阅读说明：本技术 一种建立***诱导致精神***症大鼠模型的方法及应用 (Method for establishing ketamine-induced schizophrenia rat model and application ) 是由 解润芳 韩梓逸 解继明 王欣 马旭涛 于 2019-08-08 设计创作，主要内容包括：本发明公开了一种建立氯胺酮诱导致精神分裂症大鼠模型的方法,通过给大鼠注射不同浓度的氯胺酮溶液,然后对大鼠的脑组织海马及前额叶皮质中的NRG1及ErbB4进行免疫组化试验,确定了本模型,本方法简单,可靠性强,不仅仅对阴性和阳性的区分,还能对精神分裂程度进行区分,本方法可用于探究氯胺酮与精神分裂症易感基因NRG1/ErbB4之间的相关性,最终探索精神分裂症的发病机制,并为法医精神分裂症鉴定提供理论依据。(the invention discloses a method for establishing a rat model of inducing schizophrenia by ketamine, which is characterized in that ketamine solutions with different concentrations are injected into rats, then immunohistochemical tests are carried out on NRG1 and ErbB4 in hippocampus and prefrontal cortex of brain tissues of the rats, and the model is determined.)

1. A method for establishing a rat model for ketamine-induced schizophrenia, comprising the steps of:

(1) Animal selection and grouping: 24 adult SD rats (without male and female parts, with the weight of 290-340 g) with the age of 2 months are randomly divided into 4 groups, namely K_Control、K_{15 to give}、K_{30 to}、K_{60 to}；

(2) Preparing ketamine solution by using distilled water to prepare ketamine solution with three concentrations of 15mg/kg, 30mg/kg and 60 mg/kg;

(3) feeding rats: k_Controlgroup means 2 times daily intraperitoneal administration of 1.2ml volume of physiological saline, K_{15 to give}、K_{30 to}、K_{60 to}The same volume of ketamine 15mg/kg, 30mg/kg and 60mg/kg was administered twice daily to the digital abdominal cavity, respectively, and the same volume of saline was administered to the K control digital abdominal cavity for 7 consecutive days;

(4) The animals were sacrificed: after the rat is administrated for 13 hours for the last time, the rat is deeply anesthetized by ether, fixed on an anatomical table, the left chest wall is cut to expose the heart, a perfusion needle is inserted into the left ventricle and fixed, the right auricle is cut open, normal saline is firstly perfused until the color of the liver and the lung is changed and the effluent liquid of the right atrium is clear, the head of the animal is cut off, the brain is taken, and the required hippocampus and prefrontal cortex parts in the brain tissue are quickly separated on ice;

(5) observation of immunohistochemical staining experiment: sections were immunohistochemically stained for hippocampal and prefrontal cortex sites, observed under a microscope and photographed. Randomly taking 2 high-power visual fields (400 x) from each section, and determining immune positive gray values of hippocampus and prefrontal cortex NRG1 and ErbB4 by using an Image-pro plus6.0 Image analysis system so as to convert the immune positive gray values into Optical Density (OD) values;

(6) Constructing a rat ketamine induced schizophrenia model, obtaining the ketamine induced schizophrenia rat model through the steps 1 to 5, and establishing a file to form the model.

2. The method of claim 1 for detecting a correlation between ketamine and expression of NRG1/ErbB 4mRNA in hippocampus and prefrontal cortex.

3. use of the method of claim 1 for the development of a medicament for the treatment of ketamine-induced schizophrenia.

Technical Field

The invention relates to the technical field of medicine, in particular to a method for establishing a rat model of ketamine induced schizophrenia.

Background

Currently, drug abuse is a serious social problem, and ketamine (K powder) is receiving more and more attention as a novel drug. In recent 20 years, the proportion of ketamine abuse in China has been rising year by year, and data show that ketamine is taken most seriously by people under twenty-one years of age. Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, which plays an important role in the induction, maintenance and expression of prominent plasticity, which is strongly associated with learning and memory mechanisms. NMDA receptor abnormalities can affect learning and memory functions and also cause damage to the central nervous system; the larger the dose of ketamine is, the more remarkable the toxic and side effects are, and the toxic and side effects are mainly manifested as central nervous system reactions, such as dream hallucination, delusions (illusion), separation states or schizophrenia, excitation, disorientation, cognitive disorder, delirium and the like. Chronic abuse of ketamine not only causes damage to the central nervous system but can also produce symptoms of schizophrenia (schizophrenia); drug addicts abusing ketamine have also been found to produce negative and positive symptoms of schizophrenia. Based on the research, the novel drug ketamine is used for inducing to establish an objective, stable and reliable animal model of schizophrenia. Schizophrenia is a serious disabling group of mental disorders, the main symptoms of which include positive symptoms, negative symptoms and impairment of cognitive function. Positive symptoms are manifested by hallucinations, delusions, disorganized thinking and bizarre behavior; negative symptoms are marked by low behavioral motivation, apathy emotional response, aphasia and low excitability; cognitive impairment is most common with impairment of attention, memory and planning ability. The cause of schizophrenia is unknown, the research on the pathogenesis of schizophrenia is less, clinical diagnosis mainly depends on the judgment of positive symptoms and negative symptoms, objective biological detection indexes are lacked, and the treatment of schizophrenia is a difficult problem in the medical field, so that the establishment of a reliable animal model of schizophrenia is particularly important for researching the pathogenesis and the treatment mechanism of schizophrenia.

The pathogenesis of schizophrenia is still unclear to date, and the generally accepted view is: schizophrenia is the result of the synergistic action of multiple "moderate to micro-effective" genes under the influence of environmental factors, among which neurodevelopmental factors are involved. Genetic studies have found a plurality of important candidate genes related to schizophrenia, wherein the neuregulin1 (neuroegulin 1, NRG1) and the receptor ErbB4 gene thereof are identified as susceptibility genes of schizophrenia. Clinical studies show that the NRG1 gene is associated with schizophrenia in Chinese Han population, and is particularly closely related to the positive symptoms of schizophrenia. NRG can bind to ErbB3, ErbB4 tyrosinase of the ErbB family to form ErbB homodimers and heterodimers, which in turn activate extracellular signaling pathways, resulting in a range of cellular responses including activation or inhibition of proliferation, apoptosis, migration, differentiation and adhesion. In summary, abnormalities in the NRG1/ErbB4 signaling system are associated with the onset of schizophrenia, which can increase susceptibility to schizophrenia after impairment of the signaling system; up-regulation of NRG1 and ErbB4 expression in schizophrenic patients, enhanced signal transduction; NRG1/ErbB4 mutant rats also exhibited typical symptoms of schizophrenia. Since the NRG1/ErbB4 signal pathway is also considered as the most typical pathway for the development and progression of schizophrenia, the NRG1/ErbB4 signal pathway was studied as a biological detection index.

The existing animal model of schizophrenia is difficult to establish an objective biological detection target and study the pathogenesis of schizophrenia. If the pathogenesis and treatment of schizophrenia are explored, the current model has no reliability and feasibility.

Disclosure of Invention

Aiming at the problems, the invention provides a method for establishing a rat model of ketamine induced schizophrenia, so as to explore the correlation between ketamine and schizophrenia susceptible genes NRG1/ErbB4, finally explore the pathogenesis of schizophrenia and provide theoretical basis for forensic schizophrenia identification.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for establishing a rat model for ketamine-induced schizophrenia, comprising the steps of:

(1) animal selection and grouping: 24 adult SD rats (without male and female parts, with the weight of 290-340 g) with the age of 2 months are randomly divided into 4 groups, namely K_Control、K_{15 to give}、K_{30 to}、K_{60 to}。

(2) The ketamine solution is prepared by using distilled water to prepare ketamine solution with three concentrations of 15mg/kg, 30mg/kg and 60mg/kg groups.

(3) Feeding rats: k_Controlgroup means 2 times daily intraperitoneal administration of 1.2ml volume of physiological saline, K_{15 to give}、K_{30 to}、K_{60 to}the same volumes of ketamine 15mg/kg, 30mg/kg and 60mg/kg were administered twice daily to the abdominal cavity of the fingers, respectively, and the same volumes of saline were administered to the K control finger abdominal cavity for 7 consecutive days.

(4) The animals were sacrificed: after 13 hours of the last administration of the drug, the rat is deeply anesthetized by ether, fixed on an autopsy table, the left chest wall is cut to expose the heart, a perfusion needle is inserted into the left ventricle and fixed, the right auricle is cut open, the physiological saline is firstly perfused until the color of the liver and the lung is changed and the effluent liquid of the right atrium is clarified, the head of the animal is cut off, the brain is taken, and the required hippocampus and prefrontal cortex parts in the brain tissue are rapidly separated on ice.

(5) Observation of immunohistochemical staining experiment: sections were immunohistochemically stained for hippocampal and prefrontal cortex sites, observed under a microscope and photographed. 2 high power fields (400 ×) were randomly selected from each section, and the immunopositive gray-scale values of hippocampal and prefrontal cortex NRG1 and ErbB4 were determined using an Image-pro plus6.0 Image analysis system and converted to Optical Density (OD) values.

(6) constructing a rat ketamine-induced schizophrenia model, obtaining the ketamine-induced schizophrenia rat model through the steps 1-5, and establishing a file to form the model.

The method for establishing the ketamine-induced schizophrenia rat model is used for detecting the correlation between the ketamine and the expression condition of NRG1/ErbB 4mRNA in hippocampus and prefrontal cortex.

The method for establishing the rat model of ketamine-induced schizophrenia is used for developing the application of the drug for treating ketamine-induced schizophrenia.

The invention has the beneficial effects that: the invention provides a method for establishing a rat model of inducing schizophrenia by ketamine, which is simple, strong in reliability and short in induction time. The method can be used for researching the correlation between ketamine and schizophrenia susceptible gene NRG1/ErbB4, finally exploring pathogenesis of schizophrenia, and providing theoretical basis for forensic schizophrenia identification.

drawings

FIGS. 1 a-d are diagrams of procedures for isolating hippocampal and prefrontal cortex sites in brain tissue;

FIG. 2 is a diagram of the prior stage of immunohistochemical assay: localization of ErbB4 in hippocampal and prefrontal cortex in brain tissue;

In fig. 3, a is the expression of ErbB4 in different concentrations of ketamine in the hippocampal CA3 region, and b is the expression of ErbB4 in different concentrations of ketamine in the prefrontal cortex cg1 region;

FIG. 4 is a PCR amplification curve;

FIG. 5 is a PCR dissolution curve.

Detailed Description

The invention is further described below with reference to the accompanying drawings.