: ① Normal saline group: After sham operation, each mouse was intraperitoneally injected with 10 mL / kg of normal saline twice a day for 7 consecutive days;
[0032] ②METH group: After sham operation, each mouse was intraperitoneally injected with 2 mg / kg METH solution twice a day for 7 consecutive days;
[0033] ③ HIV-1 Tat protein group: Each mouse brain was stereotactically positioned in the lateral ventricle and injected with 10 μg / kg HIV-1 Tat protein once, and then each mouse was intraperitoneally injected with 10 mL / kg of normal saline twice a day for 7 consecutive days;
[0034] ④METH+HIV-1 Tat protein group: 10 μg / kg HIV-1 Tat protein was injected into the lateral ventricle of the brain stereotaxically once, and then 2 mg / kg METH solution was injected intraperitoneally to each mouse twice a day for 7 consecutive days.
[0035] Behavioral testing: Three days before drug administration, the mice were placed in an open field box for adaptation, and the data collected on the third day were used as baseline data before drug administration. On the fourth day, the mice received a stereotaxic injection of HIV-1 Tat protein, and recovered for two days after surgery to ensure that the mice were able to resume normal activities. From the seventh day, the mice received intraperitoneal administration of METH. After 7 consecutive days of administration, behavioral testing began on the 14th day, and open field tests, new object recognition tests, elevated cross maze tests, and water maze tests were performed successively. The modeling diagram is shown in the figure. Figure 1 shown.
[0036] The present invention uses open field and elevated plus maze tests to evaluate the anxiety-like behavior of mice. Figure 2 As shown:
[0037] The open field test is used to evaluate the spontaneous activity, exploratory behavior and anxiety response of mice in a novel environment: In this example, mice are placed in an open square or circular area, and their activity trajectory, movement speed, residence time, regional preference and other behavioral parameters are recorded over a certain period of time by cameras and behavioral analysis software. These data help researchers understand the adaptability, motor ability and emotional state of mice to new environments, and are often used in neuroscience research, drug effect evaluation and the establishment of psychological disease models.
[0038] Open field test results:
[0039] The total distance that mice move in the open field is an important indicator for assessing their overall activity level. Figure 2 A. Figure 2 As shown in B, compared with the control group, the total distance traveled by mice treated with METH or HIV-1 Tat protein alone was shortened, indicating that their activity level was reduced; the central area is usually considered an unsafe area, and anxious mice tend to avoid entering it. Figure 2 C. Figure 2 As shown in D, the number of times these mice entered the central area and the time they stayed there decreased, indicating that their anxiety levels increased. When METH and HIV-1 Tat proteins acted together, the total distance traveled, the number of times they entered the central area, and the time they stayed there were further significantly reduced, indicating that the two had a synergistic enhancing effect on anxiety behavior.
[0040] Elevated plus maze test: In this example, mice are placed in a cross-shaped maze elevated above the ground, which consists of two open arms and two closed arms. The behavior of mice in the maze is recorded and analyzed. Usually, mice tend to avoid open arms and choose closed arms because open spaces increase their anxiety. The experiment evaluates the anxiety level and exploratory behavior of mice by calculating parameters such as the time they stay in the open arms and the number of times they enter the closed arms. The elevated plus maze test is widely used in neuropsychopharmacology and behavioral research because it can sensitively reflect the anxiety-like behavior and memory ability of mice.
[0041] Results of the elevated plus maze test: In the elevated plus maze, anxiety-like behavior was reflected in the mice's tendency to avoid the open arms. Compared with the control group, mice treated with METH or HIV-1Tat protein alone spent less time in the open arms. Figure 2 E. Figure 2As shown in F; after co-treatment with the two, the mice's stay time in the open arm was further shortened, showing a significant increase in anxiety levels, further confirming the synergistic effect between METH and HIV-1 Tat protein.
[0042] The above results clearly show that METH and HIV-1 Tat protein exhibit significant anxiety-like behavioral effects on mice, and when they act together, the effect is more significant. Through the changes in these behavioral indicators, we can further explore the complex relationship between anxiety and cognitive impairment, as well as the possible physiological mechanisms in disease models, which provides basic data support for the development of therapeutic strategies for anxiety-related cognitive disorders.
[0043] Anxiety and cognitive impairment have a certain relationship and can affect cognitive function in various ways. The present invention uses water maze and novel object recognition experiments to evaluate the cognitive function of mice. Figure 3 As shown:
[0044] Water maze test: In this example, mice are placed in a pool with an escape platform hidden under the water. The mice need to learn and remember the location of the platform so that they can find it faster in subsequent tests. The experiment evaluates the spatial memory ability of the mice by recording parameters such as the time (latency) it takes them to find the platform, the swimming path, and the strategy. At different stages of the experiment, platform visible training, platform hidden training, and exploration tests without the platform may be included to evaluate the animals' spatial memory and learning memory retention ability. The water maze experiment has important application value in the study of cognitive dysfunction, Alzheimer's disease, hippocampal function, and new drug development.
[0045] Water maze test results:
[0046] In this example, the mice showed a gradual improvement in their learning ability during the training process, as shown by a decrease in the time taken to find the hidden platform; the differences between the different groups became significant, especially from the fourth day. The data showed that the time taken for mice in the group treated with METH and HIV-1 Tat protein to find the platform was significantly longer than that in the group treated with either protein alone, indicating that their learning ability was significantly affected. Figure 3 As shown in B; in the platform removal test on the fifth day, the swimming time of each group of mice in the first quadrant where the original platform was located was used to evaluate memory. The movement trajectory of the mice in the water maze is shown in Figure 3 As shown in A. The analysis results showed that the mice in the METH+HIV-1 Tat protein group swam significantly less in the first quadrant than the mice treated with METH or HIV-1 Tat alone. Figure 3 C, indicating that their memory ability was more impaired; there was no significant difference in the swimming speed of mice in each group. Figure 3As shown in D, the influence of motor ability differences on the results was excluded, which further indicated that the potential mechanism of cognitive impairment originated from cognitive impairment rather than motivation deficit.
[0047] Novel object recognition test: In this example, mice are first introduced to two identical objects, and after a certain period of time, one of the familiar objects is replaced by a new object. The experiment assesses the mice's preference and recognition ability for new objects by measuring the time they explore the new and old objects. If the mouse spends more time exploring the new object, this indicates that it is able to recognize and remember the object it was previously exposed to. Novel object recognition tests are often used to study cognitive function, memory processes, and the effects of neurodegenerative diseases and psychiatric drugs.
[0048] New object recognition experimental results:
[0049] After 2 hours of training: In the short-term memory test phase, the mice in the METH+HIV-1 Tat protein group showed significantly shortened exploration time and reduced exploration times for the new object. Figure 3 E, Figure 3 As shown in F, in contrast, although the exploration of novel objects by mice treated with METH or HIV-1 Tat protein alone was also reduced, it was not as obvious as that of the co-treatment group. This performance indicates that the combined treatment of METH and HIV-1 Tat protein has a greater negative impact on the short-term cognitive ability of mice.
[0050] After 24 hours of training:
[0051] In the long-term memory test, the time and number of explorations of the new objects by mice in the METH or HIV-1 Tat protein treatment groups were reduced compared with those in the control group. Figure 3 G, Figure 3 H shows that long-term memory function is impaired. Mice in the METH+HIV-1 Tat protein group showed more obvious cognitive dysfunction, and their exploration time and frequency of new objects were significantly less than those in the single treatment group, indicating that the combined effect of the two on long-term memory is particularly prominent.
[0052] The above results indicate that the combined action of METH and HIV-1 Tat protein has a significant negative impact on the cognitive function of mice, especially long-term memory. The synergistic effect of the two suggests that they may induce cognitive impairment by jointly affecting key memory-related neural mechanisms. This provides a new direction for further studying the synergistic mechanism of METH and HIV-1 Tat in the central nervous system, and also provides a scientific basis for the development of therapeutic strategies for this cognitive dysfunction.
[0053] Data collection and analysis: The data of behavioral experiments were collected and analyzed using statistical software to evaluate the effects of METH and HIV-1 Tat protein on the cognitive function of mice. The above experimental data are expressed as mean ± standard error, *p<0.05, **p<0.01, ***p<0.001; n≥6 / group.
[0054] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.
Claims
1. A method for modeling cognitive dysfunction in mice induced by METH and HIV-1Tat protein, characterized by: Each mouse was injected once with 10 μg / kg HIV-1 Tat protein into the lateral ventricle by stereotaxic brain imaging, and then each mouse was intraperitoneally injected with 2 mg / kg METH solution for 7 consecutive days.
2. The method for modeling cognitive dysfunction in mice induced by METH and HIV-1Tat protein according to claim 1, characterized in that: The mice were 6-8 week old C57BL / 6J male mice.
3. The method for modeling cognitive dysfunction in mice induced by METH and HIV-1Tat protein according to claim 1, characterized in that: The animal model was further comprised of behavioral tests after model establishment, including an open field test, a novel object recognition test, an elevated plus maze test, and a water maze test.
4. The method for modeling cognitive dysfunction in mice induced by METH and HIV-1Tat protein according to claim 3, characterized in that: The data from behavioral experiments were collected and analyzed using statistical software to evaluate the effects of METH and HIV-1Tat protein on the cognitive function of mice.
5. The method for modeling cognitive dysfunction in mice induced by METH and HIV-1Tat protein according to claim 3, characterized in that: The open field test and the elevated plus maze test evaluate anxiety-like behaviors of mice.
6. The method for modeling cognitive dysfunction in mice induced by METH and HIV-1Tat protein according to claim 3, characterized in that: The water maze test and the novel object recognition test evaluate the cognitive function of mice.