Method for establishing subacute cerebellar ataxia animal model and application thereof

Abstract

The application provides a method for establishing a subacute cerebellar ataxia animal model and application. Specifically, a method for establishing a subacute cerebellar ataxia animal model based on a low-dose staged increasing administration mode of a compound preparation of talipexole and application thereof are provided. The application induces mice to have pathological characteristics such as movement coordination disorders, cerebellar volume atrophy and Purkinje cell loss under the premise of ensuring the survival rate of animals by intraperitoneal injection of Zoletil 50 for several weeks. The model has high stability, good repeatability and controllable damage degree, and can be used for anti-ataxia drug screening and neuroprotective mechanism research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for establishing and applying a subacute cerebellar ataxia animal model. Background Technology

[0002] Cerebellar ataxia (CA) is a neurological disorder characterized by impaired motor coordination, gait instability, abnormal postural control, and intention tremor. Its core pathological basis lies in the degeneration, loss, or dysfunction of Purkinje cells (PCs) in the cerebellar cortex, accompanied by imbalances in cerebellar neural circuits. Based on etiology, cerebellar ataxia can be divided into two main categories: hereditary and acquired.

[0003] Existing animal models mainly include genetically modified models, single high-dose toxin-induced models, inflammation-induced models, and cerebellar surgical injury models. However, all of these models have limitations to varying degrees in practical applications.

[0004] Hereditary transgenic models: These models can simulate gene mutations related to specific types of spinocerebellar ataxia (SCA) (including SCA1, SCA2, SCA3, etc.), mimicking the expression of specific mutated genes and the abnormal aggregation of proteins, demonstrating strong molecular mechanism correspondence. However, they are characterized by long construction cycles, high costs, slow disease progression, and significant inter-individual variability, making them unsuitable for short-term drug screening and evaluation of intervention effects. Furthermore, the proportion of patients with hereditary ataxia in clinical practice is relatively limited, while most ataxia cases are of acquired, metabolic, toxic, or immune-related causes. Therefore, relying solely on genetic models cannot fully reflect the actual clinical situation and has certain limitations in translational medicine and drug screening.

[0005] Single high-dose chemical toxin models: Traditional chemical toxin models, especially those induced by a single high-dose administration of neurotoxins such as 3-acetylpyridine, can produce significant behavioral impairments in a short period of time, but they generally suffer from high mortality, uncontrollable extent of damage, high pathological variability, and insufficient experimental reproducibility. High-dose acute injury often leads to simultaneous damage to the brainstem and other central nervous system structures, making it difficult to establish a relatively specific cerebellar injury pattern, simulate the progressive subacute injury process in clinical practice, and is also unfavorable for dose-dependent studies.

[0006] Inflammation or immune-induced models: Simple inflammation-induced models often induce neuroinflammatory responses through lipopolysaccharide (LPS) or immune stimulation. Their advantage is that they can be used to study inflammatory mechanisms, but they often cannot stably cause significant Purkinje cell loss or persistent motor dysfunction, and their behavioral performance fluctuates greatly, resulting in insufficient model stability.

[0007] Surgical injury models: Although surgical injury models can directly cause cerebellar tissue defects, they are complex to operate, highly invasive, inconsistent with most non-invasive clinical etiologies, and difficult to use for large-scale drug screening studies.

[0008] In summary, the existing technologies generally have the following problems: (1) the injury patterns are too extreme (acute high dose or permanent gene defects), lacking progressiveness and controllability; (2) the model stability and repeatability are insufficient; (3) the mortality rate is too high, affecting experimental efficiency; (4) it is difficult to simulate the clinically common subacute or acquired cerebellar injury process; (5) it is not conducive to the evaluation of neuroprotective drugs, anti-inflammatory drugs and neurorepair therapy.

[0009] In clinical practice, apart from a few hereditary cases, a large number of patients with cerebellar ataxia suffer from subacute or acute acquired injury, such as drug or toxin exposure, metabolic disorders, post-infectious inflammatory responses, autoimmune cerebellitis, and ischemic-hypoxic injury. These diseases typically present a pathological process of "progressive damage—functional deterioration—partial reversibility or intervention," with a clear treatment window.

[0010] Therefore, establishing an animal model that can simulate the subacute injury process, has controllable injury severity, low mortality, and good reproducibility is of great significance for drug screening and translational medicine research.

[0011] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0012] The purpose of this invention is to provide a method for constructing and applying a subacute cerebellar ataxia animal model.

[0013] Specifically, this invention provides a method for constructing a subacute cerebellar ataxia model by continuous intraperitoneal injection of Zoltil 50. This method can stably induce behavioral abnormalities, cerebellar structural atrophy, and Purkinje cell loss in mice.

[0014] In a first aspect of the present invention, a method for constructing an animal model of subacute cerebellar ataxia is provided, comprising the following steps: (1) Provide laboratory animals, which are non-human mammals (e.g., C57BL / 6 mice). (2) The experimental animals were given a compound preparation (inducer) containing NMDA receptor antagonist and GABA_A receptor modulator to obtain a subacute cerebellar ataxia animal model.

[0015] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0016] In another preferred embodiment, the animal model includes animal models that have not been genetically engineered or have been genetically engineered.

[0017] In another preferred embodiment, the non-human mammal is a rodent.

[0018] In another preferred embodiment, the rodents include rats and mice.

[0019] In another preferred embodiment, the experimental animal is an 8-week-old male C57BL / 6 mouse.

[0020] In another preferred embodiment, the C57BL / 6 mouse weighs 15–30 g, preferably 20–25 g.

[0021] In another preferred embodiment, the administration includes injection (e.g., intramuscular injection, intraperitoneal injection).

[0022] In another preferred embodiment, the experimental animals are subjected to adaptive feeding before modeling, and the conditions for adaptive feeding are: SPF-grade feeding, temperature 21–22°C, 12-hour light-dark cycle, and free access to food and water.

[0023] In another preferred embodiment, the experimental animals are kept under the following conditions during the modeling process: SPF-grade feeding, temperature 21–22°C, 12-hour light-dark cycle, and free access to food and water.

[0024] In another preferred embodiment, the NMDA receptor antagonist is telemetamine.

[0025] In another preferred embodiment, the GABA_A receptor modulator is zolpidem.

[0026] In another preferred embodiment, the compound formulation comprises tledamine and zolpidem.

[0027] In another preferred embodiment, the ratio of telexamine to zolpidem is 1:1 in the compound formulation.

[0028] In another preferred embodiment, the compound formulation is Zoltil 50.

[0029] In another preferred embodiment, the administration is carried out by intraperitoneal injection for 28 to 35 consecutive days, preferably 28 to 32 days (e.g., 30 days).

[0030] In another preferred embodiment, the intraperitoneal injection includes: (1) First stage; (2) Second stage; (4) Optional, third stage; The dosage for intraperitoneal injection is increased in stages, with the dosage D1 in the first stage being less than the dosage D2 in the second stage.

[0031] In another preferred embodiment, the method further includes (3) an evaluation step, which determines whether the modeled mouse needs to enter the third stage based on the completion time of the balance beam experiment in the modeled mouse; (i) If the completion time is more than 7 seconds, there is no need to proceed to the third stage; (ii) If the completion time is less than 7 seconds, the third stage needs to be entered.

[0032] In another preferred embodiment, the dosage D1 for the first stage is 15 mg / kg to 25 mg / kg (e.g., 20 mg / kg).

[0033] In another preferred embodiment, the duration T1 of the first stage is 5 to 9 days, for example, 7 days.

[0034] In another preferred embodiment, the dosage D2 for the second stage is 25 mg / kg to 35 mg / kg (e.g., 30 mg / kg).

[0035] In another preferred embodiment, the duration T2 of the second stage is 17 to 25 days (e.g., 21 days).

[0036] In another preferred embodiment, the dosage D3 for the third stage of administration is 25 mg / kg to 35 mg / kg (e.g., 30 mg / kg).

[0037] In another preferred embodiment, the duration T3 of the third stage is 3 to 14 days (e.g., preferably 7 days).

[0038] In another preferred embodiment, the intraperitoneal injection is administered once daily.

[0039] In another preferred embodiment, the intraperitoneal injection is administered at a fixed time from 8:00 AM to 12:00 PM daily, for example, 9:00 AM to 10:00 AM.

[0040] In another preferred embodiment, the dosage D1 for the first stage is 15 mg / kg to 25 mg / kg (e.g., 20 mg / kg), and the dosage D2 for the second stage is 25 mg / kg to 35 mg / kg (e.g., 30 mg / kg), with the dosing frequency being once daily.

[0041] In another preferred embodiment, the method further includes (3) verifying (confirming) the successful construction of the model through behavioral, imaging and / or histological examinations.

[0042] In another preferred embodiment, the behavioral detection includes: (1) Balance beam experiment; (2) Optionally, the rotator experiment; (3)Optional, CatWalk gait analysis.

[0043] In another preferred embodiment, the balance beam experiment includes the following steps: (a1) a mouse traverses a 100 cm long wooden ramp (0.8 cm in diameter) with a starting point 30 cm above the ground and a slope of 45°, and finally enters a black target box.

[0044] In another preferred embodiment, the analytical indicators of the balance beam experiment include: completion time, total number of steps, fall rate, and number of falls.

[0045] In another preferred embodiment, in the balance beam experiment, the success of modeling is determined based on the completion time of the modeling mice: (i) If the completion time is more than 7 seconds, the model is considered successful; (ii) If the completion time is less than 7 seconds, the modeling is not completely successful.

[0046] In another preferred embodiment, the rotator experiment includes the following steps: (a2) Place the mouse on the rotating cylinder; (b2) The rotational speed of the cylinder is accelerated from 4 rpm to 40 rpm within 60 s; (c2) The rotating cylinder is maintained at 40 rpm for 240 s; (d2) Record the fall latency period; (e2) The formal experiment was conducted continuously for 5 days, with 3 tests per day, and the average value of the 3 tests was taken.

[0047] In another preferred embodiment, the maximum test time during the rotator bar experiment is 300 s.

[0048] In another preferred embodiment, the rotator bar experiment is conducted with continuous training for 3 days to ensure stable performance.

[0049] In another preferred embodiment, the CatWalk gait analysis includes the steps of: (a3) Mice are allowed to pass freely through the glass tunnel. Each mouse must complete at least 3 valid passes, and each pass must include at least 5 complete gait cycles. (b3) Record footprints: left anterior (LF), left posterior (LH), right anterior (RF), right posterior (RH); (c3) Automatic software recognition + manual correction.

[0050] In another preferred embodiment, the criteria for effective passage are no pauses, no hair removal, no turning, and no upright movement.

[0051] In another preferred embodiment, the CatWalk gait analysis metrics include: diagonal support percentage, body speed, single support time, and footprint width.

[0052] In another preferred embodiment, the imaging examination is a small animal MRI structural scan.

[0053] In another preferred embodiment, the small animal MRI structural scan is performed 45 to 55 days (e.g., 30 days) after drug administration.

[0054] In another preferred embodiment, the histological examination is Calbindin immunofluorescence staining.

[0055] In a second aspect of the invention, a use of Shutai 50 is provided for preparing a drug or formulation for inducing a non-human mammalian subacute cerebellar ataxia animal model.

[0056] In another preferred embodiment, the formulation is a liquid formulation.

[0057] In a third aspect of the invention, the use of the animal model constructed by the method described in the first aspect of the invention is provided, comprising: (1) Used for screening drugs for the prevention and / or treatment of cerebellar ataxia; (2) Used to study the formation mechanism of cerebellar ataxia; (3) Used for screening novel biomarkers.

[0058] In another preferred embodiment, the cerebellar ataxia is subacute cerebellar ataxia.

[0059] In another preferred embodiment, the drug is selected from the group consisting of: small molecules, antibodies, fusion proteins, nucleic acids, and oligonucleotide molecules (oligonucleotide drugs).

[0060] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0061] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0062] Figure 1 This is a schematic diagram of the animal model construction process of the present invention.

[0063] Figure 2 The diagram shows the behavioral results of the balance beam and spinning bar exercises.

[0064] Figure 3 This is a graph showing the results of the gait analysis.

[0065] Figure 4 Image showing the MRI results of a small animal.

[0066] Figure 5 This is a graph showing the results of immunofluorescence. Detailed Implementation

[0067] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0068] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0069] Through extensive and in-depth research, the inventors have, for the first time, conceived and proposed a low-dose, staged-stage controlled subacute cerebellar ataxia animal model to address existing problems in this field. By employing escalating administration of low-dose neurotoxins, combined with inflammatory pretreatment or synergistic mechanisms, Purkinje cell damage can be gradually induced while avoiding acute high-dose toxicity, thereby establishing a stable subacute motor dysfunction model. This model can achieve graded control of damage intensity while ensuring animal survival, improving experimental reproducibility, and more realistically simulating the occurrence and development of clinically acquired cerebellar ataxia.

[0070] Specifically, this invention aims to construct a low-dose, staged induction strategy based on a telexamine compound preparation (Shutai 50), gradually inducing cerebellar nerve damage through escalating dosing to form a stable, controllable, and reproducible subacute cerebellar ataxia animal model. This model can achieve the following expected goals: ① Inducing stable cerebellar damage while avoiding the risk of acute high-dose death; ② Reducing drug resistance caused by receptor adaptation changes through dose escalation; ③ Achieving a subacute progressive injury pattern rather than an acute explosive injury; ④ Establishing a multi-dimensional validation system encompassing behavioral, imaging, and pathological dimensions to improve model reliability; ⑤ Constructing an animal model that more closely resembles the clinically acquired cerebellar ataxia course. Based on these goals, this invention has been completed.

[0071] the term

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0073] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0074] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0075] Methods for constructing an animal model of subacute cerebellar ataxia

[0076] This invention provides a method for constructing an animal model of subacute cerebellar ataxia, comprising the following steps: (1) Provide laboratory animals, which are non-human mammals (e.g., C57BL / 6 mice). (2) The experimental animals were given a compound preparation (inducer) containing NMDA receptor antagonist and GABA_A receptor modulator to obtain a subacute cerebellar ataxia animal model.

[0077] Typically, in the method of the present invention, the compound formulation is Zoltil 50.

[0078] Typically, in the method of the present invention, the intraperitoneal injection dosage is increased in stages, including: (1) First stage; (2) Second stage; (4) Optional, third stage; The dosage D1 in the first stage is less than the dosage D2 in the second stage.

[0079] Applications of animal models of subacute cerebellar ataxia

[0080] This invention provides the use of the subacute cerebellar ataxia animal model constructed by the above method, including: (1) Used for screening drugs for the prevention and / or treatment of cerebellar ataxia; (2) Used to study the formation mechanism of cerebellar ataxia; (3) Used for screening novel biomarkers.

[0081] Typically, the model constructed in this invention can be used to screen potential drugs for cerebellar atrophy or cerebellar ataxia and evaluate their efficacy in alleviating cerebellar atrophy and improving motor coordination. It can serve as an important tool for preclinical research in the drug development process.

[0082] Typically, the model constructed in this invention helps to study the mechanisms of nerve cell damage and neuroprotection during cerebellar atrophy, which provides a basis for developing treatment strategies for ataxia, especially related therapies for cerebellar function protection.

[0083] Typically, by analyzing biomarkers (such as proteins and gene expression) at different time points in the model constructed by this invention, new biomarkers can be discovered for the early diagnosis of cerebellar ataxia or related diseases or for monitoring disease progression.

[0084] The main advantages of this invention include: (1) Inducing stable cerebellar injury while avoiding the risk of acute high-dose death; (2) Reduce drug resistance caused by receptor adaptation changes by increasing the dose; (3) To achieve a subacute progressive injury pattern rather than an acute fulminant injury; (4) Establish a multi-dimensional validation system encompassing behavioral, imaging, and pathological aspects to improve model reliability; (5) The constructed animal model is closer to the clinical characteristics of the course of acquired cerebellar ataxia.

[0085] The present invention will be further explained below with reference to specific embodiments.

[0086] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0087] I. Laboratory Animals and Their Housing Conditions

[0088] Eight-week-old male C57BL / 6 mice (20–25 g) (mice manufacturer: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.; License No.: SCXK(Su)2023-0009) were selected, housed in SPF-grade conditions, at a temperature of 21–22℃, with a 12-hour light-dark cycle, and with free access to food and water. Experiments began after 7 days of acclimatization. All procedures were approved according to animal ethics guidelines.

[0089] II. Preliminary Experimental Dosage Exploration

[0090] To determine the optimal induction dose, three dose groups were set up: 30 mg / kg, 40 mg / kg, and 60 mg / kg.

[0091] Administration: Intraperitoneal injection, once daily for 7 consecutive days.

[0092] The results showed that the mortality rate was approximately 40% in the 60 mg / kg group, approximately 20% in the 40 mg / kg group, and less than 5% in the 30 mg / kg group, although mild motor abnormalities were observed. Therefore, 30 mg / kg was determined to be the safe and effective range.

[0093] III. Formal Mold Design Plan

[0094] 1. Grouping

[0095] A total of 20 mice were randomly divided into two groups: a control group (n=10) and a model group (n=10).

[0096] 2. Preparation of inducing agents

[0097] Dissolve Sutacetin 50 in sterile water to a concentration of 25 mg / mL. The dosage volume should be calculated based on body weight and controlled within the range of 0.1 mL / 10 g body weight.

[0098] 3. Phased incremental induction

[0099] Phase 1 (Week 1): Low-dose induction phase

[0100] Dosage: 20 mg / kg; Frequency: Once daily; Objective: To induce mild neurological disturbances while avoiding acute toxicity.

[0101] Phase 2 (Weeks 2–4): High-dose induction phase

[0102] Dosage: 30 mg / kg; Frequency: Once daily; Cycle: 21 consecutive days; Phase 3 (after week 5): Intensive Induction Phase The time taken to pass over the balance beam can be used to preliminarily determine whether mice have ataxia. If the time is less than 7 seconds, several additional injections of 30 mg / kg (once a day) can be given in week 5 until the time taken to pass over the balance beam exceeds 7 seconds.

[0103] Notice: 1) The administration time is fixed at 9:00-10:00 daily; 2) The control group received an equal volume of physiological saline; 3) During the experiment, the animals' general condition was observed daily, including: weight changes, activity level, coat condition, and respiratory status. Behavioral analyses were all performed by experimenters who were unaware of the group assignments (blinded method).

[0104] A schematic diagram of the animal model construction process of this invention is shown below. Figure 1 .

[0105] IV. Behavioral Testing Methods

[0106] Behavioral assessments were conducted one week after the end of drug administration.

[0107] (a) Balance Beam Test

[0108] Compared to the spinning bar, the balance beam requires a higher level of fine motor control and hind limb coordination.

[0109] 1. Experimental steps: The mouse must traverse a 100 cm long wooden ramp (0.8 cm in diameter) with a starting point 30 cm above the ground and a 45° incline, before finally entering the black target box. Figure 2 (A)

[0110] 2. Evaluation Indicators: Completion Time: The time required from the starting point to entering the target box. This is a key indicator for evaluating the success of the model. Based on the performance of the control group, the inventors used 7 seconds as the cutoff. If the completion time of the model group mice was more than 7 seconds, the model was considered to be successfully established; if the completion time was less than 7 seconds, the model was considered not to be completely successful, and the mice continued to the third stage, namely the reinforcement induction stage (see...). Figure 1 ).

[0111] Total Steps: The total number of steps taken to complete the entire wooden beam section. It reflects the sense of security and center of gravity control while walking on the beam.

[0112] Missteps: The percentage of hind limb slippages out of the total number of steps. This is a key indicator of the degree of ataxia. Mice require precise visual-motor integration when navigating narrow wooden beams; a significantly elevated missteps rate suggests impairment in proprioception of the hind limbs or fine motor error correction mechanisms in the cerebellum.

[0113] Number of Missteps: The number of times the hind limbs slip during the completion of the entire wooden beam section, which is also an indicator of the degree of ataxia.

[0114] 3. Experimental Results: See results Figure 2 B, C, D, E.

[0115] Completion time: The completion time of the modeling group was significantly longer than that of the control group; Total steps: The total number of steps in the modeling group was significantly higher than that in the control group; The failure rate and number of failures both showed an increasing trend compared to the control group.

[0116] (ii) Accelerating Rotarod Test

[0117] This experiment is primarily used to assess the motor coordination, balance, and motor learning ability of mice. Figure 2 (Middle F).

[0118] 1. Experimental steps: 1) Place the mouse on the rotating cylinder; 2) The engine speed accelerates from 4 rpm to 40 rpm within 60 seconds; 3) Maintain 40 rpm for 240 seconds; 4) Maximum test time: 300 seconds; 5) Record the fall latency period; 6) Train continuously for 3 days to ensure stable performance; 7) The formal experiment was conducted continuously for 5 days, with 3 tests per day, and the average value of the 3 tests was taken.

[0119] 2. Analytical indicators (average of 3 valid runs for each animal): Fall latency: This refers to the time it takes for a mouse to fall off the robin. The longer the latency, the better the mouse's motor coordination.

[0120] 3. Experimental Results: See results Figure 2 The latency period was significantly shortened in the G and H model groups, reflecting ataxia caused by cerebellar dysfunction.

[0121] (III) CatWalk gait analysis

[0122] CatWalk gait analysis is an advanced pressure-sensing gait analysis system that captures dynamic features that are difficult to observe with the naked eye. It utilizes the CatWalk XT (Noldus) system, which consists of: a high-speed camera, a glass walkway, a green light reflection system, and automated analysis software.

[0123] 1. Experimental steps: 1) Mice freely pass through the glass tunnel; 2) Each mouse must complete at least 3 valid passes; 3) Valid criteria: No pauses, no grooming, no turning, no standing upright; 4) Each pass must contain at least 5 complete gait cycles; 5) Record footprints: left anterior (LF), left posterior (LH), right anterior (RF), right posterior (RH); 6) Automatic software recognition + manual correction.

[0124] 2. Analytical indicators (average of 3 valid runs for each animal): Support Diagonal %: Diagonal support is the most stable gait pattern, and a decrease in this value indicates a disordered walking rhythm. Body Speed: In ataxia, mice tend to overcome their loss of balance by running fast, similar to a "frantic gait". Single Stance: This reflects a decrease in the weight-bearing capacity of the affected limb on a single leg, requiring a rapid switch to the supporting foot. Print Width: Increasing the width of the base support is a typical compensatory strategy to deal with instability.

[0125] 3. Experimental Results: See results Figure 3 The model group showed a significant decrease in diagonal support percentage, reduced single-leg support time, and a significant increase in body speed. The single-leg support time of the left hind limb was significantly shortened, and the footprint width of the right forelimb increased, indicating gait rhythm disorder.

[0126] V. Small Animal Magnetic Resonance Imaging (MRI) Brain Volume Analysis

[0127] An in vivo MRI scan was performed approximately 45 days after the completion of behavioral testing and administration of the drug.

[0128] 1. Anesthesia

[0129] Induction was performed using 3–4% isoflurane; during the scan: respiratory rate was monitored, and a heating system was used to maintain body temperature; 2. Imaging parameters T2-weighted structural scans were used to cover the entire brain; after the scan, image motion artifacts were checked to exclude data with severe artifacts. 3. Volumetric quantitative analysis Deformation-based morphometry was performed using ANTs software. Individual structural images are registered to templates; log-Jacobian values ​​are calculated; cerebellar regions are extracted; and intergroup comparison analysis is performed.

[0130] 4. Experimental Results

[0131] See results Figure 4 MRI images showed significant cerebellar structural atrophy in the model group. Statistical analysis showed that the log-Jacobian value in the experimental group was significantly lower than that in the control group, confirming the structural tissue loss.

[0132] VI. Histology and Immunofluorescence

[0133] 1. Irrigation and Fixation

[0134] Anesthesia was administered with isoflurane; PBS was perfused through the heart; followed by perfusion with 4% PFA; brain tissue was removed and fixed in 4% PFA at 4°C for 4 hours; then transferred to 20%–30% sucrose solution for dehydration until the tissue settled.

[0135] 2. Frozen slices

[0136] 30 μm coronal sections were prepared using a Leica CM1950 cryostat; the sections were then attached to an adhesive slide.

[0137] 3. Immunofluorescence staining

[0138] Animals were anesthetized and fixed with PBS and 4% PFA via cardiac perfusion. Brain tissue sections (30 μm) were stained with Calbindin immunofluorescence. Quantitative analysis was performed using ImageJ software after image acquisition. Details are as follows: 1) Blocking solution composition: 3% BSA, 5% goat serum, 0.4% Triton X-100, PBS; 2) Close for 2 hours; 3) Add primary antibody: Calbindin antibody (1:500, Huabio ET1702-54), incubate overnight at 4°C; 4) Wash 3 times with PBS; 5) Add secondary antibody: Goat Anti-Rabbit IgG (H+L) Alexa Fluor 555 conjugate (Abcam, ab150078), antibody dilution 1:1000, incubate at room temperature for 2 hours; 6) DAPI sealing; 7) Image acquisition equipment: Olympus VS200, FV3000 confocal microscope; 8) ImageJ software was used for quantitative image analysis.

[0139] 4. Experimental Results: See results Figure 5 Compared with the control group, the model group showed greater loss of Purkinje cells in the third and ninth lobes of the cerebellar vermis (indicated by white arrows). Calbindin (red) labels the cerebellar Purkinje cells, and DAPI (blue) labels the cell nuclei. As the only output neurons of the cerebellar cortex, apoptosis of Purkinje cells is the direct pathological basis for ataxia.

[0140] VII. Statistical Analysis

[0141] Data are expressed as mean ± SD. Normality and homogeneity of variance were tested using an independent samples two-tailed t-test, with P < 0.05 considered significant.

[0142] VIII. Experimental Conclusions

[0143] Based on the above comprehensive analysis of behavioral, imaging, and histological findings: 1. The completion time, number of steps, and error rate of the balance beam modeling group increased; 2. The drop latency of the spinning rod is significantly shortened; 3. Obvious rhythm disorder was observed in gait analysis; 4. MRI confirmed structural changes in the cerebellum; 5. Calbindin immunofluorescence showed a decrease in the number of Purkinje cells; This demonstrates that continuous administration of Zoltil 50 can successfully establish a stable animal model of subacute cerebellar ataxia.

[0144] discuss

[0145] This invention belongs to the field of animal model construction technology for nervous system diseases, specifically relating to a subacute cerebellar ataxia animal model constructed based on telexamine compound preparation and its application.

[0146] Tiletamine is a phenylcyclohexylpiperidine derivative, belonging to the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, and is classified as a dissociative anesthetic. Its chemical structure and pharmacological mechanism are similar to ketamine, but it is more potent (approximately 3-4 times more potent), has a longer duration of action, and possesses stronger analgesic effects.

[0147] Clinical observations have shown that telexamine has dose-dependent neurotoxic effects on the central nervous system. Repeated administration can cause cerebellar dysfunction, manifested as intention tremor, gait abnormalities, and ataxia, suggesting that it may affect cerebellar neural circuits and Purkinje cells.

[0148] In clinical and experimental settings, telexamine is often combined with the benzodiazepine zolazepam in a 1:1 ratio to form a compound anesthetic (trade name Zoltil 50). Zolazepam, as a GABA_A receptor agonist, can regulate the excitation-inhibition balance through a "balanced anesthesia" mechanism, partially alleviating the stress response caused by NMDA receptor antagonism.

[0149] However, long-term single continuous administration may lead to adaptive upregulation of NMDA receptors or compensatory changes in the neurotransmitter system, thereby generating drug resistance and affecting model stability.

[0150] Currently, there is no systematic technical solution for constructing a controllable subacute cerebellar ataxia model based on telapharmonic or salbutamol using a low-dose, phased escalation strategy.

[0151] This invention aims to solve the following technical problems: (1) Existing animal models of hereditary cerebellar ataxia have long construction cycles, high costs, slow disease progression and large individual differences, which are not conducive to drug screening and evaluation of intervention effects in a short period of time; (2) Existing single high-dose chemical toxin induction models have defects such as high mortality, uncontrollable damage, significant damage to non-specific areas such as the brainstem, and insufficient experimental repeatability, making it difficult to stably simulate the subacute cerebellar injury process. (3) Existing simple inflammation-induced models lack stable and continuous Purkinje cell damage, and the behavioral changes fluctuate greatly, resulting in insufficient model stability. (4) Existing models are generally unable to realistically simulate the occurrence and development of acquired or subacute cerebellar ataxia commonly seen in clinical practice, and lack technical means to control the intensity of damage and evaluate the treatment time window. (5) The existing technology lacks a cerebellar ataxia animal model with low mortality, short modeling cycle, gradeable and reproducible damage degree, which makes it difficult to meet the needs of anti-ataxia drug screening, neuroprotective research and translational medicine research.

[0152] Therefore, developing a low-dose, staged, controllable subacute cerebellar ataxia animal model has a clear practical need and important application value.

[0153] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A method for constructing an animal model of subacute cerebellar ataxia, characterized in that, Includes the following steps: (1) Provide laboratory animals, wherein the laboratory animals are non-human mammals; (2) The experimental animals were given a compound preparation containing NMDA receptor antagonist and GABA_A receptor modulator to obtain a subacute cerebellar ataxia animal model.

2. The method according to claim 1, characterized in that, The compound preparation is Shutai 50.

3. The method according to claim 1, characterized in that, The administration is performed by intraperitoneal injection for 28 to 35 consecutive days, preferably 28 to 32 days.

4. The method according to claim 3, characterized in that, The intraperitoneal injection includes: (1) First stage; (2) Second stage; (4) Optional, third stage; The dosage for intraperitoneal injection is increased in stages, with the dosage D1 in the first stage being less than the dosage D2 in the second stage.

5. The method according to claim 4, characterized in that, The dosage for the first phase, D1, is 15 mg / kg to 25 mg / kg, and the dosage for the second phase, D2, is 25 mg / kg to 35 mg / kg, with a dosing frequency of once daily.

6. The method according to claim 1, characterized in that, The method also includes (3) verifying the successful construction of the model through behavioral, imaging and / or histological testing.

7. The method according to claim 6, characterized in that, The behavioral tests include: (1) Balance beam experiment; (2) Optionally, the rotator experiment; (3)Optional, CatWalk gait analysis.

8. The method according to claim 6, characterized in that, The histological examination was performed using Calbindin immunofluorescence staining.

9. An application of Sutacetin 50, characterized in that, Drugs or preparations used to prepare animal models of subacute cerebellar ataxia in non-human mammals.

10. The use of the animal model constructed by the method according to any one of claims 1-8, characterized in that, include: (1) Used for screening drugs for the prevention and / or treatment of cerebellar ataxia; (2) Used to study the formation mechanism of cerebellar ataxia; (3) Used for screening novel biomarkers.

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