Bacterial endophthalmitis is a devastating ocular infection, which if not diagnosed and treated quickly, can result in vision loss within a few hours [1
]. Any ocular perforation (surgery and/or trauma) predisposes a patient to developing endophthalmitis. Cataract surgery, one of the most common surgical procedures performed among the aged population worldwide, has a higher incidence of endophthalmitis than any other type of ocular surgery [2
]. The incidence of endophthalmitis has also steadily risen over the past two decades due to the increased popularity of suture-less cataract surgery, small-gauge vitrectomy, and intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs for treating age-related macular degeneration and diabetic macular edema [4
Innate immunity provides the first line of defense against bacterial infections [6
]. However, the eye, being an immune-privileged site, provides an immunosuppressive environment for the growth and proliferation of microbial pathogens [8
]. Moreover, the eye is isolated from systemic circulation due to the presence of the blood–retinal barrier (BRB), making it difficult for the immune system to reach the site of infection [9
]. Traditionally, murine models have been used for studying innate defense mechanisms and disease pathology against bacterial endophthalmitis [10
]. However, the scientific community continues to investigate new models to reduce the cost and high demands of animals. Vertebrate fish models have been utilized as a model organism for several infectious diseases and innate immune system studies. Previous studies have demonstrated that the zebrafish (Danio rerio
) model is a valuable model for the study of host–pathogen interactions and immune mechanisms against several bacterial infections, including Streptococcus
], Vibrio cholera
], and Salmonella
]. The zebrafish has many similarities to the human immune system, and its well-developed genetics, small size, and rapid generation time has made it an easy choice as a model for several disease, developmental, and immune system studies [18
]. However, the feasibility of zebrafish as an alternative model for endophthalmitis has never been explored.
The aim of this study was to develop a zebrafish model of Staphylococcus aureus endophthalmitis and to compare the disease pathobiology to an established murine model. We report that in comparison to the murine model, zebrafish were able to rapidly clear the inoculated bacteria from the eyes and did not develop endophthalmitis.
While zebrafish is increasingly being used as an infectious disease model, to our knowledge, its susceptibility to ocular infection has not been investigated. In this study, we explored the possibility of developing a zebrafish model of bacterial endophthalmitis to provide new insights into the pathogenesis of this blinding eye disease. We report that regardless of infectious dose, zebrafish eyes were able to rapidly clear the infection (S. aureus
), resulting in no ocular/retinal tissue damage, which is typically seen in human clinical findings and a murine model of this disease. Moreover, intraocular inoculated bacteria appeared to be cleared through the retinal vasculature and phagocytic activities of infiltrated monocytes/macrophages in the zebrafish eyes. Given the close resemblance between the human and zebrafish immune systems [23
], a better understanding of the protective innate immune mechanisms operating in zebrafish could lead to the identification of new host targets.
Among bacterial pathogens, Staphylococci
remain the leading cause of bacterial endophthalmitis, with S. aureus
infection resulting in severe disease outcomes and often leading to visual disability and blindness [27
]. In clinical settings, most bacterial endophthalmitis arises due to postsurgical complications (e.g., cataract surgery), where pathogens from the ocular surface gain access to the eye and cause ocular tissue damage [28
]. To mimic this situation, studies from our [7
] and other laboratories [31
] have developed murine models where bacteria are directly inoculated in the vitreous cavity. Previous studies have demonstrated that S. aureus
caused endophthalmitis in mice when eyes were inoculated with 5000 or less CFUs [7
]. In contrast to the mice, we found that zebrafish eyes infected with 5000 CFU of S. aureus
did not cause any ocular pathology. Therefore, we postulated that zebrafish might need a higher inoculum to cause endophthalmitis. Surprisingly, our dose–response study revealed that even the intravitreal injection of a 50-fold higher dose, i.e., 250,000 CFU/eye, did not cause pathology in the zebrafish eyes, as was evident by their intact retinal architecture. These findings indicate that zebrafish are resistant to S. aureus
Being an immune-privileged organ, the eye is conducive to the proliferation of various endophthalmitis causing bacteria. Moreover, there was a time-dependent increase in bacterial growth in the infected mouse eyes. Our bacterial burden analysis revealed that after inoculation, bacterial growth increased up to 8 h followed by a decline at 24 h; and by 48 and 72 h, bacteria were cleared from the eye. This data suggests that zebrafish eyes could mount an adequate immune response to clear the infection and maintain retinal integrity. To study the potential mechanisms of bacterial clearance, we utilized GFP-labeled S. aureus
, as reported in our prior study [35
]. We discovered that within 2 h post-bacterial infection, GFP-positive S. aureus
was localized in the choroid, and at the 8-h time point, bacteria was present both in the choroid and inside the retinal blood vessels. Similarly to the bacterial plate count assay, this histological analysis revealed that bacteria were gradually cleared from the eyes within 48–72 h, as evidenced by the lack of GFP positivity. Moreover, we observed that S. aureus
within the vitreous colocalized with innate immune cells, primarily monocyte/macrophages. Collectively, these results indicate that bacteria were cleared from the zebrafish eyes via the combined action of retinal blood vessels and the phagocytic activities of infiltrated innate immune cells in the vitreous.
The presence of monocyte/macrophages in the infected zebrafish eyes indicated the induction of the innate immune response. In mouse eyes, polymorphonuclear neutrophils (PMN) are the prominent immune cells infiltrated during endophthalmitis to contain bacterial proliferation. The depletion of PMNs has been shown to increase the bacterial burden in the eye [7
]. Since the recruitment and activation of innate immune cells are regulated by the production of inflammatory mediators, [7
], we assessed their expression in infected zebrafish eyes. Our data showed a significantly increased expression of Il-1β
and no changes were observed in the levels of Tnf-α
in the control versus the S. aureus
-infected eyes. While the production of inflammatory mediators is a protective host response during infection, their excessive levels could lead to collateral tissue damage [29
]. Among the various inflammatory mediators produced, Tnf-α
has been shown to cause retinal tissue damage in several ocular diseases [37
] and has been reported to exert a protective role in Bacillus
]. Similarly, Il-1β
has been shown to protect the host from S. aureus
infection in various models [39
]. Further studies are needed to dissect the role of these individual cytokines in protecting zebrafish eyes from Staphylococcal
Zebrafish have been used as an attractive alternative model to study host–pathogen interactions and innate immunity due to several advantages, including cost-effectiveness, rapid breeding, and their close resemblance to the human immune system [23
]. In conclusion, our study demonstrates that zebrafish eyes are resistant to bacterial endophthalmitis even when challenged with a higher dose of S. aureus
in comparison to mouse models. Zebrafish have a profound ability to clear the pathogen from eyes and protect retinal tissue integrity, thus maintaining normal vision. The ability of zebrafish to mount a protective innate response is mediated via bacterial phagocytic clearance by monocytes/macrophages. Further studies are needed to determine whether similar mechanisms operate in zebrafish eyes infected with other endophthalmitis-causing pathogens, including fungi.
4. Materials and Methods
4.1. Animals and Bacterial Maintenance
Wild-type (WT) zebrafish (Danio rerio
) (AB strain, 9–12 months old) were used for this study and kept in standard laboratory conditions using a light schedule of 14 h on and 10 h off at a temperature of 28.5 °C [41
]. Fish were fed daily using a combination of dry food and brine shrimp. Following injections, zebrafish were not fed and were closely monitored for any adverse reactions to bacterial injections. C57BL/6 WT mice (6–8 weeks old) were purchased from Jackson Laboratory, maintained at the Department of Laboratory Animal Resources (DLAR) facility with a 12:12 light–dark cycle, and fed LabDiet rodent chow and water ad libitum. All animal care and experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee at Wayne State University and were in compliance with the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in vision research.
GFP-expressing Staphylococcus aureus (AL1743) (harboring chloramphenicol resistance) were maintained in tryptic soy broth/agar containing chloramphenicol. Prior to injection, bacteria were cultured in tryptic soy broth with chloramphenicol (20 µg/mL) overnight at 37 °C adjusted to a dosage of 250,000 CFU/0.5 µl in PBS.
4.2. S. aureus Intraocular Injections
Zebrafish were anesthetized, and a small incision was made at the edge of the cornea (a third of the corneal diameter long) using a scalpel (Safety Sideport Straight Knife 15°; Beaver-Vistec International). S. aureus
AL1743 suspension (250,000 CFU/0.5 µL/eye) was injected into the vitreous chamber through an incision into the eye using a blunt-end 33-gauge Hamilton syringe. Fish were then placed back into water and incubated various times. In C57BL/6 mice, endophthalmitis was induced by giving an intravitreal injection of S. aureus
(5000 CFU/eye) as described previously [7
4.3. Bacterial Burden Estimation
The bacterial burden from zebrafish eyes was estimated using serial dilution and the plate count method. At each respective time point, fish were euthanized using 0.4mg/L of 2-phenoxyethanol. The eyes were enucleated and homogenized in sterile PBS using a Dounce homogenizer. The homogenate was serially diluted in sterile PBS, plated on tryptic soy agar plates containing chloramphenicol (20 µg/mL), and incubated at 37 °C. Following growth, the bacterial colonies were counted, and the results are expressed as the mean number of CFU/eye ± standard deviation (SD).
Following euthanasia, the eyes were enucleated and fixed for 1 h in 4% paraformaldehyde solution at room temperature. Following a PBS wash for 30 min, the eyes were then cryoprotected using a sucrose gradient of 5%–20%. The eyes were embedded in Optimal Cutting Temperature (OCT) Medium and sectioned. The frozen sections were dried for 2 h at 50 °C, followed by rehydration in PBS. The cryosections were blocked using a blocking solution consisting of 2% normal goat serum, 1% dimethyl sulfoxide (DMSO), and 0.2% Triton-X 100 in PBS for 1 h at room temperature. The sections were then incubated with primary antibodies diluted in blocking solution overnight at 4 °C. The primary antibody used in this study was rabbit monoclonal anti-GFP (1:1000, Abcam). The sections were washed with PBS containing 0.05% Tween-20 (PBST) and incubated for 1 h with Alexa Flour 488-labeled secondary antibodies. Nuclei were stained using TO-PRO-3 (TP3; 1:750; Life Technologies, Grand Island, NY). Sections were washed with PBST and mounted with ProLong Gold mounting media (Molecular Probes, Eugene, OR, USA). The sections were observed and imaged using a Leica TCS SP8 confocal microscope.
All RNA were isolated from zebrafish eyes using the Trizol method per the manufacturer’s protocol (Invitrogen), cDNA were prepared, and quantitative real-time PCR (qRT-PCR) was performed for inflammatory cytokines (Tnfα, Il1β, and Il6) using SYBR green-based primers on a CFX Connect Real-Time System (Bio-Rad). The quantification of gene expression was determined via the comparative ΔΔCT method. Gene expression in the test samples was normalized to the endogenous control, gapdh, and was reported as fold change relative to gapdh gene expression.
4.6. Statistical Analysis
All data are expressed as the mean ± standard error of the mean (SEM) unless indicated otherwise. Statistical differences between experimental groups were determined using Student’s t-test. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). A value of p < 0.05 was considered statistically significant.