Construction method of post-stroke depression mouse model

Abstract

The invention discloses a construction method of a post-stroke depression mouse model. The construction method comprises the following steps: injecting a rose-bengal solution into a mouse; then carrying out light shielding treatment on the mouse; and selecting a target modeling ischemic area, and irradiating the target modeling ischemic area with laser to obtain the post-stroke depression mouse model. According to the invention, mouse platelets are induced to agglutinate to form thrombus through a photochemical embolism method, so that focal permanent cerebral ischemia is caused. The neuromotor function of the mouse model provided by the invention is basically recovered within 7-14 days after modeling, but depression-related phenotypes appear within 21-42 days after modeling and are consistent with clinical disease characteristics, and the pathological characteristics of post-stroke depression can be effectively simulated.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and in particular relates to a method for constructing a post-stroke depression mouse model. Background Art

[0002] Stroke is characterized by high morbidity, mortality, and disability rates, and it also leads to a decline in patients' quality of life, placing a heavy burden on families and society. Post-stroke depression (PSD) is a common complication of stroke that severely impacts patients' quality of life. Identifying the pathological mechanisms of PSD is crucial for improving stroke treatment and rehabilitation, promoting patient recovery, and enhancing long-term quality of life. Although several studies have extensively discussed the pathogenesis of PSD, its specific details and key points remain to be further elucidated.

[0003] Clinical research on post-stroke depression is an effective way to discover pathological symptoms and differences in treatment efficacy, and to propose mechanistic hypotheses. Preclinical experiments are crucial for explaining and validating these phenomena and mechanisms. Existing combined post-stroke depression models sequentially construct ischemic stroke and depression in the same animal, failing to fully consider the holistic nature of post-stroke depression. Furthermore, the large phenotypic differences among the model animals and the independent evaluation results of each phenotype are issues currently present in both disease models. Furthermore, compared to depression, the multiple etiological characteristics of post-stroke depression pose certain difficulties in establishing animal models.

[0004] To simulate the overall characteristics of post-stroke depression, complex models that superimpose depression modeling on brain tissue damage, such as social isolation after middle cerebral artery embolization ("MCAO+isolation"), are currently more common models of post-stroke depression. Although these models can effectively simulate the disease phenotype of post-stroke depression and have high face validity, their construct validity is low and their predictive validity has not been fully demonstrated. This poses a challenge to the exploration of the pathological mechanisms of post-stroke depression, the optimization of diagnosis and treatment, and the verification of new drugs. Middle cerebral artery embolization is currently the most commonly used model of focal ischemia. The usual approach is temporary ischemia followed by reperfusion, that is, the middle cerebral artery is occluded with a suture for 0.5-2 hours, and the suture is then removed to restore blood flow. However, this model has many defects in simulating the pathogenesis of post-stroke depression: (1) Because the model uses sutures to occlude blood vessels, thrombolytic drugs are ineffective, which is inconsistent with the actual situation that stroke patients can receive clinical treatment; (2) This model blocks the middle cerebral artery, resulting in an infarct volume of up to 20-40% of the total brain volume, while the infarct volume of stroke patients is usually less than 5% of the total brain volume, otherwise they are very likely to die. Therefore, this model is quite different from the actual clinical situation; (3) Within the model group, there are large differences between individual animals in terms of infarct volume and degree of neurological damage, resulting in varying degrees of functional impairment in multiple brain regions, which brings great interference to the study of post-stroke depression. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a method for constructing a mouse model of post-stroke depression. This method, which does not rely on traditional depression modeling methods, instead uses photochemical embolization to induce vascular embolism in mice. The resulting mice maintain normal neuromotor function but display depression-related phenotypes, effectively simulating the pathological characteristics of post-stroke depression.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides a method for constructing a post-stroke depression mouse model, comprising the following steps:

[0008] (1) Injecting red Bengal solution into mice; then protecting the mice from light;

[0009] (2) Selecting a target modeling ischemic area and irradiating the target modeling ischemic area with a laser to obtain the post-stroke depression mouse model.

[0010] As a preferred embodiment, the mice are 2 to 6 months old.

[0011] As a preferred embodiment, the rose bengal solution is a normal saline solution of rose bengal with a concentration of 10 to 15 mg / mL.

[0012] As a preferred embodiment, the injection dose of the rose bengal solution is 50-55 mg / kg.

[0013] As a preferred embodiment, the target modeled ischemic area is irradiated with laser after the light-proof treatment for 5 to 10 minutes.

[0014] As a preferred embodiment, the laser irradiation is performed for 1 to 10 minutes using a laser having an output power of 50 to 150 mW and a wavelength of 520 to 560 nm.

[0015] As a preferred embodiment, the target modeling ischemic area is located in the prefrontal cortex of the mouse; in certain specific embodiments, the target modeling ischemic area is located 1.5 mm to the left of the sagittal suture of the anterior fontanelle and 1.5 mm in front of the fontanelle as the origin, and has a diameter of 1 to 3 cm.

[0016] In another aspect, the present invention provides a post-stroke depression mouse model obtained by the above-mentioned construction method.

[0017] In another aspect, the present invention provides use of the above-mentioned post-stroke depression mouse model in preparing a drug for treating and / or preventing post-stroke depression.

[0018] The present invention has the following advantages:

[0019] The present invention uses photochemical embolization to induce platelet aggregation in mice to form thrombi, resulting in focal permanent cerebral ischemia. Compared with the middle cerebral artery embolization method using sutures to cause permanent cerebral ischemia, it is more consistent with the pathological characteristics of clinical stroke patients. The present invention can accurately control the size and location of the infarct area, and the consistency of damage between individual animals is high. The mouse model provided by the present invention basically recovers neuromotor function within 7-14 days after modeling, but develops depression-related phenotypes within 21-42 days after modeling, which is consistent with the clinical disease characteristics and can effectively simulate the pathological characteristics of post-stroke depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A This is a diagram of the construction process of the mouse model in Example 1 of the present invention.

[0021] Figure 1B Schematic diagram of the location of the mouse modeling area in Example 1 of the present invention.

[0022] Figure 2 This is a statistical graph of the modified neurological severity score (mNSS) results of mice in Example 1 of the present invention.

[0023] Figure 3 3 is a graph showing the results of the depression-related behavior assessment experiment on mice in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] The following embodiments are merely some of the embodiments of the present invention, rather than all of them. Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but rather merely represents selected embodiments of the present invention. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0025] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0026] Example 1

[0027] This example provides a mouse model of post-stroke depression. The construction process is as follows: Figure 1A As shown, the specific steps include:

[0028] (1) Prepare 5 mL of 10 mg / mL Bengal rose solution in normal saline, filter sterilize through a 0.22 μm filter, wrap in tin foil to protect from light, and store at room temperature for use.

[0029] (2) Two-month-old male C57Bl6 / J wild-type mice were selected and intraperitoneally injected with 50 mg / kg of Zotai 50 (tiletamine hydrochloride + zolazepam hydrochloride). After anesthesia, they were fixed in a brain stereotaxic apparatus, the skin at the surgical site was prepared, and eye ointment was applied to the eyeballs.

[0030] (3) After the preoperative preparation is completed, the scalp is incised from the middle of the top of the head, and 10 mg / mL Bengal rose solution is injected intraperitoneally at a dose of 50 mg / kg; after the injection, the surface of the mouse body is covered with tin foil to avoid light, and wait for 5 minutes until the Bengal rose diffuses into the blood vessels throughout the body.

[0031] (4) Cut a small hole with a diameter of 1 mm on the tin foil. The position of 1.5 mm to the left and 1.5 mm in front of the sagittal suture of the mouse's anterior fontanelle is used as the origin. The center of the small hole on the tin foil is aligned with the origin to form a circular modeling area with a diameter of 1 mm (such as Figure 1B The rest of the mouse's head and body were covered with tin foil to avoid light.

[0032] (5) Aim a 525 nm laser at 125 mW of power at the center of the small hole in the foil. Set the laser tip to 0.5 cm from the mouse's scalp. After 5 min of laser irradiation, turn off the laser. Suture the skin and apply lincomycin-lidocaine gel for antibacterial and analgesic effects. Place the mouse back in its cage after it regains consciousness on a heating pad.

[0033] Effect verification

[0034] 1. Neurological function score test

[0035] On days 1, 3, and 7 after stroke modeling, the modified Neurological Severity Score (mNSS) of the mice was scored according to the items shown in Table 1. A higher score indicates a more severe neurological deficit.

[0036] Table 1

[0037]

[0038]

[0039] Figure 2 The figures show the results of the modified neurological severity score (mNSS) of the mouse model constructed in this example on days 3, 7, 14, 21, and 42 after modeling, indicating that the neuromotor function of the modeled mice had returned to normal by day 14, with no significant difference from the sham operation group.

[0040] 2. Assessment of depression-related behaviors

[0041] (1) Open field test: Before the experiment begins, mice are allowed to acclimate to the test environment for 1 h. The experimental apparatus is then installed and debugged. Mice are placed in the central area of the open field and allowed to move freely. The animals' activity in the open field is recorded for 5 min. Evaluation indicators include total distance traveled, time spent stationary, time spent moving in the central area, and number of fecal pellets.

[0042] (2) Tail suspension test: After installing and debugging the experimental apparatus, remove the animal from the cage and secure its tail to the apparatus. The tape is fixed 1 cm from the tip of the tail. The mouse is placed in a head-down position, with the tip of the animal's nose 25 cm away from the apparatus floor to avoid force stress. The test lasts for 6 minutes. Evaluation indicator: limb immobility time.

[0043] (3) Y-maze spontaneous alternation test: Before the experiment begins, mice are allowed to acclimate to the test environment for 1 h. The experimental apparatus is then installed and debugged. Mice are placed in the center of the Y-maze and allowed to move freely. The animal's movement trajectory in the Y-maze is recorded. The experiment lasts for 5 min. Evaluation indicator: spontaneous alternation rate.

[0044] Figure 3 The figure shows the behavioral test results of the depressive phenotype on days 21, 28, and 42 after mouse modeling, indicating that the stroke model mice showed differences in open field, tail suspension, and Y-maze behavioral indicators compared with the sham operation group.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a post-stroke depression mouse model, characterized in that: The following steps are involved: (1) Injecting red Bengal solution into mice; then protecting the mice from light; (2) Selecting a target modeling ischemic area and irradiating the target modeling ischemic area with a laser to obtain the post-stroke depression mouse model.

2. The construction method according to claim 1, characterized in that The mice are 2 to 6 months old.

3. The construction method according to claim 1, wherein The rose bengal solution is a normal saline solution of rose bengal with a concentration of 10-15 mg / mL.

4. The construction method according to claim 1, characterized in that The injection dosage of the rose bengal solution is 50-55 mg / kg.

5. The construction method according to claim 1, characterized in that After the light-proof treatment for 5 to 10 minutes, the target modeled ischemic area is irradiated with laser.

6. The construction method according to claim 1, characterized in that The laser irradiation is performed for 1 to 10 minutes using a laser having an output power of 50 to 150 mW and a wavelength of 520 to 560 nm.

7. The construction method according to claim 1, characterized in that The target modeling ischemic area is located in the prefrontal cortex of mice.

8. The construction method according to claim 1, wherein: The target modeling ischemic area is located 1.5 mm to the left of the sagittal suture of the anterior fontanelle and 1.5 mm in front of the fontanelle as the origin, with a diameter of 1 to 3 cm.

9. A post-stroke depression mouse model obtained by the construction method according to any one of claims 1 to 8.

10. Use of the post-stroke depression mouse model according to claim 9 in preparing a drug for treating and / or preventing post-stroke depression.

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