X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models

All the experiments that are described were conducted under protocols approved by the Institutional Animal Care and Use Committee at Michigan State University.

1. Extended anti-inflammatory treatment

1. Prepare sucralose gel cups as oral dosing of carprofen. Provide rats with this anti-inflammatory treatment from 2 days before receiving ID injection of 70% EtOH to 7 days after the procedure.
2. Dilute a working solution of carprofen in sterile PBS for injection into the cup. From 50 mg/mL stock solution, prepare a diluted 2 mg/mL solution colored with 1% v/v sterile blue food dye and inject 500 µL into each cup. Addition of the dye aids in visualization of complete mixing of the drug within the sucralose of the cup.
3. Follow the manufacturer's recommendation to prepare the cup for the addition of carprofen. Unless otherwise indicated by the vendor, warm the cup in a water bath at 60 °C for 15 min and dry off upon removal to reduce the risk of contamination.
   1. Wipe-clean the lid of the sucralose cup with 70% EtOH in the area and introduce the needle of the syringe containing the carprofen working solution. Dispense the appropriate volume (500 µL).
   2. Cover the puncture with a sticker. Shake the cup energetically for 15 s, and then place that cup in a vortex for an additional 15 s. Visually assess the homogenous and complete mixing before storing for later use. Look for the presence of a dark blue color.
      ​NOTE: Allow the cups to come to room temperature. Store the cups at room temperature if desired but pay attention to drug efficacy guidance from the manufacturer. Alternatively, store the cups at 4 °C and use within a month. Dating the sticker is a good practice for keeping track of the injection date without the risk of a pen or sharp marker puncturing the lid.
4. Just before use, wipe down the exterior of the cup with 70% EtOH. Remove the lid before placing the cup into the animal cage. Replace cups every other day or when empty. Check the level of gel daily to ensure adequate dosing. One cup can supply carprofen for up to two rats for up to 2 days; however, rats may consume entire cup sooner.

2. Preoperative preparation

NOTE: Ensure that the animal preparation step precedes the ID injection procedure by 2-3 days.

1. Turn on the isoflurane vaporizer (2%-3% isoflurane, 1.5 L/min of oxygen) to anesthetize the rat. Move the animal to a nose cone on a warming pad. Apply eye lubricant to the rat, and then position the animal to its back. Carefully monitor the animal's respiration to ensure that the anesthetic plane is maintained at 1%-3% of isoflurane.
   NOTE: An electric razor may be used to remove excess fur before depilation. Extreme care must be taken not to damage any nipples with the razor. For this reason, this step can be skipped. Rats are more sensitive to depilatory cream than mice, so the removal of excess cream is very important. Avoid injecting an ethanol-containing solution into an area that already has an abrasion present from depilation. Some creams have added compounds such as aloe and lanolin that can help minimize the likelihood of abrasions.
2. Use a cotton-tip applicator to spread the over-the-counter depilatory cream onto the nipple area. Use the applicator to rub the cream into the area for 10-30 s. Check whether the fur has quickly loosened.
   1. Leave the cream on the rat for the shortest possible interval and remove completely to avoid burning of the skin. Rats are even more sensitive to this procedure than mice.
3. After 10-30 s of application, wet gauze with warm water and use it to rinse the cream and the loosened fur from the animal. Perform at least three rinses of the area with fresh moistened gauze and dry with dry gauze after the final rinse. Confirm good visibility and access to the area of the nipple from where the fur is removed. Repeat the depilatory procedure if necessary.
4. Place the rat in a clean cage on a heating pad and allow it to recover. Check on the rat to make sure that it is fully recovered from anesthesia before bringing it back to its permanent cage.
5. Place one carprofen-dosed (1 mg/cup) sucralose gel cup in the cage for anti-inflammatory treatment. Check the gel consumption daily and replace with a fresh cup as appropriate. Do not leave the cup for more than 2 days. Typically, cups will need to be replaced after 1 day.

3. Intraductal injection

1. Prepare the TaO_x stock solution at 333.3 mM as described¹⁹ using sterile phosphate buffered saline (PBS). Warm the solution if the powder does not fully dissolve. Stir gently. Do not vortex or shake vigorously to avoid bubble formation.
2. Mix three parts of 333.3 mM TaO_x with seven parts of 100% EtOH for a final 70% EtOH 100 mM/TaO_x solution. Optionally, add an appropriate amount of 0.5%-1.5% ethyl cellulose (EC) as a gelling agent to maximize local retention of the ablative solution. Add 1% v/v blue food dye to the ablative solution for visual examination of the delivery into the ductal tree during infusion.
3. Prepare a volume appropriate for experimental needs. Gland pairs 1 (cervical) and 6 (inguinal) can be filled with up to 100 μL of the solution while all other pairs can be filled with up to 300 μL.
4. Anesthetize the rat as in step 2.1 and move the rat to the nose cone once fully anesthetized. Apply eye lubricant to both the eyes and then place the animal on its back. Secure the rat beneath the stereoscope using tape near the nipples that will be injected, if desired. The weight of the rat is generally enough to keep it from moving substantially without taping.
5. To prepare the nipples for injection, remove any dead skin that covers the nipple opening with fine pointed forceps, if possible. Rats often have a plug protruding from the nipple opening that can prevent successful cannulation of the nipple if not removed.
   NOTE: It is important to note that larger injection volumes of ablative solutions being used in rats may be more likely to result in superficial skin wounds near the injection site(s). For this reason, injecting every other nipple in a single session is less damaging and irritating to the animal than injecting adjacent nipples. Monitoring the rats for any abrasions for 7 days post-injection helps to ensure no serious health effects from the animal scratching and introducing the possibility of infection from contamination with cage floor debris. Triple antibiotic ointment or washes with chlorhexidine solution can be used to treat any signs of injury infection that may occur (Table 1).
6. Use a 500 µL syringe with a 33 G needle to aspirate 101-301 µL of ablative solution. Aspirate an extra 1 µL of the solution for possible minor leakage when removing the cannulated needle.
   NOTE: These are recommendations for volumes aimed at fully filling the ductal tree(s): up to 100 µL in cervical and inguinal glands, and up to 300 µL in the other glands. For other applications, it may be appropriate to use smaller or larger volumes.
7. Use a tweezer to gently hold the nipple and cannulate the needle into the nipple opening. Gently continue inserting the needle until the bevel is fully inside the nipple. To accommodate the needle in the nipple, bring the nipple up toward the needle instead of pushing the needle down into the nipple. (Table 1). Take care to follow the path of the nipple opening.
   NOTE: Rat nipples are generally much easier to manipulate and cannulate successfully than those in mice due to larger size. However, the increased amount of fat surrounding the nipple opening also makes it more likely to mistakenly inject the fat pad if the needle deviates from the main duct.
8. Once the needle bevel is completely inserted, slowly infuse the solution at a constant rate of approximately 100 µL/min in rats. Abrupt changes in infusion rate can burst or damage the ductal tree. Wait for 30 s after the end of infusion before removing the needle from the cannulated tree with assistance using forceps; this ensures that the injected volume remains within the ductal tree (Figure 2) and reduces the likelihood of leakage.
9. Clean off any spilled solution with moistened gauze or an EtOH wipe to avoid extraneous contrast solution in the images.
10. Inject PBS containing 5% sucrose (10 mL/kg) intraperitoneally to mitigate the effects of alcohol intoxication if ethanol is contained within the ID injection solutions. This dose may be given at the beginning and at the end of the procedure.

4. Micro-CT imaging

1. After all the desired glands have been injected, move the animal swiftly to the micro-CT system and continue maintaining anesthesia using the incorporated isoflurane vaporizer.
2. Straighten the spine of the animal and tape each hind leg in an extended position, so that the leg bones of the animal will be further away from the lower glands of interest and not overlap with the region of interest in the scanned image.
3. Tape across the abdomen to minimize breathing artifacts if scanning the lower glands.
   NOTE: Animals can be imaged with different scanning parameters (e.g., high resolution, longitudinal scans) if care is taken to determine an appropriate acceptable lifetime dose of radiation for rats and ensuring that the cumulative dose does not exceed this level. Radiation exposure may be further reduced by acquiring fluoroscopy stills and videos without performing scans (Figure 2).
4. Perform TaO_x imaging of the rat ductal tree with good resolution and opportunity for repeated standard (2 min) acquisition scans using the following scan parameters: 90 kVp/88 µA; field of view (FOV), 72 mm; number of slices, 512; slice thickness, 72 µm; voxel resolution, 72 µm³. High-resolution scans for longer time periods (4-14 min) can also be acquired in animals that will not be scanned longitudinally using the same parameters.
5. After data acquisition, carefully take the rat away from the anesthesia cone and place in a new clean cage on a heating pad. Check on the rat to make sure it has fully recovered from anesthesia before bringing it back to its permanent cage. Place the carprofen-containing sucralose cup and appropriately replace as described in step 2.5 to ensure that the animals continue to receive anti-inflammatory treatment for the next 7 days.
6. Process the scanned images into quick renditions within the micro-CT software to better appreciate any contrast leaks, only partial filling, or overfilling (Figure 2).
7. Proceed to the next section to perform formal image processing for publication or detailed analysis of scans if desired (Figure 3).

5. Image analysis

1. Use specialized software packages to produce renderings of the filled ductal tree.
   NOTE: It is best to segment out the mammary fat pad in order to get best rendition of the injected ductal tree. Spline trace the dark boundaries of the fat pad throughout the complete thickness of the animal in order to achieve this segmentation.
2. To segment the fat pad (unlike mice, the boundaries of this compartment are not as easily distinguishable from the peritoneal cavity, femoral muscles, and skin due to similar Hounsfield units) within which the ductal tree of interest is contained, selecting the "spline trace" option from the manual menu is the first step in creating a rendering.
3. Spline trace the fat pad outline in every third slice. Click on the Propagate Objects option from the semi-automatic menu. This will propagate and connect all the slices into a single segmented object of interest.
   NOTE: Changing the threshold within the segmented region allows visualization of the signal only within a certain range of Hounsfield units (HU); for other contrast agents or imaging parameters, this range may need to be adjusted. A software package or Artificial Intelligence analysis may be used to make other measurements and images to show how much the ductal tree was filled.
4. Set HU values to a low point of 300 and a high point of 3,000 on the semi-automatic menu under the threshold volume tab. This allows for creation of a rendition only displaying the contrast (TaO_x) within the ductal tree.
5. Set the rendition as primary using the "view" button. This will change the display to only show the 3D rendition of the ductal tree.
   NOTE: Perform reconstruction of the ductal tree for further analysis.