A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

All procedures were approved by the West Virginia Institutional Animal Care and Use Committees and conducted in accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals by the National Research Council¹⁸.

1. Preparation of Bacterial Inoculum

1. Streak a Tryptic soy agar (TSA) plate with an inoculating loop for isolation of a single colony from a freezer stock of E. coli O1:K1:H7-lux that stably expresses luciferase and carries kanamycin resistance³. Incubate overnight at 37 °C.
2. The following day allow Luria broth (LB) to come to room temperature (25 °C) in a biosafety cabinet.
3. Under a biosafety cabinet hood, identify a single colony from the streaked plate and inoculate it in 3 mL of LB supplemented with kanamycin (30 μg/mL). Incubate overnight at 37 °C with shaking (220 rpm). This is the starter culture.
4. Dilute the starter culture 1:100 into a fresh 3 mL of LB under a biosafety cabinet hood and return it to the incubator for 2-3 h at 37 °C with shaking (220 rpm). This is the stock culture.
5. Read the optical density (OD) of both the blank and stock culture at 600 nm using a spectrophotometer. Add 100 µL of LB (containing no bacteria) into one well of a 96 well flat bottom assay plate; this is the blank. Then add 100 µL from the stock culture to a separate well. Repeat for two additional replicates. The absorbance is read using a plate reader.
6. Subtract the blank absorbance from the stock culture absorbance value (the OD value) and compare to a previously generated and validated growth curve to determine an approximation of the bacterial density in the stock culture for the preparation of infectious dose.
7. Generate target inoculums depending on the research question. Target inoculum of 2 x 10⁶ (low) and 7 x 10⁶ (high) colony-forming units (CFUs) per mouse (/mouse) were used for this study.
   1. Divide the target dose per mouse (Dose_T) by the estimated concentration of bacteria in the stock culture (Stock) to get the volume of bacteria needed from the stock tube (V_S).
   2. Multiply V_S by the number of mice (N_M) that need to be infected along with enough for 5-10 extras for the total amount of bacteria required for the infection plus 5-10 additional doses. Remove this volume from the stock tube and add it to a new centrifuge tube.
   3. Use the equation below:
      Dose_T/Stock = V_S x N_M = total volume (V_T) of bacteria to be removed from the stock tube.

8. Centrifuge the bacteria at 2,000 x g for 5 min at 4 °C and resuspend the bacterial pellet in 50 µL of PBS (pH 7.2-7.6) per mouse to be infected (e.g., for 10 doses of 2 x 10⁶ bacteria each dose, the pellet of 2 x 10⁷ bacteria would be resuspended in 500 μL PBS). Again, it is recommended to prepare more inoculum than is needed. Prepare an equal volume of PBS only for control inoculations. Maintain the infectious inoculum and PBS control on ice until infection.
9. Perform seven ten-fold serial dilutions into PBS in a 96 well plastic bottom dilution plate, and plate 25 μL of the dilutions in duplicate onto quadrant TSA plates supplemented with kanamycin (30 µg/mL) to enumerate the actual amount of bacteria administered. Incubate at 37 °C overnight for colony formation prior to enumeration.

2. Animal identification

1. Arrange a sufficient number of breeding pairs such that litters may be synchronized for age-matched pups. Age variability of ± 1 day is acceptable.
2. Identify a pregnant C57BL/6 female mouse and monitor for birth of the litter in advance of the planned experiment to accurately determine age.
3. To distinguish between control and infected 3- or 4-day old pups, use a pair of small, fine-tipped, iris scissors to snip the ends of the tails of the control pups only. The infected pups do not receive tail snips. Before cutting the tail, disinfect the skin with a cotton ball doused in 70% ethanol. Apply pressure to the end of the tail with a cotton ball or gauze as needed.
   NOTE: This procedure is performed under a biosafety cabinet hood. A tail snip of approximately 1/8 of an inch is sufficient.
4. To identify pups within the control and infected groups, use a 1 mL insulin syringe with a 28 G x ½’’ permanent needle to tattoo the tails of the pups. Before tattooing, disinfect the skin with a cotton ball doused in 70% ethanol. This procedure is performed under a biosafety cabinet hood.
5. To tattoo the tail, apply animal tattoo ink to the tip of the needle. Next, carefully restrain the pup with one hand, with their tail fully exposed. Gently insert the needle under the skin, while maintaining a superficial level of depth, and move the needle parallel with the skin a few millimeters until a small marking, or dot, has been created. Wait a few seconds before slowly removing the needle from under the skin, to avoid excess ink releasing from under the skin.
6. Apply pressure to the wound with a cotton ball or gauze as needed. Remove excess tattoo ink on the surface of the skin with 70% ethanol.
7. Repeat this process with subsequent mice in the infected and control groups, while adding an additional dot with each successive pup tattooed (e.g., pup 1 will have 1 dot on their tail, pup 2 will have two dots on their tail, etc.).
   NOTE: For an additional layer of identification, it is recommended to use separate colors of animal tattoo ink for the control and infected groups.

3. Subscapular inoculation

NOTE: For this study, 2 experiments were performed with a low-dose and high-dose group designated for each experiment. In the first experiment, 7 pups were given the low dose inoculum (4 pups were used as controls), and 5 pups from a separate litter were given the high dose (3 pups were used as controls). The pups from experiment 1 provided data for only the 24 h timepoint. In the second experiment, 8 pups were given the low dose inoculum (2 pups were used as controls), and 6 pups were given the high dose inoculum (2 pups were used as controls). Pups from experiment 2 provided data for the 0, 10, and 24 h timepoints.

1. Age match pups ≤ 1 day. Assign each litter as either a low dose or a high dose litter. Within a litter randomly assign pups as a control or infected pup.
2. On day 3 or 4 postnatal, record weights of all pups prior to inoculation with E. coli-lux or the PBS control. Separate the dam from the pups during this time to ensure they are not moved during the infection.
3. Within a biosafety cabinet using an insulin needle, aspirate either PBS or the E. coli-lux inoculum. For this work, inoculums of 2 x 10⁶ and 7 x 10⁶ CFUs per mouse were used. Keep both infectious inoculum and PBS on ice until administration via subscapular injection.
4. Place the neonate on a clean surface in the biosafety cabinet hood and raise the skin at the nape of the neck as if to scruff the pup.
5. In the space now created between the skin and the muscle of the animal, insert the needle, bevel up, just beneath the skin and inject 50 µL of PBS or E. coli-lux. Simultaneously release the pinched portion of skin to prevent injection backflow.
6. Remove the needle slowly and with care. Place pups back with dams after injections are finished.
   NOTE: Due their anatomical stage in development, it is technically challenging to administer a tail vein or intraperitoneal injection to neonatal pups at day 3-4. Thus, the subscapular infection route was chosen for this study due to the ease of execution.

4. Evaluation of disease and endpoint criteria

1. Monitor the pups twice daily throughout the duration of infection. Note any abnormalities in appearance.
2. Record weights as an objective measurement of morbidity.
3. In addition to weight changes, test the ability of the pups to right themselves by positioning the neonate on the dorsal side. Sick animals will be unable to turn over to the ventral side and onto the feet or will complete this action with difficulty.
4. Check for the following to mark the animals close to endpoint criteria: less than 85% of normal body weight; decreased movement and inability to right themselves; discoloration of the skin and a more grey or transparent appearance as opposed to pink; feeling cool to the touch, indicative of decreased body temperature and hemorrhagic bruising along the sides, also indicative of advance illness.
   NOTE: If the neonates have failed to gain weight over two days and fit any of the descriptions in steps 4.4, they have met endpoint criteria. Pups that receive the high dose often meet endpoint criteria by 24 h. Control pups within the low and high dose litters will be euthanized at the same time to allow for comparative analysis between the control and experimental groups. Proceed to the euthanasia section below.

5. In vivo imaging of bacterial burden

1. Use a microCT imager and software for imaging and subsequent analysis.
   NOTE: Pup skin color does not impact imaging quality.
2. Place the cage with E. coli-lux-infected neonatal mice and dam into a BSL-2 level laminar flow hood. Remove mice to be imaged, and place into a transparent isoflurane chamber within the hood. It is recommended to start with uninfected controls to gauge the amount of isoflurane needed.
3. Open the software on the computer attached to the microCT. Initialize the system and wait for the CCD temperature to lock at -90 °C.
4. Turn the isoflurane vaporizer on and adjust the dial to 5% isoflurane flow. Keep mice in the chamber with this isoflurane mixture for 20-30 s until they stop moving; longer or shorter anesthesia exposure times may be needed for some mice. Once mice stop moving, they are sufficiently anesthetized, and can be imaged.
5. Move mice into the microCT imaging chamber and place them onto the imaging box in the prone position, with noses facing perpendicular to nose cones. Use dental wax to gently restrain the feet on the imaging box to limit any movement. Up to 4 neonatal mice can be imaged at a time.
6. Turn the isoflurane vaporizer down to 2-4% flow to keep mice anesthetized during imaging. Shut the microCT imaging chamber door. Check on the mice a few seconds later. If they begin to move, douse a cotton ball in isoflurane and hold it to the nose of the animal moving for 5 seconds to anesthetize. Keep the cotton ball near the animals during imaging. Be careful not to over anesthetize and terminate the mice.
7. Using the software, choose the Luminescent option for imaging. Use an excitation filter set to Block and the emission filter set to Open, 500 nm, 520 nm, 560 nm, 580 nm, 600 nm, and 620 nm. There will be seven total emission filters set for luminescence.
8. Image the mice at each time point (0, 10, and 24 h post-infection [hpi]) and save all images to a folder for each time point. Return the pups to the cage with the dam and check that all pups have recovered from anesthesia.
9. To analyze 2D images, open images in the software. Change units to Radiance (photons); this will turn into the Total Flux (photons/second).
10. Only analyze one image set with its multiple emission filters at a time. From each image set, take note of the minimum and maximum radiance values located at the bottom right corner of each image (e.g., if there are 7 emission filters, there will be 7 images, and 7 minimum and maximum values). Repeat for each image set that is to be compared.
11. To determine a scale that will encompass the values and luminescence for all images, locate the lowest minimum value and the highest maximum value for each image set. For this study, the Open filter images were used as representative.
    1. Highlight and open the image of choice to change the scale. On the Tool Palette, click on the Image Adjust tab and change the Color Scale to the lowest minimum and highest maximum values previously identified. Save each image set as a TIFF. Individually analyze each time point in this manner to ensure the correct scale is displayed.
12. To quantify the total flux (amount of luminescent signal per mouse) for each individual mouse, open an image as previously described in step 5.9-5.10. Open the ROI Tools tab on the Tool Palette and select the circle tool. Choose 1 circle if analyzing one area of luminescence.
13. Move ROI to Overlay on the area of luminescence. Adjust the size of the ROI if necessary.
    NOTE: If adjustment is necessary, adjust ROIs in other images comparably to maintain consistency. Choose Measure ROIs. The ROI Measurements window will open displaying Total Flux (p/s), Average Radiance (p/s/cm²/sr), Standard Deviation of Radiance, Minimum Radiance and Maximum Radiance.
14. Record total flux measurements for each image set. This number is the quantified amount of luminescence in the mouse in 2D images.
15. To make 3D reconstructed microCT images, open the DLIT 3D Reconstruction panel on the Tool Palette and check all wavelengths to be included under the Analyze tab. Select Reconstruct.

6. Euthanasia

1. Prepare and label tubes for tissues/organs of interest for necropsy and appropriate downstream applications.
2. Separate the neonates from the dam in a biosafety cabinet.
3. Soak a cotton ball in veterinary-grade isoflurane and place inside of a transparent containment chamber.
4. If collecting blood, prepare a P200 micropipette with a tip and have a 1.5 mL tube with 10 µL of 5 mM EDTA as an anticoagulant. A volume of 50-200 µL of blood is expected.
5. Place a neonate in the chamber and monitor the pup until it becomes motionless.
6. Quickly, remove neonate and decapitate with scissors. If allowed to breathe fresh air for a prolonged period, the pup can regain consciousness. Neonates have reduced lung capacity relative to adult mice, and, therefore, do not breathe deeply enough for euthanasia by isoflurane alone.
7. Collect blood from the trunk at the base of the head using a P200 micropipette. To maximize the amount of blood collected, perform this step as quickly as possible following decapitation. Enumerate bacteria in the blood by serial dilution and standard plate counting as described in step 1.9.
8. Sterilize the entire neonate with 70% ethanol prior to excision of tissue samples.

7. Tissue harvest

1. Within a biosafety cabinet, douse the neonate with 70% ethanol to prevent contamination. Lay the animal on its right side.
2. Using forceps, grasp the skin at a point between the abdomen and rear left leg and make an incision with fine-tipped surgical scissors. Continue to cut the skin away moving upwards towards the back. Progress until the entire spleen is exposed.
3. Use the forceps to grasp the spleen and remove it from the abdomen, using scissors to disconnect the connective tissue. Place the spleen in the solution appropriate for its downstream application.
4. To obtain the lungs, peel back the skin of the chest completely.
5. Entering at the base of the sternum with scissors held vertically, cut upwards until the rib cage is split.
6. Use forceps to grasp the right and left lungs individually and remove them from the thoracic cavity. Remove the heart from the lung tissue by cutting with scissors.
7. Place the lung in the solution appropriate for its downstream application. For RNA isolation, use 500 µL of guanidine thiocyanate/phenol (GTCP). For histopathology, use 5 mL of 10% neutral-buffered formalin.

8. RNA isolation from lung tissue for gene expression

1. Pre-cool the microcentrifuge to 4 °C.
2. Mince the lung tissue in GTCP with scissors. Next, homogenize the tissue with a battery-powered homogenizer. Continue until the solution is as uniform as possible. Incubate at room temperature for 3-5 min.
3. Using filtered pipette tips, add 100 µL of chloroform. Invert the tube for 15 s and incubate 3-5 min at room temperature.
4. Centrifuge for 15 min at 12,000 x g.
5. During the spin, prepare 1.5 mL tubes with 500 µL of 70% ethanol. Assemble and label the columns and collection tubes from the RNA isolation kit.
6. Carefully remove the top, aqueous layer without disturbing the interphase layer that formed during centrifugation. Place the aqueous layer in the tubes containing 70% ethanol.
7. Move the ethanol and lysate mixture to the column in the collection tube.
8. From this point on, follow the RNA isolation kit commercial product protocol until the final elution of RNA.
9. Analyze the RNA for purity and quantity. Use immediately or store at -80 °C until further use.

9. cDNA synthesis

1. Label PCR tubes and set aside.
2. Add 1 µg of RNA to the cDNA reaction mixture for each sample.
3. Add the reagents and template to the PCR tube as described in the cDNA protocol. Add the enzyme to the mixture last.
4. Place PCR tubes in a thermocycler with the following run settings: 5 min at 25 °C, 40 min at 42 °C, 15 min at 85 °C and 4 °C final hold.
5. Remove PCR tubes from thermocycler and use immediately or store at -20 °C until further use.

10. Real-time quantitative PCR (qPCR) cycle

1. Prepare a reaction mix cocktail for each of the genes to be analyzed. Each 15 µL PCR reaction requires 7.5 µL of 2x reagent mix, 0.75 µL of 20X 5’-FAM-labeled gene-specific primer/probe, and 3.75 µL of nuclease-free water. Amplicons typically range from 60-120 bp.
2. Add 3 µL of cDNA template for each experimental group to the appropriate wells.
3. Add 12 µL of the gene-specific reaction mix cocktail to the appropriate wells.
4. Cover the plate with optical adhesive film and centrifuge for 1 min at 1,000 x g to remove any bubbles that may have formed in the wells.
5. Place the PCR plate in a real-time PCR thermocycler.
6. Set the run method as follows: 3 min at 95 °C, 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min.
7. Analyze data by normalizing the gene of interest to an internal control and express data from infected samples relative to uninfected control samples using the 2^-ΔΔCt formula and a log₂ transformation of the numbers.

11. Lung histopathology

1. Remove the lungs from the neonatal pup as described above.
2. Place the tissue in a volume of 10% neutral-buffered formalin so that the ratio of solution to tissue is approximately 20:1 for 3-7 days.
3. Coordinate with an appropriate histology service for paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining. For this work, the West Virginia University Histopathology Core was utilized. Alternatively, follow previously described protocols¹⁹.

12. In vitro bacterial killing assay

1. Remove the spleen from the uninfected neonatal pup as described above and place it in a 40 µm nylon basket within a sterile 60 mm Petri dish. Repeat this and pool spleens into one tube to be harvested and homogenized together.
2. Add 5 mL of PBS supplemented with 10% FBS.
3. Disaggregate the tissue using a sterile 3 mL syringe plunger until a single cell suspension is created.
4. Collect the single-cell suspension outside of the nylon basket, transfer to a 15 mL centrifuge tube, and pellet cells at 350 x g for 5 min.
5. Suspend the cells in red blood cell lysis buffer (2 mL for up to 7-8 spleens) and let it stand for 5 min at room temperature to eliminate erythrocytes.
6. Wash splenocytes with PBS and pellet as above.
7. Suspend the splenocytes in 0.25 mL of PBS supplemented with 0.5% BSA and 2 mM EDTA according to expected cell yield.
8. Count the splenocytes using a hemocytometer or other appropriate application.
9. Isolate Ly6B.2⁺ (myeloid population of granulocytes/inflammatory monocytes) cells with immunomagnetic beads according to manufacturer protocol.
10. Seed Ly6B.2⁺ cells at a density of 1 x 10⁵ cells per well in a black or white 96-well plate in a volume of 0.1 mL of DMEM that contains 10% FBS, 2 mM glutamine, and 25 mM HEPES (complete medium).
11. Enumerate bioluminescent E. coli as described in section 1 and prepare the bacterial inoculum at the desired multiplicity of infection (MOI) in a final volume of 0.1 mL. This is best done by making what is necessary for all wells at a common MOI in batch.
12. Add 0.1 mL of bacterial inoculum or complete medium alone as a control. Incubate the multi-well plate at 37 °C and 5% CO₂ for 1 h.
13. Replace the media with 0.2 mL of fresh complete media that contains gentamicin (100 µg/mL) by gently removing media with a pipette and adding fresh media with a new pipette tip. Return the culture to incubation for an additional 2 h.
14. At 3 h post-infection, measure the luminescence in each well of the lidded culture plate from the bottom using a plate reader and then return the culture to incubation.
15. Repeat measurements of luminescence at other desired time points.