Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Summary

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression.

Abstract

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 µL. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 µL of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 µL sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 °C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

Introduction

Bacterial endophthalmitis is a devastating infection that causes inflammation, and, if not treated properly, can result in loss of vision or blindness. Endophthalmitis results from the entry of bacteria into the interior of the eye^1,2,3,4,5. Once in the eye, bacteria replicate, produce toxins and other noxious factors, and can cause irreversible damage to delicate retinal cells and tissues. Ocular damage can also be caused by inflammation, due to the activation of inflammatory pathways leading to inflammato....

Protocol

All procedures were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center (protocol numbers 15-103, 18-043, and 18-087).

1. Sterile glass needles

1. Turn On the needle pipette puller.
2. Adjust the Heater knob until the display shows 12.6.
3. Open the door and manually feed the 5 μL....

Results

Generating a reproducible inoculum and accuracy of the intravitreal injection procedure are key steps in developing models of microbial endophthalmitis. Here, we demonstrated the intravitreal injection procedure using Gram-positive Bacillus cereus. We injected 100 CFU/0.5 μL of B. cereus into the mid-vitreous of five C57BL6 mice. After 10 h postinfection, we observed intraocular growth of B. cereus to approximately 1.8 x 10⁵ CFU/eye. Figure 1 demonstrate.......

Discussion

Even with the availability of potent antibiotics, anti-inflammatory drugs, and vitrectomy surgery, bacterial endophthalmitis can blind a patient. Clinical studies have been useful in studying endophthalmitis; however, experimental models of endophthalmitis provide quick and reproducible results that can be translated to progress in standard of care, resulting in better visual outcome for patients.

The vitreous volume of the mouse eye is approximately 7 µL⁴⁰. This s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-20 µL pipette	RANIN	L0696003G	NA
37oC Incubator	Fisher Scientific	11-690-625D	NA
Bacto Brain Heart Infusion	BD	90003-032	NA
Cell Microinjector	MicroData Instrument, Inc.	PM2000	NA
Fine tip forceps	Thermo Fisher Scientific	12-000-122	NA
Glass beads 1.0 mm	BioSpec	11079110	NA
Incubator Shaker	New Brunswick Scientific	NB-I2400	NA
Microcapillary Pipets 5 Microliters	Kimble	71900-5	NA
Micro-Pipette Beveler	Sutter Instrument Co.	BV-10	NA
Microscope Axiostar Plus	Zeiss		NA
Microscope OPMI Lumera	Zeiss		NA
Mini-Beadbeater-16	BioSpec	Model 607	NA
Multichannel pipette 30-300 µL	Biohit	15626090	NA
Multichannel pipette 5-100 µL	Biohit	9143724	NA
Needle/Pipette Puller	Kopf	730	NA
PBS	GIBCO	1897315	Molecular grade
Protease Inhibitor Cocktail	Roche	4693159001	Molecular grade
Reverse action forceps	Katena	K5-8228	NA