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 pipette puller.</li>
	<li>Adjust the Heater knob until the display shows 12.6.</li>
	<li>Open the door and manually feed the 5 &#956;L capillary tube through the upper clamp and heater filament until half of the tubing extends below the filament (Figure 1A).</li>
	<li>Confirm that the capillary tube is in the &#8220;V&#8221; groove in the upper clamp. Tighten the upper clamp.</li>
	<li>With the lower clamp open, manually adjust the vertical slide up until the lower clamp reaches its upper limit. Confirm that the tube is in the &#8220;V&#8221; groove in the lower clamp. Tighten the lower clamp.</li>
	<li>Close the door and confirm that the &#8216;Ready&#8217; light is on. Push the Start button to initiate the heater and pulling sequence (Figure 1B).</li>
	<li>Ensure the heater turns off automatically after heat and gravity pulls the capillary tube apart (Figure 1C), and the slide moves downward.</li>
	<li>Open the door. While holding the top of the capillary tube, untighten the top clamp. Remove the upper capillary tube and set it aside.</li>
	<li>Hold the capillary tube in the bottom slide and untighten the lower clamp. Remove the lower capillary tube and set it aside.</li>
	<li>Use a microelectrode beveler with a 0.05 &#956;m alumina abrasive grinding plate to bevel the pulled capillary tube to create the injection needles.</li>
	<li>Pipette enough water onto the grinding plate to cover the surface (Figure 1D).</li>
	<li>Turn On the beveller so that it begins to rotate at 60 rpm. Place the pulled capillary tube into the pipette clamp in the &#8220;V&#8221; groove. Tighten the clamp and adjust the angle of the pipette clamp to 30&#176;.</li>
	<li>Adjust the tip of the pointed edge of the capillary tube approximately two-thirds distance away from the center of rotation of the grinding plate.</li>
	<li>Lower the clamped capillary tube by adjusting the coarse control knob at the top of the manipulator. Continue to lower the capillary tube until the tip of the capillary tube extends below the surface of the water and contacts the abrasive surface of the grinding plate.</li>
	<li>Monitor the beveling progress using the microscope attached to the beveller. After 5 min, adjust the fine control to raise the glass needle from the grinding plate.</li>
	<li>Remove the glass needle and place it under 10x magnification to check the tip of the capillary tube (Figure 1E,F).</li>
	<li>To ensure that there are no blockages, insert the glass needle into a pipette holder on a syringe and push air into a 1 mL tube of 99% ethanol. If air bubbles form at the tip of the needle, the needle has no blockages and can be used for microinjection. This process also ensures the sterility of the glass needles.</li>
	<li>One capillary tube can hold up to 5 &#956;L fluid. Mark the capillary tube into 10 sections to calculate the distances on the needle to mark for 0.5 &#956;L volumes. Mark a scale with that distance that holds 0.5 &#956;L for future preparation. For a 5 &#956;L needle, distances of 0.7 mm apart equate to 0.5 &#956;L (Figure 1G).</li>
</ol>
2. Bacillus cereus culture
<ol>
	<li>Perform all procedures in this section under Biosafety Level 2 conditions.</li>
	<li>Streak freezer stock of B. cereus ATCC 14579 for single colony isolation on a 5% sheep blood agar plate and incubate overnight at 37 &#730;C (Figure 2A).</li>
	<li>Pipette 5 mL of sterile Brain Heart Infusion (BHI) broth into a 10 mL sterile snap-cap tube (Figure 2B). Using a sterile loop or needle, pick a single colony of B. cereus ATCC 14579 from the agar plate and inoculate the liquid BHI.</li>
	<li>Vortex the sample briefly. Place the snap-cap tube into a rotating incubator. Set the temperature at 37 &#176;C and speed at 200 rpm. After 18 h, remove the culture from the incubator (Figure 2B).</li>
</ol>
3. Bacterial dilution for intravitreal injection
<ol>
	<li>Perform all procedures in this section under Biosafety Level 2 conditions.</li>
	<li>Calculate the volume of the overnight B. cereus culture to add to 10 mL of fresh BHI to achieve 200 colony forming units per microliter (CFU/&#956;L). For example, an overnight culture of B. cereus in BHI replicates to approximately 2 x 108 CFU/mL, which can then be diluted to 200 CFU/&#956;L by pipetting 10 &#956;L of overnight culture into 10 mL of fresh BHI.</li>
	<li>Pipette 1 mL of the freshly diluted culture into a 1.5 mL microcentrifuge tube. Maintain this tube on ice until intravitreal injection. Perform the intravitreal injection within 60 min of diluting the bacterial culture.</li>
</ol>
4. Mouse intravitreal injection
<ol>
	<li>Perform all procedures in this section under Biosafety Level 2 conditions.</li>
	<li>Prepare the procedure site by placing medical underpads on the operating table.</li>
	<li>Turn On the microinjector and open the gas valve on the compressed air tank attached to the microinjector. Adjust the tank regulator until the gas delivery is approximately 60 psi.</li>
	<li>On the microinjector, press Mode 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 &#8216;Fill/Output&#8217; of the injector. This tube is always connected to the injector.</li>
	<li>Insert the capillary needle to the other end of the pipette holder by screwing tight the pipette holder connector around the needle.</li>
	<li>Turn on the ophthalmic microscope and turn on its light to an intensity of 50%. Adjust the microscope over the procedure site on the operating table and adjust the microscope to the desired focus.</li>
	<li>Before the procedure begins, confirm that the mouse is adequately anesthetized by testing the pedal withdrawal reflex. Be sure to follow your IACUC approved protocol for anesthesia.&#160;</li>
	<li>Place the anesthetized mouse on the medical underpads with its nose pointed to the right and leaning on its left side.</li>
	<li>Look at the right eye of the mouse through the ophthalmic microscope and open the lids by opening the tongs of a reverse action forceps on either side of the eye to expose the injection site (Figure 3C).</li>
	<li>Fill the capillary needle with the 200 CFU/&#956;L dilution by left clicking the mouse pad connected to the microinjector (Figure 3D).</li>
	<li>Secure the animal&#8217;s head with the left hand and place the tip of needle at the limbus of the eye. With the needle in the bevel up position and at 45&#176; angle, puncture the mouse eye, but ensure that only the sharp tip of the needle (~0.5 mm) is inserted when injecting.</li>
	<li>Once the needle tip is inserted, move the left hand from the mouse head to the mouse pad and right click on the mouse pad to inject 0.5 &#956;L of the B. cereus dilution. To prevent leakage, leave the needle tip inside the mouse eye for 2-3 seconds before removing (Figure 3E)1,19,26,27,32,34,36,37,38.</li>
	<li>Release the forceps and place the mouse into a cage that is sitting on a warming pad. Monitor these mice until they have recovered from anesthesia (Figure 3F).</li>
	<li>Once mice are recovered from the anesthesia, return the cage to its proper rack. If the mice will be subjected to retinal function analysis by electroretinography, cages should be returned to a dark room for proper dark adaptation.</li>
</ol>
5. Harvesting tube preparation
<ol>
	<li>Place 1 mm sterile glass beads into 1.5 mL screw cap tubes.</li>
	<li>Sterilize these tubes in an autoclave on a dry setting. Let the tubes cool to room temperature before use.</li>
	<li>Add 10 mL of 1x sterile phosphate-buffered saline (PBS) to a sterile 15 mL centrifuge tube.</li>
	<li>Add 1 tablet of protease inhibitor cocktail tablet into the tube. Mix by vortexing19,27,29,34,35.</li>
	<li>Pipette 400 &#956;L of 1x phosphate-buffered saline (PBS) containing protease inhibitor into each autoclaved harvest tube. Label the tubes and place on ice. (Figure 4A).</li>
</ol>
6. Harvesting the eyes
<ol>
	<li>Perform all procedures in this section under Biosafety Level 2 conditions.</li>
	<li>Euthanize the mouse by CO2 inhalation. Use a secondary method to confirm euthanasia.</li>
	<li>Hold the euthanized mouse head secure and open the fine tip forceps on either side of the infected eye. Push down towards the head to proptose the eye. Once the tongs are behind the globe of the eye, squeeze the tongs together. Pull forceps away from the head to detach the eyeball (Figure 4B).</li>
	<li>Immediately place the eyeball into a labeled harvesting tube. Place tubes on ice for no more than 60 min (Figure 4C).</li>
</ol>
7. Intraocular bacterial count
<ol>
	<li>Perform all procedures in this section under Biosafety Level 2 conditions.</li>
	<li>Confirm that all harvest tubes are tightly closed and are balanced while in the tissue homogenizer (Figure 5A). Turn on the tissue homogenizer for 1 min to homogenize the samples. Wait for 30 s, then turn on for another minute. Place tubes on ice (Figure 5B,C).</li>
	<li>Serially dilute each sample 10-fold by sequentially transferring 20 &#956;L of the homogenate into 180 &#956;L of sterile 1x PBS. Dilute until a factor of 10-7 is reached (Figure 5D).</li>
	<li>Label each row of a warm, square BHI plate with the proper dilution factors. Transfer 10 &#956;L of each dilution in a row to the top BHI plate that is tilted approximately 45&#176;. Let the sample run until it almost reaches the bottom of the plate, then lay the plate flat (Figure 5E)39.</li>
	<li>When sample is absorbed into the BHI agar, transfer the plate to a 37 &#176;C incubator. Colonies should begin to be visible 8 h after being placed in the incubator.</li>
	<li>Remove the plate from the incubator before the growth of the B. cereus colonies interferes with identifying individual colonies (Figure 5F).</li>
	<li>For an accurate representation of the concentration in the sample, count the row that has between 10-100 colonies. For example, a row with the dilution fraction of 10-4 that has 45 colonies will have a concentration of 4.5 x 105 CFU/mL.</li>
	<li>To calculate the total number of bacteria per eye, multiply the concentration by 0.4, which represents the milliliter volume of 1x PBS used to homogenize the eye. For example, the 4.5 x 105 CFU/mL concentration would translate to 1.8 x 105 CFU B. cereus per eye.</li>
</ol>
8. Preservation of samples
<ol>
	<li>Place homogenate samples in a labeled freezer box and place this box into a -80 &#176;C freezer. These samples can be used later for inflammatory mediator analysis by ELISA.</li>
</ol>

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 &amp;micro;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 &amp;micro;L</td><td>Biohit</td><td>15626090</td><td>NA</td></tr><tr><td>Multichannel pipette 5-100 &amp;micro;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 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 demonstrat...

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 ...

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

Research

JoVE Journal

Immunology and Infection

11.2K Views.  University of Oklahoma Health Sciences Center. Ons protocol maakt de reproduceerbare introductie van micro-organismen en therapeutische middelen in het oog van de muis mogelijk om de pathogenese van intraoculaire infectie en nieuwe behandelingseffectiviteit te bestuderen. Onze techniek maakt het mogelijk intraoculaire infecties te vestigen in een muismodel en vergemakkelijkt de kwantificering van micro-organismen, gastheer immuunreacties, infectie gerelateerde gastheer en pathogene genexpressie, en nieuwe behandeling effectiviteit. Visual...

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

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Biology

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.

Primed Mycobacterial Uveitis PMU as a Model for Post-Infectious Uveitis

This protocol outlines the steps for inducing&#160;Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays.

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology:  ...

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...

Neuroscience

A Murine Model of Primed Mycobacterial Uveitis to Study Post-Infectious Uveitis

Source: John, S., et al. <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021).
This video demonstrates a method to generate a mouse model of primed mycobacterial uveitis (PMU) for studying post-infectious uveitis. Upon induction of PMU by intravitreal injection of heat-killed Mycobacterium tuberculosis in a mouse previously immunized with the same immunogen, the progression of chronic inflammation in the eye is analyzed using imaging and histopathological examination

Source: John, S., et al. Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis. J. Vis. Exp. (2021).
This video demonstrates a method ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model

Source: John, S., et al. <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021)
This video demonstrates an in vivo imaging technique for detecting and quantifying eye inflammation in a mouse model of post-infectious uveitis. The technique, known as optical coherence tomography (OCT), generates high-resolution cross-sectional images of the eye for assessing disease progression.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunodiagnostics

Imaging and Visualization Techniques

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

Source: Murinello, S. et al., <a href="https://app.jove.com/v/54677">Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.

Cellular Immunodiagnostics

Bacterial Endophthalmitis Mouse Model: A Method for Generating Bacterial Endophthalmitis via Intravitreal Injection of Bacterial Suspension into Eye of Mouse

Source: Mursalin, M. H. et al., <a href="https://www.jove.com/v/61749">Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis.</a> J. Vis. Exp. (2021).
This video describes a method for generating a mouse model that mimics bacterial endophthalmitis &#8211; an intraocular infection resulting from a bacterial invasion into the eye&#8217;s vitreous cavity &#8211; via intravitreal injection of a bacterial suspension into the eye. This mouse model helps study the disease mechanism and evaluate therapeutics for it.

Source: Mursalin, M. H. et al., Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis. J. Vis. ...

Rodent Models

Adult

Bacterial Infection Quantitation Assay: A Method to Quantify Intraocular Bacterial Load in Mouse Model of Bacterial Endophthalmitis

Source: Mursalin, M. H. et al., <a href="https://www.jove.com/v/61749">Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis.</a> J. Vis. Exp. (2021).
This video describes the technique of quantifying bacterial infection from eye homogenates obtained from a murine model of bacterial endophthalmitis caused by Bacillus cereus. This technique helps study the host immune response and its dependency on the host and ocular bacterial gene expression in murine models.

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

Samenvatting

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