Construction method and application of animal model of paternal origin cognitive and affective disorders

Abstract

The application discloses a construction method of a paternal cognitive and affective disorder animal model and application thereof. The paternal cognitive and affective disorder animal model is constructed by daily intragastric perfusion of caffeine (60 mg / kg.d) to male rats at the age of 6 weeks, and the administration is continued for 8 weeks to cover a complete spermatogenesis cycle, so that a paternal pre-pregnancy caffeine exposure model is constructed. The offspring is weaned and separated into cages at the age of 4 weeks, and typical cognitive and affective behavior abnormalities of the male and female offspring appear at the age of 12 weeks and 28 weeks. Based on the paternal cognitive and affective disorder animal model, it is found that the pre-pregnancy caffeine exposure (PPCE) can cause inhibition of hippocampal neural stem cell differentiation of the offspring. The animal model constructed by the application is novel, reliable and simple, and has important significance for elucidating the occurrence mechanism of the paternal cognitive and affective disorder, and exploring early warning and intervention targets.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing an animal model of paternal cognitive and emotional disorders and its application. Background Technology

[0002] With the development of the social economy, social pressure has increased in tandem, and the incidence of cognitive and emotional disorders has been rising year by year. According to statistics from the National Health Commission in 2019, the prevalence of depression in my country reached 2.1%, the prevalence of anxiety disorder was 4.98%, and the incidence of both comorbidities exceeded 7%. Cognitive disorders are also becoming more and more serious, with a prevalence of about 5%-7.2% in my country. The cognitive and emotional neurobehavioral issues of children and adolescents deserve more social attention. A survey of more than 74,000 children and adolescents in five provinces of China showed [1] that the total prevalence of cognitive and mental disorders among school students aged 6 to 16 in China was 17.5%. Among them, the most prevalent are low intelligence index, impaired language expression ability, changes in executive function related to long-term and short-term memory, attention deficit hyperactivity disorder (ADHD), and anxiety and depression, which usually exist as comorbidities. Cognitive and emotional disorders have become major diseases that seriously endanger human health and are also public health issues that urgently need to be addressed.

[0003] The "Developmental Origins of Health and Disease (DOHaD) theory" has received widespread attention, but it focuses more on the impact of adverse intrauterine environment during maternal pregnancy on fetal development. The impact of adverse paternal environmental exposure on offspring development has not received sufficient attention. Relevant evidence shows that paternal environmental stress, malnutrition, exposure to exogenous substances, infection and other factors can all have adverse effects on offspring development, involving the growth and development and physiological functions of multiple tissues and organs in offspring. For example, paternal bisphenol A exposure can cause anxiety and abnormal social behavior in offspring[2]; paternal dietary restriction can lead to metabolic phenotype and depressive neurobehavior in offspring[3].

[0004] Currently, there are relevant animal models of maternal adverse environmental exposure during pregnancy used to study the effects of adverse maternal environments during pregnancy on fetal growth and development in utero and after birth. However, there is still no well-established animal model of paternal adverse environmental exposure leading to neurodevelopmental abnormalities in offspring. Paternal disease models are fundamentally different from maternal disease models. Exogenous interventions in maternal models, such as chronic stress, viral infections, and toxin intake, can mostly directly affect fetal development through the placental barrier, simulating fetal exposure to adverse environmental conditions during pregnancy. In contrast, exogenous interventions in paternal models can only indirectly affect the fetus through sperm, highlighting the impact of exogenous interventions during "preconception preparation" and before pregnancy on fetal growth and development.

[0005] Epigenetic regulatory changes in germ cells and their transmission are considered to be an important mechanism by which adverse parental environments program offspring for multiple organ developmental abnormalities [4]. Compared with maternal adverse environmental exposure, which can directly affect fetal growth and development through the placenta, how epigenetic regulatory changes in sperm caused by adverse paternal environments escape developmental reprogramming and thus lead to developmental abnormalities and long-term disease susceptibility in offspring remains an important scientific question that has not yet been clarified, making early clinical warning and intervention difficult. Although some animal models can simulate cognitive and emotional behavioral abnormalities, no experimental model can completely replicate cognitive and emotional dysfunctions. Moreover, most animal models have obvious drug side effects or gene defects during their establishment, and cannot simulate universal cognitive and emotional disorders. For example, animal models of anxiety and depression created using different stress environments, and animal models of cognitive and emotional disorders created using surgery, drugs, etc., all have certain limitations and cannot reflect the influence of the paternal environment on offspring before pregnancy. At present, there are no clear animal models of paternal diseases, which makes it difficult to study paternal cognitive and emotional dysfunctions. Therefore, there is an urgent need to establish a stable animal model of paternal cognitive and emotional dysfunction, and to further explore the pathogenesis, early warning and prevention strategies of paternal diseases through this model.

[0006] Caffeine is a common chronic stressor in modern life. It has the effects of refreshing the mind and relieving fatigue. It is commonly found in coffee, tea, chocolate and compound drugs. The annual growth rate of coffee consumption in my country is 15% to 20%, which is about 10 times the world average. According to data from the National Health and Nutrition Examination Survey in the United States, the average caffeine intake of men of reproductive age (240 mg / day) is about 1.5 times that of women; men over 18 years of age consume far more caffeine-rich beverages such as coffee and tea than women. Studies have shown that caffeine, as a central nervous system stimulant, can cause significant chronic stress in men with long-term exposure [5]. This suggests that the current situation of caffeine intake by men of reproductive age is serious and is a common chronic stressor. At present, most countries have proposed relevant safe doses for the caffeine intake of pregnant women. For example, the United States and Canada have stipulated that the daily caffeine intake of pregnant women should not exceed 200 mg, which is equivalent to a 300 ml cup of coffee per day. However, there are no clear regulations for men who are trying to conceive. Although moderate amounts of caffeine have no obvious toxic side effects on the nervous system, studies have shown that caffeine intake can lead to impaired reproductive function. Caffeine, as a common developmental toxin found in everyday beverages, is highly representative for constructing animal models of paternal diseases and is more relevant to guiding men's preparation for pregnancy. Therefore, using paternal pre-pregnant caffeine exposure (PPCE) to establish a phenotype of cognitive and emotional dysfunction in offspring caused by paternal germ cell epigenetic alterations is feasible, more closely mimicking daily life and clinical practice.

[0007] References

[0008] 1. Li, F., Cui, Y., Li, Y., Guo, L., Ke,

[0009] 2. Fan, Y., Tian, ​​C., Liu, Q., Zhen,

[0010] 3. Dimofski, P., Meyre, D., Dreumont, N., & Leininger-Muller, B. (2021). Consequences of paternal nutrition on offspring health and disease. Nutrients, 13(8), 2818.

[0011] 4. Yeshurun, S., & Hannan, AJ (2019). Transgenerational epigenetic influences of paternal environmental exposures on brain function and predisposition to psychiatric disorders. Molecular Psychiatry, 24(4), 536-548.

[0012] 5. Lovallo, WR, Farag, NH, Vincent, AS, Thomas, TL, & Wilson, MF (2006). Cortisol responses to mental stress, exercise, and meals following caffeine intake in men and women. Pharmacology Biochemistry and Behavior, 83(3), 441-447. Summary of the Invention

[0013] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for constructing an animal model of paternal cognitive and emotional disorders that is highly successful, effective, reliable, reproducible, simple and easy to implement.

[0014] To achieve the above objectives, the technical solution of the present invention is as follows:

[0015] In a first aspect, the present invention provides a method for constructing an animal model of paternal cognitive and emotional disorders, characterized by comprising the following steps:

[0016] S1: Healthy, sexually mature male rats were selected and administered 60 mg / kg of caffeine via gastric gavage daily for 8 weeks;

[0017] S2: The above-mentioned caffeine-treated animals were mated with healthy female mice, and the pregnant mice gave birth naturally to obtain the F1 generation;

[0018] S3: Offspring are weaned 4 weeks after birth and separated into male and female cages. They are fed a normal diet until 12 weeks after birth. Behavioral indicators are tested to comprehensively determine cognitive and emotional behavioral abnormalities.

[0019] S4: Some offspring were raised until 28 weeks after birth, and behavioral indicators were tested to comprehensively determine changes in cognitive and emotional behavior; ultimately, an animal model of paternal cognitive and emotional disorders was obtained.

[0020] As a preferred embodiment, in step S1, the male mouse is not limited to male rats; SPF-grade rodents such as SD rats, Kunming mice, C57 mice, guinea pigs, hamsters, etc. are also included.

[0021] Furthermore, in step S2, the production date is taken as day 0 after birth. One day after birth, a litter with 12 to 14 offspring is selected, and 6 male and 6 female offspring are raised in each litter for nursing.

[0022] Furthermore, in step S3, the normal diet formula is the same as the experimental animal formula feed specified in the National Standard of the People's Republic of China GB14924.1-2001.

[0023] Furthermore, in steps S3 and S4, the behavioral detection includes: novel object recognition experiment, open field experiment, and elevated cross maze.

[0024] In a second aspect, the present invention provides an animal model of paternal cognitive and emotional disorders for screening offspring for neurodevelopmental toxicity caused by paternal preconception environmental disturbances or drug exposure, characterized in that: the animal model of paternal cognitive and emotional disorders is obtained by any of the above-mentioned construction methods.

[0025] Thirdly, the present invention provides an application of an animal model of paternal cognitive and emotional disorders in screening early warning targets for paternal cognitive and emotional disorders and preparing intervention target drugs, characterized in that: the animal model of paternal cognitive and emotional disorders is obtained by any of the above-mentioned construction methods.

[0026] The technical principles and research process of this invention are as follows:

[0027] This invention detects cognitive and emotional behavioral indicators in offspring of fathers with paternal pre-pregnant caffeine exposure (PPCE), confirming that PPCE offspring exhibit cognitive and emotional behavioral abnormalities, thereby establishing a model of paternal cognitive and emotional disorders. This model simulates the disease phenotypes of paternal cognitive and emotional disorders, and is of great significance for elucidating the mechanisms of their development and exploring early warning and prevention targets.

[0028] This invention first exposed male rats to caffeine for 8 weeks, covering a complete spermatogenesis cycle, at a dose of 60 mg / kg / day, equivalent to a human exposure of 576 mg / day, a daily accessible dose. The caffeine-exposed male rats were then mated with normal-aged female rats to identify sperm in vaginal secretions as Gestational Day 0. Some pregnant rats were then sacrificed on Gestational Day 20 as intrauterine fetal mouse specimens. The remaining pregnant rats gave birth naturally, and the offspring were conventionally raised to 12 and 28 weeks for postnatal specimens. At 12 and 28 weeks, the rats underwent neurobehavioral experiments such as the elevated cruciate maze test, open field test, and novel object recognition test to assess the cognitive and emotional states of these paternally caffeine-exposed offspring, determining the success of the model.

[0029] The advantages and beneficial effects of this invention are as follows:

[0030] 1. The technical concept of this invention is relatively novel. Caffeine, as an exogenous substance, has the characteristic of daily exposure. Using caffeine to establish an animal model of pre-pregnancy paternal exposure is more in line with daily life and has wider applicability.

[0031] 2. The modeling method of this invention is simple and reliable. A PPCE model is established by administering caffeine to the paternal offspring for eight consecutive weeks after sexual maturity. Adult offspring are then subjected to behavioral tests such as novel object recognition, open field tests, and elevated cross mazes. This model can simulate the cognitive abilities, environmental exploration abilities, and emotional states such as anxiety and depression of patients with cognitive and emotional disorders. The model exhibits good stability and strong repeatability, providing a reliable method for constructing models of paternal cognitive and emotional disorders.

[0032] 3. This invention is an animal model of paternal pre-pregnancy adverse environmental exposure, which is significantly different from animal models of maternal pre-pregnancy adverse environmental exposure. This model is a PPCE model, and there are currently no related animal model construction methods or applications in neurodevelopmental toxicity. PPCE has the characteristic of indirect effects on offspring, which is of particular significance for studying pre-pregnancy reproductive developmental toxicity.

[0033] 4. The paternal cognitive and emotional disorder model constructed based on this invention can be used to guide men's rational preparation for pregnancy and rational use of drugs in clinical practice, explore the occurrence and development mechanism of paternal cognitive and emotional disorders, and be used in early warning and intervention targets. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the behavioral experimental model of the present invention and related results.

[0035] Figure 1 In the experiment: A: Open field experiment; B: Elevated cross maze experiment; C: New Thing cognition experiment. *P<0.05,**P<0.01

[0036] Figure 2 Representative trajectory diagrams and statistical results of open field behavior in offspring of different doses of PPCE

[0037] Figure 2 The image shows the trajectory of the rats in the open field experiment, along with a statistical graph of the total distance traveled by each group and the proportion of time spent in the central region (within the red box) to the total open field movement time. *P<0.05, **P<0.01

[0038] Figure 3 Representative trajectory diagrams and statistical results of elevated cross maze behavior in progeny of PPCE at different doses

[0039] Figure 3 The image shows the trajectory of the elevated cross maze experiment and the statistical graphs of the percentage of time spent in the open arm and the number of entries by rats in each group. *P<0.05,**P<0.01

[0040] Figure 4 Representative trajectory plots and statistical results of novel object recognition behavior in offspring of different doses of PPCE

[0041] Figure 4 In the novel object recognition experiment, representative trajectory maps were plotted over a 24-hour period, and statistical analysis was performed on the exploration time and cognitive coefficients of rats in each group for both new and old objects. *P<0.05, **P<0.01

[0042] Figure 5 Inhibition of expression of neurogenesis-related markers in PPCE offspring

[0043] Figure 5In the figure: A: mRNA expression levels of neural stem cell marker Nestin, neuron marker Tuj1, and newborn neuron marker DCX in fetal and adult rats of PPCE offspring rats compared with those in the Control group (A); B: Detection of protein expression levels of Nestin and Tuj1. *P<0.05, **P<0.01

[0044] Figure 6 Abnormal proliferation and differentiation of neural stem cells in PPCE offspring

[0045] Figure 6 In the figure: A: Double immunofluorescence staining of Nestin / Ki67 in the hippocampus of offspring rats represents the proliferation status of neural stem cells; B: Double immunofluorescence staining of Mcm2 / DCX in the hippocampus of offspring rats indicates abnormal differentiation / neurogenesis of neural stem cells. *P<0.05, **P<0.01 Detailed implementation manners

[0046] The technical content of the present invention will be further elaborated in detail below in combination with specific embodiments and drawings.

[0047] Example 1: Construction of an animal model of paternal cognitive and emotional disorder diseases

[0048] 1. Experimental animals:

[0049] SPF-grade healthy Wistar rats were purchased from Beijing Speywood Biotechnology Co., Ltd., and the animal license number was: SCXK (Beijing) 2019-0010. This study was approved by the Ethics Committee of the Medical Faculty of Wuhan University and was strictly carried out in accordance with the relevant treatment guidelines of the international experimental animal protection certification and evaluation agency.

[0050] The experimental animals were housed in a barrier environment at a temperature of 22-25°C, a humidity of 50%, and a 12-hour day-night cycle. [[ID= 27]]

[0051] 2. Experimental methods:

[0052] Male rats at 6 weeks of age were given caffeine exposure (60 mg / kg.d, equivalent to the exposure amount of 576 mg / d in humans) for 8 weeks (to cover a complete spermatogenic cycle) to establish a PPCE rat model. Then they were mated with normal适龄female rats. Every night at 6 o'clock, the female: male ratio was 2:1 for co-housing. Pregnancy was checked the next morning, and if sperm were found in the vaginal secretion, it was (gestational day 0, GD0) GD0.

[0053] Some pregnant rats were anesthetized and sacrificed at GD20 to obtain fetal rats, weighed, and the hippocampal tissues were collected for further testing. Another part of the pregnant rats gave birth naturally, and the offspring were conventionally raised until 12 weeks and 28 weeks after birth. A series of behavioral tests such as the elevated plus maze test, open field test, and novel object recognition test were performed on the offspring to observe changes in their cognitive and emotional behaviors.

[0054] The feed was purchased from Wuhan Wanqianjiaxing Biotechnology Co., Ltd., and the feed formula was the same as that for mice and rats as specified in the National Standard of the People's Republic of China GB14924.3-2001.

[0055] 3. Detection indicators and methods:

[0056] Before the behavioral tests began, rats were placed in the laboratory for at least 30 minutes to acclimatize. Less stressful behavioral tests were performed first, followed by more stressful ones. The testing order was: open field test, novel object recognition test, and elevated cross maze test.

[0057] 3.1 Open Field Experiment

[0058] The experimental setup measures 100×100×50cm in length, width, and height. 3 An open wooden box was used, with all sides and bottom painted black. After wiping the box clean with alcohol, the rat was placed in the central square of the box. Using the Smart V 3.0 intelligent video tracking system, the ground was automatically divided into 25 squares, and the rat's exploration trajectory and behavioral parameters were recorded over 5 minutes. Observation indicators: ① Central movement time (movement time in the central 9 squares); ② Total movement distance (movement distance across all 25 squares).

[0059] 3.2 New Object Recognition Experiment

[0060] The number of times, time, and distance that rats explored new and old objects were recorded, i.e., the number of times, time, and distance the mice moved around the new and old objects, to assess the mice's cognitive abilities. If the rats had poor cognitive abilities, there would be no difference in the exploration of new and old objects; if the rats had normal cognitive abilities, they would explore new objects for longer than old objects. The recognition index (RI) is calculated as follows: RI (%) = new object / (new object + old object) × 100. Before each experiment, the three arms of the maze were wiped clean with alcohol and then with a clean paper towel. The experimental setup measures 100 × 100 × 50 cm. 3 The rats used an open wooden box, with all sides and bottom painted black. The objects to be identified were approximately 5-8 cm in diameter. Three types of objects, A, B, and C, were required. The objects were odorless and would not be moved by the rats. Objects A and B were identical in appearance, while object C was different from both objects A and B.

[0061] To begin training, firstly... Figure 1As shown, objects A and B are placed at opposite ends of the center of a square box. A rat is placed in the testing area with its back to both objects, ensuring the distance from its nose to each object is the same. The rat is placed for 10 minutes, and immediately after placement, the recording equipment is turned on. The experimenter immediately leaves the testing room and records the rat's contact with the two objects, including the number of times its nose or mouth touches the objects and the time spent exploring within 2-3 cm of the objects. After 10 minutes, the rat is immediately returned to its original enclosure and allowed to rest for 24 hours before the next test (during which time the rat remains in the testing room). After 24 hours of rest, the test begins again. Object B is replaced with object C. The rat is still placed with its back to both objects, ensuring the distance from its nose to each object is the same. The rat is placed for 10 minutes, and immediately after placement, the recording equipment is turned on. The experimenter immediately leaves the testing room and records the rat's contact with the two objects, including the number of times its nose or mouth touches the objects and the time spent exploring within 2-3 cm of the objects.

[0062] 3.3 Elevated Cross Maze

[0063] The device consists of two open arms and two closed arms connected by a central platform area. Before the experiment, the device was wiped clean with 75% ethanol to avoid the influence of odors and rat secretions on the test results. At the start of the experiment, the rats were placed in the central platform area and allowed to move freely for 5 minutes. The Smart V 3.0 intelligent video tracking system automatically recorded the rats' movement trajectories and time proportions in the open and closed arms over those 5 minutes.

[0064] 4. Experimental Results

[0065] PPCE can induce cognitive and emotional behavioral changes in offspring rats, specifically: In the elevated cruciate maze test, compared to the control group, PPCE offspring took significantly less time to enter the open arms; in the open field test, compared to the control group, PPCE offspring entered the central area significantly less frequently and for a shorter time, suggesting anxiety-like behavior accompanied by a reduced desire to explore. In the novel object recognition test, compared to the control group, PPCE offspring showed a decreased ability to distinguish between new and old objects, suggesting a decline in cognitive abilities.

[0066] 4.1 Anxiety-like behaviors in offspring

[0067] The results of anxiety-like behavioral changes in offspring are shown in Table 1. Compared with the control group, the proportion of male and female offspring entering the open arms at 12 weeks of age was significantly lower (P<0.01, Table 1); in addition, the time spent in the open arms by male and female offspring at 12 weeks of age was significantly shorter than that in the control group (P<0.01, Table 1), suggesting that male and female offspring of PPCE exhibit anxiety characteristics at 12 weeks of age. However, there was no significant difference in the total number of times male and female offspring entered the open and closed arms compared with the control group at 12 weeks of age, suggesting that there was no significant difference in activity level between male and female offspring of PPCE and the control group at 12 weeks of age. At 28 weeks of age, the offspring showed changes consistent with those at 12 weeks of age, but the total number of times male offspring entered the open and closed arms was significantly lower than that in the control group at 28 weeks of age (P<0.01, Table 1), suggesting that male and female offspring of PPCE exhibited reduced activity level at 28 weeks of age compared with the control group.

[0068] Table 1: 12-week-old offspring of the elevated cross maze experiment (mean ± SD, n = 12)

[0069]

[0070] 28-week-old offspring (mean±SEM, n=12)

[0071]

[0072] 4.2 Changes in offspring's ability to explore freely

[0073] The results of changes in the free exploration ability of offspring rats are shown in Table 2. Compared with the Control group, the proportion of time spent in the center was significantly lower in both male and female PPCE offspring at 12 weeks of age (P<0.01, Table 2). In addition, the total distance traveled by female offspring was shorter than that in the Control group (P<0.01, Table 2), while no significant change was observed in male offspring. The experimental results suggest that 12-week-old PPCE offspring exhibit anxiety behavior and reduced free exploration ability. Furthermore, the mobility of 12-week-old females was lower than that in the Control group. The 28-week-old offspring showed consistent changes with the 12-week-old offspring. Moreover, the total distance traveled by both male and female offspring at 28 weeks of age was significantly lower than that in the Control group (P<0.01, Table 2). These results suggest that PPCE offspring have reduced free exploration ability and exhibit anxiety.

[0074] Table 2: Open field experiments of 12-week-old offspring (mean ± SEM, n = 12)

[0075]

[0076] Offspring at 28 weeks of age (mean±SEM, n = 12)

[0077]

[0078] 4.3 Changes in offspring cognitive ability

[0079] The results of the changes in the cognitive ability of offspring rats are shown in Table 3. Compared with the Control group, the cognitive coefficient of 12-week-old male offspring of PPCE was significantly decreased (P < 0.01, Table 3). However, there was no significant change in the cognitive coefficient of 12-week-old female offspring. There was a decrease in the cognitive coefficient in both male and female offspring of PPCE at 28 weeks of age (P < 0.01, Table 3), suggesting that compared with the Control group, PPCE offspring had cognitive impairment.

[0080] Table 3: Offspring at 12 weeks of age in the novel object recognition experiment (mean±SEM, n = 12)

[0081]

[0082] Offspring at 28 weeks of age (mean±SEM, n = 12)

[0083]

[0084] The method of the present invention constructs a PPCE model by intragastric perfusion of 60 mg / kg of caffeine to male rats daily for 8 weeks, and mates with age-matched females to obtain offspring. It is found that they have changes in cognitive and emotional behaviors, indicating that an animal model of paternal cognitive and emotional disorder diseases has been successfully established. The modeling method of the present invention is simple, and the indexes such as anxiety behavior, free exploration ability, and cognitive ability in the model have good stability, indicating that the modeling method of the present invention is stable, effective, reliable, and highly reproducible.

[0085] Example 2: Application of the model of the present invention to discover the pathogenesis of paternal cognitive and emotional disorder diseases

[0086] 1. Experimental animals

[0087] SPF-grade healthy Wistar rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal license number was: SCXK (Beijing) 2021-0011. This study was approved by the Ethics Committee of the Medical Faculty of Wuhan University and was strictly carried out in accordance with the relevant treatment guidelines of the International Laboratory Animal Protection Certification and Evaluation Agency. The experimental animals were housed in a barrier environment at a temperature of 22-25 °C, a humidity of 50%, and a 12-hour day-night cycle.

[0088] 2. Experimental methods

[0089] A PPCE rat model was established by exposing 6-week-old male rats to caffeine for 8 weeks (to cover a complete spermatogenesis cycle) (15, 30, 60 mg / kg / day, equivalent to human exposures of 144, 288, and 576 mg / day). The rats were then mated with normal-aged female rats at a female:male ratio of 2:1 at 6 PM each evening. Pregnancy testing the following morning revealed GD0 sperm in vaginal secretions. Feed was purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., and the feed formula was identical to the mouse and rat feed formula specified in the People's Republic of China National Standard GB14924.3-2001.

[0090] Some pregnant mice were euthanized under GD20 anesthesia, and fetal tissue was collected, weighed, and collected for analysis. Other pregnant mice gave birth naturally, with the birth date designated as day 0 of the offspring. One day after birth, litters of 12-14 offspring were selected from each group, with 6 male and 6 female offspring per litter for nursing to ensure balanced nutrition. Offspring were weaned at 4 weeks of age and separated into male and female cages, and were routinely raised until 28 weeks of age. The 28-week-old male offspring underwent a series of behavioral tests, including the open field test, elevated cruciate maze test, and novel object recognition test, to observe changes in cognitive and emotional behavior. The expression of Nestin (a neural stem cell marker), Tuj1 (a neuronal marker), and DCX (a marker of newly formed neurons) in hippocampal tissue was detected, and hippocampal neurogenesis was observed to explore the mechanisms of hippocampal developmental damage in paternal cognitive and emotional disorders.

[0091] 3. Detection indicators and methods:

[0092] 3.1 The open field test, elevated cross maze test, and novel object recognition test were performed on male offspring rats according to the method in Example 1.

[0093] 3.2 Detection of hippocampal developmental damage and related mechanisms in animal models of paternal cognitive and emotional disorders

[0094] Behavioral changes in male offspring exposed to different doses of PPCE were examined to further confirm the correlation and dose-response between paternal caffeine exposure and cognitive and emotional disorders in offspring. Hippocampal tissues were collected from GD20 and 28-week-old male offspring, and the expression changes of neural stem cell marker Nestin, neuronal marker Tuj1, and neonatal neuron marker DCX were detected by RT-qPCR and Western blotting. Changes in hippocampal neurogenesis were observed by immunofluorescence double staining of Nestin / Ki67 and Mcm2 / Dcx. The potential mechanisms of hippocampal dysplasia in animal models of paternal cognitive and emotional disorders were explored.

[0095] 4. Experimental Results

[0096] PPCE can induce cognitive and emotional disorders in 28-week-old male offspring rats. The underlying mechanism is related to the inhibition of hippocampal neural stem cell differentiation, specifically manifested as follows: compared to the Control group, male offspring in the caffeine group exhibited anxiety-like behavior and reduced free exploration ability, accompanied by decreased cognitive function. This is due to impaired hippocampal neural stem cell differentiation and inhibition of neurogenesis.

[0097] 4.1 Male offspring of PPCE exhibit cognitive and emotional dysfunction.

[0098] Paternal caffeine exposure was administered using low (15 mg / kg / day), medium (30 mg / kg / day), and high (60 mg / kg / day) caffeine doses, respectively. Behavioral changes were observed in male offspring (results are shown below). Figure 2-4 In the open field experiment, compared with the control group, the paternal pre-pregnant caffeine exposure group (PPCE_L) showed significantly lower efficacy (P<0.05). Figure 2 The paternal pre-pregnant caffeine exposure with mid-dose (PPCE_M) group (P<0.01) was significantly lower than that of the paternal pre-pregnant caffeine exposure group. Figure 2 The groups with high-dose paternal pre-pregnant caffeine exposure (PPCE_H) and those with high-dose paternal pre-pregnant caffeine exposure (P<0.01) were compared (P<0.01). Figure 2 The male offspring of the PPCE_M and PPCE_H groups spent significantly less time in the central area of ​​the open field, and the total distance traveled by the animals in both groups was significantly reduced (P<0.05). Figure 2 In the elevated cross maze experiment, compared with the control group, the PPCE_M group (P<0.01) showed significantly lower performance. Figure 3 ) and PPCE_H group (P<0.01, Figure 3 The male offspring of the PPCE_H group spent less time in the open arm, while there was no significant difference in the time spent in the open arm between the PPCE_L group and the Control group. Statistical analysis of the number of times animals crossed the open arm showed that male rats in the PPCE_H group entered the open arm significantly less frequently (P<0.01). Figure 3 The PPCE_L and PPCE_M groups showed no significant difference from the Control group in the number of times they entered the open arm. Figure 4 As shown, in addition, the new object cognition experiment found that compared with the control group, the PPCE_M group (P<0.01, Figure 4) and PPCE_H group (P<0.01, Figure 4 The cognitive abilities of male offspring in the Control and PPCE_L groups were slightly decreased, while those in the PPCE_L groups showed no significant change. Statistical analysis of exploration time showed that the cognitive abilities of male rats in both groups were normal, but they spent significantly more time exploring new objects than old ones (P<0.01). Figure 4 In conclusion, PPCE can lead to anxiety-like behaviors and cognitive impairment in male offspring, especially with high-dose paternal preconception caffeine exposure.

[0099] 4.2 PPCE-induced differentiation disorder of male progeny hippocampal neural stem cells

[0100] Abnormal neurobehavior is closely related to the state of neurons. This invention quantitatively measures the neuronal marker (Tuj1). qPCR results showed that, compared with the Control group, the hippocampus of fetal rats in the PPCE_H group had significantly lower levels of this marker (P<0.01). Figure 5 (A) and adult rat hippocampus (P<0.01, Figure 5 Tuj1 expression was significantly reduced in the PPCE_H group (A); Nestin, a marker of neural stem cells, was measured, and qPCR results showed that Nestin expression in male offspring of the PPCE_H group was significantly lower than that in the Control group, regardless of whether they were fetal or adult mice (P<0.01). Figure 5 In the study of neonatal neurons (CdX), quantitative measurements of markers of newborn neurons (DCX) were performed, and the results showed that male offspring fetal mice in the PPCE_H group (P<0.01) showed better results. Figure 5 In mice (A) and adult mice (P<0.01), Figure 5 The mRNA expression of DCX in the A) group was lower than that in the Control group; Western blot analysis confirmed this, and the expression levels of Nestin and Tuj1 proteins in the hippocampus of male offspring in the PPCE_H group during fetal rat period were significantly lower than those in the Control group (P<0.01). Figure 5 In adult mice (P<0.05), the expression levels of Nestin and Tuj1 proteins in the hippocampus of male offspring in the PPCE_H group were significantly lower than those in the Control group (P<0.05). Figure 5 (B) This suggests that the insufficient number of neurons may be due to damage to neural stem cells.

[0101] Hippocampal neural stem cells were co-labeled with Nestin and Ki67 to observe their proliferation. Results showed that the Nestin / Ki67 co-localized proliferative neural stem cells in the male offspring of the 28-week-old PPCE_H group were significantly lower than those in the Control group (P<0.01). Figure 6Further investigation using Mcm2 / DCX fluorescence co-localization revealed impaired differentiation of neural stem cells into neurons. Results showed a reduction in Mcm2 / DCX co-localized cells in the hippocampus of male progeny in the 28-week-old PPCE_H group (P<0.01). Figure 5 This suggests impaired differentiation of neural stem cells and inhibition of neurogenesis.

[0102] In summary, PPCE can inhibit the proliferation and differentiation of hippocampal neural stem cells and reduce neurogenesis, thereby causing cognitive and emotional dysfunction in offspring rats. This demonstrates that this method is an effective approach for establishing animal models of paternal cognitive and emotional disorders, and can be used to study the mechanisms of these models. Furthermore, it can guide the screening of neurodevelopmental toxicities caused by pre-pregnancy environmental disturbances or drugs, and the discovery of early warning and intervention targets for paternal cognitive and emotional disorders.

Claims

1. A method for constructing an animal model of paternal cognitive and emotional disorders, characterized in that: Includes the following steps: S1: Healthy, sexually mature male mice were administered 60 mg / kg of caffeine via gastric gavage daily for 8 weeks. S2: The above-mentioned caffeine-treated animals were mated with healthy females, and the pregnant mice gave birth naturally to obtain the F1 generation; S3: Offspring are weaned 4 weeks after birth and separated into male and female cages. They are fed a normal diet until 12 weeks after birth. Behavioral indicators are tested to comprehensively determine cognitive and emotional behavioral abnormalities. S4: Some offspring were raised until 28 weeks after birth, and behavioral indicators were tested to comprehensively determine cognitive and emotional behavioral abnormalities. Ultimately, an animal model of paternal cognitive and emotional disorders was obtained; In step S1, the male rat is SPF grade, including SD rat, Kunming mouse, C57 mouse, guinea pig, or hamster; In step S2, the production date is taken as day 0 after birth. One day after birth, a litter with 12 to 14 offspring is selected, and 6 male and 6 female offspring are raised in each litter for nursing. In step S3, the normal diet is formulated in the same way as the experimental animal feed formula specified in the National Standard of the People's Republic of China GB14924.1-2001. In steps S3 and S4, the behavioral detection includes: novel object recognition experiment, open field experiment, and elevated cross maze.

2. The application of an animal model of paternal cognitive and emotional disorders in screening offspring for drugs that cause neurodevelopmental toxicity due to paternal preconception environmental disturbances or drug exposure, characterized in that: The animal model of paternal cognitive and emotional disorders is obtained by the construction method in claim 1.

3. The application of an animal model of paternal cognitive and emotional disorders in screening early warning targets for paternal cognitive and emotional disorders and preparing intervention target drugs, characterized in that: The animal model of paternal cognitive and emotional disorders is obtained by the construction method in claim 1.

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