| Objective:This article aims to prepare a zebrafish tuberculosis meningitis model and verify its preparation effect.Using the constructed zebrafish Mycobacterium tuberculosis meningitis model to test and compare the efficiency of first-line anti-tuberculosis drugs penetrating the blood-brain barrier(BBB),in order to evaluate the effectiveness of the model in screening and developing anti-tuberculosis brain infection drugs.Methods:1.Two models of zebrafish tuberculous meningitis were prepared by microinjection of 3dpf(3 day-post-fertilization,3dpf)via the duct of Cuvier of zebrafish juveniles and infection of Mycobacterium marinum via the hindbrain of 2dpf juveniles.Observing the dynamic changes of Mycobacterium marunum in zebrafish through high-resolution microscopy and laser confocal microscopy.Simultaneously,experimental methods such as acid fast staining,HE staining,and survival curve evaluation were used to evaluate the effectiveness of the above two models,confirming the successful preparation of the models.Thus,laying the foundation for subsequent experiments in evaluating brain anti-tuberculosis drugs.2.In the preparation of two models,we found that the zebrafish tuberculous meningitis model prepared by posterior brain injection infection showed "localized"growth of Mycobacterium marinum in the zebrafish brain within 4 days after injection.We believe that the model prepared by this method has unique advantages in studying the effects of drug specificity through the blood-brain barrier.Therefore,in the second part of this paper,a model prepared by injecting Mycobacterium marinum infection via the hindbrain was selected to study the effects of first-line anti-tuberculosis drugs crossing the blood-brain barrier,in order to evaluate the application value of this model.The drug was administered by soaking in solution:after soaking in the same concentration of isoniazid(INH)and ethambutol(ETH)respectively,the brain was injected with zebrafish infected with 1dpi(1 day-post-injection,1dpi)/3dpf of the green fluorescent plasmid Mycobacterium marinum.The drug was soaked for three consecutive days and the solution was changed every day.At the same time,a group of zebrafish infected with Mycobacterium marinum but not treated with medication were set as blank controls.At the same time,we soaked 2dpf zebrafish infected via the duct of Cuvier injection in the same way with isoniazid and ethsarnbutol solution of the same concentration,and set a group of blank controls.Because in the preparations of tuberculous meningitis models,we found that zebrafish infected with 1dpi via the duct of Cuvier injection had less presence of marine bacteria in the brain.Therefore,the use of continuous immersion of 1dpi/3dpf zebrafish in this section is considered to study the therapeutic effects of drugs on systemic infection.After continuous immersion in the medicinal solution for 3 days,the fluorescence of Mycobacterium marinum in zebrafish was captured by using a high-resolution microscope.The fluorescence intensity of Mycobacterium marinum in zebrafish was quantified by using ImageJ image processing software,and the fluorescence intensity was used as the bacterial load.The above experimental methods are used to demonstrate whether the presence of the zebrafish blood-brain barrier affects the of anti-tuberculosis drugs through the blood-brain barrier.3.Using the micro double dilution method to test the minimum inhibitory concentration(MIC)of isoniazid and rifampicin(RIF)against the green fluorescent plasmid Mycobacterium marinum strains used in the experiment.The zebrafish infected with 1 dpi of green fluorescent Mycobacterium marinum was administered once through a total intravenous injection of isoniazid and rifampicin at an equal dose gradient.After 3 days of injection treatment,the fluorescence of Mycobacterium marinum in zebrafish was captured by a high-intensity microscope.ImageJ image processing software was used to quantify the fluorescence intensity of Mycobacterium marinum in zebrafish,and the fluorescence intensity was used as the bacterial load.Immerse zebrafish infected with 1dpi in different MIC concentrations of isoniazid and rifampicin solution and inject it into the brain for 3 consecutive days.Using the same high-intensity microscope to capture Mycobacterium marinum in zebrafish on day 3 and ImageJ solftware to quantify the bacterial count in zebrafish,the bacterial load in zebrafish brain after drug action was detected.Results:1.Injecting Mycobacterium marinum via the duct of Cuvier can spread to the brain of young zebrafish with the growth of blood circulation,and pathological reactions such as inflammation,local tissue necrosis,and proliferation of inner epithelial cells can be observed in the brain of zebrafish.Proving that this infection modeling method can simulate the infection pathway of Mycobacterium tuberculosis entering the brain with the blood circulation system.Injection of Mycobacterium marinum into the posterior brain can trigger immune responses such as inflammatory cell aggregation,local tissue necrosis,and epithelial cell proliferation in zebrafish brains,and it has been found that there is a trend of granuloma formation in zebrafish brains.In addition,when injected into the posterior brain to infect zebrafish with 4 dpi,Mycobacterium marinum still showed "localized" and "aggregated" growth in the brain,which is beneficial for studying and evaluating the effectiveness of drug specificity through the blood-brain barrier.2.After being soaked with ethambutol,which is clinically considered to be unable to cross the blood-brain barrier,zebrafish infected by brain injection did not show significant differences in the bacterial load of zebra brain compared with the blank control group infected with Mycobacterium marinum injected into the back brain but not treated with drugs after 3 days of treatment.Compared with the ethambutol treatment group and the blank control group,the brain bacterial load of the experimental group soaked in isoniazid significantly decreased.The zebrafish infected with 1dpi by total main intravenous injection were soaked in the same concentration of isoniazid and ethambutol solution.After 3 days of continuous immersion,the bacterial load in zebrafish in isoniazid treatment group and ethambutol treatment group was significantly lower than that in the blank control group.The presence of the blood-brain barrier in zebrafish affects the passage and effectiveness of drugs.3.The in vitro MIC test results showed that the MIC results of rifampicin was 0.4mg/L,isoniazid was 50mg/L,and isoniazid had a higher MIC than rifampicin.Giveing 1dpi zebrafish a total intravenous injection of the drug.After 3 days,in both the isoniazid and rifampicin treated groups,it was found that as the dosage increased,the fluorescence intensity of Mycobacterium marinum in the zebrafish brain decreased,and the bacterial load obtained from quantitative treatment decreased.Zebrafish infected with 3 dpi were injected into the brain after soaking with MIC concentration of isoniazid and rifampicin solution,and soaked continuously for 3 days.In the isoniazid treatment group,the experimental results on the third day showed that when the concentration of isoniazid solution was higher than 1 × MIC,the bacterial count in the brain of 3dpt zebrafish was lower than the initial bacterial count 2 hours after injection(P<0.05).The concentration of isoniazid solution is less than 1×MIC,the bacterial load in the brain of zebrafish was lower than that of the blank control group,but higher than the initial bacterial load after 2 hours of injection(P<0.05),In the rifampicin treatment group:when the concentration of the medicine solution is 0.5×MIC、1×MIC、5×MIC,there was no significant difference in the brain bacterial load between the experimental groups and the blank control group after soaking for 3 days(P>0.05).Compared to the anti-tuberculosis drug rifampicin,which is believed to partially pass through the blood-brain barrier,the first-line drug isoniazid is more likely to achieve effective concentration in the brain region through the zebrafish blood-brain barrier,thereby effectively controlling and killing the growth of Mycobacterium marinum in the brain.This is also consistent with the fact that isoniazid can pass through the blood-brain barrier in the human body,while rifampicin can only partially pass through the blood-brain barrier.Conclusions:1.Two different models of zebrafish tuberculous meningitis can be successfully prepared by injecting Mycobacterium marinum via the duct of Cuvier and hindbrain to infect young zebrafish.2.The injection and immersion administration experiments of a hindbrain injection infection model can reproduce the situation where existing anti-tuberculosis drugs pass through the blood-brain barrier in the human body.This indicates that the zebrafish tuberculosis meningitis model is reliable in drug screening applications and can be used for the screening and development of related potential drugs in the future. |
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