Parkinson's disease rat model based on cerebral microcirculation disturbance as well as construction method and application of Parkinson's disease rat model

Abstract

The invention discloses a Parkinson's disease rat model based on cerebral microcirculation disturbance and a construction method and application thereof, and belongs to the technical field of biomedical experimental models. According to the method, cerebral microcirculation disturbance is induced by controlling a suppository blocking position (MCA starting part, propulsion 18-22 mm) and a blood flow standard, 2 mg / ml of 6-OHDA is injected in a targeted manner according to nigra positioning coordinates that the fontangle A / P is equal to-5.2 mm, the outer side L is equal to 2.2 mm, and the ventral side V is equal to-8.0 mm, the injection amount is 10 microliters, the injection speed is 0.5 microliter / min, screening is performed through a blood flow analysis technology and behavioral neurological function evaluation, and a target model is obtained. The rat model capable of simulating the pathological characteristics of blood-brain barrier damage and dopaminergic neuron progressive damage of the Parkinson's disease is obtained. The death rate of the model is lower than 5%, the phenotype consistency reaches 90%, and the model is remarkably superior to a traditional rotenone and gene editing model and is suitable for Parkinson's disease mechanism research and drug development.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical experimental models, and in particular relates to a Parkinson's disease rat model based on cerebral microcirculation disorder, and a construction method and application thereof. Background Art

[0002] At present, the research on Parkinson's disease uses animal models with normal microcirculation status, but this animal model is very different from the actual pathophysiological condition of Parkinson's patients, and its experimental results cannot truly reflect the actual situation of Parkinson's patients. Studies have found that cerebral microcirculation disorders can increase the permeability of the blood-brain barrier, and a large amount of inflammatory factors in the systemic circulation infiltrate into 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 excessive reactive oxygen is produced, further aggravating the apoptosis of dopaminergic neurons. These evidences suggest that cerebral microcirculation disorders can cause necrosis of dopaminergic neurons in the substantia nigra and striatum. This animal model is closer to the actual pathophysiological condition of Parkinson's patients. The use of microcirculatory disorder animal models for the study of Parkinson's disease is more in line with the actual pathophysiological environment of Parkinson's patients, and the experimental results are more valuable for reference.

[0003] The existing technology mainly adopts the following two types of models, but they have the following 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] In order to solve the above technical problems, the present invention provides a method for constructing a Parkinson's disease rat model by inducing cerebral microcirculation disorders and coordinating targeted neurotoxin injections, so as to solve the problems in the prior art that the pathophysiological environment of the model deviates from the real patient, the phenotype is unstable, and the experimental repeatability is poor.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide a material composition for constructing a Parkinson's disease rat model, the material composition comprising a thread plug and a 6-hydroxydopamine (6-OHDA) solution; the thread plug is a nylon thread wrapped with silica gel, with a diameter of 0.33-0.35 mm; the 6-OHDA solution is a 6-OHDA solution dissolved in 0.8-1.0% physiological saline with a concentration of 1.5-2.5 mg / ml.

[0009] The second object of the present invention is to provide a method for constructing a Parkinson's disease rat model, which comprises using the material composition.

[0010] The third object of the present invention is to provide an application of the substance composition or the method in constructing a Parkinson's disease rat model based on cerebral microcirculation disorder, wherein the method of using the substance composition is to first use the thread plug to induce cerebral microcirculation disorder, and then inject the 6-OHDA solution at the substantia nigra location coordinates.

[0011] Furthermore, the application specifically includes the following steps:

[0012] (a) blocking the origin of the middle cerebral artery of rats by means of the thread plug to induce cerebral microcirculatory disorder; wherein the advancement distance of the thread plug is 18-22 mm, and the blood flow decreases by ≥70%;

[0013] (b) One week after surgery, the 6-OHDA solution was injected into the substantia nigra localization coordinates; wherein the injection volume of the 6-OHDA solution was 8-12 μl, and the injection speed was 0.4-0.6 μl / min.

[0014] Furthermore, the rats are 7-9 week old male SD rats, weighing 200-250 g.

[0015] Furthermore, the step (a) specifically comprises the following steps:

[0016] (1) After the rats were anesthetized, the bilateral common carotid arteries (CCA), internal carotid arteries (ICA) and external carotid arteries (ECA) were exposed;

[0017] (2) Ligate the distal end of the ECA, insert a suture plug through the ECA incision, and push the suture plug along the ECA→CCA→ICA path to 18–22 mm into the skull to block blood flow at the origin of the MCA;

[0018] (3) Monitor blood flow until it drops by ≥70% to confirm successful occlusion, maintain ischemia for 30-60 minutes, and restore blood flow to more than 80% of the baseline after reperfusion.

[0019] Furthermore, in step (b), the substantia nigra is located at the anterior bregma A / P=-5.2 mm, the lateral L=2.2 mm, and the ventral V=-8.0 mm; and the 6-OHDA solution contains 0.1-0.3 mg / ml ascorbic acid.

[0020] Furthermore, the application also includes screening steps of blood flow analysis and neurological function assessment.

[0021] A fourth object of the present invention is to provide a Parkinson's disease rat model based on cerebral microcirculation disorder, wherein the Parkinson's disease rat model is constructed by using any of the methods described.

[0022] A fifth object of the present invention is to provide an application of the substance composition and / or the rat model in neurodegenerative disease mechanism research or drug screening.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Pathological authenticity: By simulating blood-brain barrier destruction and inflammatory factor infiltration through microcirculatory disturbances, 6-OHDA can be used to induce dopaminergic neuron apoptosis, which is closer to the pathophysiological characteristics of Parkinson's disease patients;

[0025] (2) Model stability: The thread plug advancement distance (18-22 mm) and blood flow monitoring standard (decline ≥ 70%) ensure that the degree of microcirculatory disorder is controllable, the 6-OHDA injection coordinate error is <0.1 mm, and the phenotypic consistency is improved by 30%;

[0026] (3) Experimental efficiency: The mortality rate is reduced to below 5% (the traditional rotenone model is 20-30%), and the modeling cycle is shortened to 5 weeks (the gene editing model requires 6-8 months). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of the line plug insertion path and MCA blocking in the present invention;

[0028] Figure 2 is the percentage of cerebral blood flow of rats in each treatment group compared with the baseline value in Example 1 of the present invention;

[0029] Figure 3 Longa score for each treatment group in Example 1 of the present invention;

[0030] Figure 4 The 30-minute rotation behavior of rats in each treatment group in Example 1 of the present invention;

[0031] Figure 5 The immunofluorescence staining results of rats in each treatment group in Example 1 of the present invention are shown;

[0032] Figure 6 It is the percentage of cerebral blood flow of rats injected with 6-OHDA only in Comparative Example 1 compared with the baseline value. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention are within the scope of the present invention. The reagents, products and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0034] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0035] Example 1

[0036] This embodiment provides a method for constructing a rat model of Parkinson's disease based on microcirculatory disorder and neurotoxin induction, and the specific steps are as follows:

[0037] 8-week-old male Sprague-Dawley rats (Wei Tong Li Hua Company, SD rat SPF grade) were selected to make their weight in the range of 200-250g. Before the experiment, the rats were placed in a temperature of 22±2℃, relative humidity of 50%-60%, followed by a 12-hour light / 12-hour dark circadian rhythm, and an environment with sufficient feed and drinking water for one week. 2%-4% isoflurane inhalation induced anesthesia was used, and after entering the anesthesia state, they were taken out and connected to the anesthesia maintenance device to maintain the concentration at 1.5%-2.5%. The body temperature (37±0.5℃) was maintained by a heating pad throughout the operation to prevent low temperature from affecting cerebral blood flow. Erythromycin eye ointment was applied to the cornea of ​​the rat for glomerular protection, and vital signs such as respiratory rate and corneal reflex were closely monitored. The neck hair was shaved and the skin was disinfected with iodine. A longitudinal incision of about 3cm long was made along the midline of the rat neck to separate the subcutaneous tissue and muscle. Use micro forceps to gently separate the sternocleidomastoid muscle and expose the left common carotid artery, avoiding damage to the vagus nerve (located in the carotid sheath and accompanying the carotid artery). Repeat the same steps to expose the right common carotid artery. Separate the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA), ligate the distal end of the ECA, and electrocoagulate its branches (superior thyroid artery and occipital artery). Use microvascular clamps to temporarily clamp the proximal end of the CCA and the distal end of the ICA. Cut a small hole (about 0.5mm) near the bifurcation of the ECA and insert a wire plug (silicone-coated nylon wire, diameter 0.33-0.35mm, purchased from Rayward, catalog number MSRC37B250PK50). Along the ECA→CCA→ICA path, gently advance the wire plug until the tip of the wire plug enters the ICA about 18-22mm into the brain and reaches the beginning of the MCA. The laser Doppler probe is fixed to the temporal bone (MCA blood supply area), and a decrease in blood flow ≥70% indicates successful occlusion. Use sutures to ligate the ECA stump and fix the suture plug to prevent displacement. Maintain the ischemia time for about 30-60 minutes. During this period, pay attention to the detection of vital signs, maintain body temperature (37±0.5℃), respiratory rate and blood oxygen saturation. After the ischemia period, slowly withdraw the suture plug to the ECA stump to restore ICA blood flow. Laser Doppler shows that the blood flow has recovered to more than 80% of the baseline, confirming that reperfusion is successful. Ligate the ECA stump, remove the vascular clamp, and restore normal blood flow to the CCA. Suture the muscles and skin layer by layer, and disinfect them twice with iodine. During the recovery period, place the animal in a warm and quiet environment until it is fully awake. Pay attention to daily observation of body weight, activity and wound healing after surgery for 1 week. One week later, a laser speckle blood flow imaging system was used to detect changes in the distribution of cranial and cerebral 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. To improve the signal-to-noise ratio, a dental drill was used to grind the skull thin until it was transparent. Care should be taken to preserve the intact dura mater 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 brain vertically and the bilateral cortex is displayed completely. Use the real-time preview mode of the software to 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 cerebral 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 used for secondary disinfection. During the recovery period, the animal is placed in a warm and quiet environment until it is fully awake. After the data is saved, it is analyzed using RFLSIAnalysis Software to detect the blood flow intensity of the main cortical vessels and microcirculation areas respectively. At the behavioral level, Longa score and Bederson score were used to analyze the motor function and neurological function of the experimental animals 2 days after surgery. Rats with significantly lower blood flow in the main cortical vessels than the baseline were excluded, and rats with microcirculatory disorders and no obvious damage to motor and neurological functions were selected to continue the experiment. One week later, the rats were anesthetized using the aforementioned anesthesia method, and the rats' vital signs were observed. The ambient temperature was maintained and the rats were kept warm. Subsequently, the anesthetized rats were fixed to 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 stereotaxic atlas of the rat brain, 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 and 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 0.5-1.0 mm / min according to the coordinates. After reaching the target point, pause for 1-2 minutes, slowly inject at a speed of 0.5 μl / min. After the injection, the needle tip stays for 15 minutes, and then slowly withdraws the needle at a speed of 0.5-1.0 mm / min. After withdrawing the needle, observe whether there is bleeding, suture the muscles and skin of the skull top layer by layer, and disinfect it twice with iodine. During the recovery period, place the animal in a warm and quiet environment until it is fully awake. Four weeks after surgery, apomorphine (0.2 mg / ml) is injected intraperitoneally at a dose of 0.5-1.0 mg / kg body weight, and the rat is placed in a transparent rotating test box. The rotation behavior analysis system is used to record the rotation behavior for 30 minutes. If the number of ipsilateral rotations is ≥7 times / min, it indicates that the animal model of Parkinson's disease was successfully constructed and successfully used in subsequent studies. If the standard is not met, the reasons for analysis should be considered for re-modeling or exclusion.

[0038] The schematic diagram of the plug insertion is as follows Figure 1 As shown in the figure, the insertion process is: separate the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA), then ligate the distal end of the ECA, and gently advance the suture plug along the ECA→CCA→ICA path until the tip of the intracranial suture plug enters the ICA and reaches the origin of the MCA.

[0039] A total of 42 rats were included in the experiment, including 6 in the control group (sham operation group), 22 in the microcirculation disorder group, and 14 in the circulation defect group. The duration of ischemia was different when the microcirculation disorder group and the circulation defect group were constructed. The ischemia time in the microcirculation disorder group was about 30 minutes, and the ischemia time in the circulation defect group was more than 60 minutes. Both groups were grouped by blood flow analysis, and behavioral neurological function assessment was further verified. The purpose of setting up these two groups is to reflect the necessity of blood flow analysis and neurological function assessment for model acquisition, which is also an important part of the establishment of this model.

[0040] The method in this embodiment is used to extend the ischemia time to about 30 minutes, with the intention of preventing extensive infarction caused by trunk ischemia from occurring after the blood vessels are reopened, but rather achieving the goal of not significantly affecting the trunk circulation and nerve function, but damaging the microcirculation, thereby forming the basis for cerebral microcirculation disorders, which is the manifestation mentioned in the present invention that is closer to the pathological process of Parkinson's disease.

[0041] The percentage of cerebral blood flow in each group of rats compared with the baseline value is as follows Figure 2 As shown, the results showed that there were significant differences in cerebral blood flow between rats with microcirculatory damage (MI) and rats with circulatory ischemia (CI) (P < 0.01), and there were significant differences compared with the control group (CON), both of which had reduced cerebral blood flow to varying degrees (P < 0.01).

[0042] The Longa score (a type of neurological function score, mainly used to evaluate the neurological dysfunction of animals after neurological damage such as cerebral ischemia) of rats 2 days after surgery is as follows Figure 3 As shown in the results, it can be seen that the Longa score of rats with microcirculatory impairment (MI) was not significantly different from that of the control group (CON), while the Longa score of rats with circulatory ischemia (CI) was significantly higher than that of the control group and the microcirculatory impairment group, with a significant difference (P < 0.01).

[0043] The 30-minute rotation behavior of rats in the sham operation group and Parkinson's disease model group Figure 4 As shown in the figure, 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, about 12 times per minute, and the difference between the two groups was significant (P<0.01). This fully demonstrates 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.

[0044] In terms of histological examination, the results of immunofluorescence staining of rats in the sham operation group and Parkinson's disease model group were as follows Figure 5The results showed that compared with the sham operation group (left), the positive staining area or intensity of specific markers in the substantia nigra and other related regions in the Parkinson's disease model group (right) was reduced, reflecting the damage or loss of dopaminergic neurons in the substantia nigra region under the action of 6-OHDA.

[0045] Comparative Example 1

[0046] This comparative example is a rat model in which only 6-OHDA injection is performed routinely. The model construction method is as follows: strictly according to the rat brain stereotaxic atlas, the substantia nigra positioning coordinates are clearly defined as the anterior fontanelle (A / P) = -5.2mm, the lateral side (L) = 2.2mm, and the ventral side (V) = -8.0mm. Use a microsyringe to draw 10μl of a 6-hydroxydopamine (6-OHDA) solution with a concentration of 2mg / ml and dissolved in 0.9% saline containing 0.2mg / ml ascorbic acid, install the syringe on the injection arm, and insert the needle vertically at a speed of 0.5-1.0mm / min according to the coordinates. After pausing for 1-2 minutes at the target point, slowly inject at a speed of 0.5μl / min, stay at the needle tip for 15 minutes after the injection, and then slowly withdraw the needle at a speed of 0.5-1.0mm / min. After withdrawing the needle, observe whether there is bleeding, suture the skin of the skull top layer by layer, and disinfect it twice with iodine tincture. During the recovery period, the animal is placed in a warm and quiet environment until it is fully awake.

[0047] The percentage of cerebral blood flow in this model rat compared with the baseline value is as follows Figure 6 As shown. Figure 6 It can be seen that the cerebral blood flow of rats injected with 6-OHDA alone was not significantly different from that of the control group (CON). Compared with the rat model constructed in the present invention, the rat model in the present invention has the characteristics and basis of microcirculatory disorders.

[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. A material composition for constructing a Parkinson's disease rat model, characterized in that: The material composition comprises a thread plug and a 6-hydroxydopamine (6-OHDA) solution; the thread plug is a nylon thread wrapped with silica gel, with a diameter of 0.33-0.35 mm; the 6-OHDA solution is a 6-OHDA solution dissolved in 0.8-1.0% physiological saline with a concentration of 1.5-2.5 mg / ml.

2. A method for constructing a Parkinson's disease rat model, characterized in that: The method comprises using the composition of matter of claim 1.

3. Use of the composition of matter according to claim 1 or the method according to claim 2 in constructing a Parkinson's disease rat model based on cerebral microcirculatory disorders, characterized in that: The method of using the material composition in the application is to first use the thread plug to induce cerebral microcirculation disorder, and then inject the 6-OHDA solution into the substantia nigra location coordinates.

4. The use according to claim 3, characterized in that: The application specifically comprises the following steps: (a) blocking the origin of the middle cerebral artery of rats by means of the thread plug to induce cerebral microcirculatory disorder; wherein the advancement distance of the thread plug is 18-22 mm, and the blood flow decreases by ≥70%; (b) One week after surgery, the 6-OHDA solution was injected into the substantia nigra localization coordinates; wherein the injection volume of the 6-OHDA solution was 8-12 μl, and the injection speed was 0.4-0.6 μl / min.

5. The use according to claim 4, characterized in that: The rats are male SD rats aged 7-9 weeks and weighing 200-250 g.

6. The use according to claim 4, characterized in that: The step (a) specifically comprises the following steps: (1) After anesthetizing the rat, the bilateral common carotid arteries (CCA), internal carotid arteries (ICA) and external carotid arteries (ECA) were exposed; (2) Ligate the distal end of the ECA, insert a suture plug through the ECA incision, and push the suture plug along the ECA→CCA→ICA path to 18–22 mm into the skull to block blood flow at the origin of the MCA; (3) Monitor blood flow until it drops by ≥70% to confirm successful occlusion, maintain ischemia for 30-60 minutes, and restore blood flow to more than 80% of the baseline after reperfusion.

7. The use according to claim 4, characterized in that: In the step (b), the coordinates of the substantia nigra are located at the anterior bregma A / P=-5.2 mm, the lateral L=2.2 mm, and the ventral V=-8.0 mm; the 6-OHDA solution contains 0.1-0.3 mg / ml ascorbic acid.

8. The use according to claim 4, characterized in that: The application also includes screening steps of blood flow analysis and neurological function assessment.

9. A Parkinson's disease rat model based on cerebral microcirculation disorder, characterized in that: The invention is constructed by using the method in any one of claims 3-8.

10. Use of the composition of matter according to claim 1 and / or the rat model according to claim 9 in neurodegenerative disease mechanism research or drug screening.

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