A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

Procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and as part of an approved protocol by the University of Washington Institutional Animal Care and Use Committee.

1. Preparation and LPS Administration

NOTE: Refer to Figure 1 for a timeline of the procedures.

1. Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgical drapes. Prepare pre-operative medications in sterile vials. Calculate the flow rate required to replace the air in the hypoxia/hyperoxia chamber with the experimental gas in 8–10 min.
2. Prepare the lipopolysaccharide (LPS from E. coli 055:B5) in sterile saline to produce a concentration of 1 mg/mL. Remove P17 ferret kits from their jills. Weigh and number them. Randomize animals by litter and sex to the control or injured (or treatment) groups.
3. Using a 300 µL insulin syringe, administer 3 mg/kg of LPS intraperitoneally to kits in the injury group, and an equivalent volume of sterile saline vehicle (3 µL/g) to control animals.
4. Place animals in a chamber within a water bath at 37–40 °C in order to maintain a target rectal temperature of 36–37 °C throughout the surgical procedures.

2. Anesthesia

1. During the procedure, continually monitor temperature, respiration rate, and heart rate of the animal.
2. Administer buprenorphine (0.05 mg/kg) subcutaneously 30 min before the surgical procedure. Induce anesthesia in a mixture of 3% isoflurane balanced with 100% oxygen. Remove the kit from the induction chamber and place it supine on a draped surgical water blanket set to 37 °C. Transfer anesthesia to the nose cone and reduce isoflurane level to 2–3%.

3. Surgical Preparation

1. Using small animal clippers remove all hair on the ventral neck region. Shave in a rectangular pattern with care to avoid nicking the skin or generating razor rash. Administer local anesthesia to the shaved area using intradermal lidocaine (4 mg/kg) and bupivacaine (2.5 mg/kg).
2. Prepare the neck by alternating application of povidone-iodine and 70% ethanol scrub with sterile cotton swabs. Repeat the scrub such that povidone-iodine and 70% ethanol are each applied 3x in alternating fashion.
3. Confirm depth of anesthesia via absence of toe-pinch reflex. Maintain level of isoflurane at the minimum percentage required for a surgical plane of anesthesia. Using sterile disposable cut-out drapes that expose the neck region, drape the animal.

4. Bilateral Carotid Artery Ligation

1. With a single-use #11 scalpel blade, make a 1.5 cm midline incision in the center of the neck. Using fine hemostats and curved forceps, bluntly dissect down to the left carotid artery. Dissect the artery away from the associated neurovascular bundle.
2. Using a pair of curved fine forceps, pass a looped 10 cm length of sterile 5-0 silk suture under the artery. Cut the suture in half. Ligate the artery by securely tying both lengths of suture, leaving at least 2 mm between the knots. Transect the left carotid artery between the sutures, taking care to leave the nerve intact.
3. Repeat the dissection on the right side. Reversibly ligate the right carotid artery with a single sterile 1/8 inch umbilical tie. Close the wound with surgical skin clips.
4. Allow the animal to recover in a temperature-controlled water bath for at least 30 min before hypoxia.
   NOTE: If the arteries are not fully isolated from the rest of the neurovascular bundle, increased mortality may be seen before or during later hypoxia.

5. Sequential Hypoxia, Hyperoxia, and Hypoxia

1. During hypoxia and hyperoxia, alter water bath temperature as necessary to maintain rectal temperature during hypoxia at 37 °C in the sentinel animal(s).
2. Place animals in the injury group in an airtight chamber within a water bath. Continuously monitor oxygen concentration within the chamber, as well as rectal temperature in at least one sentinel animal. Flush the chamber with humidified 9% oxygen (91% nitrogen) then maintain a flow rate of 3–5 L/min, depending on chamber size. Once the concentration of oxygen in the chamber has reached 9%, continue for 30 min.
3. After 30 min, switch gas supply to 80% humidified oxygen (20% nitrogen), and allow the chamber to reach the target concentration based on flow rate and chamber size. Continue for 30 min of hyperoxia. Open the chamber to allow it to more rapidly reach normoxia by equilibrating with room air.
4. Seal the chamber, and flush with 9% humidified oxygen. Continuously monitor all animals visually, taking note of animals that display bradypnea. Once the concentration of oxygen in the chamber has reached 9%, continue for 30 min. If intra-hypoxic mortality (respiratory arrest) in any of the animals is seen before the end of the 30 min period, terminate hypoxia immediately.

6. Reversal of Right Carotid Artery Ligation

1. Return animals to the surgical area, and induce anesthesia in a mixture of 3% isoflurane balanced with 100% oxygen. Transfer anesthesia to nose cone and reduce isoflurane level to 2–3%. Remove the surgical wound clips and re-prepare the wound area with povidone-iodine. Confirm depth of anesthesia via absence of toe-pinch reflex. Maintain the level of isoflurane at the minimum percentage required for a surgical plane of anesthesia. Using sterile disposable cut-out drapes that expose the neck region, drape the animal.
2. Using curved forceps, identify and untie the umbilical tape from the right carotid artery. Close the wound with surgical skin clips.

7. Recovery and Temperature Management

1. Return all kits to their jills for 60 min, for nursing and recovery. After 60 min, return injured animals to the water baths at 37–40 °C for 6 h, adjusting water temperature as needed in order to maintain rectal temperature at 36–37 °C. Return kits to their jills.
2. Remove surgical clips 10–14 days after surgery (P27–P31).

8. Reflex Testing

1. Perform all the reflex tests daily from P21–P28, and at least 3x per week from P28–P42, whilst remaining blinded to exposure (or treatment) group. Prior to reflex testing, place kits in a chamber with heat assistance (37 °C water bath, heat pad, etc.) for 1 h. For each test, complete all trials per kit before testing the next kit.
2. Negative geotaxis (25°)
   1. Place a flat board (16 ½ in. x 12 in.) wrapped in an absorbent benchtop protector against an object so that the board forms a 25° angle with the table. Place a kit on the board prone and facing downhill, approximately 75% of the way up the board.
   2. Ensure the kit’s body is straight and that it has all four paws gripped against the board before releasing it. As soon as the kit is placed, start the time assessment.
   3. Record the time when the kit manages to rotate its body 90° relative to its starting position. Record the time at which the kit rotates its body 180° and takes a full step towards the top of the board. Perform 3 trials at the 25° incline before moving onto the next test.
3. Negative geotaxis (45°)
   1. Perform 3 trials of the previously described negative geotaxis test again, this time with the board set to a 45° angle.
4. Cliff aversion
   1. Place a padded platform around 1 foot below the ledge to minimize injury to kits if they fall.
   2. Place a kit facing, and perpendicular to, the edge of the lab bench. Ensure the kit's body is straight, with its front paws flush with the edge. Begin time assessment from the moment the kit is placed. Take care to differentiate between conscious movement away from the cliff and other spontaneous movements that do not involve coordinated walking.
   3. Record the time when the kit moves its body away from the edge (defined as the kit backing up, turning its body, or moving its front limbs away from the edge). Record the time the kit completes its first step in the opposite direction of the edge (defined as any direction or angle past a 90° rotation from its starting position facing the edge).
   4. Perform 3 cliff aversion trials per kit before moving onto the next test.
5. Righting reflex
   1. Place a kit supine on the bench, holding it gently in that position before releasing it and simultaneously beginning the stopwatch. Record the time that the kit brings itself to rest with all four paws simultaneously flat against the bench in weight-bearing positions. Record the time when the kit takes a full step in any direction (defined as the placement of all four paws to achieve progress in a given direction with no spinning or dragging of the body).
   2. Perform 5 trials of the righting reflex test per animal.
   3. After every kit has completed the 5 righting reflex trials, return the litter to the jill.

9. Catwalk Testing

1. On P42, remove kits from the jill. Place kits in plastic cages approximately 10 min before testing, so they can acclimate to the environment. Turn the lights off in the testing room to ensure ambient light does not affect catwalk function.
2. Create a new experiment in the relevant software. Adjust experimental settings so that the maximum run duration does not exceed 10.00 s and the minimum run duration is not less than 1.50 s. Set the maximum speed variation so it does not exceed 60%. Set a minimum requirement of three compliant runs for each animal.
3. Adjust the width of the walkway relative to the size of the animal so that it is able to freely locomote without touching the walls while remaining narrow enough to discourage turning. Add a new detection setting in the Detection settings profiles tab using auto detection. Use the same detection settings for all litters and animals at a given age.
4. Thoroughly clean the walkway with a low-lint paper tissue and 70% ethanol before and after each animal. Clean the ferret's paws regularly to improve accuracy of detection and classification. Once the walkway and animal are prepared, begin trial acquisition.
5. Pause acquisition to clean the catwalk if footprints accumulate on the glass, or if animals pass urine or feces. Stop acquisition once the catwalk software has recognized three compliant runs based on the pre-determined experiment settings.

10. Open Field Testing (P42)

1. Use a non-porous acrylic box (55 cm x 55 cm x 40 cm high) painted matte-white. Position the camera so that it is centered directly above box and all four walls are captured. Clean the testing arena with 70% ethanol before first use and between animals.
2. In the relevant software, select New From Template and Apply a Pre-defined Template. Continue the set-up procedure by sequentially selecting Subject type: Other; Arena template: Open field, square; Zone template: Center, border, corners; Features to track: Center-point.
3. Open Arena Settings and grab the background image from the camera input, making sure that the tops of the arena walls are visible. Calibrate the dimensions of the arena using the scale tool.
4. Adjust the pre-defined arena zones by resizing the outlines to fit the wall zones (NW, NE, SW, SE) and floor zones (left top, middle top, right top, left middle, center, right middle, left bottom, middle bottom, right bottom). Validate the set-up to confirm that no zones overlap.
5. Open the Acquisition window and press Start Acquisition. Place the ferret at the center of the testing arena oriented in such a way that is consistent for every test subject. Allow the ferret to move freely throughout the arena for a period of 5 min. At the end of the testing period, press Stop Acquisition. Repeat the procedure with the next ferret.
   NOTE: All experimenters in the room should position themselves to be unobservable by the ferret and remain quiet during the testing period.

11. Fixation-perfusion

1. On P42, deeply anesthetize kits with 5% isoflurane. Administer a pentobarbital overdose (120–150 mg/kg i.p.). Ensure deep anesthesia by lack of response to toe pinch and loss of respiratory movements.
2. Transfer the animal to a fume hood. Open the thorax and clamp the descending aorta using fine hemostats. Cut the right atrium. Using a perfusion pump, perfuse the left ventricle with 60 mL of sterile saline at a rate of 30 mL/min. Perfuse with 60 mL of formalin (10% formaldehyde) at a rate of 30 mL/min.
3. Decapitate the carcass, and remove the brain from the skull using scissors, forceps, rongeurs, and a spatula. Take high-resolution photographs of the dorsal, ventral, and lateral aspects of each brain. Post-fix the brain in formalin for at least 48 h.

12. Ex Vivo Brain Measurement

1. Remove the brain from formalin (step 11.3) and place onto a paper towel to absorb excess liquid.
2. Using an electronic caliper, measure the height of the brain by placing the tips of the caliper at the dorsal and ventral aspects of the brain. Measure the length of the brain by placing the tips of the caliper at the olfactory bulb and the most posterior border of the occipital lobe. Measure the width of the brain by placing the tips of the caliper at the most lateral portions of the temporal lobes. Weigh the brain.
3. Measure the longitudinal fissure (anterior and posterior to the cruciate sulcus), lateral sulci, suprasylvian sulci, coronal sulci, pseudosylvian sulci, ansinate sulci, cruciate sulci, presylvian sulci, lateral gyri, suprasylvian gyri, sigmoid gyri (anterior and posterior), coronal gyri, ectosylvian gyri (anterior and posterior), and orbital gyri. Measure all sulci from the beginning and end of the most distinct portion of the corresponding sulcus. Measure all gyri from the widest aspect of each corresponding gyrus.
4. Measure the amount of cerebellum exposed by placing one tip of the caliper at the most posterior point of the longitudinal fissure and placing the other tip of the caliper at the most posterior part of the cerebellum.
   NOTE: The ferret brain atlas, as found in Biology and Diseases of the Ferret¹⁴, was used to develop the ex vivo ferret brain measurements.

13. Gross Injury Scoring

1. Using the photographs taken in step 11.3., apply the scoring criteria in steps 13.2–13.4 to assess gross brain injury (0–9 scale) whilst remaining blinded to exposure (or treatment) groups.
2. Assess the longitudinal fissure. If it appears normal, assign a score of 0. If it is mildly widened (approximately 2x normal width), but the increase in width is incomplete along length of fissure, assign a score of 1. If it is moderately widened (approximately 2–3x normal), assign a score of 2. If it is markedly widened, with a visible gap >3x normal width along most of the length of fissure, apply a score of 3.
3. Assess the lateral sulci. If they show normal definition, with separation of the lateral gyri and suprasylvian gyri, assign a score of 0. If mild unilateral or bilateral reduced definition of sulcus is seen, particularly in the caudal portion, with minimal narrowing of frontal and temporal lobes relative to the occipital lobes, assign a score of 1.
   1. If moderately reduced definition of the sulci is seen, with depression of the suprasylvian gyri, narrowing of the coronal and ectosylvian gyri, and mild narrowing of frontal and temporal lobes relative to occipital lobes, assign a score of 2. If unilateral cystic degeneration is seen, with minimal change of the contralateral hemisphere, assign a score of 3. If poor definition of the lateral sulci is present, with bilateral cystic or severe degeneration of the occipital and temporal lobes, assign a score of 4.
4. Assess the visible portion of the cerebellum. If it appears normal, with (<75% of the vermis and <66% of the hemispheres visible, assign a score of 0. If 75–90% of the vermis and ≥66% of the hemispheres are visible, assign a score of 1. If most of the cerebellum is visible, showing all of the vermis and ≥66% of hemispheres, assign a score of 2.

14. Data Analysis

1. For reflex testing data, assign failures a score of 61 s to allow them to be compared to successes at the end of time (60 s), but with a worse ranking in the statistical analysis. Calculate an area under the curve for each animal over time in each of the reflex tests.
2. Adjust catwalk data that involves paw size of pressure by the weight of the animal.
3. Analyze data using non-parametric statistical methods, describing data using the median and interquartile range (IQR).