Neurodevelopmental Reflex Testing in Neonatal Rat Pups

The Animal Care and Use Committee, Health Sciences at the University of Alberta approved all animal studies.

Note: While this protocol may be adapted to other species and strains, this protocol is written for Long-Evans rats. These rats have been shown to have superior motor performances and visual acuity compared to other rodent strains. ^25,26 The protocol for timed pregnancies, dietary supplementation, maternal inflammation, and neurodevelopmental reflexes is as follows.

1. Experimental Animals

1. When rats arrive to the animal housing facility, refrain from any interaction for a day or two to allow them to acclimate to their new environment. Following acclimatization, handle the rats every day for approximately five days, or when the rats are no longer showing signs of stress. Human-induced stress can alter test results and lead to pregnancy loss.
2. Once the rats are comfortable, breeding begins. Place two females and one male rat in a double decker breeding cage (h= 38 cm, 1,800 cm² floor area) overnight (3 PM – 8 AM). These cages prevent stress and overcrowding. In the morning, using a transfer pipette, flush the vagina of the female rats with approximately 0.25 mL of saline. Transfer the solution into a microcentrifuge tube.
3. Place the two females and male back into their respective conventional cages.
4. View the solution collected in section 1.2 under a light microscope (100X) to determine which stage of the estrous cycle the rat is experiencing and whether or not sperm are present. If there are sperm, record the date as embryonic day (E1) (Figure 1). If pregnant, give the female her own cage on E14.
   NOTE: The cells in the vaginal smear are clear, therefore use a low light setting. Ensure that the condenser is adjusted to its lowest setting. Adjust the brightness control to dim the light. If a brightness dial is available, set it to 1.

2. Dietary Supplementation

NOTE: This protocol is intended to assess the therapeutic effects of dietary broccoli sprout consumption during the last week of pregnancy. Grow the sprouts according to Wu et al. ²⁷.

1. Soak the broccoli sprouts seeds for 2 – 3 h. Spread the seeds out on a countertop seed sprouter box and set on a warm surface for germination.
2. Water the seeds twice daily, once in the AM (8 – 9 AM) and once in the PM (3 – 4 PM).
3. On day five, place the sprouts by a window for the day (sprouts should have two leaves each and will become green over the day).
4. At the end of the day, harvest the sprouts by gently pulling the broccoli sprouts out of the sifted box and lay on a flat surface to dry.
5. Once the sprouts are completely dried, weigh out 200 mg and place in a sealed plastic bag.
6. Beginning on E14, feed each pregnant rat 200 mg of dried broccoli sprouts daily until PD21 (the day pups are weaned).

3. Inflammation

1. To induce inflammation and reproduce white matter injury in offspring, inject pregnant rats with lipopolysaccharide (LPS) every 12 h on E19 and E20. On the day of injection, bring the cage into the lab from the housing facility. Allow the pregnant rat to acclimate and become calm for at least 1 h. If a rat shows clinical signs of permanent systemic or kidney damage, it will be euthanized immediately and removed from the study. 
2. Weigh the pregnant dam.
3. Dissolve 200 µg/kg of LPS (serotype 0127:B8) in 100 µL of saline. Aspirate the solution into a 1 mL syringe equipped with a 30 G ½ needle.
   1. Use a new needle for each injection. Allow the solution to warm up for a few min to minimize discomfort.
4. Record the time and inject the pregnant dam intraperitoneally. Alternate injections between sides to ensure equal distribution.
   NOTE: Machholz et al. describes proper restraint and injection procedures. ²⁸

4. Developmental Reflex Tests

1. On PD1 (day of birth), remove pups from the dam and record birth weights. Place the pups back into their respective cages and move them to the animal facility until PD3. This ensures maternal bonding between dam and pup.
   1. Record the reflex testing for reliable assessment. Adjust the video camera so that it is at maximum shutter speed for frame-by-frame analyses (1/1,000 s). Position the video camera so that it is directly above or beside the rat pup and all materials used for each reflex. For example, for hindlimb placing, place the camera beside the rat pup to ensure the lifting and placing of the hindlimb is captured.
   2. For accelerated righting, place the camera above the rat pup and landing pad in order to record the ability of the pup to right in midair.
2. Record weights daily until the end of the experiment. On PD3, begin neurodevelopmental reflex testing (Figure 4). Move cages into a quiet room for a minimum of 1 h prior to testing to allow for acclimatization to the environment.
3. Perform neurodevelopmental reflex testing at a consistent time each day as rats are nocturnal. For example, test between 9 AM – 12 PM daily. Keep the pups directly under a heating lamp or on a heat pad at all times, to maintain a stable body temperature of 36.5 °C; rat pups of this age lose heat easily. Measure with a rectal temperature probe. If done efficiently, pups body temperatures do not change because they are not removed from the dam and litter long enough. NOTE: Monitor the temperature of heat lamp to ensure it does not exceed 37 °C.
   1. Record the score of each reflex each day until a positive response is observed. A positive reflex response occurs when the pup is able to perform a task on two consecutive days. The date of the first occurrence of a positive response is used for quantification. No further testing is required following the positive response. Hence the end date for each reflex is variable.
4. Forelimb Grasping
   1. Beginning on PD3, conduct the forelimb grasp reflex test. Place a blunt rod against the palm of each forepaw and apply light pressure manually. The light pressure should slightly displace the forepaw to ensure that contact is made and the pups can feel the rod. Grasping will appear as flexion of all digits around the rod. Successful acquisition of this reflex occurs when both forepaws grasp the rod for two days in a row.
   2. Score the forelimb grasp:
      0 for no grasping
      1 for successful grasping by one forepaw (left or right forepaw can be specified here)
      2 for successful grasping by both forepaws
5. Hindlimb Grasping
   1. Beginning on PD3, conduct the hindlimb grasp reflex test. Place a blunt rod against the sole of each hindpaw and apply light pressure manually. The light pressure should slightly displace the hindpaw to ensure that contact is made and the pups can feel the rod. Successful grasping of the rod appears as flexion of the digits around the rod. Successful acquisition of this reflex occurs when both hindpaws grasp the rod for two days in a row.
   2. Score the hindlimb grasp:
      0 for no grasping
      1 for successful grasping by one hindpaw (left or right hindpaw can be specified here)
      2 for successful grasping by both hindpaws
6. Righting
   1. Beginning on PD3, begin the righting reflex test. Firmly hold the pup in a supine position, with all four paws upright. Let go of the pup and immediately start the timer. Righting is achieved when the pup is able to flip/roll over onto all four paws, and each paw is perpendicular to the body. Give each pup a maximum of 15 s to achieve this goal.
   2. Score the righting:
      0 for lying on the back (or 15 s for the maximum allocated time)
      1 for lying on the side (left or right side can be specified here) or the ability of the pup to right but in the wrong posture (or 15 s for the maximum allocated time)
      2 for successful righting and appropriate posture
7. Hindlimb placing
   1. Beginning on PD4, begin the hindlimb placing reflex test. Hold the pup vertically in the air, by the torso. Gently stroke the dorsum of the hindpaw with a blunt surface (such as the edge of a table). A correct placing reflex is when the rat pup withdraws the stimulated hindlimb, followed by placement of the hindlimb down on that surface.
   2. Score the hindlimb placing reflex:
      0 for no successes
      1 for placing of one hindpaw (left or right hindpaw can be specified here)
      2 for successful hindlimb placing for both hindpaws
8. Cliff Avoidance
   1. Beginning on PD4, begin the cliff avoidance testing. Place the rat pup at the edge of a flat surface, such that the forepaws and snout of the pup are over the edge. The correct outcome is a protective response, where the rat pup turns away from the edge of the cliff. The experimenter’s hand or a foam landing is placed beneath the cliff to catch the pup from the fall.   
   2. Score the cliff avoidance:
      0 for no movement or falling off the edge
      1 for attempts to move away from the cliff but with hanging limbs
      2 for successful movement away from the cliff
9. Gait
   1. Begin the evaluation of gait on PD6. Place pups in the center of a 15 cm diameter circle and allow the pup 30 s to complete the task.¹⁴ A successful gait is performed when the rat pup is able to move both forepaws outside the circle in less than 30 s.
   2. Score the gait as the time in seconds it takes the rat pup to move outside the circle. Record the time it takes the pup to move both forepaws outside the circle. Record 30 s if the pup is unable to complete the task within the given time
10. Auditory Startle
    1. Begin the analysis of auditory startle on PD10. Present a loud noise (bell) directly over the pup to assess whether or not a startle response is present. A positive startle response is observed when the pup displays a 'jerking' movement, away from the sound.
    2. Score the auditory startle:
       (or no) for no startle response
       (or yes) for a positive startle response
11. Posture
    1. Begin posture analysis on PD12. Place pups on a non-slippery open surface, and observe the posture the pup has when moving. An immature posture is reflected by dragging of the abdomen when moving, and perpendicular pointing of both forepaws and hindpaws relative to the body. A mature posture is acquired when the pup can lift the abdomen from the surface and both forepaws and hindpaws are pointed straight, or parallel to the body, when moving.
    2. Score the posture:
       0 for no movement
       1 for immature posture when moving
       2 for mature posture while moving
12. Eye Opening
    1. Begin eye-opening analysis on PD12. Record the day both eyelids open:
       0 for no visible eyes
       1 for one visible eye (left or right side can be specified here)
       2 for two visible eyes
13. Accelerated Righting
    1. Beginning on PD14, begin accelerated righting testing. Place the pup in a supine position 30.48 centimeters above a foam landing. Drop the pup and observe its ability to right itself. Righting refers to when the pup is able to turn over to the prone position and land on its paws.
    2. Score the accelerated righting:
       0 for falling on its back
       1 for falling on its side (left or right side can be specified here)
       2 for landing on its paws

Note: Different types of quantification of the reflexes are available depending on the question of the investigators. Quantification of the reflexes can be done as initial observation of a reflex, first day of appearance up until the disappearance of the reflex, time it takes to successfully perform the task, speed of performance, and/or improvement of performance over time. ^{13, 14, 29,30,31} For the current study, the first day of appearance was used as a score. When using multiparous models, pups from the same litter typically behave similarly due to their genetic makeup, in utero and postpartum environment, and nutritional availability.³² Therefore, a single pup cannot be accounted for as an n = 1 due to litter bias. Pups examined within a litter can be averaged so that each litter represents an n = 1. ^{32, 33} Alternatively, several pups within the same litter can be analyzed using the mixed effects model, which takes into account pups within the same litter. ^{32, 33}