A method for constructing a parkinson's disease animal model

Abstract

The present application relates to a kind of Parkinson's disease animal model construction method, which is directly injected into the brain of animal by MPTP in the substantia nigra, and then the characteristic index score of animal is judged according to the characteristic index score standard, one Parkinson's disease animal model only needs to be injected once by MPTP for one animal, which is simple in operation process and short in time, greatly reduces the difficulty of modeling and animal care, which can achieve the purpose of modeling with a small amount of MPTP, thereby improving the modeling efficiency, and since the neurotoxicity response induced by MPTP is irreversible, MPTP is directly injected into the brain to act, which can also avoid the hindrance when MPTP is transmitted to the substantia nigra from other tissues of animal, thereby improving the success rate and stability of modeling.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and particularly relates to a construction method of a Parkinson's disease animal model. BACKGROUND

[0002] As a common neurodegenerative disease, Parkinson's disease is mainly common in the elderly. As a world problem, all treatments for Parkinson's disease in the world are to control symptoms, and it is currently impossible to cure it. The Parkinson's disease animal models induced by different modeling methods cannot completely match the clinical pathogenesis.

[0003] Therefore, a Parkinson's disease animal model with higher matching degree is being sought as a preclinical reference.

[0004] The current modeling technology of animal models is to continuously and long-term inject MPTP into the vein or to inject MPTP into the internal jugular through multiple operations. In the invention patent application (application number: 202210300121.2, name: a construction method of a Parkinson's disease cynomolgus monkey model), MPTP solution is injected into the internal carotid artery of the animal. MPTP needs to pass through multiple barriers such as blood, body tissue and brain tissue during transmission. Due to the characteristics of animal insensitivity to MPTP, the utilization rate of MPTP is low when it reaches the action point, and multiple injections and long-term observation are required. The modeling time is long, which leads to a low success rate of the model and poor stability. SUMMARY

[0005] The present application aims to provide a construction method of a Parkinson's disease animal model to solve the technical problems mentioned in the background.

[0006] In order to achieve the above-mentioned purpose, the present application discloses a construction method of a Parkinson's disease animal model, which comprises the following steps: determining the spatial coordinates of the substantia nigra in the brain of the animal, injecting MPTP into the substantia nigra in the brain of the animal, and judging the characteristic index score of the animal according to the characteristic index scoring standard, wherein the characteristic index includes one or more of consciousness, gait and posture, and vision.

[0007] Preferably, the MPTP is a solution.

[0008] Optionally, the method for determining the spatial coordinates of the substantia nigra in the brain of the animal comprises the following steps: obtaining the head image of the animal by MRI and CT joint scanning, and determining the spatial coordinates of the substantia nigra in the brain of the animal according to the image by using open source analysis software.

[0009] Preferably, the animal is anesthetized before injecting MPTP.

[0010] Optionally, during the anesthesia process, ketamine and xylazine are first used for induction anesthesia, and then isoflurane is used for continuous anesthesia.

[0011] Optionally, the ketamine dose is 5-15 mg / kg and the xylazine dose is 0.2-2 mg / kg.

[0012] Preferably, the ketamine dose is 8.75 mg / kg and the xylazine dose is 0.75 mg / kg.

[0013] Preferably, the ketamine and xylazine are administered by intramuscular injection.

[0014] Optionally, the anesthesia is maintained by intubation with 1.5-3% isoflurane gas.

[0015] Preferably, the anesthesia is maintained by intubation with 2% isoflurane gas.

[0016] Optionally, the MPTP is prepared by dissolving MPTP hydrochloride in normal saline, with 0.2-0.4 mg of MPTP hydrochloride per 1 ml of normal saline.

[0017] Preferably, the MPTP is prepared by dissolving MPTP hydrochloride in normal saline, with 0.3 mg of MPTP hydrochloride per 1 ml of normal saline.

[0018] Optionally, the MPTP is injected at a rate of 0.5-2 μL / min.

[0019] Preferably, the MPTP is injected at a rate of 1 μL / min.

[0020] Optionally, the MPTP is injected in a dose of 10-20 μL.

[0021] Preferably, the MPTP is injected in a dose of 15 μL.

[0022] Optionally, the consciousness scale is alert and responsive, scored as 3, depressed and / or comatose but arousable, scored as 2, comatose and not arousable, scored as 1, and no vital signs, scored as 0, the gait and posture scale is normal gait and posture, scored as 3, abnormal gait and / or posture but able to walk independently, scored as 2, abnormal gait and / or posture but requires assistance to walk, scored as 1, and complete loss of ability to walk and posture, scored as 0, and the vision scale is normal vision, rapid avoidance of light source to eye and normal light reflex, scored as 3, decreased vision, relatively rapid avoidance of light source to eye and normal light reflex, scored as 2, low vision, response to light source after a long time, scored as 1, and no response to light source, scored as 0.

[0023] Optionally, the animal is a dog or a monkey.

[0024] Preferably, the dog is a beagle and the monkey is a cynomolgus monkey or a rhesus monkey.

[0025] Optionally, the animal weighs 10.0-15.0 kg.

[0026] Optionally, the animal is 7-8 months old.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application can obtain a Parkinson's disease animal model by injecting MPTP into the substantia nigra of the brain of an animal once, and the operation process is simple and short, which greatly reduces the difficulty of modeling and animal care, can achieve the purpose of modeling with a small amount of MPTP, thereby improving the modeling efficiency, and since the neurotoxicity reaction induced by MPTP is irreversible, the MPTP is directly injected into the substantia nigra of the brain to act, which can also avoid the hindrance of MPTP transmission from other tissues of the animal to the substantia nigra, thereby improving the success rate and stability of modeling. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the simulated injection path of an embodiment of the present application.

[0030] 1, needle insertion port, 2, injection path, 3, intracerebral substantia nigra, 4, injection point. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application scheme will be described in detail below by combining the drawings and specific embodiments. It should be understood that the following description is only to explain the present application and does not limit its content.

[0032] 1, modeling animal information

[0033] Species Strain Beagle dog Grade Common grade Number 4 Gender Male Supply unit Beijing Mass Biotechnology Co., Ltd. Animal production license SCXK(Jing)2021-0002 Animal use license SYXK(Su)2017-0041

[0034] 2, modeling animal feeding management

[0035] The above experiment uses 4 beagle dogs, the ages of which are selected to be 7 months old, 7.5 months old, 7.5 months old and 8 months old, and the weights of which are selected to be 11.8 kg, 12.5 kg, 12.8 kg and 13.7 kg, and are numbered as 1001, 1002, 1003 and 1004 in turn. The period of 14 days or more of physical examination, quarantine and domestication is started, wherein the quarantine mainly checks the luster of the fur and whether there is a phenomenon of hair loss, checks whether there is secretion around the mouth, eyes, nose and ears, checks whether there is trauma and inflammation in each part of the body, checks whether the behavior and gait are normal, checks whether the autonomous activity is normal, checks whether the urination, defecation, food intake and water drinking are normal, etc.

[0036] At the same time, the temperature of the feeding room for the Beagle is 18-26℃, and the humidity is 40-70%, each Beagle is placed in a stainless steel cage, the size of which can be selected as 100x85x100cm, and the feeding room and the cage are cleaned once a day, the cage is replaced every two weeks, and the floor and wall of the feeding room are disinfected once every two weeks.

[0037] In addition, except for the fasting required by the test, the Beagle can drink purified water freely every day, and about 300g of full-value granular feed is fed to each Beagle, and the feed needs to be tested to not contain pollutants that interfere with the experimental results and animal health, and the drinking water needs to be tested for pollutant content that does not affect the experimental results.

[0038] After 14 days, it is checked that the above four Beagles meet the modeling requirements this time.

[0039] 3. Experimental equipment and reagent information

[0040] Instrument / reagent name Model / CAS number Manufacturer CT ScintCare CT16 Mingfeng Medical System Co., Ltd. MRI Panion Premier 1.5T Jiangsu Meisheng Medical Technology Co., Ltd. Injection pump R452 Shenzhen Ruivode Life Science and Technology Co., Ltd. Brain stereotaxic apparatus 68916 Shenzhen Ruivode Life Science and Technology Co., Ltd. MPTP hydrochloride 23007-85-4 Sigma

[0041] 4. Modeling method

[0042] Take 1001 Beagle as an example.

[0043] Firstly, the spatial coordinates of the substantia nigra 3 in the brain of the Beagle are determined.

[0044] Through MRI and CT co-location, the Beagle's head image is scanned, and the MRI and CT images are fused and paired by using the open source analysis software 3Dslicer, and the Beagle's head and the substantia nigra 3 in the brain are three-dimensionally reconstructed, so as to determine the spatial coordinates of the substantia nigra 3 in the brain of the Beagle, and at the same time, the injection path 2 is simulated, and the position of the needle insertion port 1 and the injection point 4 is simulated, and the simulated injection path 2 is as shown in Figure 1 , that is, the spatial coordinates of the Beagle injected with MPTP can be determined.

[0045] Among them, MRI does T22D FSE Ax, T22D FSE Dor and T22D FSE Sag three layers, and the scanning layer thickness is determined by the machine field strength, the thinner the better, this modeling adopts 1.5T field strength, and the scanning layer thickness is 3mm, CT does conventional head spiral scanning, and the scanning layer thickness is 0.58mm and the layer spacing is 0.58mm.

[0046] It should be noted that during the CT and MRI scanning process, the Beagle's head should be properly positioned as much as possible, so as to effectively improve the efficiency of the software processing the image.

[0047] Secondly, the Beagle is first induced with ketamine and xylazine, and then continuously anesthetized with isoflurane.

[0048] According to the body weight of the beagle, first, 8.75 mg of ketamine and 0.75 mg of xylazine per 1 kg of beagle are injected intramuscularly to induce anesthesia, and after the beagle's limbs are observed to be relaxed and the limbs are not responsive to pinching, 2% isoflurane gas is continuously anesthetized through tracheal intubation.

[0049] Finally, the beagle is fixed on the brain positioning instrument and the position is adjusted, combined with the simulated injection path 2, the actual injection path 2 of the beagle is determined with the front skull as the spatial zero point, the head operation site is disinfected, the skin is cut to expose the skull mark needle entry port 1, a surgical port is drilled at the needle entry port 1 using a skull drill, and 15 μL of a 0.3 mg MPTP hydrochloride / ml saline MPTP solution is configured. The 15 μL of MPTP solution can be injected into the injection point 4 of the nigra 3 in the brain of the beagle through the micro-injection needle at a speed of 1 μL / min.

[0050] 1002, 1003 and 1004 are performed according to the steps of 1001, which will not be repeated here.

[0051] 5, modeling results

[0052] For the above-mentioned 4 beagles injected with MPTP, the observation of 10 days or more is started, the characteristic index of each beagle is observed, the characteristic index score of the beagle is judged according to the characteristic index score standard, and finally the 4 Parkinson's disease beagle models are successfully completed. The characteristic index scores of the 4 beagles are shown in Table 1.

[0053] Table 1: Characteristic index score of beagle after injection of MPTP

[0054] Cycle / day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1001 9 8 9 9 7 6 6 6 6 4 4 3 3 3 3 1002 9 8 8 9 8 7 6 6 5 5 5 4 4 3 3 1003 9 8 9 9 7 6 5 4 4 4 4 4 4 3 3 1004 9 9 9 9 9 7 6 6 5 4 4 3 3 3 3

[0055] From the above score trend, the characteristic index scores of the 4 beagles before being injected with MPTP, i.e. on day 0, are all "9" points. After being injected with MPTP, i.e. after day 1, the characteristic index scores start to be lower than "9" points, and after day 5, the scores of the 4 beagles tend to gradually decrease, and after day 9, the scores of the 4 beagles tend to be stable.

[0056] Since the neurotoxicity reaction induced by MPTP is irreversible, after the above-mentioned model is successfully obtained, MPTP injection is not required.

[0057] The above-mentioned is only the preferred embodiment of the present application, and is not used to limit the application. Any modification, equivalent replacement, improvement, etc. within the design concept of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing an animal model of Parkinson's disease, characterized in that, The construction method includes: Determine the spatial coordinates of the substantia nigra in the animal's brain, and then inject MPTP into the substantia nigra. The animal's characteristic index score is determined according to the characteristic index scoring criteria, wherein the characteristic index includes one or more of consciousness, gait and posture, and vision; The MPTP is prepared by dissolving MPTP hydrochloride in physiological saline, wherein 0.2-0.4 mg of MPTP hydrochloride is added to each 1 ml of physiological saline. The injection rate of the MPTP is 0.5-2 µL / min; The injection dose of the MPTP is 10-20 µL; The consciousness indicators are: alertness and quick responsiveness, scored as 3 points; depression and / or coma but waking up, scored as 2 points; coma and unawakable, scored as 1 point; and absence of vital signs, scored as 0 points. The gait and posture indicators are as follows: normal gait and posture, scored as 3 points; abnormal gait and / or posture but able to walk independently, scored as 2 points; abnormal gait and / or posture but requiring assistance to walk, scored as 1 point; and complete loss of walking ability and posture, scored as 0 points. The visual acuity indicators are as follows: Normal vision: The eyes can quickly avoid light stimulation and have a normal light reflex, which is scored as 3 points; Weak vision: The eyes can avoid light stimulation relatively quickly and have a normal light reflex, which is scored as 2 points; Low vision: The eyes only react after a long time to light stimulation, which is scored as 1 point; No reaction to light stimulation, which is scored as 0 points.

2. The construction method as described in claim 1, characterized in that, Methods for determining the spatial coordinates of the substantia nigra in an animal's brain include: obtaining images of the animal's skull through a combination of MRI and CT scans, and then using open-source analysis software to determine the spatial coordinates of the substantia nigra based on the images.

3. The construction method as described in claim 1, characterized in that, The animals were anesthetized before MPTP injection.

4. The construction method as described in claim 3, characterized in that, During the anesthesia process, ketamine and xylazine were first used to induce anesthesia, followed by isoflurane for continuous anesthesia.

5. The construction method as described in claim 4, characterized in that, Continuous anesthesia is administered via intubation using 1.5-3% isoflurane gas.

6. The construction method according to any one of claims 1-5, characterized in that, The animal in question is either a dog or a monkey.

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