The authors thank Dr. Feng Li and Mark Dittmar (OUHSC P30 Live Animal Imaging Core, Dean A. McGee Eye Institute, Oklahoma City, OK, USA) for their assistance. Our research has been supported by National Institutes of Health grants R01EY028810, R01EY028066, R01EY025947, and R01EY024140. Our research has also been supported by P30EY21725 (NIH CORE grant for Live Animal Imaging and Analysis, Molecular Biology, and Cellular Imaging). Our research has also been supported by the NEI Vision Science Pre-doctoral Trainee program 5T32EY023202, a Presbyterian Health Foundation Research Support grant, and an unrestricted grant to the Dean A. McGee Eye Institute from Research to Prevent Blindness.

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
<ol>
	<li>Turn On the needle p...

The authors have no financial conflicts to disclose.

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 &#181;L40. This s...

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 eye1,2,3,4,5. 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 inflammatory cell influx into the interior of the eye1,5,6. Endophthalmitis can occur following intraocular surgery (post-operative), a penetrating injury to the eye (post-traumatic), or from metastatic spread of bacteria into the eye from a different anatomical site (endogenous)7,8,9,10. Treatments for bacterial endophthalmitis includes antibiotics, anti-inflammatory drugs, or surgical intervention3,4,11. Even with these treatments, vision or the eye itself may be lost. The visual prognosis after bacterial endophthalmitis generally varies depending upon the treatment effectiveness, the visual acuity at presentation, and the virulence of the infecting organism.
Bacillus cereus (B. cereus) is one of the major bacterial pathogens that causes post-traumatic endophthalmitis7,12. A majority of B. cereus endophthalmitis cases have a rapid course, which can result in blindness within a few days. The hallmarks of B. cereus endophthalmitis include quickly evolving intraocular inflammation, eye pain, rapid loss of visual acuity, and fever. B. cereus grows rapidly in the eye compared to other bacteria which commonly cause eye infections2,4,12 and possesses many virulence factors. Therefore, the window for successful therapeutic intervention is relatively short1,&#160;2,&#160;3,&#160;4,&#160;5,&#160;6,&#160;7,&#160;11,&#160;12,&#160;13,&#160;14,&#160;15,&#160;16,&#160;17,&#160;18,&#160;19,&#160;20,&#160;21,&#160;22,&#160;23,&#160;24,&#160;25. Treatments for this infection are usually successful in treating endophthalmitis caused by other less virulent pathogens, but B. cereus endophthalmitis commonly results in greater than 70% of patients suffering from significant vision loss. About 50% of those patients undergo evisceration or enucleation of the infected eye7,16,22,23. The destructive and rapid nature of B. cereus endophthalmitis calls for immediate and proper treatment. Recent progress in discerning the underlying mechanisms of disease development have identified potential targets for intervention19,26,27. Experimental mouse models of B. cereus endophthalmitis continue to be useful in discerning the mechanisms of infection and testing potential therapeutics that may prevent vision loss.
Experimental intraocular infection of mice with B. cereus has been an instrumental model for understanding bacterial and host factors, as well as their interactions, during endophthalmitis28. This model mimics a post-traumatic or post-operative event, in which bacteria are introduced into the eye during an injury. This model is highly reproducible and has been useful for testing experimental therapies and providing data for improvements in standard of care1,6,19,29,30. Like many other infection models, this model allows for independent control of many parameters of infection and enables efficient and reproducible examination of infection outcomes. Studies in a similar model in rabbits over the past few decades have examined the effects of B. cereus virulence factors in the eye2,4,13,14,31. By injecting B. cereus mutant strains lacking individual or multiple virulence factors, the contribution of these virulence factors to disease severity can be measured by outcomes such as the concentration of bacteria at different hours of postinfection or the loss of visual function13,14,27,31,32. In addition, host factors have been examined in this model by infecting knockout mouse strains lacking specific inflammatory host factors26,29,33,34,35. The model is also useful for testing potential treatments for this disease by injecting novel compounds into the eye after infection30,36. In this manuscript, we describe a detailed protocol which includes infecting a mouse eye with B. cereus, harvesting the eye after infection, quantifying intraocular bacterial load, and preserving specimens to assay additional parameters of disease severity.

<table>
	<tbody>
		<tr>
			<td>2-20 &#181;L pipette</td>
			<td>RANIN</td>
			<td>L0696003G</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>37oC Incubator</td>
			<td>Fisher Scientific</td>
			<td>11-690-625D</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>90003-032</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Cell Microinjector</td>
			<td>MicroData Instrument, Inc.</td>
			<td>PM2000</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Fine tip forceps</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-000-122</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Glass beads 1.0 mm</td>
			<td>BioSpec</td>
			<td>11079110</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>NB-I2400</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microcapillary Pipets 5 Microliters</td>
			<td>Kimble</td>
			<td>71900-5</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Micro-Pipette Beveler</td>
			<td>Sutter Instrument Co.</td>
			<td>BV-10</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microscope Axiostar Plus</td>
			<td>Zeiss</td>
			<td></td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microscope OPMI Lumera</td>
			<td>Zeiss</td>
			<td></td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Mini-Beadbeater-16</td>
			<td>BioSpec</td>
			<td>Model 607</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Multichannel pipette 30-300 &#181;L</td>
			<td>Biohit</td>
			<td>15626090</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Multichannel pipette 5-100 &#181;L</td>
			<td>Biohit</td>
			<td>9143724</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Needle/Pipette Puller</td>
			<td>Kopf</td>
			<td>730</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>1897315</td>
			<td>Molecular grade</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Molecular grade</td>
		</tr>
		<tr>
			<td>Reverse action forceps</td>
			<td>Katena</td>
			<td>K5-8228</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

intravitreal injection and quantitation of infection parameters in a mouse model of bacterial endophthalmitis

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 &#181;L. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 &#181;L 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 &#181;L 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 &#176;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.

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 &#956;L 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 demons...

Watch this Scientific Journal Video about Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis at JoVE.com

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

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.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Our protocol allows for the reproducible introduction of microorganism and therapeutic agents into the mouse eye to study the pathogenesis of intraocular infection and novel treatment effectiveness. Our technique allows intraocular infections to be established in a mouse model and facilitates the quantitation of microorganisms, host immune responses, infection related host and pathogen gene expression, and novel treatment effectiveness. Visualization of the injection site, needle angle, and delivery are critical for successful execution of this technique and a proper eye harvest is essential for quantification of the infection parameters.In a biosafety level two facility, add five milliliters of BHI broth to a 10 milliliter snap cap tube and use a sterile loop to transfer a single B.cereus colony from an auger plate culture into five milliliters of broth. After brief vortexing, incubate the sample in a 37 degrees Celsius rotating incubator at 200 revolutions per minute for 18 hours. At the end of the incubation, dilute the culture to a 200 colony forming units per microliter concentration in 10 milliliters of fresh BHI, and after vortexing, add one milliliter of the freshly diluted culture to a 1.5 milliliter microcentrifuge tube on ice.For intravitreal injection, turn on the microinjector and open the gas valve on the compressed air tank attached to the microinjector. Press Mode on the microinjector until the screen shows Balance. Press Balance and place the computer mouse on the operating table.Connect the stainless steel pipette holder to the tubing attached to the fill output on the injector and tightly screw the pipette holder connector around a beveled glass capillary pipette tip needle to allow insertion of the capillary needle into the other end of the pipette holder. Turn on the ophthalmic microscope and set the light intensity to 50%Position the microscope over the procedure site and adjust the microscope to the desired focus. After confirming a lack of response to pedal reflex, place the anesthetized mouse on its left side on the medical underpads with the nose pointed to the right.Locate the right eye through the ophthalmic microscope objective and place the tongs of reverse action forceps on either side of the eye to expose the injection site. Left click the mouse connected to the microinjector to fill the prepared capillary needle with the diluted bacteria solution, and securing the head with the left hand, place the tip of the needle, bevel side up, at the limbus of the eye. With the needle at a 45 degree angle, puncture the eye, taking care that only 0.5 millimeters of the sharp tip of the needle is inserted.Once the needle tip has been inserted, use the left hand to right click on the mousepad to inject 0.5 microliters of the B.cereus solution. To prevent leakage, leave the needle tip inside the mouse eye for two to three seconds before removing, then release the forceps and the eye will go back into the socket by itself. Transfer the mouse to a recovery cage on a warming pad with monitoring until full recumbency.At the appropriate experimental endpoint, add 400 microliters of PBS supplemented with protease inhibitor in one labeled, autoclaved harvest tube per eye on ice and place open fine tip forceps on either side of the infected eye. Push the tips down towards the head to proptose the eye. Once the tongs are behind the eye globe, squeeze the tongs together and pull the forceps away from the head to detach the eyeball.Then immediately place the eyeball into the appropriately labeled harvesting tube. Within 60 minutes of sample collection, place the closed harvesting tubes into a tissue homogenizer and homogenize the samples for two, one minute homogenization periods with a 30 second period of rest in between. After homogenization, place the samples on ice and use 20 microliter aliquots of homogenate to serially dilute the samples in 180 microliters of PBS per dilution, until a factor of one times 10 to the negative eight is reached.Next, label each row of a square pre-warmed BHI plate with the appropriate dilution concentration, and with the plate tilted at a 45 degree angle, add 10 microliters of each dilution into individual rows at the top of the plate. Let each sample run until it almost reaches the bottom of the plate before laying the plate flat. When the samples have been absorbed into the agar, transfer the plate to a 37 degrees Celsius incubator.Colonies should begin to be visible after eight hours of incubation. For an accurate representation of the concentration in the sample, count the row that has between 10 to 100 colonies. The construction of a beveled glass needle tip is critical for delivering the bacteria into the mid-vitreous of the mouse eye.In these images, Bacillus cereus growing on a blood agar plate and in broth culture tubes are shown. As illustrated in this graph of interocular bacterial counts from five different mouse eyes at 10 hours post-infection between one times 10 to the fifth and one times 10 to the seventh colony forming units of bacteria can typically be recovered from a single infected eye. The most important thing to remember when attempting this procedure is to inject that appropriate volume of bacteria into the mouse eye.Following this procedure, we can study inflammation by histology, inflammatory mediators by ELISA, and gene expression by high throughput RNA sequencing to facilitate a better understanding of the pathogenesis of this disease.

intravitreal-injection-quantitation-infection-parameters-mouse-model

Research

JoVE Journal

Immunology and Infection

10.6K visualizações.  University of Oklahoma Health Sciences Center. Nosso protocolo permite a introdução reprodutível de microrganismos e agentes terapêuticos no olho do rato para estudar a patogênese da infecção intraocular e a eficácia do novo tratamento. Nossa técnica permite que infecções intraoculares sejam estabelecidas em um modelo de camundongos e facilita a quantitação de microrganismos, respostas imunes hospedeiras, expressão genética de hospedeiro e patógeno relacionada à infecção, e nova eficácia do tratamento. A visualização...