CN115500315B - Construction method and application of father-source cognitive disorder animal model - Google Patents
Construction method and application of father-source cognitive disorder animal model Download PDFInfo
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- CN115500315B CN115500315B CN202210861165.2A CN202210861165A CN115500315B CN 115500315 B CN115500315 B CN 115500315B CN 202210861165 A CN202210861165 A CN 202210861165A CN 115500315 B CN115500315 B CN 115500315B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The application relates to the technical field of animal model construction, and discloses a construction method and application of a father cognitive disorder animal model. The application has the beneficial effects that: through combined treatment of father mice high-fat diet and cadmium water exposure, offspring mice have reduced learning and memory capacity, hippocampal pathological damage and synaptic related protein level, the father cognitive disorder model is successfully established, the father cognitive disorder model can be used for simulating a composite exposure mode of human environmental poison and diet, the action mechanism and treatment targets of the father cognitive disorder are explored, targeted treatment drugs of the father cognitive disorder are screened, and important basis is provided for formulating early prevention strategies of the father cognitive disorder in the future.
Description
Technical Field
The application relates to the technical field of animal model construction, in particular to a construction method and application of a parent cognitive impairment animal model.
Background
Cognitive impairment is a neurodegenerative disease that manifests as reduced learning and memory, and mainly includes dementia, reactive psychosis, and the like. In recent years, global dementia has increased year by year. This not only brings great disease burden to the family of patients, but also brings heavy burden to the public health and the old health care system in China. Thus, cognitive impairment has become a significant medical and public health problem in China and even worldwide.
Currently, the major risk factors for cognitive impairment include environmental poison exposure, altered eating patterns, and the like [5] . Cadmium is a toxic heavy metal that is widely present in the environment. Epidemiological studies have shown that urinary cadmium concentration is inversely related to cognitive function; animal studies have further demonstrated that contemporary environmental cadmium exposure can cause cognitive dysfunction [6-7] . Furthermore, with the increasing level of our daily lives, high-fat diets are becoming more common, and population and animal studies indicate that contemporary high-fat diets may lead to reduced cognitive function [8-9] . However, most of the past studies were single factor exposure in contemporary environments. In a real world environment, it is common for a variety of environmental factors to coexist and be exposed to the body. And in recent years, the theory of health and disease father origin (POHaD) believes that exposure of father-to-bad factors may increase the risk of adult chronic disease in offspring [10-11] . A recent study showed that father high-fat diet caused a reduction in cognitive function levels in offspring [12] . This study suggests that cognitive impairment may present with a parent etiology.
However, current animal models of cognitive disorders are contemporary and single factor treatments, such as mice exhibiting reduced spatial memory capacity following exposure to contemporary dioctyl phthalate or 27-hydroxycholesterol [13-14] . Although these modeling methods can simulate disease models in which cognitive impairment occurs, the models are poor in stability and fail to truly reflect the human complex exposure pattern, which is inconsistent with the parental pathogenesis. Therefore, the existing research is urgent to establish a stable and father-source model of cognitive impairment of mice consistent with the complex exposure mode of human environmental toxicants and diets, and to screen possible risk factors therefrom, thereby performing early prevention and control on the risk factors and promoting human beingsAnd (5) health-like.
[ reference ] to
1.Ritchie K,Lovestone S.The dementias.The Lancet.2002；360(9347):1759-1766.
2.Jia L,Quan M,Fu Y,Zhao T,Li Y,Wei C,Tang Y,Qin Q,Wang F,Qiao Y,Shi S,Wang YJ,Du Y,Zhang J,Zhang J,Luo B,Qu Q,Zhou C,Gauthier S,Jia J；Group for the Project of Dementia Situation in China.Dementia in China:epidemiology,clinical management,and research advances.Lancet Neurol.2020；19(1):81-92.
3.Gale SA,Acar D,Daffner KR.Dementia.Am J Med.2018；131(10):1161-1169.
4.Deng Y,Zhao S,Cheng G,et al.The Prevalence of Mild Cognitive Impairment among Chinese People:A Meta-Analysis.Neuroepidemiology.2021；55(2):79-91.
5.Silva MVF,Loures CMG,Alves LCV,de Souza LC,Borges KBG,Carvalho MDG. Alzheimer's disease:risk factors and potentially protective measures.J Biomed Sci.2019； 26(1):33.
6.Gustin K,Tofail F,Vahter M,Kippler M.Cadmium exposure and cognitive abilities and behavior at 10years of age:A prospective cohort study.Environ Int.2018；113:259-268.
7.Wang H,Abel GM,Storm DR,Xia Z.Adolescent cadmium exposure impairs cognition and hippocampal neurogenesis in C57BL/6 mice.Environ Toxicol.2022；37(2):335-348.
8.Hoscheidt S,Sanderlin AH,Baker LD,et al.Mediterranean and Western diet effects on Alzheimer's disease biomarkers,cerebral perfusion,and cognition in mid-life:A randomized trial.Alzheimers Dement.2022；18(3):457-468.
9.Yang Y,Zhong Z,Wang B,et al.Early-life high-fat diet-induced obesity programs hippocampal development and cognitive functions via regulation of gut commensal Akkermansia muciniphila.Neuropsychopharmacology.2019；44(12):2054-2064.
10.Soubry A.Epigenetics as a Driver of Developmental Origins of Health and Disease:Did We Forget the FathersBioessays.2018；40(1):10.
11.Soubry A.POHaD:why we should study future fathers.Environ Epigenet.2018；4(2): dvy007.
12.Zhou Y,Zhu H,Wu HY,et al.Diet-Induced Paternal Obesity Impairs Cognitive Function in Offspring by Mediating Epigenetic Modifications in Spermatozoa.Obesity(Silver Spring).2018；26(11):1749-1757.
13.Wang Y,An Y,Ma W,et al.27-Hydroxycholesterol contributes to cognitive deficits in APP/PS1 transgenic mice through microbiota dysbiosis and intestinal barrier dysfunction.J Neuroinflammation.2020；17(1):199.
14.Lv J,Li Y,Chen J,et al.Maternal exposure to bis(2-ethylhexyl)phthalate during the thyroid hormone-dependent stage induces persistent emotional and cognitive impairment in middle-aged offspring mice.Food Chem Toxicol.2022；163:112967.
Disclosure of Invention
The technical problem to be solved by the application is how to provide a construction method of a father source cognitive impairment animal model, which has the advantages of high success rate, effectiveness, reliability, strong repeatability, simplicity and easiness.
The application solves the technical problems by the following technical means:
the first aspect of the application provides a construction method and application of a parent cognitive impairment animal model, wherein the construction method comprises the following steps:
(1) Selecting healthy male mice of 4-6 weeks old, randomly grouping, respectively carrying out combined treatment of low-fat diet, cadmium water exposure, high-fat diet and cadmium water exposure, and mating with normal female mice after 9-11 weeks;
(2) The female pregnant mice which are successfully mated are normally eaten and naturally delivered, the produced offspring mice are fed to the lactation period of 4 weeks, then the female pregnant mice are subjected to male-female cage separation and weaning, and are naturally fed according to the normal eating;
(3) Naturally breeding the offspring mice to 14-15 weeks old, and performing a behavioural experiment on the offspring mice; resting for 1-2 weeks after the behavioural experiment is finished, and carrying out anesthesia and sacrifice on offspring mice, taking sea horse tissues for pathological examination and molecular biology experiment, so as to judge the occurrence of cognitive dysfunction.
The beneficial effects are that: the modeling method is simple and easy to implement, the occurrence of the cognitive impairment of the mice is verified together through a plurality of indexes of a behavioral experiment, a pathological examination and a molecular biological experiment, the repeatability is high, and a reliable method is provided for the construction of a father cognitive impairment model.
Preferably, the low fat diet group mice in the step (1) consume 10% kcal of low fat feed, and the high fat diet group mice consume 60% kcal of high fat feed.
Preferably, the cadmium water in the cadmium water exposure group in the step (1) adopts a cadmium chloride aqueous solution, and the concentration of the cadmium chloride is 95-105 mg/L.
Preferably, the male mice and female mice in the step (1) are SPF class C57BL/6N mice; and the male mice and the female mice are mated according to the proportion of 2:3-5.
Preferably, the offspring mice produced in the step (2) are adjusted to have the same number of male mice and female mice per litter on days 3-5 after birth and are fed in a lactation mode so as to ensure balanced nutrition of the mice.
Preferably, the behavioral test is Morris water maze, the pathological examination and molecular biological test comprises mouse hippocampal hematoxylin-eosin staining, nile staining, golgi staining and detection of levels of synapse-related proteins including PSD95, synagin-1 and BDNF, and the cognitive impairment of the mice is determined comprehensively by the behavioral test and the pathological examination and molecular biological test.
The second aspect of the application provides application of the parent cognitive impairment animal model constructed by the construction method.
Preferably, the parent cognitive impairment animal model is applied to simulate the composite exposure mode of human environmental poison and diet and explore the action mechanism and treatment targets of the parent cognitive impairment, and the application aims at diagnosis or treatment of non-diseases.
Preferably, the parenchymal cognitive disorder animal model is applied to the future establishment of an early prevention strategy of the parenchymal cognitive disorder, and the application aims at diagnosis or treatment of non-diseases.
Preferably, the parenchymal cognitive disorder animal model is applied to targeted therapeutic drugs for screening the parenchymal cognitive disorder in the future, and the application aims at diagnosis or treatment of non-diseases.
The beneficial effects are that: the parental cognitive impairment model constructed by the application can be further used for simulating a composite exposure mode of human environmental poison and diet, exploring the action mechanism and the treatment target of the parental cognitive impairment, screening the targeted therapeutic drugs of the parental cognitive impairment, and providing important basis for formulating an early prevention strategy of the parental cognitive impairment in the future.
The application has the advantages that:
1. the modeling method is simple and easy to implement, the occurrence of the cognitive impairment of the mice is verified together through a plurality of indexes of a behavioral experiment, a pathological examination and a molecular biological experiment, the repeatability is high, and a reliable method is provided for the construction of a father cognitive impairment model;
2. the parental cognitive impairment model constructed by the application can be further used for simulating a composite exposure mode of human environmental poison and diet, exploring the action mechanism and the treatment target of the parental cognitive impairment, screening the targeted therapeutic drugs of the parental cognitive impairment, and providing important basis for formulating an early prevention strategy of the parental cognitive impairment in the future.
Drawings
FIG. 1 is a graph showing the results of Morris water maze test on offspring mice in example 1 of the present application.
FIG. 2 is a graph showing the result of hematoxylin-eosin staining of hippocampal tissue of a offspring mouse in example 1 of the present application.
FIG. 3 is a graph showing the result of Nib staining of hippocampal tissue of a offspring mouse in example 1 of the present application.
FIG. 4 is a graph showing the result of Golgi staining of hippocampal tissue of a offspring mouse in example 1 of the present application.
FIG. 5 is a graph showing the results of detection of levels of synapse-associated proteins in hippocampal tissue of a offspring mouse in example 1 of the present application.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described in the following in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1
Experimental animals: all animal experimental procedures of the study were approved by the ethical committee of animal experiments at university of Anhui medical science, SPF grade C57BL/6N mice were purchased from Experimental animal technologies, inc. of Lehua, beijing, and all mice were kept in a strictly controlled environment with the ambient humidity maintained at 50% -60% and the temperature maintained at 25+ -2deg.C for 12 hours with circadian alternation.
Experiment feed: the low-fat feed and the high-fat feed for feeding the male mice of the ancestors in the study are purchased from Nantong Talaofe feed technology Co., ltd, wherein the calorie of the low-fat feed is 10kcal, and the calorie of the high-fat feed is 60% kcal; the feed for feeding female pregnant mice and offspring mice was purchased from the company of the company australia synergetic feed, beijing, and was a common grade mouse breeding feed. The cadmium water adopts a cadmium chloride aqueous solution, and the concentration of the cadmium chloride is 100mg/L.
The implementation provides a construction method of a parent cognitive impairment animal model, which comprises the following steps:
(1) After the purchased SPF-grade C57BL/6N mice are adaptively fed for one week, 80 healthy male mice with the age of 5 weeks are selected and randomly divided into four groups, and respectively subjected to low-fat diet (Ctrl), cadmium water exposure (Cd), high-fat diet (HFD) and combined treatment of high-fat diet and cadmium water exposure (HFD+Cd); 10 weeks later mating with 10 week old female mice normally fed with normal grade mouse breeding feed at a ratio of 2:4, and the next morning, female mice with vaginal embolism were scored as pregnant for 0 days.
(2) The female pregnant mice which are successfully mated are normally eaten and naturally delivered, and the generated first generation offspring mice are adjusted to 3 male mice and female mice in each nest on the 3 rd day after birth and are fed in a lactation mode so as to ensure balanced nutrition of the mice; after the offspring mice are fed to 4 weeks of age, female and male cage separation and weaning are carried out, and each group of 20 mice are naturally fed with common grade mouse breeding feed.
(3) Naturally breeding the offspring mice to 15 weeks old, and performing Morris water maze experiment on the offspring mice; the Morris water maze experiment was stopped for 2 weeks, the offspring mice were anesthetized and sacrificed, and their hippocampal tissues were taken for pathological examination and molecular biology experiments.
1) Morris water maze experiment
The Morris water maze experiment lasted seven days in total, with the first six days being the mouse spatial learning phase and the seventh day being the mouse spatial memory phase. The specific operation is as follows: the water maze experimental operation method is established in Smart animal behavioural analysis software, the water maze area is divided into four fan-shaped areas with equal area, the platform is placed at one quadrant fixed position (the water surface is 1 cm higher than the platform), the quadrant is marked as the quadrant where the platform is located, and the other three quadrants are marked as the outer quadrants of the platform. At the beginning of the experiment, one quadrant was randomly chosen as the starting quadrant into which the mouse was placed facing the wall. After the completion, the mice are placed in the remaining three quadrants in turn, the next quadrant is selected as the starting quadrant in the clockwise direction beginning the next day, the mice are placed in the four quadrants in turn, and so on. Each quadrant was timed for 1min, the mice stopped the quadrant experiment after boarding the platform, and the next quadrant experiment was continued. After six days, the platform was removed on day seven and the mice were placed in four quadrants in sequence facing the wall, each quadrant timed for 1min. The upper platform latency, the activity course of the offspring mice in the seventh day spatial memory experiment, the upper platform latency, the activity time in the target quadrant region, the number of traversals on the platform, and the activity distance on the platform of the previous six sky-space learning experiment of the offspring mice in each experimental group were observed.
In the Morris water maze experiment process, water is replaced every two days so as to avoid the influence of water quality on the experiment result; the water temperature is detected at the beginning of each experiment, and the water temperature is ensured to be 21-22 ℃. Each mouse is dried on an electric blanket after the experiment is finished, so that the cold is avoided; the laboratory illuminance and noise were measured and recorded after the end of the experiment.
The observation results of the offspring mice are shown in fig. 1, wherein fig. 1-a is a graph showing the variation of the upper platform latency of the learning experiment between the six sky before the female offspring mice in each experimental group, and fig. 1-B, 1-C, 1-D, 1-E, 1-F are the activity route, the first platform latency crossing, the activity time in the target quadrant region, the number of times of crossing on the platform, and the activity distance on the platform of the offspring mice in the seventh day spatial memory experiment of the female offspring mice in each experimental group, respectively. FIG. 1-G is a graph showing the variation of the upper plateau latency of the study experiment between the six sky preceding the male offspring mice in each experimental group, and FIGS. 1-H, 1-I, 1-J, 1-K, 1-L are the course of activity of the offspring mice in the seventh day spatial memory experiment of the male offspring mice in each experimental group, the first crossing plateau latency, the activity time in the target quadrant region, the number of crossings on the plateau, and the distance of activity on the plateau, respectively. In fig. 1-B and 1-H, the active route pattern is the path of the mice swimming in water in the seventh day of spatial memory experiment, and the small circle in the second quadrant of the route pattern is the position where the platform is placed. Since the mouse is naturally offensive to the water environment, it will strive to find the platform and board up when it is in the water environment, after the first six days of boarding training on the platform, the normal memory mouse has developed a long-term memory for the specific location of the platform. Thus, after the platform is removed on the seventh day, the normal memory mouse's active roadmap should be immediately around the quadrant in which the platform is located (second quadrant) or repeatedly across the platform. Thus, the platform latency, the course of activity, the first time the platform latency is traversed, the time of activity in the target quadrant region, the number of traversals on the platform, and the time of traversals on the platform can be traversed by the mouseAnd comprehensively judging whether the memory of the mouse is damaged or not under the conditions of the activity distance on the table and the like. The data in FIGS. 1-C-1-F and 1-I-1-L are expressed as mean ± standard error, a P<0.05, representing comparison to a low fat diet (Ctrl) group; b P<0.05, representing a comparison with a cadmium water exposure (Cd) group.
As can be seen from the fact that the father low-fat diet (Ctrl) is taken as a control group and combined with the figures 1-A and 1-G, in the spatial learning experiment of the first six days, the combined treatment of father high-fat diet and cadmium water exposure (HFD+Cd) obviously prolongs the upper platform incubation period of female and male offspring mice. As can be seen in conjunction with fig. 1-B and 1-H, the aquatic route of the offspring mice of the father low-fat diet (Ctrl) group immediately surrounds the second quadrant and repeatedly crosses the platform, while the route of the combined treatment of father high-fat diet and cadmium water exposure (hfd+cd) group is very disordered, which illustrates that the combined exposure of father high-fat diet and cadmium water significantly impairs the memory of the offspring mice. In combination with figures 1-C-1-F and figures 1-I-1-L, it can be seen that the combined treatment of father high fat diet plus cadmium water exposure (hfd+cd) significantly increases the first pass platform latency of female and male offspring mice and reduces their time of activity in the target quadrant region, number of passes on the platform, and distance of activity on the platform in the seventh day spatial memory experiment. And the above index damage effect of the father high fat diet plus cadmium water exposure combined treatment (hfd+cd) group is further aggravated compared with the simple cadmium water exposure (Cd), high Fat Diet (HFD) group. The above experimental results demonstrate that the combination treatment of father high fat diet plus cadmium water exposure significantly reduced learning and memory in female and male offspring mice.
2) Mouse hippocampal histopathological examination and molecular biology experiments include hematoxylin-eosin (HE) staining, nikose staining, golgi staining, and synaptic related protein level detection, wherein synaptic related proteins include PSD95, synapsin-1 and BDNF.
(1) The specific operation steps of hematoxylin-eosin (HE) staining are as follows: the offspring mice are dewaxed in an oven at 55-60 ℃ for 30-35min, soaked in xylene for 3 times and 10-12min each time, hydrated by 100%, 95%, 80%, 70%, 50% and 30% alcohol each time, and dyed after soaking in distilled water for 5-6 min. The sections were stained with hematoxylin and eosin for 10-15min, then dehydrated sequentially with 30%, 50%, 70%, 80%, 95%, 100% alcohol, each stage for 2-3min, and transparent in xylene twice for 4-5min each. Finally, the plates were blocked with neutral resin, and the hippocampal neurons were observed under a microscope and counted, and the results are shown in fig. 2.
FIG. 2-A is a representative picture of hematoxylin-eosin (HE) staining of hippocampal tissue of female and male offspring mice; FIGS. 2-B, 2-C are the numbers of neurons in the hippocampal CA1, DG regions, respectively, of female offspring mice; FIGS. 2-D, 2-E are the numbers of neurons in the hippocampal CA1, DG region of male offspring mice, respectively. In fig. 2-a, the darker colored areas with a certain outline are the aggregation areas of the hippocampal neurons, the neurons after HE staining can take on dark round shapes, and the number of the hippocampal neurons can be known by quantitatively counting the neurons, and the number of the hippocampal neurons has a close relationship with the cognitive function and the neurogenesis. FIGS. 2-B, 2-C, 2-D, 2-E are results of quantifying the number of neurons in each of the groups of FIG. 2-A; and the data in figures 2-B, 2-C, 2-D, 2-E are expressed as mean + standard error, a P<0.05, representing comparison to a low fat diet (Ctrl) group; b P<0.05, representing a comparison with a cadmium water exposure (Cd) group. It can be seen that the reduction of the number of neurons in CA1 and DG regions of the hippocampus of the offspring mice is achieved by the combined treatment of the father high-fat diet and the cadmium water exposure (HFD+Cd) by taking the father low-fat diet (Ctrl) as a control group, which shows that the combined exposure of the father high-fat diet and the cadmium water damages the neurons of the hippocampus of the female and male offspring mice, thereby damaging the cognitive function.
(2) The specific operation steps of the Nib dyeing are as follows: the offspring mice are dewaxed in an oven at 55-60 ℃ for 30-35min, soaked in xylene for 3 times and 10-12min each time, hydrated by 100%, 95%, 80%, 70%, 50% and 30% alcohol each time, and dyed after soaking in distilled water for 5-6 min. Sea horse slices were stained with 0.1% cresyl violet at 37 ℃ for 10-12min; then sequentially dehydrating with 30%, 50%, 70%, 80%, 95% and 100% ethanol for 2-3min, and transparent in xylene for 4-5min each time. The method comprises the steps of carrying out a first treatment on the surface of the Finally, the pellet was sealed with neutral resin, and the hippocampal nuclei of the mice were observed under a microscope and counted, and the results are shown in fig. 3.
FIG. 3-F is a representative picture of Nile staining of hippocampal tissue of female and male offspring mice; FIGS. 3-G, 3-H are the number of nuclear-contracted neurons in the CA1 region and DG region, respectively, of the hippocampus of female offspring mice; FIGS. 3-I and 3-J show the numbers of nuclear-contracted neurons in the CA1 region and DG region, respectively, of the hippocampus of male offspring mice. In fig. 3-F, the area with a certain outline and darker color is the aggregation area of the hippocampal neurons, the neurons after being dyed by nikose also take on a dark round shape, but the neurons with the nuclear shrinkage take on a darker oval or strip shape, the number of the hippocampal nuclear shrinkage neurons can be known by quantitatively counting the neurons, and the number of the nuclear shrinkage neurons, namely the damaged neurons, is also closely related to the cognitive function and the neurogenesis. FIGS. 3-G, 3-H, 3-I, 3-J are results of quantifying the number of nuclear contractile neurons of each group of FIG. 3-F; and the data in figures 3-G, 3-H, 3-I, 3-J are expressed as mean + standard error, a P<0.05, representing comparison to a low fat diet (Ctrl) group; b P<0.05, representing a comparison with a cadmium water exposure (Cd) group. It can be seen that the combined treatment of father low-fat diet (Ctrl) and cadmium water exposure (HFD+Cd) obviously increases the number of nuclear-contracted neurons in the CA1 and DG areas of the hippocampus of the offspring mice.
(3) The specific operation steps of the Golgi dyeing are as follows: placing fresh brain tissue into a freshly prepared Golgi dye solution, and dyeing for one week; soaking brain tissue soaked in Golgi dye solution in 30% sugar water for 2 days; cutting hippocampal tissue with the thickness of 200 mu m by using an oscillation slicer, and placing the hippocampal tissue on an adhesion glass slide for subsequent staining; and sealing the slices after dyeing, finding out the neurons under a low power microscope, shooting in an enlarged mode, observing the dendritic spine density and the dendritic branch number of the neurons in different areas of the hippocampus to evaluate the development condition of the neurons of the hippocampus, and the observation result is shown in figure 4.
FIG. 4-K is a representative picture of the dendritic spines of neurons in the CA1 and DG regions of hippocampal tissue of female and male offspring mice; FIG. 4-L is the density of the dendritic spines of neurons in the CA1 and DG regions of female progeny hippocampal tissue; FIG. 4-M is the density of the neuronal dendrites of the hippocampal CA1 and DG regions of male progeny.In fig. 4-K is a dendritic spine on the secondary dendritic branch of the hippocampal neuron, which is capable of receiving and transmitting neural signals, the number density of which is closely related to brain learning and memory functions and central nervous system diseases. The density of dendritic spines is quantified as the number of dendritic spines per 10 μm length of dendritic branches. FIGS. 4-L, 4-M are results of quantifying the density of dendritic spines for each of the sets of neurons of FIG. 4-K; and the data in figures 4-L, 4-M are represented as mean + standard error, a P<0.05, representing comparison to a low fat diet (Ctrl) group; b P<0.05, representing a comparison with a cadmium water exposure (Cd) group. It can be seen that the combined treatment of father low-fat diet (Ctrl) and cadmium water exposure (HFD+Cd) obviously reduces the density of the neurons dendritic spines in the CA1 and DG areas of the hippocampus of the offspring mice, which indicates that the brain learning and memory functions of the offspring mice are reduced.
Hematoxylin-eosin (HE) staining, nikohlrabi staining and golgi staining showed that the combination treatment of father high fat diet plus cadmium water exposure significantly damaged the number and structure of hippocampal neurons in offspring mice.
(4) The protein levels of PSD95, synapsin-1 and BDNF are detected by a protein immunoblotting method, and the method comprises the following specific steps: mixing the offspring mouse hippocampal tissue with the lysate, homogenizing the mixture into liquid by using a homogenizer, centrifuging for 15min by using an ultracentrifuge 15000g, and collecting the supernatant as the total protein. Total proteins were then separated using 12.5-15% SDS-PAGE polyacrylamide gel electrophoresis and transferred to PVDF membrane. After 1-1.5h incubation with 5% skim milk, PVDF membranes were treated with PSD95, synapsin-1 and BDNF primary antibodies for 1-2 h. After the first antibody is incubated, the membrane is washed three times with membrane washing liquid for 8-10min each time, and after the membrane washing, the PVDF membrane is incubated for 1-2 hours by the second antibody. Finally, PSD95, synapsin-1 and BDNF protein levels were detected by the Bio-Rad ChemicdocTM Touch imaging system. The results are shown in FIG. 5.
FIGS. 5-A and 5-C are graphs showing the levels of PSD95, symplin-1 and BDNF proteins expressed in hippocampal tissues of female and male offspring mice, respectively, and FIGS. 5-B and 5-C are results of quantifying PSD95, symplin-1 and BDNF proteins in each of the groups of FIGS. 5-A and 5-C, respectively. The color intensity of the bands in FIGS. 5-A and 5-C represents the abundance of protein expression, and the darker the color isThe higher the protein level is shown. Three proteins, PSD95, synapsin-1 and BDNF, can influence synaptic plasticity of neurons, and are the biomolecular basis of learning and memory capacity. Thus, a decrease in the levels of these three proteins would also lead to a decrease in cognitive function. The data in figures 5-B and 5-D are expressed as mean + standard error, a P<0.05, representing comparison to a low fat diet (Ctrl) group; b P<0.05, representing a comparison with a cadmium water exposure (Cd) group. It can be seen that, by taking the father low-fat diet (Ctrl) as a control group, the combined treatment of father high-fat diet and cadmium water exposure (HFD+Cd) obviously reduces the levels of PSD95, synapsin-1 and BDNF proteins in the hippocampal tissues of female and male offspring mice, which indicates that the combined treatment of father high-fat diet and cadmium water exposure obviously damages the functions of hippocampal neurons of offspring mice.
According to the method, the father mice are subjected to combined treatment of high-fat diet and cadmium water exposure, the offspring mice are routinely bred to 15 weeks and subjected to Morris water maze test and various hippocampal pathology examination and molecular biology experiments, and as a result, the situation that the high-fat diet and cadmium water exposure combined treatment group mice have reduced learning and memory capacity, hippocampal pathology damage and reduced synaptic related protein level is found, which indicates that the father cognitive disorder model is successfully established. The modeling method is simple and easy to implement, and has strong repeatability through common verification of a plurality of indexes such as Morris water maze, golgi staining, hematoxylin-eosin (HE) staining, nishi staining, synapse-related proteins and the like.
Example 2
The present embodiment provides an application of a parental cognitive impairment animal model, in which environmental toxicants and dietary patterns are changed and applied to the animal model of example 1, the influence of candidate environmental toxicants and dietary patterns on various indexes of the animal cognitive impairment is observed, the environmental toxicants and dietary patterns with potential harm are evaluated, the mechanism of action and therapeutic targets of the parental cognitive impairment are explored, and early prevention strategies of the corresponding parental cognitive impairment are formulated.
Example 3
The present embodiment provides an application of a parental cognitive impairment animal model, wherein a candidate drug for treating cognitive impairment is applied to the animal model of example 1, and a drug capable of treating cognitive impairment is selected.
According to the method, the father mice are subjected to combined treatment of high-fat diet and cadmium water exposure, the offspring mice are routinely fed for 15 weeks and subjected to Morris water maze test and various hippocampal pathology examination and molecular biology experiments, and the result shows that the high-fat diet and cadmium water exposure combined treatment group mice have the conditions of reduced learning and memory capacity, pathological damage of the hippocampus and reduced level of synapse-related proteins, which means that a father cognitive disorder model is successfully established, the father cognitive disorder model can be used for simulating a composite exposure mode of human environmental poison and diet, the action mechanism and treatment targets of the father cognitive disorder are explored, and targeted treatment drugs of the father cognitive disorder are screened, so that important basis is provided for formulating early prevention strategies of the father cognitive disorder in future.
The above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims (9)
1. A construction method of a father-source cognitive disorder animal model is characterized by comprising the following steps: the method comprises the following steps:
(1) Selecting healthy male mice of 4-6 weeks old, randomly grouping, performing high-fat diet and cadmium water exposure combined treatment on the grouped male mice, and mating the grouped male mice with normal diet female mice after 9-11 weeks;
(2) The female pregnant mice which are successfully mated are normally eaten and naturally delivered, the produced offspring mice are fed to the lactation period of 4 weeks, then the female pregnant mice are subjected to male-female cage separation and weaning, and are naturally fed according to the normal eating;
(3) Naturally breeding the offspring mice to 14-15 weeks old, and performing a behavioural experiment on the offspring mice; resting for 1-2 weeks after the behavioural experiment is finished, and carrying out anesthesia and sacrifice on offspring mice, taking sea horse tissues for pathological examination and molecular biology experiment, so as to judge the occurrence of cognitive dysfunction.
2. The method for constructing a parent cognitive impairment animal model according to claim 1, wherein: the mice exposed to the high fat diet plus cadmium water in step (1) eat 60% kcal of high fat diet.
3. The method for constructing a parent cognitive impairment animal model according to claim 1, wherein: the cadmium water in the cadmium water exposure in the step (1) adopts a cadmium chloride aqueous solution, and the concentration of the cadmium chloride is 95-105 mg/L.
4. The method for constructing a parent cognitive impairment animal model according to claim 1, wherein: the male mice and the female mice in the step (1) are SPF-grade C57BL/6N mice; and the male mice and the female mice are mated according to the proportion of 2:3-5.
5. The method for constructing a parent cognitive impairment animal model according to claim 1, wherein: and (3) 3-5 days after birth of the offspring mice generated in the step (2), adjusting the number of male mice and female mice in each nest to be the same, and feeding the offspring mice in a lactation way so as to ensure balanced nutrition of the mice.
6. The method for constructing a parent cognitive impairment animal model according to claim 1, wherein: the behavioral tests are Morris water maze, the pathological examination and molecular biology tests comprise mouse hippocampal hematoxylin-eosin staining, nile staining, golgi staining and synaptic related protein level detection, the synaptic related proteins comprise PSD95, synapsin-1 and BDNF, and the cognitive disorder of the mice is comprehensively judged through the behavioral tests and the pathological examination and molecular biology tests.
7. Use of the construction method according to claims 1-6 for simulating the complex exposure pattern of human environmental toxicants and diets and for exploring the mechanism of action and therapeutic targets of parent cognitive impairment, characterized in that: the use is for the diagnosis or treatment of non-diseases.
8. Use of the construction method according to claims 1-6 for future establishment of early prevention strategies for parent cognitive impairment, characterized in that: the use is for the diagnosis or treatment of non-diseases.
9. Use of the construction method according to claims 1-6 for the future screening of targeted therapeutic drugs for parental cognitive impairment, characterized in that: the use is for the diagnosis or treatment of non-diseases.
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