Experimental material for constructing animal model of cerebral microcirculation disturbance Parkinson's disease and application of experimental material

Abstract

The invention discloses an experimental material for constructing a cerebral microcirculation disturbance Parkinson's disease animal model and application of the experimental material, and belongs to the technical field of animal model construction. The invention particularly relates to an experimental material for constructing a model, application of the experimental material, a model construction method and application of the model. Research finds that cerebral microcirculation disturbance can cause increase of blood brain barrier permeability, and neuronal damage aggravation caused by penetration of a large amount of inflammatory factors in systemic circulation into the brain; meanwhile, glial cells at the nerve and blood vessel coupling position are activated, tight connection between endothelial cells is damaged, and excessive active oxygen is generated, so that apoptosis of dopaminergic neurons is further aggravated. The research on the Parkinson's disease by adopting the microcirculation disturbance animal model is more in line with the actual pathophysiological environment of the Parkinson's disease patient, and the experimental result has higher reference value.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and in particular relates to an experimental material for constructing an animal model of Parkinson's disease with cerebral microcirculation disorder and an application thereof. Background Art

[0002] Current studies on Parkinson's disease all use animal models with normal microcirculation conditions. This animal model is very different from the actual pathophysiological conditions of Parkinson's patients, and its experimental results cannot truly reflect the actual conditions of Parkinson's patients.

[0003] Existing technology defects:

[0004] Rotenone model: Rats have different metabolic abilities for rotenone, resulting in large fluctuations in the degree of neuronal damage and the rate of pathological progression. For example, Lewis rats are more sensitive to rotenone, while other strains of rats require higher doses, and the comparability of results between different strains is low. About 20%-30% of animals die from damage to peripheral organs such as liver and kidney, requiring frequent dose adjustments or combined supportive treatment. Chronic administration often causes animals to lose more than 15% of their weight, which may interfere with behavioral test results (e.g., decreased motor ability may be caused by weakness rather than PD pathology).

[0005] Rat model involving gene editing technology: It is difficult to complete gene editing and establish a stable animal model in rats. The plasma membrane and nuclear membrane of rat egg cells are thick and elastic, which can easily lead to embryo damage or decreased survival rate during microinjection. In addition, the in vitro culture conditions of rat embryos are demanding, superovulation technology (such as embryo acquisition after hormone treatment) is inefficient, and the proportion of deformed embryos is high, which further increases the failure rate of gene editing. Although the CRISPR / Cas9 system simplifies the gene editing process in rats, there are still risks of off-target effects and abnormal chromosome structure (such as large fragment deletion and translocation). Studies have shown that Cas9 may cause the insertion of exogenous DNA fragments in rats (accounting for 5.2%), and the new Cas12f tool is safer, but the editing activity is low, making it difficult to balance efficiency and safety. Although rats have a high degree of gene homology with humans, there are significant differences in the functional regulation mechanisms of some genes. For example, the expression pattern of α-synuclein, a Parkinson's disease-related gene, in rats is not completely consistent with that in humans, making it difficult for the edited phenotype to simulate the characteristics of human diseases. There is high genetic heterogeneity between rat strains, and phenotypic instability may occur after gene editing due to differences in genetic background. For example, the same gene knockout may show different degrees of pathological changes in different strains, requiring purification of the genetic background through multiple generations of backcrossing, which is time-consuming and costly. Summary of the invention

[0006] The invention provides an experimental material for constructing an animal model of Parkinson's disease with cerebral microcirculation disorder, comprising a suture for forming cerebral microcirculation disorder; and an injection for forming Parkinson's disease, wherein the injection is a 6-OHDA solution.

[0007] The present invention also provides a method for constructing a Parkinson's disease animal model with cerebral microcirculation disorder, which is constructed using the above experimental materials. The method comprises the following steps:

[0008] 1) Establish a cerebral microcirculatory disorder model by inserting bilateral carotid artery sutures;

[0009] 2) After the above state lasts for 1-2 weeks, 5-15 μL of 1-3 mg / mL 6-OHDA solution is stereotaxically injected into the substantia nigra;

[0010] 3) Verify the effectiveness of the model.

[0011] Furthermore, the state in step 2) lasts for 1 week before subsequent operations are performed.

[0012] The present invention also provides the application of the above experimental materials in constructing a Parkinson's animal model with cerebral microcirculatory disorder.

[0013] Furthermore, the construction method is:

[0014] 1) Establish a rat model of cerebral microcirculatory disorder by cuffing bilateral carotid arteries;

[0015] 2) After the above state lasts for 1-2 weeks, 5-15 μL of 1-3 mg / mL 6-OHDA solution is stereotaxically injected into the substantia nigra;

[0016] 3) Verify the effectiveness of the model.

[0017] Furthermore, the substantia nigra positioning coordinates are bregma = -5.2 mm, lateral = 2.2 mm, ventral = -8.0 mm.

[0018] Furthermore, the 6-OHDA solution was dissolved in 0.9% saline containing 0.2 mg / mL ascorbic acid.

[0019] Furthermore, the injection method is to vertically insert the needle at a speed of 0.5-1.5 mm / min according to the coordinates, pause for 1-2 minutes at the target point, and then slowly inject at a speed of 0.3-0.8 μl / min. After the injection is completed, the needle tip stays for 13-18 minutes, and then slowly withdraws the needle at a speed of 0.5-1.5 mm / min.

[0020] The present invention also provides a Parkinson's disease animal model with cerebral microcirculation disorder, and the animal model is obtained by the above-mentioned construction method.

[0021] In addition, the present invention also provides the use of the animal model in screening or preparing drugs for treating Parkinson's disease caused by cerebral microcirculation disorders.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Studies have found that cerebral microcirculatory disorders can increase the permeability of the blood-brain barrier, and a large amount of inflammatory factors in the systemic circulation infiltrate the brain, causing aggravated neuronal damage; at the same time, glial cells at the neurovascular coupling are activated, tight junctions between endothelial cells are damaged, and reactive oxygen species are excessively produced, further aggravating the apoptosis of dopaminergic neurons. These evidences suggest that cerebral microcirculatory disorders can cause necrosis of dopaminergic neurons in the substantia nigra and striatum. This animal model is closer to the actual pathophysiological conditions of patients with Parkinson's disease. Compared with the current routine rats that only undergo 6-OHDA injections, this protocol has the characteristics and basis of microcirculatory disorders. The use of microcirculatory disorder animal models for the study of Parkinson's disease is more in line with the actual pathophysiological environment of patients with Parkinson's disease, and the experimental results are more valuable for reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the anatomy of bilateral carotid artery ligation in Example 1;

[0025] Figure 2 This is a comparison chart of blood flow between the model group and the control group in Example 1;

[0026] Figure 3 The figure is a graph of the neurological function scoring results in Example 1 (wherein, Figure a is the Longa score and Figure b is the Bederson score);

[0027] Figure 4 The statistical diagram of the rat rotation behavior in Example 1;

[0028] Figure 5 The immunohistochemical staining results in Example 1 (the left side is the sham operation group and the right side is the model group); DETAILED DESCRIPTION

[0029] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0030] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0031] Example 1

[0032] 1. Experimental Methods

[0033] Eight-week-old male Sprague-Dawley rats were selected to have a body weight in the range of 200-250 g. Before the experiment, the rats were placed in an environment with a temperature of 22±2°C, a relative humidity of 50%-60%, a 12-hour light / 12-hour dark daytime rhythm, and sufficient feed and water for one week to adapt to the environment.

[0034] Induce anesthesia using 2%-4% isoflurane inhalation. After entering the anesthetized state, remove the patient and connect the anesthesia maintenance device to maintain the concentration at 1.5%-2.5%. Maintain body temperature (37±0.5℃) with a heating pad throughout the operation to prevent hypothermia from affecting cerebral blood flow.

[0035] Apply erythromycin eye ointment to the cornea of ​​the rat to protect the cornea, and pay close attention to vital signs such as respiratory rate and corneal reflex. Shave the neck hair and disinfect the skin with iodine. Make a longitudinal incision of about 3 cm along the midline of the rat's neck to separate the subcutaneous tissue and muscle. Use microtweezers to gently separate the sternocleidomastoid muscle and expose the left common carotid artery to avoid damaging the vagus nerve (located in the carotid sheath and accompanying the carotid artery). Repeat the same steps to expose the right common carotid artery. Temporarily clamp the proximal end of the blood vessel to reduce intraoperative bleeding. Use a non-absorbable surgical suture with a diameter of 0.08 mm to firmly ligate a metal wire with a diameter of 0.18 mm to the CCA, slowly withdraw the wire, observe blood flow, maintain for 2 hours, then loosen the ligature, remove the vascular clamp, observe for bleeding, and use an electrocoagulation pen to stop bleeding if necessary. Suture the muscles and skin layer by layer, and disinfect with iodine for a second time. During the recovery period, place the animal in a warm and quiet environment until it is fully awake. After the operation, pay attention to daily observation of body weight, activity range and wound healing for 1 week (this operation reduces the blood flow of bilateral carotid arteries by about 70%).

[0036] One week later, the laser speckle blood flow imaging system was used to detect changes in the distribution of cranial blood flow in rats. The rats were anesthetized using the above steps. After anesthesia, the hair on the head of the rats was shaved, the skin was disinfected with iodine, and a longitudinal incision of about 2 cm was made along the center of the rat's skull to separate the periosteum. In order to improve the signal-to-noise ratio, the skull was thinned and polished to transparency using a dental drill. It was necessary to keep the dura mater intact to avoid damage to the brain tissue. After the skull was translucent, the rat's head was fixed on a stereotaxic instrument to keep the olfactory fissure level to avoid motion artifacts. When collecting images, pay attention to adjusting the position of the imaging probe so that the laser beam irradiates the rat's skull vertically so that the bilateral cortex is displayed intact. Use the real-time preview mode of the software and adjust the focus until the vascular texture is clearly visible. During the process, turn off the indoor lighting to avoid interference from ambient light sources, maintain the room temperature at 25-28°C, and prevent low temperature from causing vasoconstriction. Collect cranial blood flow images in a resting state (lasting 30-60 seconds). After the image acquisition is completed, the muscles and skin of the skull are sutured layer by layer, and iodine is disinfected for the second time. During the recovery period, the animals are placed in a warm and quiet environment until they are fully awake. After the data were saved, they were analyzed using RFLSI Analysis Software to detect the blood flow intensity in the main vessels of the cortex and the microcirculation area.

[0037] At the behavioral level, Longa score and Bederson score were used to analyze the motor and neurological functions of experimental animals 2 days after surgery. Rats with significantly decreased cortical main vessel blood flow compared with the baseline were excluded, and rats with microcirculatory disorders and no obvious damage to motor and neurological functions were selected to continue the experiment.

[0038] One week later, the rats in the group (set as the microcirculation disorder group) were anesthetized using the above-mentioned anesthesia method, with 6 rats in the control group (sham operation group) and 21 rats in the experimental group (microcirculation disorder group). The rats' vital signs were observed, the ambient temperature was maintained, and they were kept warm. Subsequently, the anesthetized rats were fixed under the frame of the stereotaxic instrument using ear bars and incisor fixators, and the head position was adjusted so that the brain plane was parallel to the coordinate axis. Strictly based on the rat brain stereotaxic atlas, the substantia nigra localization coordinates were clearly defined as bregma (A / P) = -5.2 mm, lateral (L) = 2.2 mm, and ventral (V) = -8.0 mm. Use a microsyringe to draw 10 μl of 6-hydroxydopamine (6-OHDA) solution with a concentration of 2 mg / ml dissolved in 0.9% saline containing 0.2 mg / ml ascorbic acid. Install the syringe on the injection arm and insert the needle vertically at a speed of 1.0 mm / min according to the coordinates. After reaching the target point, pause for 1 minute and then slowly inject at a speed of 0.5 μl / min. After the injection, the needle tip stays for 15 minutes and then injects at 1.0 mm / min.

[0039] The needle was slowly withdrawn at a speed of 1.0 mm / min. After the needle was withdrawn, the bleeding was observed, and the muscles and skin of the skull top were sutured layer by layer, and iodine was used for secondary disinfection. During the recovery period, the animal was placed in a warm and quiet environment until it fully woke up.

[0040] Four weeks after surgery, apomorphine (0.2 mg / ml) was intraperitoneally injected at a dose of 0.5-1.0 mg / kg body weight, and the rats were placed in a transparent rotating test box, and the rotation behavior was recorded for 30 minutes using a rotation behavior analysis system. If the number of ipsilateral rotations was ≥7 times / minute, it indicated that the Parkinson's disease animal model was successfully constructed and successfully used for subsequent studies. If the analysis reasons did not meet the standards, re-modeling or exclusion was considered.

[0041] Histological examination:

[0042] Take rat brain tissue slices with a thickness of 4 microns, spread them at 42℃ water temperature, and bake them in a 60℃ oven for 30 minutes. Xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, anhydrous ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water for 5 minutes. EDTA microwave heat repair for 5-8 minutes, cool to room temperature. Draw circles with an immunohistochemistry pen to prevent the reagent from flowing out, add endogenous peroxidase blocking solution, incubate at room temperature for 10 minutes, and wash 3 times with PBS buffer, 5 minutes each time. Add blocking serum and incubate at 37℃ for 30 minutes, shake off excess serum, add primary antibody, incubate in a 37℃ wet box for 2h, and wash 3 times with PBS buffer, 5 minutes each time. Add HRP-labeled goat anti-rabbit, incubate at 37℃ for 30 minutes, and wash 3 times with PBS buffer, 5 minutes each time. Add DAB colorimetric solution, and observe under the microscope until the positive is significantly enhanced and the background is clean to stop color development. Add Mayer's hematoxylin for 30 seconds, wash with distilled water, soak in bluing solution for 1 minute, wash with water. Dehydrate with 75%-95%-100% alcohol gradient, 1 minute per cylinder, clear with three cylinders of xylene, 2 minutes per cylinder, and seal with neutral gum.

[0043] 2. Experimental results

[0044] In the microcirculation disorder determination process described in the present application, the above-mentioned rats used to construct microcirculation disorders were evaluated for microcirculation disorders based on the blood flow baseline, and used in the subsequent construction of the Parkinson's disease model. The experimental group rats can be divided into a microcirculation disorder group and a circulation defect group based on the blood flow baseline expression results, including 21 cases in the microcirculation disorder group and 16 cases in the circulation defect group. The microcirculation disorder group and the circulation defect group can be distinguished based on the definitions of the two. The blood vessels of the rats in the circulation defect group were damaged, the blood flow baseline changed significantly, and there were obvious neurological abnormalities; the blood vessels of the rats in the microcirculation disorder group were not obviously damaged, the microvascular system was significantly affected (slowed blood flow, insufficient microvascular perfusion), and the blood flow baseline was affected to a certain extent, but there were no obvious neurological abnormalities. By the following Figure 2The percentage of cerebral blood flow of the rats shown compared with the baseline value shows that there is a significant difference in cerebral blood flow between the rats with damaged microcirculation, namely the microcirculatory disorder group (MI) and the rats with circulatory ischemia, namely the circulatory defect group (CI) (P < 0.01), and there is a significant difference compared with the control group (CON), both of which have different degrees of reduced cerebral blood flow (P < 0.01).

[0045] Similarly, the rats used to construct microcirculatory disorders were evaluated for microcirculatory disorders based on neurological function, and were used in the subsequent construction of Parkinson's disease models. The experimental group rats were also divided into a microcirculatory disorder group and a circulatory defect group based on the neurological function assessment results. There were 21 cases in the microcirculatory disorder group and 16 cases in the circulatory defect group. Figure 3 The Longa score and Bederson score (a type of neurological function score, mainly used to evaluate the neurological dysfunction of animals after neural damage such as cerebral ischemia) of the rats shown 2 days after surgery showed that the Longa score and Bederson score of the rats with microcirculation damage (MI) were not significantly different from those of the control group (CON), while the Longa score and Bederson score of the rats with circulatory ischemia (CI) were significantly higher than those of the control group and the microcirculation damage group, with a significant difference (P < 0.01).

[0046] Down Figure 4 The results showed that the rats in the sham operation group had very few rotations, while the rats in the Parkinson's disease model group had a significant increase in rotations, reaching about 12 times per minute. The difference between the two groups was significant (P<0.001), which fully demonstrated that the injection of 6-OHDA into the substantia nigra successfully established an animal model of Parkinson's disease. The model rats had damaged dopaminergic neurons and showed obvious ipsilateral rotations after injection of apomorphine.

[0047] Regarding histological examination, Figure 5 Immunohistochemical staining showed that compared with the sham operation group, the positive staining area or intensity of specific markers in the substantia nigra and other related regions in the Parkinson's disease model group was reduced, reflecting the damage or loss of dopaminergic neurons in the substantia nigra region under the action of 6-OHDA.

[0048] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An experimental material for constructing an animal model of Parkinson's disease with cerebral microcirculatory disorder, characterized in that: The invention comprises a suture for forming cerebral microcirculation disorder; and an injection for forming Parkinson's disease, wherein the injection is a 6-OHDA solution.

2. A method for constructing an animal model of Parkinson's disease with cerebral microcirculation disorder, characterized in that: The experimental material described in claim 1 is used for construction, and the construction method comprises the following steps: 1) Establish a cerebral microcirculatory disorder model by inserting bilateral carotid artery sutures; 2) After the above state lasts for 1-2 weeks, 5-15 μL of 1-3 mg / mL 6-OHDA solution is stereotaxically injected into the substantia nigra; 3) Verify the effectiveness of the model.

3. The construction method according to claim 2, characterized in that: The state in step 2) lasts for 1 week before subsequent operations are performed.

4. Use of the experimental material according to claim 1 in constructing an animal model of Parkinson's disease with cerebral microcirculatory disorders.

5. The use according to claim 4, characterized in that: The construction method is: 1) Establish a rat model of cerebral microcirculatory disorder by cuffing bilateral carotid arteries; 2) After the above state lasts for 1-2 weeks, 5-15 μL of 1-3 mg / mL 6-OHDA solution is stereotaxically injected into the substantia nigra; 3) Verify the effectiveness of the model.

6. The use according to claim 5, characterized in that: The substantia nigra localization coordinates are bregma = -5.2 mm, lateral = 2.2 mm, ventral = -8.0 mm.

7. The use according to claim 5, characterized in that: The 6-OHDA solution was dissolved in 0.9% saline containing 0.2 mg / mL ascorbic acid.

8. The use according to claim 5, characterized in that: The injection method is to vertically insert the needle at a speed of 0.5-1.5 mm / min according to the coordinates, pause for 1-2 minutes at the target point, and then slowly inject at a speed of 0.3-0.8 μl / min. After the injection is completed, the needle tip stays for 13-18 minutes, and then slowly withdraws the needle at a speed of 0.5-1.5 mm / min.

9. A Parkinson's disease animal model with cerebral microcirculatory disorder, characterized in that: The animal model is constructed by the method described in any one of claims 2-3.

10. Use of the animal model according to claim 9 in screening or preparing drugs for treating Parkinson's disease caused by cerebral microcirculatory disorders.

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