A method for constructing a model of hydrocephalus with primary ciliary motor disorder
A primary ciliary motor disorder hydrocephalus model was constructed by immersing zebrafish models in benzimidazole solution, which solved the problems of model construction stability and operability, and provided an effective experimental platform for research and treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TIANJIN UNIV
- Filing Date
- 2025-04-28
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies make it difficult to construct stable and operable animal models of primary ciliary motor disorder hydrocephalus, and traditional methods may cause harm to animals, lacking effective research and treatment methods.
A non-invasive method was used to screen benzimidazole solution by immersing zebrafish models in the solution, and benzimidazole was selected as a modeling agent to construct a primary ciliary motor disorder hydrocephalus model. The success of the model was verified by pathological morphology and behavioral studies.
A stable and reproducible animal model was successfully established, reducing harm to animals and providing an experimental basis for studying the mechanism and treatment of primary ciliary motor disorder hydrocephalus. It can also monitor ventricular dilation in real time.
Smart Images
Figure CN120392749B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal model technology, and in particular to a method for constructing a primary ciliary motor disorder hydrocephalus model. Background Technology
[0002] Hydrocephalus is a neurological disorder caused by impaired cerebrospinal fluid production or absorption. Its high disabling nature and insidious course make it a critical challenge for public health systems. For example, idiopathic normal pressure hydrocephalus (iNPH) has a prevalence as high as 2.9% in people over 65 years of age. Its clinical treatment faces significant challenges. On the one hand, its pathogenesis and therapeutic targets remain unclear. On the other hand, treatment options are limited; current treatment primarily relies on surgery, which carries high risks and numerous complications, and targeted therapies are lacking.
[0003] Primary ciliary dyskinesia is a hereditary disease whose core pathology involves abnormalities in the structure and function of cilia. Cilia are widely distributed in various organs and tissues of the human body, including the ependymal cells of the respiratory tract, reproductive system, and ventricular system. Normally, these cilia rhythmically propel cerebrospinal fluid (CSF) through the circulation of CSF within the ventricular system, maintaining a dynamic balance of intraventricular pressure. When ciliary function is impaired, the cilia of the ependymal cells within the ventricles may be unable to effectively propel CSF circulation, leading to CSF accumulation. The persistent accumulation of CSF causes increased intraventricular pressure, gradually leading to ventricular dilation, or hydrocephalus. Long-term hydrocephalus and increased intracranial pressure can further compress the brain parenchyma, affecting neurological development and even causing irreversible brain damage. Currently, the pathogenesis of hydrocephalus is unclear; animal models can provide new insights into the disease's pathogenesis and lay the foundation for developing new treatments.
[0004] Using gene knockout of the EFcab2 gene (expressing the DRC8 protein) to construct animal models results in animals carrying a lifelong genetic defect, often presenting a technical challenge of homozygous lethality. However, by targeting the EFcab2 gene expression protein with drugs and administering them at a specific time, interference with early animal development can be avoided, achieving the gene knockout effect at a specific time. Therefore, developing inhibitors targeting the DRC8 protein and constructing PCD (polycephalus cerebrospinal fluid) animal models has significant scientific and clinical value.
[0005] Zebrafish, as a model organism, has wide applications in constructing disease models due to its characteristics such as in vitro fertilization, embryonic transparency, low breeding cost, and strong regeneration ability. This invention proposes a novel method for constructing an animal model of hydrocephalus with primary ciliary dyskinesia using benzimidazole. Summary of the Invention
[0006] The purpose of this invention is to provide a method for constructing a primary ciliary motor disorder hydrocephalus model. A zebrafish hydrocephalus model is constructed using benzimidazole solution, enabling a simple and stable establishment of this animal model. This non-invasive method minimizes harm to the animal model. By immersing the animal model in benzimidazole, the model is successfully established with good stability, repeatability, objectivity, simplicity, and operability, reducing potential interference factors. This provides a necessary experimental foundation for studying the mechanisms and developing treatments for primary ciliary motor disorder hydrocephalus.
[0007] To achieve the above objectives, the present invention provides a method for constructing a primary ciliary motor disorder hydrocephalus model, comprising the following steps:
[0008] Step 1: Modeling agent compounds were screened based on DRC8, a substructural protein of the inin-dyne regulatory complex (N-DRC). The database used for screening was the CNS central nervous system drug database. Compounds were screened based on their free binding energy. The top 100 compounds were further screened using medical and toxicological methods to determine benzimidazole as the modeling agent.
[0009] Step 2: Under normal feeding conditions, administer benzimidazole solution to the experimental animals and set up a blank control group;
[0010] Step 3: After treating the experimental animals with benzimidazole solution, the experiments were verified through pathological morphology and behavioral methods.
[0011] Step four: Compared with the control group, the experimental animals showed significantly enlarged ventricles and significantly reduced motor function in terms of pathological morphology and behavioral indicators. If this condition is met, the primary ciliary motor disorder hydrocephalus model is successfully constructed.
[0012] Preferably, in step one, the benzimidazole is 4,5-dimethyl-1H-benzimidazole.
[0013] Preferably, in step two, the rearing conditions are: temperature 28.5℃, 14 hours of light / 10 hours of darkness.
[0014] Preferably, in step two, the experimental animal is a zebrafish.
[0015] Preferably, in step two, the concentration of benzimidazole solution administered is 200 ug / ml or 300 ug / ml.
[0016] Preferably, the concentration of benzimidazole solution administered is 300 ug / ml.
[0017] Preferably, in step two, the benzimidazole solution is administered via immersion.
[0018] The advantages and beneficial effects of the method for constructing a primary ciliary motor disorder hydrocephalus model described above are as follows:
[0019] 1. This invention employs a non-invasive method to construct a primary ciliary motor disorder hydrocephalus model. By immersing the animal model in benzimidazole, a primary ciliary motor disorder hydrocephalus model was successfully established with minimal harm to the animal model.
[0020] 2. The animal model constructed in this invention has good stability and repeatability, is objective and simple, and is highly operable, reducing other possible interfering factors. It provides the necessary experimental basis for studying the mechanism of primary ciliary motor disorder hydrocephalus and developing treatment methods.
[0021] 3. The primary ciliary motor disorder hydrocephalus animal model prepared by this invention can monitor the degree of hydrocephalus in real time by measuring the size of the ventricles and detecting the level of motor activity during the research process. Therefore, the modeling process of this invention is a dynamic process, which can observe the expansion of the ventricles at different time points, providing a good experimental platform for the study of primary ciliary motor disorder hydrocephalus. At the same time, this model also provides a good tool for developing new treatment methods.
[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0023] Figure 1 This is a predicted structure diagram of the zebrafish DRC8 protein according to the present invention;
[0024] Figure 2 The results of computer simulation of the interaction between DRC8 and 4,5-dimethyl-1H-benzimidazole in Example 1 of the present invention are shown, where A is the surface and molecular structure of DRC8 protein and B is the protein molecular interaction relationship.
[0025] Figure 3 This is a flowchart of the zebrafish experiment in Embodiment 2 of the present invention;
[0026] Figure 4 These are images of the ventricle detection results of zebrafish according to the present invention, where A is the ventricle image of zebrafish in the experimental group (300 μg / mL) (n=20) and B is the ventricle image of zebrafish in the control group (n=20). Detailed Implementation
[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0028] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0029] Unless otherwise defined, the instruments, equipment, reagents, and materials used in this invention are all commercially available.
[0030] Experimental animals: zebrafish embryos 20 hours after fertilization.
[0031] Experimental materials: CNS central nervous system drug database (from Enamine), benzimidazole; PTU; stereomicroscope.
[0032] Example 1
[0033] A method for constructing a model of hydrocephalus with primary ciliary motor disorder includes the following steps:
[0034] Step one: N-DRC (intin-dynein regulatory complex) is an important structure in motor cilia. The research group screened compounds based on the N-DRC sub-structural protein DRC8 using the CNS central nervous system drug database (from Enamine), which contains 47,360 compounds. Compounds were screened based on their free binding energy, and the top 100 compounds were further screened using medical and toxicological methods, ultimately identifying benzimidazole as the modeling agent.
[0035] DRC8 protein structure: predicted using alphafold, while also predicting interaction sites based on other homologous proteins.
[0036] Virtual screening: Screening is performed using Autodockvina based on estimated binding sites.
[0037] Dataset selection: The dataset used is the Enamine CNS dataset, which contains 47,360 small molecules and is a database of small molecule compounds with high CNSMPO scores. This implies that the molecules have high blood-brain barrier permeability.
[0038] Scoring Assessment: Overall, molecules with larger molecular weights have higher upper limits for docking scores. However, for CNS drugs, we generally prefer smaller molecular weights. Therefore, we choose to use the average heavy atom binding energy, as shown in the following formula:
[0039] Score = Docking score / Number of heavy atoms
[0040] It means that the docking score is divided by the number of heavy atoms in the molecule. Using this score for sorting helps to find the structure with the highest bonding efficiency.
[0041] Post-processing: The molecules were sorted according to their average heavy atom binding energy, and the residue numbers of the target protein interactions with the molecules were counted. Molecules binding to residues 150-162, which had low confidence levels, were excluded. The pharmacological and toxicological properties of the remaining molecules were investigated.
[0042] The final modeling agent, benzimidazole, was determined to be the parent structure of 4,5-dimethyl-1H-benzimidazole. The chemical structural formula of benzimidazole is as follows:
[0043]
[0044] The chemical structural formula of the parent structure of 4,5-dimethyl-1H-benzimidazole is as follows:
[0045]
[0046] Step 2: Under the rearing conditions of 28.5℃ and 14 hours of light / 10 hours of darkness, zebrafish were administered benzimidazole solution at a concentration of 200-300 ug / ml. The benzimidazole solution was administered by immersion, and a blank control group was set up.
[0047] Step 3: After treating the experimental animals with benzimidazole solution, the experiments were verified through pathological morphology and behavioral methods.
[0048] Step four: Compared with the control group, the experimental animals showed significantly enlarged ventricles and significantly reduced motor function in terms of pathological morphology and behavioral indicators. If this condition is met, the primary ciliary motor disorder hydrocephalus model is successfully constructed.
[0049] Example 2
[0050] (1) Preparation of zebrafish fry
[0051] Wild-type zebrafish were all sourced from the China Zebrafish Resource Center and raised according to standard protocols. The zebrafish were maintained at 28°C under 14-hour light / 10-hour dark conditions. Zebrafish embryos were obtained through natural mating, further washed and preserved in a culture medium containing 2 mg / L methylene blue (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4). The culture medium pH was maintained at 7.1 and changed daily.
[0052] (2) Experimental grouping and treatment
[0053] a. Model group: Zebrafish embryos 20 hours after fertilization were randomly transferred to 24-well cell culture plates, with 10 eggs per well. The embryos were exposed to benzimidazole solutions of 200 ug / ml and 300 ug / ml, which were prepared from E3 solution (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2 and 0.33 mM MgSO4).
[0054] b. Blank control group: Zebrafish embryos 20 hours after fertilization were randomly transferred to 24-well cell culture plates, with 10 eggs per well, and the embryos were placed in E3 solution.
[0055] (3) Imaging of zebrafish ventricles using a stereomicroscope
[0056] Embryos were developed to 54 hours and then embedded in low-melting-point agarose for later use. After adjusting the stereomicroscope, images of the zebrafish ventricles were acquired. The size of the ventricles was measured using ImageJ.
[0057] In summary, the animal model of primary ciliary motor disorder hydrocephalus prepared by this invention allows for real-time monitoring of the degree of hydrocephalus by measuring ventricular size and detecting motor level during the research process. Therefore, the modeling process of this invention is a dynamic process, which can observe the ventricular expansion at different time points, providing an excellent experimental platform for studying primary ciliary motor disorder hydrocephalus. At the same time, this model also provides a valuable tool for developing novel treatment methods.
[0058] Therefore, this invention employs the aforementioned method for constructing a primary ciliary motor disorder hydrocephalus model. This non-invasive method constructs the model, minimizing harm to the animal model. By administering benzimidazole to the animal model through immersion, a primary ciliary motor disorder hydrocephalus model is successfully established. The model exhibits good stability and repeatability, is objective and simple, and is highly operable, reducing other possible interfering factors. This provides a necessary experimental foundation for studying the mechanisms and developing treatment methods for primary ciliary motor disorder hydrocephalus.
[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for constructing a model of primary ciliary motor disorder hydrocephalus, characterized in that, Includes the following steps: Step 1: Modeling agent compounds were screened based on DRC8, a substructural protein of the inlinin-dynein regulatory complex. The database used for screening was the CNS central nervous system drug database. Compounds were screened based on their free binding energy. The top 100 compounds were further screened using medical and toxicological methods to identify benzimidazole as the modeling agent. The benzimidazole is 4,5-dimethyl-1H-benzimidazole. Step 2: Under normal rearing conditions, the experimental animals were administered benzimidazole solution, and a blank control group was set up; the experimental animals were zebrafish; the rearing conditions were: temperature 28.5℃, 14 hours of light / 10 hours of darkness. Step 3: After treating the experimental animals with benzimidazole solution, the experiments were verified through pathological morphology and behavioral methods. Step four: Compared with the control group, the experimental animals showed significantly enlarged ventricles and significantly reduced motor function in terms of pathological morphology and behavioral indicators. If this condition is met, the primary ciliary motor disorder hydrocephalus model is successfully constructed.
2. The method for constructing a primary ciliary motor disorder hydrocephalus model according to claim 1, characterized in that: In step two, the concentration of benzimidazole solution administered is 200 ug / ml or 300 ug / ml.
3. The method for constructing a primary ciliary motor disorder hydrocephalus model according to claim 1, characterized in that: The benzimidazole solution was administered at a concentration of 300 ug / ml.
4. The method for constructing a primary ciliary motor disorder hydrocephalus model according to claim 1, characterized in that: In step two, the benzimidazole solution is administered via immersion.