Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Summary

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Abstract

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

Introduction

Neurodevelopmental reflexes, or developmental milestones, are one of the earliest assessments used on human newborns and infants. Neurological reflexes are involuntary and repetitive movements that demonstrate brain stem and spinal cord reflexes. Maturation of higher cortical networks characterized by evolving migration, myelination, and synaptogenesis promote voluntary control and cortical inhibition. Alterations in the normal progression of the central nervous system evolution can disrupt brain development, resulting in abnormal cortical wiring, functioning, and myelination, causing neurodevelopmental reflex delays or absences. Human infants at high risk for neurodevelopmental disability often display abnormal early reflexes. Abnormal reflexes can present as a delay in acquisition, absence, prolonged presence, or reappearance later in life, and are predictive of developmental disabilities.^1,2 Therefore, it is important to mimic reflex delays in experimental models of neurodevelopmental disabilities.

Rodents are commonly employed as experimental models. Rat pups are altricial when born, and therefore too immature to undertake specific or complex motor, sensory and/or cognitive behavioral tasks. In this regard, their developmental immaturity relates to both their physical and organ development. Rats are born hairless with an inability to thermoregulate, are blind, and unable to walk. With reference to brain development, substantial cortical maturation occurs postnatally. Newborn rat pups (day of birth referred to as postnatal day 1; PD1) have been suggested to reach a brain maturation level that is similar to a preterm human brain of 23 - 28 weeks gestation, whereas PD7-10 pups are equivalent to near-term human brain. ^3,4,5,6 This correlation is based on gross anatomical analyses, however, other measures of brain maturation such as myelination and amplitude integrated electroencephalograms have also been described. ^5,7 For example, pre-oligodendrocytes are the predominant cells in the developing human fetal brain from 23 - 32 weeks in utero, and this maturational stage corresponds to a PD1-3 rodent. ^5,8,9,10 Moreover, myelination begins in utero in humans whereas in rat pups it appears in the forebrain around PD7-10; the newborn rodent brain remains largely un-myelinated. ^11,12 Tucker et al. found that the amplitude integrated electroencephalogram pattern of a P1 rat to be similar to a 23-week gestation human fetus, whereas a PD7 and PD10 pup is akin to a 30 - 32 week and term infant, respectively.⁷ For these reasons, newborn reflex testing in neonatal rat pups provides an opportunity for capturing the ontogeny and/or disruption of brain development.

The battery of reflexes described below are adapted from studies by W.M. Fox and A. Lubics^13,14 W.M. Fox was one of the earliest investigators with respect to the ontogeny of reflexes in the mouse. ¹³ These reflexes include, but are not limited to, limb grasping and placing, cliff avoidance, righting, accelerated righting, gait, auditory startle, posture, and eye opening. Both forelimb and hindlimb grasp (referred to as palmar and plantar grasp in humans, respectively) are facilitated by spinal reflexes and corticospinal inhibition from non-primary motor areas. ^15,16 Hindlimb placing (plantar reflex) reflects maturation of the corticospinal tract. ^16,17,18 Cliff avoidance (protective responses), righting (labyrinth), and accelerated righting involve integration and communication between sensory input and motor output (such as those involved with the vibrissae and vestibular systems). ^19,20,21 Gait reflects locomotion.¹⁴ Auditory startle assesses acoustic stimulation and synaptic connections of giant neurons in the nucleus reticularis pontis caudalis.²¹ Posture involves appropriate cortical-spinal/spinal-cortical projections, muscle strength, and neuromuscular innervation. ^22,23 Maturation of the gamma-aminobutyric acid receptors may correlate with eye opening.²⁴ It is important to keep in mind that the reflexes reflect a much more complicated network and provided here is a general correlation. Moreover, these reflexes provide a quick and easy method of assessing neurological development at very young ages where more complex behavioral testing is not feasible.

The objective of this paper is to provide a general guideline for neurodevelopmental reflex testing that can be easily incorporated into experimental neonatal rat studies. The methodology described was carried out in Long-Evans neonatal rat pups and quantification of the results was based on first day of appearance. The day that reflex testing is initiated and the equipment utilized may be modified to better suit a different experimental model (such as for different strains and species). By establishing the normal physiological progression of reflex maturation in a specific animal model, investigators can evaluate the effects of external stressors, endogenous manipulations, and/or therapeutic interventions on neurodevelopment in neonatal rat models. Overall, the use of reflexes as a determination of brain maturity is advantageous in predicting perinatal brain injury, and is reflective of later neurodevelopmental outcomes.

Protocol

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}

Results

The timeline of this experimental design is presented in Figure 2. ³⁰ The methods and results have previously been published. ³⁰ The objective of the study was to assess whether dietary supplementation with broccoli sprouts during gestation and the preweaning period protected the offspring from neurodevelopmental delay induced by in utero exposure to LPS. Timed pregnant rats were given intraper...

Discussion

Neurodevelopmental reflex testing is a predictive measure of abnormal cortical development and maturation, which may be of significance under circumstances where overt neuropathology is not evident. During neurodevelopmental testing, it is critical to ensure that pups are examined at the same time daily. Rats are nocturnal and therefore, their circadian rhythm may alter performance if testing is performed at different times during the day.³⁴ Testing should be completed in a quiet room as loud nois...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Breeding
Transfer pipettes	Fisherbrand	12-711-9AM	Used for vaginal flushes.
Sterile Saline	Hospira	7983254	The solution used to collect cells during vaginal flushes.
400 µL Microcentrifuge tubes	Fisherbrand	05-408-120	Used to hold the saline solution.
Light microscope	Leica	Leica ATC 2000	For observation of the saline solution. Can be any light microscope used in the lab.
Slides	Fisherbrand	12-552-5	The saline solution is placed on the slide. Can be any slides used in the lab.
Coverslips	Fisherbrand	12-545-F	To coverslip the slides. Can use any coverslips used in the lab.
Dietary  Supplementation
Broccoli Sprouts seeds	Mumm's Sprouting Seeds		Broccoli sprouts seeds are ordered and grown in the lab.
Countertop Seed Sprouter Box	Mumm's Sprouting Seeds		A box is used to germinate and grow the seeds prior to harvest.
250 mL beaker			The beaker is used to soak the seed. Any size beaker that would fit can be used.
Maternal Inflammation
Lipoplysaccharide (LPS)	Sigma	L3129	The endotoxin used to mimic maternal inflammation.
1 mL Syringe	BD Syringe	309659	Used to inject the pregnant rat.
Gauge (30 G x 1/2)	BD PrecisionGlide Needle	305106	Use the smallest needle to avoid pain and discomfort.
Sterile Saline (0.9% Sodium Chloride, USP)	Hospira		Saline is used to dissolve LPS.
Weights
Scale	Denver Instrument		For recording the weights. Can be any scale with 2 decimal places used in the lab.
Neurodevelopmental Reflexes
Thin blunt rod			Can be a paperclip or toothpick. This is for forelimb and hindlimb grasping.
Round filter paper	Whatman	1001 150	15 cm diameter paper used for gait analysis.
Timer	Fisher Scientific	06-662-51	For timing the time allocated to righting and gait.
Blunt surface			Can be an edge of a table. This is for hindlimb placing and cliff avoidance.
Foam landing			For when the pups perform accelerated righting.
Video recorder	Sony	VCT-D580RM	To record all reflexes tested. Must be able to record at 1/1,000 fps
Bell			For auditory startle.
Heat lamp or pad			To maintain the body temperature of the pups underoing examination.