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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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, 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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.

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Protokół

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 capillary tube through the upper clamp and heater filament until half of the tubing extends below the filament (Figure 1A).
  4. Confirm that the capillary tube is in the “V” groove in the upper clamp. Tighten the upper clamp.
  5. 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 “V” groove in the lower clamp. Tighten the lower clamp.
  6. Close the door and confirm that the ‘Ready’ light is on. Push the Start button to initiate the heater and pulling sequence (Figure 1B).
  7. Ensure the heater turns off automatically after heat and gravity pulls the capillary tube apart (Figure 1C), and the slide moves downward.
  8. Open the door. While holding the top of the capillary tube, untighten the top clamp. Remove the upper capillary tube and set it aside.
  9. Hold the capillary tube in the bottom slide and untighten the lower clamp. Remove the lower capillary tube and set it aside.
  10. Use a microelectrode beveler with a 0.05 μm alumina abrasive grinding plate to bevel the pulled capillary tube to create the injection needles.
  11. Pipette enough water onto the grinding plate to cover the surface (Figure 1D).
  12. Turn On the beveller so that it begins to rotate at 60 rpm. Place the pulled capillary tube into the pipette clamp in the “V” groove. Tighten the clamp and adjust the angle of the pipette clamp to 30°.
  13. 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.
  14. 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.
  15. 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.
  16. Remove the glass needle and place it under 10x magnification to check the tip of the capillary tube (Figure 1E,F).
  17. 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.
  18. One capillary tube can hold up to 5 μL fluid. Mark the capillary tube into 10 sections to calculate the distances on the needle to mark for 0.5 μL volumes. Mark a scale with that distance that holds 0.5 μL for future preparation. For a 5 μL needle, distances of 0.7 mm apart equate to 0.5 μL (Figure 1G).

2. Bacillus cereus culture

  1. Perform all procedures in this section under Biosafety Level 2 conditions.
  2. Streak freezer stock of B. cereus ATCC 14579 for single colony isolation on a 5% sheep blood agar plate and incubate overnight at 37 ˚C (Figure 2A).
  3. 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.
  4. Vortex the sample briefly. Place the snap-cap tube into a rotating incubator. Set the temperature at 37 °C and speed at 200 rpm. After 18 h, remove the culture from the incubator (Figure 2B).

3. Bacterial dilution for intravitreal injection

  1. Perform all procedures in this section under Biosafety Level 2 conditions.
  2. 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/μL). 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/μL by pipetting 10 μL of overnight culture into 10 mL of fresh BHI.
  3. 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.

4. Mouse intravitreal injection

  1. Perform all procedures in this section under Biosafety Level 2 conditions.
  2. Prepare the procedure site by placing medical underpads on the operating table.
  3. 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.
  4. 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 ‘Fill/Output’ of the injector. This tube is always connected to the injector.
  5. Insert the capillary needle to the other end of the pipette holder by screwing tight the pipette holder connector around the needle.
  6. 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.
  7. 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. 
  8. Place the anesthetized mouse on the medical underpads with its nose pointed to the right and leaning on its left side.
  9. 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).
  10. Fill the capillary needle with the 200 CFU/μL dilution by left clicking the mouse pad connected to the microinjector (Figure 3D).
  11. Secure the animal’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° angle, puncture the mouse eye, but ensure that only the sharp tip of the needle (~0.5 mm) is inserted when injecting.
  12. 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 μL 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.
  13. 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).
  14. 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.

5. Harvesting tube preparation

  1. Place 1 mm sterile glass beads into 1.5 mL screw cap tubes.
  2. Sterilize these tubes in an autoclave on a dry setting. Let the tubes cool to room temperature before use.
  3. Add 10 mL of 1x sterile phosphate-buffered saline (PBS) to a sterile 15 mL centrifuge tube.
  4. Add 1 tablet of protease inhibitor cocktail tablet into the tube. Mix by vortexing19,27,29,34,35.
  5. Pipette 400 μL of 1x phosphate-buffered saline (PBS) containing protease inhibitor into each autoclaved harvest tube. Label the tubes and place on ice. (Figure 4A).

6. Harvesting the eyes

  1. Perform all procedures in this section under Biosafety Level 2 conditions.
  2. Euthanize the mouse by CO2 inhalation. Use a secondary method to confirm euthanasia.
  3. 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).
  4. Immediately place the eyeball into a labeled harvesting tube. Place tubes on ice for no more than 60 min (Figure 4C).

7. Intraocular bacterial count

  1. Perform all procedures in this section under Biosafety Level 2 conditions.
  2. 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).
  3. Serially dilute each sample 10-fold by sequentially transferring 20 μL of the homogenate into 180 μL of sterile 1x PBS. Dilute until a factor of 10-7 is reached (Figure 5D).
  4. Label each row of a warm, square BHI plate with the proper dilution factors. Transfer 10 μL of each dilution in a row to the top BHI plate that is tilted approximately 45°. Let the sample run until it almost reaches the bottom of the plate, then lay the plate flat (Figure 5E)39.
  5. When sample is absorbed into the BHI agar, transfer the plate to a 37 °C incubator. Colonies should begin to be visible 8 h after being placed in the incubator.
  6. Remove the plate from the incubator before the growth of the B. cereus colonies interferes with identifying individual colonies (Figure 5F).
  7. 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.
  8. 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.

8. Preservation of samples

  1. Place homogenate samples in a labeled freezer box and place this box into a -80 °C freezer. These samples can be used later for inflammatory mediator analysis by ELISA.

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Wyniki

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...

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Dyskusje

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...

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Ujawnienia

The authors have no financial conflicts to disclose.

Podziękowania

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.

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Materiały

NameCompanyCatalog NumberComments
2-20 µL pipetteRANINL0696003GNA
37oC IncubatorFisher Scientific11-690-625DNA
Bacto Brain Heart InfusionBD90003-032NA
Cell MicroinjectorMicroData Instrument, Inc.PM2000NA
Fine tip forcepsThermo Fisher Scientific12-000-122NA
Glass beads 1.0 mmBioSpec11079110NA
Incubator ShakerNew Brunswick ScientificNB-I2400NA
Microcapillary Pipets 5 MicrolitersKimble71900-5NA
Micro-Pipette BevelerSutter Instrument Co.BV-10NA
Microscope Axiostar PlusZeissNA
Microscope OPMI LumeraZeissNA
Mini-Beadbeater-16BioSpecModel 607NA
Multichannel pipette 30-300 µLBiohit15626090NA
Multichannel pipette 5-100 µLBiohit9143724NA
Needle/Pipette PullerKopf730NA
PBSGIBCO1897315Molecular grade
Protease Inhibitor CocktailRoche4693159001Molecular grade
Reverse action forcepsKatenaK5-8228NA

Odniesienia

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