A method for constructing a parkinson's disease animal model with improved survival rate and application thereof

Abstract

The application discloses a method for constructing a Parkinson's disease animal model with improved survival rate and application thereof, and relates to the biomedical field, and comprises the following steps: S1, injecting a gepefron solution into the abdominal cavity of an experimental mouse; S2, performing brain stereotactic injection on the experimental mouse by using a 6-OHDA solution; S3, injecting meloxicam into the abdominal cavity of the experimental mouse, and dynamically adjusting the dosage of meloxicam based on the daily weight change of the experimental mouse; S4, performing postoperative nursing on the experimental mouse, including nutritional support and graded gavage; and S5, performing behavior testing, pathological verification and inflammation evaluation on the experimental mouse, and obtaining a Parkinson's disease animal model through the above steps. The application has the advantages of high postoperative survival rate of the animal and high success rate of modeling.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for constructing and applying an animal model of Parkinson's disease that improves survival rate. Background Technology

[0002] The 6-hydroxydopamine (6-OHDA) model is a commonly used animal model for studying Parkinson's disease. It is an animal model that simulates the pathological characteristics of Parkinson's disease by directly injecting the neurotoxin 6-OHDA into a specific area of ​​the animal's brain using stereotactic injection technique to damage central dopaminergic neurons.

[0003] Traditional methods for constructing 6-OHDA models typically involve stereotactic injection of 6-OHDA into animals to lesion dopaminergic neurons. This model is widely used due to its directness and relatively well-defined phenotype.

[0004] However, existing methods for constructing 6-OHDA models have a significant technical drawback: low postoperative survival rates in model animals, particularly in rodent mice. More than half of these mice experience a "cytokine storm" within two weeks of 6-OHDA injection into the substantia nigra of the midbrain, leading to difficulty eating and drinking, severe emaciation, and ultimately death. This results in high animal attrition and costs, and the insufficient number of surviving samples severely impacts the reproducibility and reliability of the experiments, limiting its application in long-term research and drug screening. Summary of the Invention

[0005] This invention aims to address one of the technical problems in related technologies to a certain extent. Therefore, this invention provides a method for constructing and applying an animal model of Parkinson's disease with improved survival rates, offering advantages such as high postoperative animal survival rates and high model establishment success rates.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for constructing an animal model of Parkinson's disease with improved survival rate includes the following steps:

[0008] S1, intraperitoneal injection of desipramine solution into experimental mice;

[0009] S2, stereotactic injection of 6-OHDA solution into the brains of the experimental mice;

[0010] S3, inject meloxicam into the experimental mice via intraperitoneal injection, and dynamically adjust the meloxicam dosage based on the daily weight changes of the experimental mice.

[0011] S4, Postoperative care including nutritional support and graded gavage was performed on the experimental mice.

[0012] S5. The experimental mice underwent behavioral tests, pathological verification, and inflammation assessment. Once all tests were passed, a Parkinson's disease animal model was obtained.

[0013] In existing technologies, the method of creating an animal model of Parkinson's disease by injecting 6-OHDA often results in a high mortality rate due to the inability to effectively suppress the acute inflammatory response after surgery, leading to high experimental costs and poor reproducibility. This application, however, establishes an "anti-inflammatory strategy based on dynamic adjustment of meloxicam dosage according to daily weight changes in experimental mice," achieving precise control of the inflammatory response. Meloxicam is an enol nonsteroidal anti-inflammatory drug with anti-inflammatory, analgesic, and antipyretic effects. Therefore, this drug can directly target the key pathological links leading to animal death, namely acute inflammatory response and multiple organ dysfunction, thereby improving the postoperative survival rate of model animals and fundamentally solving the problems of resource waste and experimental interruption caused by mass animal deaths. Furthermore, in existing modeling methods, even if the model animals survive, the degree of dopaminergic neuron damage is often uncontrollable due to individual differences in the inflammatory response, resulting in significant fluctuations in model success rates. Before stereotactic injection of 6-OHDA solution into the brains of experimental mice, this application first administers a low-dose injection of desipramine to selectively protect the noradrenergic neurons of the mice, ensuring that the toxic effects of 6-OHDA on dopaminergic neurons are effectively exerted, thus resulting in a high success rate of model establishment. Furthermore, existing modeling methods cause damage to dopaminergic neurons too acutely, failing to simulate the progressive neurodegeneration process of human Parkinson's disease that lasts for several years, and the intense inflammation they induce does not match the chronic inflammation characteristic of human Parkinson's disease. This invention, by delaying anti-inflammatory intervention to 36–48 hours post-surgery, allows for the establishment of necessary initial damage signals while effectively suppressing subsequent excessive inflammatory responses, thereby prolonging the dopaminergic neuron damage process and better simulating progressive pathological evolution. In addition, postoperative care, including nutritional support and graded gavage, reduces the interference of craniotomy during stereotactic injection on the physiological state of the experimental mice, avoiding additional variations caused by dehydration and malnutrition.

[0014] The core technical principle of the construction method of this invention has species universality and can be applied in principle to other mammalian experimental animals with mature brain stereotaxic technology, such as rats and hamsters.

[0015] Optionally, in step S1, the desipramine solution is prepared by dissolving desipramine powder in sterile saline, the injection dose of the desipramine solution is 1~2 mg / kg, and step S1 is performed 30 minutes to 12 hours before the stereotactic injection in step S2.

[0016] In existing modeling methods, 6-OHDA causes non-targeted damage to noradrenergic neurons, easily leading to acute postoperative death in animals. This application addresses this issue by injecting a low dose of desipramine (1-2 mg / kg), utilizing its pharmacological properties as a norepinephrine transporter inhibitor to pre-block the toxin uptake pathway of noradrenergic neurons before toxin attack, achieving specific protection for this type of neuron. Furthermore, the low dose of 1-2 mg / kg desipramine ensures effective protection while avoiding potential autonomic nervous system side effects (such as drastic fluctuations in heart rate and blood pressure) caused by desipramine itself, thereby reducing early mortality due to the combined effects of non-targeted damage and pretreatment drug toxicity, and improving the survival rate of experimental mice. It should be noted that the injection dose of 1-2 mg / kg refers to injecting 1-2 mg of the active ingredient of desipramine per kilogram of mouse. The applicant's research found that the optimal administration time is 30 minutes to 12 hours before surgery, as early administration may lead to a decrease in drug concentration at the time of surgery, weakening the protective effect; while late administration fails to establish effective protection before the toxin arrives. This timing ensures that during 6-OHDA injection, the NET of norepinephrine neurons is continuously and effectively inhibited, while the DAT function of dopaminergic neurons remains unaffected. As a result, the damage rate of dopaminergic neurons is stably limited within an ideal range, avoiding the pathological background confounding and excessive behavioral variation caused by simultaneous damage to the norepinephrine system in traditional models, thereby improving the success rate of animal models.

[0017] Optionally, in step S2, the target point for stereotactic injection is the medial forebrain tract, and the stereotactic coordinates are: posterior to the anterior fontanelle A / P=-1.2mm, lateral to the midline M / L=1.3mm, subdural D / V=-4.8mm.

[0018] This application uses the medial forebrain fasciculus (PFMF) as the injection target. The PFMF is the main fiber tract from which dopaminergic neurons in the substantia nigra project upwards to the striatum. Injecting a small amount of 6-OHDA at this site allows the toxin to be taken up by the axonal terminals of dopaminergic neurons passing through this area and delivered directly to the cell bodies in the substantia nigra via retrograde axoplasmic transport, inducing concentrated apoptosis. Compared to directly attacking the cell bodies or terminals, using the PFMF as the injection target allows for more precise destruction of the target neuronal population with less drug, thereby consistently improving the model's success rate. Furthermore, the coordinates provided in this application (A / P = -1.2 mm posterior to the anterior fontanelle, M / L = 1.3 mm para-midline, D / V = -4.8 mm subdurally) ensure that the injection needle tip reaches the same biological location across different experimental batches and under different operators, thus avoiding data fluctuations caused by positioning deviations and achieving standardization of the animal model.

[0019] Optionally, the 6-OHDA solution is prepared and used immediately, and becomes ineffective 2 hours after preparation; the 6-OHDA solution is placed in a light-protected environment at 4°C within 2 hours.

[0020] By preparing and using the solution immediately, uncertain degradation caused by storage is avoided; the oxidation reaction rate is inhibited by using a low temperature of 4°C; photocatalytic oxidation is avoided by avoiding light; and the chemical stability of the 6-OHDA solution is ensured by using it within 2 hours, ensuring that the 6-OHDA solution maintains high activity with each injection, which facilitates the output of stable pathological results (i.e., damage rate).

[0021] Optionally, in step S2, the concentration of the 6-OHDA solution is 12 mg / mL and contains 0.02% ascorbic acid; when performing stereotactic injection into the brain, the injection rate is 0.1~0.3 μL / min, and the needle is left in place for 5 minutes after injection.

[0022] A concentration of 12 mg / mL ensures sufficient 6-OHDA for effective neurotoxicity when injecting small volumes of solution, while avoiding chemical damage caused by excessively high concentrations. Ascorbic acid, as a potent antioxidant, inhibits the oxidation of 6-OHDA during preparation and before injection, ensuring that the injected drug is the biologically active original drug (in conjunction with the aforementioned "prepared and used immediately" approach). Furthermore, ascorbic acid is consumed very quickly after injection, protecting 6-OHDA from premature oxidation as it diffuses along the needle path. An injection rate of 0.1–0.3 μL / min allows the drug to penetrate the tissue at a very slow rate, reducing mechanical damage to the tissue caused by injection pressure and unintended diffusion of the drug, thus confining the drug effect to the area around the target site. Retaining the needle for 5 minutes after injection prevents backflow of the drug.

[0023] Optionally, in step S3, the dynamic adjustment of meloxicam dosage based on the daily weight changes of the experimental mice includes: weighing the experimental mice before stereotactic brain injection to obtain their original weight; weighing the experimental mice daily starting 24 hours after stereotactic brain injection to obtain their real-time weight; if the real-time weight decreases by more than 20% compared to the original weight, immediately injecting the experimental mice intraperitoneally with 5 mg / kg of meloxicam, followed by daily injections of 3 mg / kg until the weight returns to the original weight; if the real-time weight decreases by less than or equal to 20% compared to the original weight, starting 36 to 48 hours after stereotactic brain injection, injecting the experimental mice intraperitoneally with 3 mg / kg of meloxicam daily for at least 3 consecutive days.

[0024] Rapid weight loss is an early marker of severe inflammatory response in laboratory mice. For high-risk individuals, meloxicam should be administered immediately at a dose of 5 mg / kg to save their lives. For individuals with a milder response (weight loss ≤20%), meloxicam should be administered at a dose of 3 mg / kg during the expected peak of inflammation (36–48 hours) to prevent inflammation and ensure that each mouse receives anti-inflammatory support appropriate to its actual physiological state, thereby improving overall survival. If the real-time weight loss compared to the original weight is less than or equal to 20%, administration should be delayed until 36–48 hours post-surgery to avoid the initial stage of the inflammatory response and allow necessary early inflammation, which serves as a signal to initiate neuronal damage, to develop. Subsequently, intervention should be initiated before the peak of inflammation (36–48 hours) with a low dose (3 mg / kg) of meloxicam to moderately suppress the inflammation.

[0025] Optionally, in step S4, the nutritional support includes replacing the daily drinking water of the experimental mice with a glucose solution with a mass-volume concentration of 5%, and changing the feed to breeding feed.

[0026] Intracerebral injection of 6-OHDA and the accompanying neuroinflammation trigger a stress response, often leading to acute anorexia, lethargy, and reduced water intake in mice. Without intervention, mice rapidly dehydrate, exacerbating systemic inflammation and metabolic disorders, and even causing death. A 5% glucose solution provides a direct energy source; its slight sweetness effectively stimulates appetite and prevents dehydration. Compared to ordinary drinking water, glucose solution can directly supplement basal energy through drinking water when mice's food intake is insufficient. Breeding feed is a specialized type of mouse feed, referring to feed with higher energy density, protein content, and fat content than ordinary feed. In situations where spontaneous food intake in mice may decrease, breeding feed ensures that every bite provides richer nutrition to support basic physiological functions, wound healing, and combat inflammatory depletion.

[0027] Optionally, in step S4, the graded gavage includes: recording the average daily food intake of the experimental mice before stereotactic brain injection as the basal food intake; monitoring the real-time food intake of the experimental mice daily after stereotactic brain injection; if the reduction in real-time food intake compared to the basal food intake is less than 50%, the experimental mice are determined to have mild anorexia, and a 5% glucose solution is provided for them to drink freely; if the reduction in real-time food intake compared to the basal food intake is 50% to 80%, the experimental mice are determined to have moderate anorexia, and the experimental mice are gavaged with glucose solution at a dose of 0.2 mL per gram of body weight twice a day, with a glucose solution concentration of 5%; if the reduction in real-time food intake compared to the basal food intake is greater than 80%, the experimental mice are determined to have severe anorexia, and the experimental mice are gavaged with glucose solution at a dose of 0.2 mL per gram of body weight three times a day, with a glucose solution concentration of 5%.

[0028] Postoperative anorexia is a major cause of dehydration, energy depletion, and even death in mice. This invention, through daily monitoring, can promptly detect declining food intake trends and initiate intervention before severe physiological reactions occur. Different levels of nutritional support (free access to water, twice-daily gavage, or three times-daily gavage) are matched according to the severity of anorexia (mild <50%, moderate 50%–80%, severe >80%). This ensures that the energy received by each mouse matches its actual needs, avoiding over-intervention in mice with mild anorexia while providing sufficient energy for mice with severe anorexia, directly preventing anorexia-induced death.

[0029] Optionally, in step S5, the passing criteria for behavioral testing, pathological verification, and inflammation assessment are as follows:

[0030] (a) Behavioral tests: Apomorphine-induced rotation greater than 7 revolutions / minute, rotarod test dwell time decreased by more than 50% compared with before stereotactic brain injection, and the limb coordination test showed a limb preference of more than 70% on the injured side;

[0031] (b) Pathological verification: Immunofluorescence staining of tyrosine hydroxylase in the brains of experimental mice showed that the destruction rate of TH-positive neurons in the striatum or substantia nigra region reached 75% to 85%;

[0032] (c) Inflammation assessment: Microglia activation was 2 to 3 times higher than that of the undamaged side of the brain, as detected by ionized calcium linker protein staining.

[0033] Existing technologies often rely solely on behavioral tests as the standard for model success, neglecting pathological verification and inflammation assessment. Mice that pass behavioral tests may still have insufficient or excessive neuronal damage, or be in a state of severe inflammation, resulting in inconsistent quality of animal models. The behavioral tests in this application comprehensively assess the motor functions (rotation, coordination, endurance) of mice, preventing the randomness that may exist with a single indicator. By unilaterally injecting 6-OHDA into the mouse brain, most of the dopaminergic nerve endings from the substantia nigra to the striatum on that side are damaged, leading to a sharp decrease in striatal dopamine levels on that side. The severe asymmetry in striatal dopamine levels during apomorphine-induced rotation drives the mouse to continuously rotate towards the undamaged side (i.e., the side without 6-OHDA injection). The greater the rotation speed, the more severe the unilateral dopaminergic neuronal damage, indicating a more successful establishment of the Parkinson's disease animal model. In the accelerated rotarod test, mice exhibited significantly reduced time spent on the rotarod compared to their pre-operative state due to abnormal muscle tone regulation and delayed motor initiation on the injured side (i.e., the side injected with 6-OHDA). A decrease of more than 50% in time indicated that the damage to dopaminergic neurons had severely affected the mouse's ability to regulate movement, signifying a more successful establishment of the Parkinson's disease animal model. It's important to note that the motor nervous system is cross-controlled, meaning the right side of the brain primarily controls movement on the left side of the body, and the left side primarily controls movement on the right side. Therefore, the injured limb refers to the limb on the same side as the side of brain injury. If the injection is given to the left side of the brain, the injured limb is the left side of the body, i.e., the healthy limb. Because the motor function of the limb controlled by the side of brain injury is impaired, mice will instinctively prioritize using their functional, healthy limb (i.e., the injured limb) when exploratory standing. When the preference for using the healthy limb exceeds 70%, it indicates that this spontaneous motor asymmetry has reached a highly significant level, meaning that the spontaneous motor selection defect caused by the loss of unilateral dopaminergic neurons has been established, and the animal model has successfully simulated the "spontaneous motor asymmetry" behavioral phenotype of Parkinson's disease. A 75% to 85% TH-positive neuron destruction rate is sufficient to induce clear core motor symptoms of Parkinson's disease, while simultaneously allowing mice to tolerate the injury and survive, and the pathological process is relatively controllable. In the traditional 6-OHDA model, microglia are violently activated, reaching 5 to 8 times the level of the undamaged side. Overactivation releases large amounts of toxic pro-inflammatory factors, directly exacerbating neuronal death, damaging the blood-brain barrier, and often triggering fatal systemic inflammatory responses. This invention, through individualized meloxicam intervention based on weight monitoring, suppresses the activation level of microglia on the damaged side of the brain to 2 to 3 times the activation level of microglia on the undamaged side. This indicator is closer to the neuroinflammatory characteristics of human Parkinson's disease; therefore, the mouse model assessed through inflammation is more realistic in terms of pathological mechanisms.

[0034] Furthermore, this invention also provides an application of an animal model of Parkinson's disease, which is obtained by the construction method described in any of the foregoing claims. The application of the animal model of Parkinson's disease provided by this invention is similar to the reasoning process of the beneficial effects of the aforementioned construction methods, and will not be repeated here.

[0035] These features and advantages of the present invention will be disclosed in detail in the following specific embodiments and accompanying drawings. The preferred embodiments or means of the present invention will be shown in detail in conjunction with the accompanying drawings, but are not intended to limit the technical solutions of the present invention. In addition, each of these features, elements and components appearing in the following text and drawings has multiple components and is labeled with different symbols or numbers for convenience, but all represent parts with the same or similar construction or function. Attached Figure Description

[0036] The present invention will be further described below with reference to the accompanying drawings:

[0037] Figure 1 This is a flowchart illustrating the method for constructing an animal model of Parkinson's disease to improve survival rate in this invention.

[0038] Figure 2 This is a schematic diagram of the coordinate points for stereotactic injection into the brain of experimental mice in this embodiment of the invention;

[0039] Figure 3 The above are the statistical results of behavioral tests between the Parkinson's group mice and the healthy group mice in the examples;

[0040] Figure 4 The image shows the immunofluorescence staining results of tyrosine hydroxylase in the striatum region of brain slices from experimental mice in this example.

[0041] Figure 5 The image shows the immunofluorescence staining results of tyrosine hydroxylase in the substantia nigra region of a brain slice from an experimental mouse in this example. Detailed Implementation

[0042] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described are intended to explain the present invention and should not be construed as limiting the invention.

[0043] The terms "an embodiment," "example," or "trademark" used in this specification refer to a particular feature, structure, or characteristic described in connection with the embodiment itself that may be included in at least one embodiment disclosed in this patent. The phrase "in an embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment.

[0044] The applicant's research revealed several key issues that need to be addressed in the application of the 6-OHDA model in existing technologies:

[0045] 1) High mortality rate due to acute inflammatory response:

[0046] The most prominent problem with existing 6-OHDA models is the poor postoperative survival rate, which typically fluctuates between 50% and 70%, sometimes even falling below 30%. This phenomenon is mainly attributed to the dual pathological processes triggered by 6-OHDA injection: on the one hand, 6-OHDA competitively enters dopaminergic neurons, generating reactive oxygen species within the cells and inhibiting mitochondrial complex I activity, directly leading to neuronal death; on the other hand, damaged neurons release damage-associated molecular patterns (DAMPs), activating microglia and triggering a neuroinflammatory cascade. Studies have shown that within 24–48 hours after 6-OHDA injection, microglia rapidly activate and excessively release pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These inflammatory mediators not only exacerbate local neuronal damage but may also trigger systemic inflammatory response syndrome (SIRS). Especially in striatal injection models, because the injection site is close to the ventricular system, inflammatory factors are more likely to diffuse into the systemic circulation, leading to multiple organ dysfunction, which is one of the main causes of death in model animals.

[0047] 2) The uncontrollability of the degree of nerve inflammation and damage:

[0048] The second key problem facing existing technologies is the difficulty in precisely controlling the degree of damage, manifested in huge fluctuations in model success rates (30%–90%) and significant variability in behavioral performance (coefficient of variation often exceeding 30%). This uncontrollability stems primarily from two factors: individual differences in neuroinflammatory responses and the lack of fine-tuning of the damage process. In traditional models, 6-OHDA-induced neuroinflammation exhibits a typical "bimodal" dynamic characteristic: the initial phase (0–24 hours) is characterized by rapid microglial activation, releasing small amounts of pro-inflammatory factors; the peak phase (36–72 hours) is accompanied by extensive macrophage infiltration and inflammasome activation, with cyclooxygenase-2 expression reaching its peak. This uncontrolled inflammatory response leads to significant differences in the degree of dopaminergic neuronal damage among different individuals, resulting in huge variations in phenotypic indicators such as rotational behavior.

[0049] 3) Acute injury and postoperative care deficiencies:

[0050] Furthermore, the applicant found that the neuronal damage induced by 6-OHDA was too acute (completed within 72 hours), failing to simulate the progressive pathological characteristics of human diseases (such as complex mechanisms lasting several years, like α-synuclein aggregation and mitochondrial dysfunction). In addition, the severe neuroinflammation triggered by acute damage does not match the chronic low-grade inflammation in humans, limiting neuroimmunological research and the evaluation of anti-inflammatory drugs. Simultaneously, neglect of postoperative care further affected model stability. After 6-OHDA injection, animals often exhibited stress responses such as decreased appetite and dehydration, activating the HPA axis and exacerbating neuroinflammatory variations. The lack of standardized support measures led to fluctuations in animal condition, reducing experimental reliability.

[0051] Based on this, the present invention conducts in-depth research on the above-mentioned problems and proposes a systematic solution.

[0052] Example: This example provides a method for constructing an animal model of Parkinson's disease with improved survival rate, such as... Figure 1 As shown, it includes the following steps:

[0053] S1, intraperitoneal injection of desipramine solution into experimental mice.

[0054] Fifty experimental mice of similar weight were selected, and desipramine powder was dissolved in 0.9% sterile saline to prepare a solution with a concentration of 0.2 mg / mL. Thirty minutes before stereotactic injection, each experimental mouse was intraperitoneally injected (IP) at a dose of 2 mg / kg, i.e., 0.2 mL of the desipramine solution was injected into a 20g experimental mouse. During injection, the needle was inserted at an angle of about 30° and slowly injected at a rate of about 0.1 mL / 10 seconds to reduce peritoneal irritation.

[0055] S2, 6-OHDA solution was used to stereotactically inject brain tissue into 50 experimental mice.

[0056] (1) Anesthesia and fixation: Each experimental mouse was anesthetized by inhalation using isoflurane gas. The induction concentration of isoflurane was 4% to induce anesthesia in the experimental mice, and the maintenance concentration of isoflurane was 1.5% to maintain the depth of anesthesia during the operation. The head of the anesthetized experimental mice was fixed in a stereotaxic apparatus. The bilateral ear rods and incisor clamps of the stereotaxic apparatus were adjusted to control the height difference between the bilateral ear rods within 0.1 mm.

[0057] (2) Localization and Craniotomy: Using the anterior fontanelle as the origin, the injection target point is determined to be the medial forebrain fasciculus, with the following stereotactic coordinates: 1.2 mm posterior to the anterior fontanelle, 1.3 mm lateral to the midline, and 4.8 mm subdural. A hole is drilled at the corresponding skull location to expose the dura mater. For example... Figure 2 The figure shown is a schematic diagram of the three-dimensional positioning coordinates during craniotomy in mice.

[0058] (3) Drug injection: A 6-OHDA solution with a concentration of 12 mg / mL and containing 0.02% (w / v) ascorbic acid was injected stereotactically into the brain using a microinfusion pump at an injection rate of 0.1 μL / min. After injection, the needle was left in place for 5 minutes before being withdrawn to prevent drug reflux and allow for sufficient drug diffusion. It should be noted that the 6-OHDA solution should be prepared fresh and stored away from light, and is effective for 2 hours at 4°C.

[0059] (4) Postoperative management: Before suturing the scalp, 0.5% lidocaine was dripped for local anesthesia. After suturing the skin, the mouse was placed on a 37°C warming pad until it woke up.

[0060] S3, 50 experimental mice were intraperitoneally injected with meloxicam, and the dosage of meloxicam was dynamically adjusted based on the daily weight changes of the experimental mice.

[0061] (1) The experimental mice were weighed before the stereotactic injection to obtain their original weight. They were weighed at the same time every day starting 24 hours after the injection to obtain their real-time weight.

[0062] (2) If the real-time weight loss is greater than 20% compared to the original weight, administer meloxicam 5 mg / kg intraperitoneally immediately (i.e., acute rescue), followed by 3 mg / kg daily (i.e., maintenance) until the weight returns to the original weight. If the weight loss is less than or equal to 20%, administer meloxicam 3 mg / kg intraperitoneally daily starting 36 to 48 hours after injection, and continue for at least 3 days. This is routine anti-inflammatory treatment. Avoid abdominal organs during injection; tilt the needle 45° towards the pelvis; gently massage the abdomen after injection to promote absorption. When preparing the meloxicam injection solution, first dissolve the meloxicam powder in a small amount of DMSO, then dilute with physiological saline to the final volume, ensuring a final DMSO concentration of 5%, thus obtaining a clear and stable meloxicam injection solution.

[0063] S4. Postoperative care (i.e., care after stereotactic injection of the brain) was provided to the experimental mice, including nutritional support and graded gavage.

[0064] (1) Nutritional support:

[0065] The mice's daily drinking water was completely replaced with a 5% (w / v) glucose solution. Simultaneously, their regular diet was replaced with breeding feed.

[0066] (2) Graded gavage:

[0067] The average daily food intake of the experimental mice before stereotactic brain injection was recorded as their basal food intake. After stereotactic brain injection, their real-time food intake was monitored daily.

[0068] ① If the reduction in real-time food intake compared to the basic food intake is less than 50%, provide the above-mentioned glucose solution for free drinking.

[0069] ② If the reduction ratio is 50% to 80%, administer the above glucose solution by gavage at a dose of 0.2 mL per gram of experimental mouse body weight (i.e., 4 mL for 20 g mice), twice a day.

[0070] ③ If the reduction rate is greater than 80%, administer the above glucose solution by gavage at a dose of 0.3 mL / g body weight, three times a day.

[0071] (3) Environment and monitoring:

[0072] Following stereotactic injection into the brain, the mice were provided with a stable temperature and humidity environment. They were weighed daily, and their weight changes were recorded. Mice whose weight consistently increased for more than two days were considered to be in stable condition.

[0073] S5, after passing behavioral tests, pathological verification and inflammation assessment of experimental mice, a Parkinson's disease animal model was obtained.

[0074] On day 14 after stereotactic injection into the brain, the following tests were performed on experimental mice that had recovered well in terms of weight and health. Experimental mice that passed all indicators were qualified animal models of Parkinson's disease.

[0075] (1) Behavioral tests:

[0076] Apomorphine-induced rotation test: After intraperitoneal injection of apomorphine (0.5 mg / kg) into experimental mice, their rotational behavior was recorded over 30 minutes. A rotational speed greater than 7 revolutions per minute was considered a pass.

[0077] Rotador test: The fall latency of mice on an accelerated rotador is recorded. The test is considered passed if the fall latency decreases by more than 50% compared to before stereotactic injection.

[0078] Asymmetric use of limbs test: Record the use of the left and right forelimbs of the mouse while it is exploring upright. If the preference for using the limb corresponding to the side of the brain that was injected with the lesion is greater than 70%, the test is considered passed.

[0079] If all three tests are passed, the experimental mouse is deemed to have passed the behavioral test.

[0080] (2) Pathological verification:

[0081] Brain tissue was harvested from experimental mice via cardiac perfusion, and brain sections were prepared for tyrosine hydroxylase (TH) immunofluorescence staining. The substantia nigra is the production center of dopaminergic neurons, while the striatum is the working terminal of dopaminergic neurons. Therefore, if the TH-positive neuron destruction rate in the striatum or substantia nigra reaches 75% to 85%, pathological verification is considered successful. TH immunofluorescence staining in the striatum and substantia nigra is as follows: Figure 4 and Figure 5 As shown, Figure 4 The green TH signal in the left striatal region is almost invisible, indicating that almost all TH-positive neurons have been destroyed. Figure 5 The green TH signal in the left substantia nigra region was significantly reduced compared to the right side (the undamaged side), indicating that the destruction of TH-positive neurons reached approximately 75% to 85%.

[0082] (3) Inflammation assessment:

[0083] Immunofluorescence staining of ion-dependent calcium linker proteins was performed on adjacent brain slices to assess microglia morphology. If the activation level of microglia on the injection side was 2 to 3 times that of the undamaged control area on the contralateral side, the inflammation assessment was considered successful.

[0084] Multiple healthy mice that had not undergone modeling were selected as the healthy group, and behavioral tests were performed on the healthy group. In this embodiment, the experimental mice were used as the Parkinson's group. The statistical differences in behavioral tests between the Parkinson's group and the healthy group are as follows: Figure 3 As shown, the behavior of the Parkinson's disease animal model mice established using the method of this embodiment shows significant changes in motor symptoms compared to healthy mice. The left bar chart represents the results of the apomorphine-induced rotation test, indicating that the Parkinson's group rotated significantly fewer times than the healthy group. The middle bar chart represents the results of the rotarod test, showing that the Parkinson's group spent significantly less time on the rotarod, indicating significantly poorer limb coordination in the Parkinson's disease model mice. The right bar chart represents the results of the limb coordination test, showing that the Parkinson's group mice showed a significantly increased preference for the injured side of the limb, indicating significantly poorer limb coordination in the Parkinson's group. It should be noted that the healthy group mice did not have an injured side; therefore, when recording limb preferences, it is only necessary to record the preference for the limbs on the same side as the Parkinson's group mice (e.g., both left or right sides).

[0085] Table 1 shows the weight and survival monitoring data of the 50 mice in this embodiment. The survival rate of the 50 mice reached 94%, and all surviving mice exhibited significant behavioral changes, indicating that they all showed obvious symptoms of Parkinson's disease. The overall modeling success rate was 94%. Through the combined intervention of desipramine and meloxicam, the fatal contradiction of "high success rate" inevitably accompanied by "extremely high mortality rate" in traditional 6-OHDA modeling was successfully resolved, achieving efficient model construction and high animal survival rate.

[0086] In addition, this embodiment also provides an application of an animal model of Parkinson's disease, which is obtained by the aforementioned construction method.

[0087] Table 1: Weight and survival monitoring data of experimental mice in this example:

[0088] ;

[0089] Comparative Example 1: This comparative example provides a method for constructing an animal model of Parkinson's disease. The difference between this method and the aforementioned embodiments is that this comparative example does not include steps S1, S3, and S4 of the aforementioned embodiments, but only includes steps S2 and S5. Fifty experimental mice were injected into the brain using 6-OHDA solution, and each mouse was anesthetized by inhalation. The anesthetized mice's heads were fixed in a stereotaxic apparatus. The bilateral ear rods and incisor clamps of the stereotaxic apparatus were adjusted, with the height difference between the bilateral ear rods controlled within 0.1 mm. Using the anterior fontanelle as the origin, the injection target point was determined to be the medial forebrain fasciculus, with stereotaxic coordinates of: 1.2 mm posterior to the anterior fontanelle, 1.3 mm lateral to the midline, and 4.8 mm subdural. A hole was drilled at the corresponding skull position to expose the dura mater. A 12 mg / mL solution of 6-OHDA containing 0.02% (w / v) ascorbic acid was administered via stereotactic injection into the brain using a microinfusion pump at a rate of 0.1 μL / min. After injection, the needle was left in place for 5 minutes to prevent drug reflux and allow for sufficient drug diffusion. It should be noted that the 6-OHDA solution should be freshly prepared and used immediately, and stored away from light; it is effective for 2 hours at 4°C. Before suturing the scalp, 0.5% lidocaine was applied for local anesthesia. After suturing the skin, the mice were placed on a 37°C warming pad until they awoke. In this comparative example, 50 experimental mice were first stereotactically injected into the brain with the 6-OHDA solution. Then, behavioral tests, pathological verification, and inflammation assessments were performed on the mice. Once all tests were passed, a Parkinson's disease animal model was obtained. Furthermore, the mouse breed, 6-OHDA solution concentration, and stereotactic injection procedure were the same as in the aforementioned examples. The body weight and survival monitoring data of the 50 mice in this comparative example are shown in Table 2. Mice numbered 4, 7, 9, 10, 14, 15, 18, 21, 23, 26, 37, 38, 39, 42, 45, 48, and 50 ultimately survived, but their behavioral manifestations were not obvious. Mice numbered 2, 11, 32, 35, 41, and 47 were the only mice that ultimately survived and met the apomorphine-induced rotation rate of >7 revolutions per minute. Of the total 50 mice, 23 survived, a survival rate of 46%. Of the surviving mice, only 6 exhibited behavioral manifestations that met the apomorphine-induced rotation rate of >7 revolutions per minute, resulting in an overall modeling success rate of 12%.

[0090] Table 2: Weight and survival monitoring data of experimental mice in Comparative Example 1:

[0091] ;

[0092] Comparative Example 2: This comparative example provides a method for constructing an animal model of Parkinson's disease. The difference between this comparative example and the aforementioned embodiments is that step S3 in the aforementioned embodiments is not included. For example: First, 50 experimental mice of similar weight were selected. Desipramine powder was dissolved in 0.9% sterile saline to prepare a solution with a concentration of 0.2 mg / mL. Thirty minutes before stereotactic injection, each experimental mouse was intraperitoneally injected (IP) at a dose of 2 mg / kg, i.e., 0.2 mL of the desipramine solution was injected into a 20g experimental mouse. During injection, the needle was inserted at approximately a 30° angle and slowly injected at a rate of approximately 0.1 mL / 10 seconds to reduce peritoneal irritation. Subsequently, 6-OHDA solution was used to perform stereotactic brain injection on the 50 experimental mice. Then, a 5% (w / v) glucose solution was used to completely replace the mice's daily drinking water. Simultaneously, the regular diet was replaced with breeding feed. The average daily food intake of the experimental mice before the stereotactic brain injection was recorded as their basal food intake. Following stereotactic injection into the brain, real-time food intake was monitored daily. If the reduction in real-time food intake compared to the basal food intake was less than 50%, the aforementioned glucose solution was provided for free drinking. If the reduction was 50% to 80%, the aforementioned glucose solution was administered by gavage at a dose of 0.2 mL per gram of mouse body weight (i.e., 4 mL for a 20g mouse), twice daily. If the reduction was greater than 80%, the aforementioned glucose solution was administered by gavage at a dose of 0.3 mL / g body weight, three times daily. In this comparative example, 50 mice were first intraperitoneally injected with desipramine solution, followed by stereotactic injection into the brain using 6-OHDA solution. Postoperative care, including nutritional support and graded gavage, was then provided. Finally, behavioral tests, pathological verification, and inflammation assessments were performed on the mice, and a Parkinson's disease animal model was obtained after all tests were passed. Furthermore, the mouse breed, 6-OHDA solution concentration, and stereotactic injection procedure were the same as in the aforementioned examples. Table 3 shows the weight and survival monitoring data of the 50 mice in this comparative example. The survival rate of the 50 mice after injection of 6-OHDA was only 14%, but the surviving mice all had obvious behavioral changes, indicating that the surviving mice had obvious symptoms of Parkinson's disease. The overall modeling success rate was 14%.

[0093] Table 3: Weight and survival monitoring data of experimental mice in Comparative Example 2:

[0094] ;

[0095] Based on the data from the comprehensive examples, Comparative Example 1, and Comparative Example 2, it is evident that this invention, through the combined intervention of desipramine and meloxicam, successfully resolves the fatal contradiction in traditional 6-OHDA modeling where "high success rate" inevitably comes with "extremely high mortality rate," achieving efficient model construction and high animal survival rates. Furthermore, this invention is the first to simultaneously increase the success rate and postoperative survival rate of the 6-OHDA Parkinson's model to over 90%, transforming a high-risk, low-efficiency modeling method into a stable, efficient, and reproducible standardized technique.

[0096] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that the present invention includes, but is not limited to, the contents described in the accompanying drawings and the specific embodiments above. Any modifications that do not depart from the functional and structural principles of the present invention will be included within the scope of the claims.

Claims

1. A method for constructing an animal model of Parkinson's disease with improved survival rate, characterized in that, Includes the following steps: S1, intraperitoneal injection of desipramine solution into experimental mice; S2, stereotactic injection of 6-OHDA solution into the brains of the experimental mice; S3, inject meloxicam into the experimental mice via intraperitoneal injection, and dynamically adjust the meloxicam dosage based on the daily weight changes of the experimental mice. S4, Postoperative care including nutritional support and graded gavage was performed on the experimental mice; S5. The experimental mice were subjected to behavioral tests, pathological verification, and inflammation assessment. Once all tests were passed, a Parkinson's disease animal model was obtained. In step S3, the dynamic adjustment of meloxicam dosage based on the daily weight changes of the experimental mice includes: weighing the experimental mice before stereotactic brain injection to obtain their original weight; weighing the experimental mice daily starting 24 hours after stereotactic brain injection to obtain their real-time weight; if the real-time weight decreases by more than 20% compared to the original weight, immediately injecting the experimental mice intraperitoneally with meloxicam 5 mg / kg, followed by daily injections of 3 mg / kg until the weight returns to the original weight; if the real-time weight decreases by less than or equal to 20% compared to the original weight, starting 36 to 48 hours after stereotactic brain injection, injecting the experimental mice intraperitoneally with meloxicam 3 mg / kg daily for at least 3 consecutive days.

2. The construction method according to claim 1, characterized in that, In step S1, the desipramine solution is prepared by dissolving desipramine powder in sterile saline. The injection dose of the desipramine solution is 1~2 mg / kg, and step S1 is performed 30 minutes to 12 hours before the stereotactic injection in step S2.

3. The construction method according to claim 1, characterized in that, In step S2, the target point for stereotactic injection of the brain is the medial forebrain tract, and the stereotactic coordinates are: posterior to the anterior fontanelle A / P=-1.2mm, para-midline M / L=1.3mm, subdural D / V=-4.8mm.

4. The construction method according to claim 1, characterized in that, The 6-OHDA solution is to be prepared and used immediately, and becomes ineffective 2 hours after preparation; the 6-OHDA solution should be placed in a light-protected environment at 4°C within 2 hours.

5. The construction method according to claim 4, characterized in that, In step S2, the concentration of the 6-OHDA solution is 12 mg / mL and contains 0.02% ascorbic acid; when performing stereotactic injection into the brain, the injection rate is 0.1~0.3 μL / min, and the needle is left in place for 5 minutes after injection.

6. The construction method according to claim 1, characterized in that, In step S4, the nutritional support includes replacing the daily drinking water of the experimental mice with a glucose solution with a mass-volume concentration of 5%, and changing the feed to breeding feed.

7. The construction method according to claim 6, characterized in that, In step S4, the graded gavage includes: recording the average daily food intake of the experimental mice before stereotactic brain injection as the basal food intake; monitoring the real-time food intake of the experimental mice daily after stereotactic brain injection; if the reduction in real-time food intake compared to the basal food intake is less than 50%, the experimental mice are determined to have mild anorexia, and a 5% glucose solution is provided for them to drink freely; if the reduction in real-time food intake compared to the basal food intake is 50% to 80%, the experimental mice are determined to have moderate anorexia, and they are gavaged with glucose solution at a dose of 0.2 mL per gram of body weight twice a day, with a glucose solution concentration of 5%; if the reduction in real-time food intake compared to the basal food intake is greater than 80%, the experimental mice are determined to have severe anorexia, and they are gavaged with glucose solution at a dose of 0.2 mL per gram of body weight three times a day, with a glucose solution concentration of 5%.

8. The construction method according to claim 1, characterized in that, In step S5, the passing criteria for behavioral testing, pathological verification, and inflammation assessment are as follows: (a) Behavioral tests: Apomorphine-induced rotation greater than 7 revolutions / minute, rotarod test dwell time decreased by more than 50% compared with before stereotactic brain injection, and the limb coordination test showed a limb preference of more than 70% on the injured side; (b) Pathological verification: Immunofluorescence staining of tyrosine hydroxylase in the brains of experimental mice showed that the destruction rate of TH-positive neurons in the striatum or substantia nigra region reached 75% to 85%; (c) Inflammation assessment: Microglia activation was 2 to 3 times higher than that of the undamaged side of the brain, as detected by ionized calcium linker protein staining.

9. An application of an animal model of Parkinson's disease, characterized in that, The Parkinson's disease animal model is obtained by the construction method described in any one of claims 1 to 8.

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