CN112931397B

CN112931397B - Construction method of parkinsonism animal model

Info

Publication number: CN112931397B
Application number: CN202110096076.9A
Authority: CN
Inventors: 申延琴; 石芸; 崔春; 乔晨萌; 赵炜疆; 黄术兵; 贾雪冰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2024-02-27
Anticipated expiration: 2041-01-25
Also published as: CN112931397A

Abstract

The invention discloses a construction method of an animal model for parkinsonism, and belongs to the technical field of animal model construction. The method is to use 5-HT ₄ The receptor antagonist is administered to the animal in combination with an environmental neurotoxin, said combination being administered at 5-HT ₄ 3 days after administration of the receptor antagonist, the environmental neurotoxin is administered on day 4 and the 5-HT is administered on day 5 and 8 consecutive days ₄ Receptor antagonists. The animal model prepared by the method of the invention better mimics the characteristics of PD patients, including brain lesions and intestinal tract changes. Can be used for screening medicaments for delaying and treating parkinsonism, and has good application prospect.

Description

Construction method of parkinsonism animal model

Technical Field

The invention belongs to the technical field of animal model construction, and particularly relates to a construction method of an animal model for parkinsonism.

Background

Parkinson's Disease (PD) is the most common motor nervous system degenerative disease, the second most common neurodegenerative disease in the world, and the prevalence of elderly people 65 years and older is as high as 2-3%. The main lesions of the brain of PD patients are substantia nigra and striatum, wherein degeneration and death of dopaminergic neurons of substantia nigra compacta (DA) is a direct cause of dopamine reduction projected into striatum. The clinical manifestations are mainly as follows: resting tremor, bradykinesia, standing instability, postural reflex disorders, and non-motor symptoms. Non-motor symptoms include gastrointestinal dysfunction, cognitive dysfunction, mood disorders, and the like. These symptoms are present before the classical motor symptoms of parkinson's disease appear and may contribute to the occurrence of motor symptoms. At present, the pathogenesis of PD is not known.

In addition to transgenic knockout animal models for studying familial inherited PD, there are three main approaches currently accepted to model PD: firstly, a model made of Dopamine (DA) energy neurons of a rat substantia nigra-striata system is destroyed by injecting 6-hydroxydopamine (6-OHDA) into the substantia nigra striata system, and the model requires three-dimensional stereotactic injection, has higher technical operation requirements and has higher failure rate; secondly, the PD rats or mice are prepared by subcutaneous or gastric administration of rotenone, which has the disadvantage that rotenone, because of its lipophilicity, can enter all cells and is not selectively accumulated in DA energy neurons, animals are susceptible to PD-independent manifestations such as cardiovascular toxicity and nonspecific brain injury due to systemic toxic effects, and models have large individual variability; thirdly, primate models such as monkeys and the like and C57BL/6 mice PD models are prepared by intraperitoneal injection of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP), but the primate Parkinson disease model established by MPTP at present mainly takes an acute model as a main part, and cannot simulate the chronic progress of human Parkinson disease. And none of the three methods can accurately simulate the pathological process of parkinsonism, such as non-motor symptoms before the occurrence of common motor symptoms, such as gastrointestinal symptoms like constipation. The shortcomings of the above models have greatly limited the use of these models, and there is an urgent need in the art to develop more reliable animal models of parkinson's disease.

Common behavioural tests are open field experiments, roller experiments, swimming experiments, nest making experiments, pole climbing experiments, hanging experiments, holding rod balance experiments and the like, and the behavioural detection method is suitable for detecting animal dyskinesia, limb coordination functions and other aspects of dyskinesia.

Tyrosine hydroxylase (Tyrosine hydroxylase, TH) is the rate limiting enzyme for dopamine synthesis, and under the catalysis of tyrosine hydroxylase, the L-tyrosine in the cytoplasm of neurons is hydroxylated into L-dopa, and subsequently, the L-aromatic amino acid decarboxylase catalyzes the decarboxylation of L-DOPAC to form DA. Changes in Tyrosine Hydroxylase (TH) are closely related to the development of PD.

5-hydroxytryptamine (5-HT) is mainly involved in exercise and painModulation of the sense and autonomous functions. 5-HT must be mediated through the corresponding receptor to function. 5-HT ₄ The receptor is taken as one of the receptors, is an important therapeutic target of intestinal dyskinesia, can regulate gastrointestinal motility, and has research on 5-HT ₄ Receptor agonists can improve constipation in PD patients. In addition, 5-HT ₄ The receptor can regulate release of rat striatum DA, regulate neuronal apoptosis, regulate inflammatory response and regulate motor capacity, 5-HT ₄ Receptors are also involved in cognitive and mood regulation. Prompt for 5-HT ₄ The receptor may modulate PD non-motor symptoms.

At present, no research on inhibiting 5-HT by using chemical small molecules at home and abroad is available ₄ The activity of the receptor was further examined for the extent of the behavioral effect of the neurotoxic substance MPTP.

Disclosure of Invention

[ technical problem ]

The invention aims to solve the technical problem that the existing parkinsonism animal model can not accurately simulate the pathological process of parkinsonism, such as non-motor symptoms before common motor symptoms appear, such as gastrointestinal symptoms like constipation.

Technical scheme

The invention provides a preparation method of an animal model of parkinsonism, which adopts 5-HT ₄ The receptor antagonist is administered in combination with the environmental neurotoxin to an animal. The 5-HT ₄ The receptor antagonist is one or two of GR125487 and GR 113808. The environmental neurotoxin is one or more than two of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine, 6-hydroxydopamine and rotenone.

The co-administration is at 5-HT ₄ 3 days after administration of the receptor antagonist, the environmental neurotoxin is administered on day 4 and the 5-HT is administered on day 5 and 8 consecutive days ₄ Receptor antagonists. 5-HT ₄ The receptor antagonist was administered once a day and the environmental neurotoxin was administered four times a day with 2 hours intervals. The 5-HT ₄ The single dose of the receptor antagonist is 1-10mg/kg of mice, and the single dose of the environmental neurotoxin is 15-20mg/kg of mice. Optionally, the 5-HT ₄ The single dose of receptor antagonist was 1mg/kg of mice,the single dose of the environmental neurotoxin is 20mg/kg of mice.

Alternatively, the co-administration is at 5-HT ₄ After 3 days of administration of the receptor antagonist GR125487, the environmental neurotoxin administration is continued and the 5-HT is continued ₄ The receptor antagonist GR125487 was administered for 8 days.

The 5-HT ₄ The frequency of administration of the receptor antagonist was 1 time/day. The environmental neurotoxin was administered 4 times/day at 2 hour intervals for 1 day.

The 5-HT ₄ The total duration of administration of the receptor antagonist and the environmental neurotoxin is between 8 and 11 days.

The administration adopts an intraperitoneal injection mode. Further, upon intraperitoneal injection, the 5-HT ₄ The solvent of the injection of receptor antagonist and environmental neurotoxin is physiological saline.

The animal is a mouse or macaque, e.g., a C57BL/6 mouse.

The construction method further comprises the step of verifying the constructed parkinsonism animal model, and specifically comprises the following steps: kinematic assays, gastrointestinal transport assays, and assays for TH expression in striatum.

[ advantageous effects ]

Typically, there are gastrointestinal symptoms, such as constipation, prior to the onset of PD. In the method for constructing an animal model of Parkinson's disease, 5-HT4R antagonists are administered to simulate pathological symptoms of the gastrointestinal tract before MPTP mimics appear in the brain pathology. The invention provides a method for treating 5-HT in animals ₄ The administration of the receptor antagonist in combination with the environmental neurotoxin results in an animal model of parkinson's disease. On one hand, the brain pathological changes can be simulated, and on the other hand, the non-exercise symptoms such as constipation and the like of PD patients before the typical exercise symptoms appear can be better simulated. Intestinal symptoms such as constipation cannot be simulated by other models.

Common motor symptoms of PD are resting tremor, myotonia, bradykinesia, abnormal gait, etc. According to the invention, the motor delay and muscle strength of the PD mice are researched by a pole climbing experiment and a suspension experiment to evaluate the motor ability of the mice. Suspension experiments are a method for measuring the muscular strength and balance of PD mice.

Drawings

Fig. 1: dosing experiments for each group of mice.

Fig. 2: experimental results graphs of the effects of body weight and food intake of each group in experimental example 1; (A): weight of the body; (B): food intake.

Fig. 3: experimental results of the kinematic effects of each group in experimental example 1; (A): performing pole climbing experiments; (B): hanging experiments.

Fig. 4: experimental results of gastrointestinal tract transport effect of each group in experimental example 1.

Fig. 5: experimental example 1 shows TH and GAPDH expression results in each group of striatum; (A): western blot experimental gel patterns of TH and GAPDH expressed in the striatum of each group; (B): the ratio of TH to GAPDH expressed in the striatum of each group.

Detailed Description

Example 1: animal model creation and grouping

Male C57BL/6J mice, 20-25g in body weight, 7 weeks old, were randomly divided into Saline groups (intraperitoneal injection of Saline), GR125487 groups (intraperitoneal injection of GR125487 and Saline), MPTP groups (intraperitoneal injection of Saline and MPTP), GR 125487+MPTP groups (intraperitoneal injection of GR125487 and MPTP), 15 groups each.

Mice were placed at 23±2 ℃ for 12 hours/day of illumination to fit the environment for one week.

As shown in FIG. 1, the GR125487 group and the GR 125487+MPTP group were first intraperitoneally injected for three days with 1mg/kg GR125487, the Saline group and the MPTP group with physiological saline. Molding was performed on the fourth day (0 d in fig. 1), and the MPTP and GR 125487+MPTP groups were intraperitoneally injected with MPTP (20 mg/kg) four times in succession, each time at 2-hour intervals; saline was injected four times into Saline and GR125487 groups. The fifth to twelfth days, GR125487 and GR 125487+mptp groups were again given for continuous 8 days of intraperitoneal injections of GR 125487. The body weight changes and food intake changes of each group of mice were recorded during the experiment. The motor ability of the mice was then evaluated by pole-climbing and suspension experiments, gastrointestinal motility was evaluated by gastrointestinal transit time, and then the striatal tyrosine hydroxylase (Tyrosine hydroxylase, TH) content was detected by western blotting. The establishment of the model was evaluated by the above method.

1. Body weight and food intake

As shown in fig. 2 (a) and 2 (B), the body weight and the feeding amount of the mice suddenly decreased on the day of modeling.

2. Behavioural assessment

Each group of mice was trained three days before the administration of the behavioural test, once a day, and the behavioural test was performed on the last day of the administration (1 h after the end of the administration was started).

The climbing rod used by the climbing rod experimental mouse is 55cm in height, 1cm in diameter and 2cm in diameter, and is erected on a rectangular metal base. In the experiment, the tail of the mouse is pinched by an index finger and a thumb, so that the front two limbs of the mouse touch the metal ball at the top end of the climbing pole, timing is started when the two rear limbs touch the metal ball at the top end of the climbing pole, timing is stopped when the front limbs of the mouse touch the metal base, the time is the climbing pole time, 3 climbing pole tests are carried out on each mouse, and 15 minutes are separated each time. Mice were trained 3 days prior to formal testing, once daily on pole climbing for a total of 3 exercises.

The motor ability of the mice was tested using a pole-climbing experiment, as shown in fig. 3. The test results showed that the average pole-climbing times of mice in Saline group, GR125487 group, MPTP group, GR 125487+MPTP group were 3.678s, 4.156s, 5.067s, 6.725s, respectively. Compared with Saline group mice, MPTP group mice have the advantages that the time required for climbing poles is increased, and the movement speed is reduced. The results demonstrate that MPTP-injected mice have reduced locomotor activity. Mice in the GR 125487+mptp group required further more time and slower speed than the model group, with significant differences (p < 0.001) compared to the MPTP group. The results demonstrate that GR125487 can exacerbate motor capacity in parkinson's disease mice.

Hanging experiment device: two fixed metal rods are selected, and a metal wire is pulled between the two fixed metal rods, wherein the diameter of the metal wire is about 1mm. The wire must be tight and remain level with the ground at a distance of 30cm from the ground. In the experiment, the tail of the mouse is pinched by an index finger and a thumb, so that the metal wire is grabbed by the two forelimbs of the mouse, and the rear limbs of the mouse are naturally suspended downwards after the tail of the mouse is released. The score was 4 if the mouse had both hind limbs caught the wire, 3 if the single hind limb caught the wire, 2 if only both forelimbs caught the wire, 1 if the single forelimb caught the wire, and 0 if the mouse had neither forelimb caught the wire. Each mouse was subjected to a total of 3 suspension tests, 15min apart. Mice were trained 3 days prior to formal testing, with one suspension per day for a total of 3 exercises.

Mice were tested for motor ability using a suspension experiment, as shown in figure 3. The results showed that the average suspension scores of mice from Saline group, GR125487 group, MPTP group, GR 125487+MPTP group were 3.767, 3.700, 2.800, and 1.667, respectively. Compared with Saline group mice, MPTP group mice have low suspension score and reduced locomotor flexibility. The results demonstrate that MPTP-injected mice have reduced locomotor activity compared to the Saline group. Mice in the GR 125487+mptp group had lower suspension scores (p < 0.001) than those in the MPTP group, and were flexible. The experimental results demonstrate that GR125487 can exacerbate motor capacity in parkinson's disease mice.

3. Evaluation of gastrointestinal tract function

Each group of mice was subjected to intragastric 200 μl of purified water adaptation two days before intragastric 6% carmine sterile solution, once daily, and gastrointestinal transit time detection was performed on the last day of dosing.

Each mouse was perfused with 200 μl of a 6% sterile solution of carmine in 0.5% sterile solution of methylcellulose and the time T0 of the lavage was recorded. Mice were placed in new cages and observed for the discharge of red feces. The time to first discharge of red feces was recorded as T1. T1-T0 was calculated as the gastrointestinal transit time. As shown in FIG. 4, the gastrointestinal transit times of mice in Saline group, GR125487 group, MPTP group, GR 125487+MPTP group were 113.25min, 127.50min, 137.75min, 157.63min, respectively. The results indicate that GR125487 further increases the gastrointestinal transit time in MPTP mice, indicating even more insufficient gastrointestinal motility.

4. Striatal TH expression

Pathological tissue material selection: on the last day of injection, the experimental animals were anesthetized by inhalation with anesthetic (isoflurane), the striatal region was rapidly removed from the whole brain on ice, and stored at-80 ℃.

1. The reagents used for immunoblotting were as follows:

(1) 10% isolation gel (20 mL):

dd H ₂ O	30％Acr-Bis	1.5M Tris-HCl(pH 8.8)	10％SDS	10％APS	TEMED
						7.9mL	6.7mL	5.0mL	0.2mL	0.2mL	0.008mL

(2) 5% gum concentrate (6 mL):

dd H ₂ O	30％Acr-Bis	1.5M Tris-HCl(pH6.8)	10％SDS	10％APS	TEMED
						4.1mL	1.0mL	0.75mL	0.06mL	0.06mL	0.006mL

(3) Running buffer (1L):

glycine 14.4g

Tris 3.03g

SDS 1.0g

Add dd H ₂ O to 1L, and preserving at 4 ℃ for standby after uniformly mixing.

(4) Transfer buffer (1L):

glycine 14.4g

Tris 3.03g

Methanol 200mL

(5) TBS solution (10X) (1L): tris 24.2g,NaCl 80g,dd H ₂ O900 mL, then adjust pH to 7.5-7.6 with HCl, add dd H ₂ O to 1L and storing at 4 ℃ for later use.

(6) TBST solution (500 mL): 50mL of 10 XTBS solution, 0.5mL of Tween-20, and dd H were added ₂ O to 500mL, and preserving at room temperature for standby.

2. Western blotting detection of TH protein expression

1) Extraction of striatal total protein

Taking out striatum on one side of a mouse from a refrigerator at the temperature of minus 80 ℃ and weighing, adding 100 mu l of RIPA and 10 mu l of PMSF into each 10mg of tissue, firstly carrying out physical crushing by using a syringe, then further crushing the tissue by using a cup type ultrasonic cell crusher, keeping the state of an ice-water mixture in an instrument cavity all the time, timely supplementing crushed ice after the instrument generates and melts ice, after the tissue crushing is finished, centrifuging at the temperature of 13000rpm at the high speed of 4 ℃, carefully sucking the supernatant after centrifugation, subpackaging into a sterilizing centrifuge tube, and preserving at the temperature of minus 20 ℃ for later use.

2) Quantification of total protein in striatum (BCA kit)

(1) Preparing a BSA protein standard: after the standard was taken out from the-20℃refrigerator and left at room temperature until it was completely dissolved, 20. Mu.l of 5mg/mL of the standard was taken, 80. Mu.l of PBS solution was added thereto, and the final concentration was diluted to 1mg/mL.

(2) BSA standard assay solutions were formulated as follows:

(3) Mu.l of each sample to be tested (extracted striatal protein) was added to the corresponding well of the 96-well plate, followed by 17. Mu.l of PBS solution.

(4) BCA working solution preparation: the BCA dosage is calculated in advance according to the required hole number, the reagent A and the reagent B are mixed according to the volume ratio of 50:1 under the light-shielding condition, and the following steps are carried out under the light-shielding condition.

(5) 200 μl BCA working solution was added to the 96-well plate in the same order as that of the samples, the plate cover of the 96-well plate was covered and covered with a tinfoil paper to avoid light, and the reaction was carried out at 37℃for 30min.

(6) The absorbance of the sample was measured (562 nm) using a microplate reader and the reading was recorded.

(7) The absorbance of the standard and sample was obtained by subtracting the absorbance of the wells without BSA samples from the original absorbance of the standard and sample. And drawing a standard curve by taking the light absorption value obtained by calculation as an ordinate and the concentration of the BSA standard substance as an abscissa, and calculating the protein concentration according to the light absorption value.

3) Western blot experiments

(1) SDS-PAGE electrophoresis

(1) The 10% separating gel and the 5% concentrating gel were prepared separately according to the formulation shown in the experimental reagent formulation.

(2) The glued glass plate was loaded into an electrophoresis tank and 1X running buffer was added to it.

(3) Taking out the protein sample to be tested from the refrigerator at the temperature of minus 20 ℃, balancing by RIPA, adding a corresponding amount of loading buffer solution, and placing in a metal bath at the temperature of 100 ℃ for 15min.

(4) Samples were added sequentially to lanes at 40. Mu.g total protein per sample, and 4.5. Mu.l and 1.5. Mu.l protein maker were added to lanes at both ends of the sample.

(5) Electrophoresis conditions: electrophoresis was performed at a constant pressure of 80V for 20min, and then at a constant pressure of 120V until electrophoresis was completed.

(2) Transfer film

(1) PVDF membrane of the same size as the gel was cut out with scissors and activated with methanol for 1min, followed by dd H ₂ O was washed for 1min.

(2) After electrophoresis is completed, the separating gel is taken down by a tool, and is placed in the membrane transferring clamp from bottom to top in the order of black sponge-double-layer filter paper-separating gel-PVDF membrane-double-layer filter paper-black sponge, and air bubbles must be removed and clamped in the placing process.

(3) And placing the transfer film clamp in a transfer film groove, and adding 1X transfer film buffer solution to the scale shown on the transfer film groove. And placing an ice bag on one side of the film transferring groove with a larger gap, and changing the ice bag after 1h to prevent the temperature in the groove from being too high.

(4) Transfer conditions: the film transfer was performed at a constant current of 200mA for 2 hours.

(5) After the film transfer process is completed, PVDF film is taken out from the film transfer clamp by using tweezers and then is quickly put into sealing liquid, so that the PVDF film cannot be exposed to air for a long time.

(3) Blocking and incubation of antibodies

(1) Closing: a5% skim milk solution prepared from TBST solution was used as a blocking solution, which was poured into a blocking box until the PVDF membrane was completely immersed, and blocked on a shaker at room temperature for 2h.

(2) Incubating primary antibodies: washing the sealed PVDF membrane with TBST solution for 3 times for 5min each time, then cutting out strips with corresponding sizes of target proteins, adding antibody working solutions of mouse anti-GAPDH (1:8000) and mouse anti-TH (1:1000) into an antibody incubation bag respectively, completely immersing the strips, and incubating overnight at 4 ℃.

(3) Incubating a secondary antibody: after the primary incubation was completed, the strips were removed from the refrigerator at 4 ℃, TBST solution was poured into the incubation box, and the strips were repeatedly washed 3 times for 10min each. HRP-labeled secondary antibody working solution (1:10000) was added to the secondary antibody incubation cassette to completely submerge the strips and incubated for 2h at room temperature on a shaker. Subsequently, the secondary antibody working solution was discarded, TBST solution was added until the strip was gone, and washing was repeated 3 times for 10min each.

(4) Imaging: and under the condition of avoiding light, placing the strips into a gel imager, opening software, adjusting the positions of the strips in the instrument according to image display in the software to enable the strips to be in the center, then dripping luminous color development liquid on each strip, wherein each strip is about 200 mu l, standing for 30s, and then executing relevant programs of a gel imaging system to observe and save the images.

(5) Analysis: gray scale analysis was performed with Image J software.

4) Experimental results: the expression level of TH in striatum was analyzed by Western blot experiments, and the results showed that TH expression in MPTP mice was reduced by 50.2% compared to Saline mice (p < 0.001), and GR125487 further reduced TH expression in MPTP-induced PD mice (p < 0.05) (FIG. 5). The above results indicate that GR125487 is able to exacerbate the decrease in striatal TH expression in PD mice, but has no effect on normal mice.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for constructing an animal model of Parkinson's disease, comprising the steps of using 5-HT ₄ The receptor antagonist is administered in combination with the environmental neurotoxin to the animal; the 5-HT ₄ The receptor antagonist is GR125487. One or two of GR 113808; the environmental neurotoxin is one or two of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine, 6-hydroxydopamine and rotenone;

the combined administration is 5-HT ₄ Receptor antagonist administration is performed for 3 days, ambient neurotoxin administration is performed for the fourth day, and 5-HT is continued for the fifth day ₄ Receptor antagonist administration until day twelve; 5-HT ₄ The dosing frequency during the dosing period of the receptor antagonist was 1 time/day, the dosing frequency during the dosing period of the environmental neurotoxin was 4 times/day, each time 2 hours apart;

the method also comprises the step of verifying the constructed parkinsonism animal model, and specifically comprises the following steps: and detecting the kinematic experiments such as pole climbing, hanging and the like, detecting the gastrointestinal transit time and detecting the content change of tyrosine hydroxylase in the striatum of the brain.

2. The method of claim 1, wherein the 5-HT is ₄ The receptor antagonist is GR125487 and the environmental neurotoxin is 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine.

3. The method of claim 2, wherein 5-HT is used as a target ₄ The single dose of receptor antagonist GR125487 is 1-10mg/kg in mice and the single dose of environmental neurotoxin is 15-20mg/kg in mice.

4. A method of constructing as claimed in any one of claims 1 to 3 wherein said administration is by intraperitoneal injection.

5. A method of construction according to any one of claims 1 to 3, wherein the animal is cynomolgus monkey or C57BL/6 mouse.

6. Use of an animal model of parkinson's disease constructed by the method of any one of claims 1-3 for screening or preparing a medicament for treating parkinson's disease.