动物造模 z-bio 2021-10-21 1046

　　Methods: The isolated rabbit and human corneas and scleral rings were placed in organ culture medium, and 108 strains of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Fusarium solani were used to infect the cornea. This infection is stabbed with a scalpel. Inject a suspension of infectious bacteria into the exposed cornea or stroma. After inoculation, the cornea was kept at 37°C for 24 and 48 hours. After incubation, the cornea is homogenized to confirm colony formation (CFU) or normal staining is used for histological examination. Compare single and mixed infections.

　　Result: After 24 hours, compared with Staphylococcus aureus and Pseudomonas aeruginosa, CFU increased significantly. However, this increase was not observed in corneas infected with Candida albicans or Fusarium solani. In most infected corneal tissues, injection-induced infection causes an increase of approximately 2 to 100 times. The female histological injury and injection model showed extensive infiltration of Pseudomonas aeruginosa throughout the cornea, while less infiltration of Staphylococcus aureus, Candida albicans and Fusarium solani. The model also supports super infection. Conclusion: Both female and injection methods are suitable for in vitro rabbit and human corneal infections. These simple and reproducible models provide useful alternative models for the detection and treatment of microbial keratitis, especially in vitro and in vivo, which may be caused by two infectious microorganisms.

　　Introduction: Microbial keratitis is a major problem worldwide and an important cause of vision loss and blindness. In vitro cell culture and in vitro models have been used to study various aspects of diseases, including pathogenicity and treatment strategies. In vivo studies require the use of multiple animal models. The welfare of these animals has become an important ethical issue. One way to solve these problems is to use in vitro cell monolayer cultures containing immortal or primary corneal epithelial cells. However, this does not represent physical condition. Different epithelial cells, ring stem cells and corneal stromal cells lack interconnected three-dimensional (3D) structures.

　　Recently, people are using corneal models to study keratitis. However, these models have no immune components. These models have been used to study wound healing, microbial attachment and molecular microbial pathogenicity. In our laboratory, we are studying corneal epithelial regeneration and inflammation models using in vitro corneal models. By gently shaking the medium on the cornea, they can be maintained in culture for at least 4 weeks. To our knowledge, the in vitro corneal model cannot compare bacteria, fungi and mixed infections. According to reports, single and mixed infections of rabbit and human corneas were compared to better understand the application of these models in microbial keratitis.

　　Materials and methods: Materials: We used two types of rabbits: wild brown rabbits and New Zealand rabbits. There was no difference in corneal performance between the two rabbits. The human cornea comes from the corneal bank. Rabbit corneal separation: After disinfection with povidone iodine, separate the corneal valve annulus from the corneal valve annulus, and immediately put it in phosphate buffer.

　　Isolated corneal organ culture: The organ culture is as above. Place the human and rabbit corneas in a 35 mm petri dish with the epithelial side closer to the bottom of the petri dish. Transfer the agarose (0.5% w/v) solution in 500 μLDMEM to the endothelial side of the cornea. After the solution has solidified, turn the petri dish over so that the epithelial side is facing up. Moderate DMEM: Ham's F12 contains 10% fetal bovine serum, 100U/ML penicillin, 100U/ML streptomycin, 2.5μg/L amphotericin B, 5μg/L insulin and 10g/mL epidermal growth factor (EGF) supplements. In the cornea underwater. Before infection, wash the cornea with PBS 3 times, and incubate in antibiotic-free and fungus-free medium for at least 24 hours to remove residual antibiotics. All experimental work was performed on rabbit corneas in the UK and human corneas in India.

　　Bacterial and fungal culture: Staphylococcus aureus (s-235), Pseudomonas aeruginosa (som-1), Candida albicans (SC5314), Fusarium solani (NCPF2699) for rabbit cornea. The strains used for human cornea are Staphylococcus aureus (25923), Pseudomonas aeruginosa (27853) and Candida albicans (90028). All bacterial and fungal strains were cultured overnight at 37°C on Brainheart Infusion Agar (BHI), and then stored at 4°C. Due to the need for experiments, the individual clones in the agar were placed in a broth medium and incubated overnight at 37°C. Stationary phase microorganisms are used in rabbit corneal experiments. For human cornea experiments, please inoculate fresh broth on the day of cornea inoculation, and inoculate according to the prescribed growth curve.

　　Extracorporeal corneal infection: The scalpel damages the cornea (3 vertical diagonals and 3 horizontal diagonals). Place a metal ring on the cornea to create a waterproof and airtight space. Inoculate the central area of the metal ring with 108 Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa or Fusarium solani. Or, inject the same amount of bacteria. The infected cornea was cultured at 37°C for 24 or 48 hours, the resulting suspension was homogenized and diluted, and then colonies were counted on an agar plate. The infected cornea was also treated, and the sections (bacteria) were stained with grams and (fungi) with periodic acid Schiff (PAS). The cornea that was not exposed to microorganisms was used as a control. Microbial imaging of the corneal surface: Staphylococcus aureus or Pseudomonas aeruginosa was labeled with 1 mg/ml fluorescein isothiocyanate (FITC) at 4°C, and washed 4 times with PBS. For fungi, cover the entire cornea with 1:1 fluorescent whitening agent and 10% potassium hydroxide (v/v) for 10 minutes, and then wash 3 times with PBS. Use a fluorescence microscope to image the cornea infected with bacteria and fungi. Results: Macroscopic observation of rabbit and human cornea: Compared with uninfected cornea, the turbidity of bacterial and fungal infection increased significantly. There are wounds on all corneas, but the infected cornea is more pronounced than the uninfected cornea. FITC-labeled bacteria were found in infected rabbits and human corneas. It was found that Staphylococcus aureus covered the corneal surface within 24 and 48 hours. Bacterial clusters with a diameter of 5 to 25 μm were detected in some parts. On the other hand, compared with the cornea observed in Staphylococcus aureus, the cornea infected with Pseudomonas aeruginosa after 24 hours and 48 hours has less aggregation. The surface of the cornea infected by Candida albicans stained with fluorescent whitening agent showed a more uniform spread, and a small amount of hyphae appeared on the surface of the cornea after 24 hours. However, after 48 hours, the distribution of Candida albicans and hyphae increased. Fusarium solani showed more hypha distribution in different positions of rabbit and human cornea within 24 hours. After 48 hours, the surface of the cornea was covered with a layer of fungus, and the hyphae were observed to move away from the fungal body, scraping and extending in all directions. The coverage rates of bacteria and fungi on the cornea of rabbits and humans are similar.

　　Rabbit and human corneal single infection: The injection method involves the introduction of bacteria and fungi into the matrix. Compared with the scalpel method, single biological injection produced higher CFU/cornea (P\u003c0.05) after 24 hours, except for Candida albicans, other differences were not significant. It can be seen that Pseudomonas aeruginosa covers the epithelium and penetrates the entire posterior elastic membrane. This has nothing to do with the vaccination method. The distribution of Candida albicans in human and rabbit corneas is similar. Yeast cells and mycelial elements are close to the scratched area and have not penetrated the matrix above 150μm. The number of hyphae and Candida albicans cells that entered the interstitium after 48 hours were not significantly different from those observed after 24 hours. The tissue permeability of Fusarium solani is lower than that of Candida albicans within 24 hours, and the matrix permeability of the inoculation site does not exceed 10 μm. However, the depth and number of invasions increased after 48 hours.

　　Two bacterial infections on the cornea: Two bacterial models can cover the bacterial species of the infected rabbit and human cornea. Compared with a single infection, Pseudomonas aeruginosa cells infiltrate the matrix containing the posterior elastic membrane, while Staphylococcus aureus appears to have spread beyond the injection site. The mixed infection of Candida albicans and Pseudomonas aeruginosa is the most common clinical infection. In this in vitro model of mixed infection, the two corneas were covered by bacteria within 24 hours after the female was injured, and Pseudomonas aeruginosa showed advantages in colony number and histology.

　　Conclusion: We have achieved the goal of establishing regenerative corneal infections in humans and animals. There is no doubt that models (including animals) are not ideal substitutes for natural human transmission. Nevertheless, the data obtained is still useful. Reproducible in vitro and fungal infection models can be established, showing that the final number of recovered bacteria/fungi is comparable to natural in vivo experiments.