CN113491251B - Preparation method of kidney-yang deficiency type Parkinson disease rat model - Google Patents
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Abstract
The invention relates to a preparation method of a kidney-yang deficiency type Parkinson disease rat model, and aims to solve the technical problems that the existing Parkinson disease rat model is unstable in symptoms, easy to generate toxic reaction, not beneficial to practical application and the like. The invention adopts a fatigue swimming test and a same cage test to establish a kidney-yang deficiency animal model which completely follows the classic traditional Chinese medicine theory, and then, the model is evaluated to screen out qualified rats with kidney-yang deficiency for the next PD model building. After the model is made, five aspects of evaluation are carried out through appearance observation, ethology detection, serum alpha-syn content detection, HE staining analysis of substantia nigra and striatum tissues and TH positive immunohistochemical detection of the substantia nigra and striatum tissues; besides appearance observation, any three other tests are needed, namely, the molding is successful. The invention truly reproduces the symptom of kidney-yang deficiency by simulating the internal cause of Parkinson disease, and provides a real, stable and reliable simulation basis for the next PD model establishment.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a preparation method of a kidney-yang deficiency type Parkinson disease rat model.
Background
Parkinson's Disease (PD) is a common degenerative disease of the central nervous system. Epidemiological investigation shows that PD becomes the most common degenerative disease of the nervous system in the middle-aged and elderly people except Alzheimer Disease (AD), and the incidence rate of industrialized countries is close to 0.3%. In China, PD patients tend to increase year by year, and the number of the patients is over 200 ten thousand at present. PD, while not directly shortening patient life, as with AD, severely impacts patient quality of life. In addition, the patient and the family are burdened and worried due to long course of disease and high disability rate.
The pathogenesis of PD is not completely understood, and western medicine suggests that its pathogenesis may be related to genetic susceptibility, oxidative stress, mitochondrial defects, neuroimmune inflammation, neuroexcitotoxicity, apoptosis, and abnormal protein accumulation. Recent studies have found that the proportion of Prevotella in the intestine of PD patients is significantly reduced, and therefore scholars believe that PD is either onset or associated with gut dysbacteriosis.
The theory of traditional Chinese medicine holds that kidneys store essence and produce marrow, brain is the marrow sea, and the kidneys govern mind and thinking and drive physical movement. The marrow is transformed by kidney essence and qi and blood to nourish brain essence, while the kidney deficiency can lead to deficiency of brain marrow and brain, and malnutrition of brain orifices can lead to brain loss. PD is just such a Central Nervous System (CNS) disorder of limb motor dysfunction characterized primarily by tremor, myotonia, postural abnormalities, and the like. Since PD is prevalent in the middle-aged and elderly, high-frequency people are grouped at age 55 and above. From the perspective of traditional Chinese medicine, this age group is the stage of hypofunction of the zang-fu organs of the human body, especially the physiological feature of the kidney-middle essence developing from excess to deficiency.
Although there have been many advances in the study of parkinson's disease, there is a long way to get away from a complete cure. Therefore, there is a need for further studies on the pathogenesis and therapeutic approaches of PD, and a stable and efficient animal model is a prerequisite for further studies on the disease.
The existing PD animal model is mainly prepared by western medicine stimulation, such as: 6-hydroxydopamine (6-OHDA) model, paraquat model, MPTP model, rotenone model, etc.
The 6-OHDA model is one of the most applied models at present, the animal behavior change of the model is lasting and stable, the observation is facilitated, the quantitative detection can be carried out, and the model has more similarities with PD patients in physiology and pathology, but the main clinical manifestation and the change of physical characteristics of PD are not simulated to be a lewy body.
The herbicide paraquat used in the paraquat model can damage dopaminergic neurons in the substantia nigra striatum and promote functional degenerative change of the nervous system, but the exposure mode of the model is different from the action mode of actual environmental factors during modeling, so the model is still to be further researched.
The MPTP model is a 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropymetrozine (MPTP) model that induces PD symptoms and dopaminergic neuronal damage, but without the formation of lewy bodies.
Rotenone is a chemical substance extracted from derris elliptica of genus leguminous of family inebriidae, is an organic pesticide with the characteristics of high efficiency, low toxicity and short residual period, has strong lipid solubility, can penetrate through a blood brain barrier, inhibits mitochondrial complex enzyme I in brain cells, generates a large amount of oxygen free radicals and releases apoptosis inducing factors to initiate oxidative stress and apoptosis, and can generate Lewy corpuscle. The model rat may also exhibit flexion posture and dyskinesia in PD patients.
However, these models are obtained by injecting chemical drugs into young rats in a short period and strong stimulation, and only simulate the external symptoms after the onset of parkinson disease as much as possible, but cannot truly reflect the internal cause of the onset of parkinson disease, so that the pathogenesis of parkinson disease cannot be revealed. In addition, the diseased symptoms of the rats are unstable, and toxic reaction is easy to occur, which is not beneficial to practical application. Therefore, research and development of a Parkinson disease animal model which accords with the pathogenic characteristics of Parkinson and has stable and reliable symptoms are urgently needed.
Disclosure of Invention
The invention aims to solve the technical problems that the existing Parkinson rat model is unstable in symptoms, easy to generate toxic reaction, not beneficial to practical application and the like, and provides a reliable Parkinson animal model preparation method which accords with the Parkinson disease morbidity characteristics and is stable in symptoms. The invention selects the old rats, applies the method of overstrain and sexual disorder, establishes a kidney-yang deficiency animal model which completely follows the classic traditional Chinese medicine theory, and then is stimulated by western medicines, thereby obtaining the Parkinson's disease animal model with stable and reliable symptoms.
In order to solve the technical problems, the invention adopts the technical scheme that:
a preparation method of a kidney-yang deficiency type Parkinson disease rat model comprises the following steps:
step one, animal selection: selecting a plurality of male rats with 18-20 months of age, and selecting a plurality of female rats with the same 2-3 months of age;
step two, preparing a rat kidney-yang deficiency model:
2.1 grouping: dividing male mice into a kidney-yang deficiency group and a blank group according to a random principle;
2.2 model preparation: carrying out a fatigue swimming test on rats in a kidney-yang deficiency group every morning; performing the same-cage experiment on the rats and the female rats in the group with forced kidney-yang deficiency every afternoon, performing the same cage experiment on 1 male rat and 3 female rats in estrus, and taking out the male rat in the next morning; alternately rotating the fatigue swimming experiment and the same cage experiment for 2-4 weeks; feeding the rats in the blank group normally;
2.3 evaluation of rat Kidney Yang deficiency model: carrying out appearance observation and diet and defecation monitoring on rats in the kidney-yang deficiency group and the blank group, comparing the two groups, and carrying out comprehensive scoring evaluation on the rat kidney-yang deficiency model to achieve 70% of the total score, namely successful modeling; the specific evaluation method is as follows:
2.3.1 appearance observation: observing the glossiness of fur, hair erection phenomenon, intolerance of cold or not, liking of crowding together, response sensitivity, autonomous activity condition, shock resistance, shrinkage of scrotum and increase of defecation frequency of two groups of rats; and comparing the kidney-yang deficiency group with the rats in the blank group, scoring for 5 points if one of the indexes appears, or scoring for 0 point if the other index appears, and counting the accumulated scores of 10 indexes in the table 1, wherein the full score is 50 points.
TABLE 1 evaluation chart for kidney yang deficiency
2.3.2 diet and fecal monitoring:
monitoring 24h water intake/weight, 24h food intake/weight and 2h fecal water content indexes of two groups of experimental rats, comparing a kidney-yang deficiency group with a blank group of rats, and scoring according to the change degree of each index, wherein 1 is recorded for every 3% increase of the 24h water intake/weight, and the like, and capping is carried out for 15 minutes; recording 1 minute for every 2 percent reduction of the food consumption/body weight 24, and sealing the top by analogy with 15 minutes; recording 1 point for every 2 percent increase of the water content of the excrement after 2 hours, and sealing the top by repeating 20 points in the same way; the full score is 50 and the scoring results are shown in Table 2.
TABLE 2 Drinking, eating and fecal water content monitoring table
And (4) scoring according to the appearance observation index and the diet and excrement monitoring index, wherein the total score is more than 70 points, which indicates that the kidney-yang deficiency model modeling is completed and can participate in the next model preparation.
Step three, establishing a rat PD model:
3.1 grouping: dividing the successfully modelled kidney-yang deficiency rats into three groups, namely a PD model group, a kidney-yang deficiency control group and a positive drug control group;
3.2 model preparation: the PD model group is given rotenone with the final injection concentration of 0.5mg/mL every day, 1mL/kg every time, the kidney-yang deficiency control group is given a blank solvent with the same injection amount, the positive drug control group is given rotenone with the final injection concentration of 0.5mg/mL, and is supplemented with levodopa with the final injection concentration of 2.5mg/mL, 1mL/kg every time; all three groups were stopped 7 days after injection;
preferably, the solvent of rotenone in the preparation of the model in the third step is dimethyl sulfoxide and normal saline in a volume ratio of 7:1, and (b) preparing the mixed solvent.
Step four, evaluating a rat PD model:
and (3) evaluating the three groups of modeled experimental rats in five aspects of appearance observation, ethological detection, serum alpha-syn content detection, HE (human embryonic development) staining analysis of substantia nigra and striatum tissues and TH (TH) positive immunohistochemical detection of the substantia nigra and striatum tissues. Besides appearance observation, any three other tests are needed, namely, the molding is successful. And after the appearance observation and the behavioral testing experiment are finished, collecting serum and tissue samples for the three subsequent tests. The specific evaluation process is as follows:
4.1 appearance observation:
the evaluation is a must-pass item, and if the evaluation does not pass, the molding fails. If the evaluation passes, other evaluation experiments can be continued. Taking the kidney-yang deficiency control group as reference, observing whether the rats in the PD model group have symptoms of slow reaction, inflexible movement, uncoordinated movement, difficult raising of head and inflexible hind legs;
the number of terms showing any symptom in each rat is counted, and four or more terms appear, so that the evaluation can be carried out. The results are shown in Table 3.
TABLE 3 evaluation table for PD shape observation
4.2 behavioral testing:
the water maze experiment can detect the learning, memory and cognitive abilities of rats and is an important basis for observing whether the rats have the Parkinson's disease movement balance disorder. The specific method comprises the following steps:
(1) Training experiment: carrying out continuous water maze training experiments on experimental rats for 6 days, 2 times a day, 1min each time, keeping the water temperature at about 24 ℃, putting the heads of the rats into water by the same person at each time towards the wall of a pool, randomly marking the heads of the rats in east, west, south and north directions by using paperboard with different colors, recording the time for the rats to find an underwater platform, recording the time for the rats to find the platform and stay for 20s, and guiding the rats to the platform and stay for 20s if the time is not found;
(2) Formal experiments: on the 7 th day of the water maze experiment, removing the platform, putting the rat into the pool from the same position, and recording the times of the rat passing through the original platform position and the residence time in the quadrant of the platform within 3 min;
(3) Evaluation: and (3) performing statistical analysis on the two recorded data, namely the times of passing through the original platform position and the residence time in the first quadrant, and comparing the two values of the PD model group with a kidney-yang deficiency control group, if the two values are reduced and the difference of the two groups of data has statistical significance, judging that the evaluation is passed. The experimental data of the positive drug control group was used as a validation index. If both values are reduced and the difference between the two data is statistically significant, the PD model group passes the verification, otherwise the evaluation fails.
And after appearance observation and a behavior detection experiment are finished, collecting serum and tissue samples for the later three detections.
4.3 serum alpha-syn content detection
The main pathological changes of PD are degeneration death and loss of midbrain substantia nigra and striatum Dopamine (DA) energy neurons and abnormal aggregation of alpha-syn, so that whether the Parkinson disease occurs can be judged by detecting the content of the alpha-syn in rat serum.
The specific detection method comprises the following steps:
anesthetizing a rat, taking blood from a vein, standing the blood for 30min, keeping the blood at 4 ℃,3500r/min, centrifuging for 10min, separating supernatant into serum, and storing the serum at 4 ℃ for later use. Measuring the content of alpha-syn protein in the serum of three groups of experimental rats by adopting an Elisa kit; and then carrying out statistical analysis on the experimental data, judging whether the content of the serum alpha-syn is increased or not in the PD model group compared with the kidney-yang deficiency control group, and if the content is increased and the data has statistical significance, judging that the evaluation is passed. The experimental data of the positive drug control group was used as a validation index. And comparing the PD model group with the positive drug control group, judging whether the content of the serum alpha-syn is increased, if so, passing the verification, and otherwise, failing to evaluate.
4.4 HE staining analysis of substantia nigra and striatum tissues
The pathological features of PD patients are represented by the selective loss of substantia nigra and striatal energy neurons in the brain and the appearance of alpha-syn inclusion bodies in the residual neuronal cells. Alpha-syn is a small soluble monomeric protein consisting of 140 amino acids, normally present primarily in the presynaptic terminal of neurons, and is used for the transport, recirculation and promotion of exocytosis fusion pores of synaptic vesicles; however, under disease conditions, α -syn misfolds to form insoluble β -sheet rich toxic aggregates that are deposited on neurons.
The analysis method comprises the following steps: pathological sections are prepared on the substantia nigra and striatum tissues of an experimental rat, then a kidney-yang deficiency control group is used as a reference, and the number and the structure of neuron morphologies of the substantia nigra and striatum tissues of the rat of a PD model group are observed under a microscope to determine whether the neuron morphologies are abnormal, the nuclear and cytoplasmic boundary is fuzzy, the cell staining is deepened, and obvious inflammation is generated or not, and the evaluation is passed when the three or more indications appear. The experimental data of the positive drug control group was used as a validation index.
4.5 positive immunohistochemical detection of TH in substantia nigra and striatal tissues
The sites of PD attack are mainly in the substantia nigra and striatum. Tyrosine Hydroxylase (TH) in substantia nigra and striatum is a specific marker of dopaminergic neurons, and the TH positive number of substantia nigra and striatum of midbrain can represent the number of dopaminergic neurons, thereby reflecting whether parkinsonism appears.
The detection method comprises the following steps: pathological sections are made on the substantia nigra and striatum tissues of experimental rats, an Eclipse Ci-L photographing microscope is used for selecting a target area of brain tissues for imaging by 200 times, tissues are filled in the whole visual field as much as possible during imaging, and the consistency of background light of each picture is ensured. After the imaging was completed, the number of TH positive neurons in the substantia nigra and striatal tissues of each group of rats was judged by performing an analysis process using Image-Pro Plus 6.0 analysis software and calculating the areal density. And (4) judging whether the TH surface density in the substantia nigra and striatum tissues of the rats in the PD model group is obviously reduced or not by taking the kidney-yang deficiency control group as reference, and if so, judging that the TH surface density is passed. The experimental data of the positive drug control group was used as a validation index.
The invention also provides a rat model of the kidney-yang deficiency type Parkinson disease, which is prepared by adopting the method.
The invention has the beneficial effects that:
1. the invention provides a rat model which accords with the Parkinson disease occurrence characteristics, is simple and convenient to operate, has stable symptoms, is real and reliable and can truly simulate the Parkinson disease cause, thereby providing assistance for pathological research and drug research and development of Parkinson diseases.
2. The invention selects the old rats, applies the method of overstrain and sexual intercourse, establishes an animal model with kidney-yang deficiency completely following the classic traditional Chinese medicine theory through the fatigue swimming test and the same cage test, and then is stimulated by western medicines, thereby obtaining the animal model of the Parkinson's disease with stable and reliable symptoms. The internal cause of the Parkinson disease is truly simulated through the pathogenesis, the kidney-yang deficiency symptom is more truly reproduced, a true, stable and reliable simulation basis is provided for the next PD model establishment, the toxic reaction is not easy to occur in the simulation method, and the actual application is facilitated.
3. The evaluation mechanism is introduced, the evaluation is comprehensively carried out on the rat kidney-yang deficiency model through appearance observation and diet and defecation monitoring during the preparation of the kidney-yang deficiency model, the successfully modeled kidney-yang deficiency rats are accurately screened out, the unsuccessful rats are rejected, and the success rate of the next modeling is improved.
4. The invention also introduces an evaluation mechanism after PD modeling, and after a rat PD model is prepared, five aspects of evaluation of appearance observation, ethological detection, serum alpha-syn content detection, HE staining analysis of substantia nigra and striatum tissues and TH positive immunohistochemical detection of the substantia nigra and striatum tissues are respectively carried out; besides appearance observation, any three other tests are needed, namely, the molding is successful. The evaluation index can accurately evaluate the PD model, comprehensively evaluates from aspects such as appearance, behavior, blood index and the like, and has high accuracy and more comprehensive evaluation.
Drawings
FIG. 1 is a representation of pathological sections of rat substantia nigra and striatum tissues (HE staining) from various groups during HE staining analysis of substantia nigra and striatum tissues in an example of the present invention.
FIG. 2 is a graph showing pathological sections (TH positive test) of substantia nigra and striatum tissues of rats in each group during TH positive immunohistochemical detection of the substantia nigra and striatum tissues in the example of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and examples. However, it will be appreciated by those of ordinary skill in the art that numerous technical details are set forth in the embodiments of the present invention in order to provide a better understanding of the present application. However, the technical solutions claimed in the claims of the present application can be implemented without these technical details and with various changes and modifications based on the following embodiments.
The preparation method of the rat model of the Parkinson disease in the embodiment comprises the following steps:
step one, animal selection: 30 SD male rats with the age of 18-20 months are selected, the weight of the SD male rats is about 400g, and 45 female rats with the same age of 2-3 months are selected, and the weight of the SD male rats is about 400 g.
Step two, preparing a rat kidney-yang deficiency model:
2.1 grouping: dividing SD male mice into kidney-yang deficiency group and blank group according to random principle, wherein each group comprises 15 mice;
2.2 model preparation: performing fatigue swimming test on 15 rats in the group with kidney yang deficiency in the morning every day, namely putting the rats in a plastic barrel with the diameter of 100cm, the water depth of 60cm and the water temperature of 30 ℃ for swimming for 30 minutes each time until the rats are found to be powerless to continue swimming, and quickly fishing out the rats when the rats sink;
performing the same-cage experiment on the rats and the female rats in the group with forced kidney-yang deficiency every afternoon, performing the same cage experiment on 1 male rat and 3 female rats in estrus, and taking out the male rat in the next morning;
alternately rotating the fatigue swimming experiment and the same cage experiment, and continuously carrying out 2-4 weeks;
feeding the rats in the blank group normally;
2.3 evaluation of rat Kidney Yang deficiency model: because the model is carried out step by step, in the first-step evaluation of the kidney-yang deficiency model, a treatment method of overexposure to rats such as blood sampling detection and the like cannot be adopted, only the appearance observation and diet and defecation monitoring are carried out on the rats in the kidney-yang deficiency group and the rats in the blank group, the two groups are compared, the comprehensive scoring evaluation is carried out on the rat kidney-yang deficiency model, the total score is more than 70, and the completion of the model building of the kidney-yang deficiency model is indicated. The specific evaluation method is as follows:
2.3.1 appearance observation: observing the glossiness of fur, hair erection phenomenon, whether the rats are intolerant of cold or not, whether the rats like to be crowded together or not, reaction sensitivity, autonomous activity condition, anti-shock capability, whether scrotum is shrunk or not and whether the frequency of urination and defecation is increased or not; 15 rats in the kidney-yang deficiency group are compared with rats in the blank group, and once one of the indexes is found, the rate is 5, otherwise, the rate is 0, and the indexes are counted in the table 1, the cumulative score of 10 indexes is calculated, and the full score is 50.
TABLE 1 evaluation chart for kidney yang deficiency
2.3.2 diet and fecal monitoring:
monitoring 24h water intake/weight, 24h food intake/weight and 2h fecal water content indexes of two groups of experimental rats, calculating the average value of three indexes of 15 blank groups of rats, comparing the average value of 15 rats in a kidney-yang deficiency group with the average value of the blank groups of rats, and scoring according to the change degree of each index, wherein 1 point is marked for every 3 percent increase of the 24h water intake/weight, and the like, 15 points are used for capping; recording 1 minute for every 2 percent reduction of the food consumption/body weight 24, and sealing the top by analogy with 15 minutes; recording 1 point for every 2 percent increase of the water content of the excrement after 2 hours, and sealing the top by repeating 20 points in the same way; the score of the patients with full score of 50 was scored for each kidney yang deficiency group and shown in Table 2.
TABLE 2 Water content monitoring table for drinking water, diet and feces
The 15 rats with kidney-yang deficiency in this example were scored comprehensively according to appearance observation, diet and fecal monitoring, and the results are shown in table 3:
TABLE 3 general score chart for appearance observation and diet and defecation monitoring
As can be seen from table 3, the total score of 12 rats in the group with kidney-yang deficiency was greater than 75 minutes, which indicates that the model building of the kidney-yang deficiency model was successful and the next drug stimulation could be performed, and the total score of the other three rats was less than 75 minutes and did not participate in the next model building, and the success rate of model building of the rats with kidney-yang deficiency was 80%.
Step three, establishing a rat PD model:
3.1 grouping: dividing 12 successfully modelled rats with kidney yang deficiency into three groups, namely a PD model group, a kidney yang deficiency control group and a positive drug control group, wherein each group comprises 4 rats;
3.2 model preparation: the PD model group is administered with rotenone with the final concentration of 0.5mg/mL per day injection, 1mL/kg per time, and the solvent of the rotenone is dimethyl sulfoxide and normal saline according to the volume ratio of 7:1, and (b) preparing the mixed solvent. The control group with kidney yang deficiency is given injection of equal amount of blank solvent, namely dimethyl sulfoxide and normal saline according to the volume ratio of 7:1, and (b) preparing the mixed solvent. The positive drug control group is given with rotenone with the final injection concentration of 0.5mg/mL, and is also supplemented with levodopa with the final injection concentration of 2.5mg/mL, 1mL/kg each time; all three groups were stopped 7 days after injection;
step four, evaluating a rat PD model:
and (3) sequentially evaluating five aspects of appearance observation, ethological detection, serum alpha-syn content detection, HE staining analysis of substantia nigra and striatum tissues and TH positive immunohistochemical detection of the substantia nigra and striatum tissues of the three groups of experimental rats after the modeling by taking the kidney-yang deficiency control group as reference. Besides the appearance observation, any three other tests are required, namely the molding is successful. And after appearance observation and a behavior detection experiment are finished, collecting serum and tissue samples for the later three detections. The specific evaluation process is as follows:
4.1 appearance observation: the evaluation is a must-pass item, if the evaluation is not passed, the molding fails, and if the evaluation is passed, the other four evaluation experiments can be continued. Taking the kidney-yang deficiency control group as reference, observing whether the rats in the PD model group have symptoms of slow reaction, inflexible movement, uncoordinated movement, difficult raising of head and inflexible hind legs;
the number of terms showing any symptom in each rat is counted, and four or more terms appear, so that the evaluation can be carried out. The results are shown in Table 4.
TABLE 4 evaluation Table for PD profile observation
Compared with the kidney-yang deficiency control group, the PD model group has 4 rats with at least four symptoms such as slow response, inflexible movement, uncoordinated movement, difficult raising of head, inflexible hind legs and the like, while the positive drug control group has little or no symptoms, so the appearance observation evaluation is passed and the next evaluation is performed.
4.2 behavioral testing:
the water maze experiment can detect the learning, memory and cognitive abilities of rats and is an important basis for observing whether the rats have the Parkinson's disease movement balance disorder. The specific method comprises the following steps:
(1) Training experiment: carrying out continuous water maze training experiments on experimental rats for 6 days, 2 times a day, 1min each time, keeping the water temperature at about 24 ℃, putting the heads of the rats into water by the same person at each time towards the wall of a pool, randomly marking the heads of the rats in east, west, south and north directions by using paperboard with different colors, recording the time for the rats to find an underwater platform, recording the time for the rats to find the platform and stay for 20s, and guiding the rats to the platform and stay for 20s if the time is not found;
(2) Formal experiment: on the 7 th day of the water maze experiment, the platform is removed, the rat is placed into the pool from the same position, and the times of the rat passing through the original platform position and the residence time in the quadrant of the placement platform within 3min are recorded;
(3) Evaluation: the two recorded data, i.e. the number of times of passing through the original platform position and the residence time in the first quadrant, were statistically analyzed, and the results are shown in table 5. Compared with the control group with kidney-yang deficiency, the PD model group has reduced values of the two items, and the difference of the two groups of data has statistical significance (P < 0.01). The two values of the rats in the PD model group are reduced compared with those in the positive drug control group, and the difference of the two groups of data has statistical significance (P < 0.05), and the evaluation is passed. Meanwhile, compared with a positive drug control group, the two values of the PD model group and the positive drug control group are both reduced, and the difference of the two groups of data has statistical significance and passes the verification. The behavioral test evaluation was passed, and the next evaluation was performed.
TABLE 5 number of times three groups of rats passed the original platform and residence time in the first quadrant
4.3 serum alpha-syn content detection
The main pathological changes of PD are degeneration death and loss of midbrain substantia nigra and striatum Dopamine (DA) energy neurons and abnormal aggregation of alpha-syn, so that whether the Parkinson disease occurs can be judged by detecting the content of the alpha-syn in rat serum.
The specific detection method comprises the following steps:
anesthetizing a rat, taking blood from a vein, standing the blood for 30min, centrifuging for 10min at 3500r/min at 4 ℃, separating supernatant into serum, and storing at 4 ℃ for later use. Measuring the content of alpha-syn protein in the serum of three groups of experimental rats by adopting an Elisa kit; and then carrying out statistical analysis on the experimental data, judging whether the content of the serum alpha-syn is increased or not in the PD model group compared with the kidney-yang deficiency control group, and if the content is increased and the data has statistical significance, judging that the evaluation is passed. The experimental data of the positive drug control group were used as a validation index and the results are shown in table 6. And (4) comparing the PD model group with the positive drug control group, judging whether the content of the serum alpha-syn is increased or not, and if the content is increased and the data has statistical significance, passing the verification. The experimental results are as follows:
TABLE 6 serum alpha-syn levels in three groups of rats
As shown in Table 6, compared with the kidney-yang deficiency control group, the content of the alpha-syn in the serum of the PD model group is remarkably increased (P is less than 0.01), and the statistical significance is achieved; compared with a positive drug control group, the content of the alpha-syn in the serum of the PD model group is obviously increased (P < 0.05), and the statistical significance is achieved. Thus, serum alpha-syn content was assessed.
4.4 HE staining analysis of substantia nigra and striatum tissues
The pathological features of PD patients are represented by the selective loss of substantia nigra and striatal energy neurons in the brain and the appearance of alpha-syn inclusion bodies in the residual neuronal cells. Alpha-syn is a small soluble monomeric protein consisting of 140 amino acids, normally present primarily in the presynaptic terminal of neurons, and is used for the transport, recirculation and promotion of exocytosis fusion pores of synaptic vesicles; however, under disease conditions, α -syn misfolds to form insoluble β -sheet rich toxic aggregates that are deposited on neurons.
The analysis method comprises the following steps: pathological sections are made on the substantia nigra and striatum tissues of an experimental rat, and then the substantia nigra and striatum tissues of a PD model group rat are observed under a microscope by taking a kidney-yang deficiency control group as a reference, whether the symptoms such as neuron number reduction, neuron shrinkage, cell staining deepening, cytoplasm boundary unclear nucleus, neuron vacuolation and the like exist, and the three or more indexes appear, and the evaluation passes.
The experimental results are as follows:
as shown in FIG. 1, the rats in the kidney-yang deficiency control group have abundant black neurons, normal neuronal morphological structure, clear nuclear and cytoplasmic boundary, obvious nucleoli and no obvious inflammation. The striatum neurons are abundant and regularly arranged. Compared with a kidney-yang deficiency control group, the PD model group rat has the advantages that a small amount of neurons shrink in substantia nigra, the cell staining is deepened, the cytoplasm of nucleus is unclear, a small amount of neurons vacuole, a single neuron shrink locally in striatum, the cell staining is deepened, the cytoplasm of nucleus is unclear, five indications appear, and the evaluation is passed.
Compared with the PD model group, the positive drug control group has rich neurons, regular arrangement, normal neuron morphological structure, clear nucleus cytoplasm boundary and obvious nucleolus, and passes the verification.
In this embodiment, three groups of experimental rats subjected to modeling are subjected to appearance observation, ethology detection, serum alpha-syn content detection and HE (black and striatum tissue) staining analysis in sequence, wherein the appearance observation evaluation passes through the ethology detection, the serum alpha-syn content detection and the HE staining analysis of the black and striatum tissues, which indicates that 4 rats in the experiment all show Parkinson-like performance, the modeling experiment is successful without performing a fifth TH (positive immunohistochemical) detection of the black and striatum tissues, and the success rate of the modeling is 80%.
In another embodiment, in the evaluation of the four-rat PD model in step, the three groups of modeled experimental rats are subjected to shape observation in sequence, and after the shape observation evaluation passes, the behavioral detection, the serum α -syn content detection, and the black and striatal tissue HE staining analysis are performed in sequence, where the behavioral detection fails, and the other two passes, and then the positive immunohistochemical detection of the black and striatal tissue TH is required in the next step, and the specific detection process is as follows:
4.5 positive immunohistochemical detection of TH in substantia nigra and striatal tissues
The sites of PD attack are mainly in the substantia nigra and striatum. Tyrosine Hydroxylase (TH) in substantia nigra and striatum is a specific marker of dopaminergic neurons, and the TH positive number of substantia nigra and striatum of midbrain can represent the number of dopaminergic neurons, thereby reflecting whether parkinsonism appears.
The detection method comprises the following steps: after the sample to be detected is fixed by 4% paraformaldehyde and the fixed state is good, trimming, dehydrating, embedding, slicing, dyeing, sealing and finally microscopic examination of the qualified sample are carried out strictly according to the SOP program of pathological experiment detection. And (3) selecting a target area of the brain tissue by using an Eclipse Ci-L photographing microscope to perform 200-time imaging, wherein the tissue is filled in the whole visual field as much as possible during imaging, and the consistency of background light of each picture is ensured. As shown in fig. 2, after completion of imaging, analysis was performed using Image-Pro Plus 6.0 analysis software, and the change in the number of TH positive neurons in each group of rats was determined by calculating the area density. The experimental results are as follows:
TABLE 7 immunohistochemical detection results of substantia nigra and striatum TH of three groups of rats
As can be seen from Table 7: the results of rat substantia nigra and striatum TH immunohistochemical detection show that compared with a kidney-yang deficiency control group, TH surface density in two tissues of a PD model group is obviously reduced, and the significant difference P is less than 0.05. Compared with the positive drug control group, the TH surface density of two tissues of the PD model group of the kidney-yang deficiency control group is obviously reduced, and the significant difference P is less than 0.05, so the number of TH positive neurons of the PD model group rats is obviously reduced in the two tissues.
Therefore, after five evaluations of appearance observation, ethological detection, serum alpha-syn content detection, HE staining analysis of the substantia nigra and striatum tissues and TH positive immunohistochemical detection of the substantia nigra and striatum tissues, the model prepared in the embodiment passes through three index evaluations besides the appearance observation, which indicates that the modeling experiment is successful.
Claims (6)
1. A preparation method of a rat model of Parkinson's disease with kidney-yang deficiency is characterized by comprising the following steps:
step one, animal selection: selecting a plurality of male rats with 18-20 months of age, and selecting a plurality of female rats with the same 2-3 months of age;
step two, preparing a rat kidney-yang deficiency model:
2.1 grouping: dividing male mice into a kidney-yang deficiency group and a blank group according to a random principle;
2.2 model preparation: carrying out fatigue swimming test on rats with kidney-yang deficiency in morning every day; performing the same-cage experiment on the rats and the female rats in the group with forced kidney-yang deficiency every afternoon, performing the same cage experiment on 1 male rat and 3 female rats in estrus, and taking out the male rat in the next morning; alternately rotating the fatigue swimming experiment and the same cage experiment, and continuously carrying out 2-4 weeks; feeding the rats in the blank group normally;
2.3 evaluation of rat Kidney Yang deficiency model: by carrying out appearance observation and diet and defecation monitoring on rats in the kidney-yang deficiency group and the blank group, comparing the two groups, and carrying out comprehensive scoring evaluation on the rat kidney-yang deficiency model, wherein 70% of the total score is reached, namely the model is successfully constructed, and the model can participate in the preparation of the next model; the appearance observation refers to observing the glossiness of fur, the phenomenon of fur erection, whether the fur is intolerant to cold or not, whether the fur is like to be crowded together or not, the response sensitivity, the autonomous activity condition, the anti-shock capability, whether scrotum is shriveled or not and whether the frequency of urination and defecation is increased or not of two groups of rats; the diet and defecation monitoring refers to monitoring 24-hour water intake/weight, 24-hour food intake/weight and 2-hour excrement water content indexes of two groups of experimental rats;
step three, establishing a rat PD model:
3.1 grouping: dividing the successfully modelled kidney-yang-deficient rats into three groups, namely a PD model group, a kidney-yang-deficient control group and a positive drug control group;
3.2 model preparation: the PD model group is given rotenone with the final injection concentration of 0.5mg/mL every day, 1mL/kg every time, the kidney-yang deficiency control group is given a blank solvent with the same injection amount, the positive drug control group is given rotenone with the final injection concentration of 0.5mg/mL, and is supplemented with levodopa with the final injection concentration of 2.5mg/mL, 1mL/kg every time; all three groups were stopped 7 days after injection;
step four, evaluating a rat PD model:
the three groups of experimental rats after model building are evaluated in five aspects of appearance observation, ethology detection, serum alpha-syn content detection, HE staining analysis of substantia nigra and striatum tissues and TH positive immunohistochemical detection of the substantia nigra and striatum tissues; besides appearance observation, any three other tests are required, namely mold making is successful;
the appearance observation refers to observing whether the rats in the PD model group have symptoms of slow response, inflexible movement, uncoordinated movement, difficult raising of the head and inflexible hind legs;
the behavioral testing refers to testing the learning, memory and cognitive abilities of the rat through a water maze experiment;
the serum alpha-syn content detection is to adopt an Elisa kit to determine the alpha-syn protein content in the serum of three groups of experimental rats; then carrying out statistical analysis on the experimental data, judging whether the alpha-syn content of the serum is increased or not in the PD model group compared with the kidney-yang deficiency control group, and if the alpha-syn content is increased and the data has statistical significance, judging that the evaluation is passed;
the HE staining analysis of the substantia nigra and striatum tissues is to prepare pathological sections of the substantia nigra and striatum tissues of an experimental rat, and then observe whether the neuron number and the structure of the substantia nigra and striatum tissues of the rat of a PD model group are abnormal, the nucleus and cytoplasm boundary is fuzzy, the cell staining is deepened, and obvious inflammation exists under a microscope by taking a kidney-yang deficiency control group as a reference, and the evaluation is passed when the three or more indications appear;
the TH positive immunohistochemical detection of the substantia nigra and striatum tissues is to prepare pathological sections of the substantia nigra and striatum tissues of experimental rats, then use an Eclipse Ci-L photographing microscope to Image the tissues, use Image-Pro Plus 6.0 analysis software to analyze and process after imaging, and calculate the surface density, thereby judging the change of the number of TH positive neurons in the substantia nigra and striatum tissues of each group of rats; and (4) judging whether the TH surface density in the substantia nigra and striatum tissues of the rats of the PD model group is obviously reduced or not by taking the kidney-yang deficiency control group as reference, and if so, evaluating to pass.
2. The method for preparing a rat model of Parkinson's disease due to kidney-yang deficiency according to claim 1, wherein the method comprises the following steps: and in the second step, when the rat kidney-yang deficiency model is evaluated, comparing the rat in the kidney-yang deficiency group with the rat in the blank group through appearance observation, and if one of the indexes appears, marking the score for 5, otherwise, marking the score for 0, and performing cumulative scoring on 10 indexes to obtain the full score of 50.
3. The method for preparing a rat model of Parkinson's disease due to kidney-yang deficiency according to claim 1, wherein the method comprises the following steps: when the rat kidney-yang deficiency model is evaluated in the second step, the kidney-yang deficiency group is compared with a blank group of rats through diet and defecation monitoring, and the rats are scored according to the change degree of each index, wherein 1 is counted for each 3% increase of the water intake/body weight of 24 hours, and the rest is done for 15 minutes for capping; 24, recording 1 point for every 2 percent of the food consumption/weight reduction, and sealing by analogy with 15 points; recording 1 minute when the water content of the excrement increases by 2 percent every 2 hours, and sealing the top by repeating the steps for 20 minutes in this way; full 50.
4. The method for preparing a rat model of Parkinson's disease due to kidney-yang deficiency according to claim 1, wherein the method comprises the following steps: during the preparation of the model in the third step, the solvent of rotenone is dimethyl sulfoxide and normal saline according to the volume ratio of 7:1, and (b) preparing the mixed solvent.
5. The method for preparing a rat model of Parkinson's disease due to kidney-yang deficiency according to claim 1, wherein the method comprises the following steps: and in the fourth step, when the PD model of the rat is evaluated, comparing the PD model group with the rat of the kidney-yang deficiency control group through appearance observation, counting the number of terms of any symptom appearing in each rat, and performing the next evaluation if four or more terms appear, or not performing the next step, wherein the index is an exclusiveness index, and the next step can be performed only if the condition of the index is satisfied.
6. The method for preparing a rat model of Parkinson's disease due to kidney-yang deficiency according to claim 1, wherein the method comprises the following steps: in the fourth step, when the rat PD model is evaluated, the adopted ethological detection method specifically comprises the following steps:
(1) Training experiment: carrying out continuous water maze training experiments on experimental rats for 6 days, 2 times a day, 1min each time, keeping the water temperature at about 24 ℃, putting the heads of the rats into water by the same person at each time towards the wall of a pool, randomly marking the heads of the rats in east, west, south and north directions by using paperboard with different colors, recording the time for the rats to find an underwater platform, recording the time for the rats to find the platform and stay for 20s, and guiding the rats to the platform and stay for 20s if the time is not found;
(2) Formal experiments: on the 7 th day of the water maze experiment, removing the platform, putting the rat into the pool from the same position, and recording the times of the rat passing through the original platform position and the residence time in the quadrant of the platform within 3 min;
(3) Evaluation: performing statistical analysis on the two recorded data, namely the times of passing through the original platform position and the residence time in the first quadrant, comparing the PD model group with a kidney-yang deficiency control group, and if the two numerical values are reduced and the difference of the two groups of data has statistical significance, the evaluation is passed; experimental data of the positive drug control group was used as a validation index; and comparing the PD model group with the positive drug control group, if the two values are reduced and the difference of the two groups of data has statistical significance, the evaluation is passed, otherwise, the evaluation is failed.
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