Neurodevelopmental Reflex Testing in Neonatal Rat Pups

The overall goal of this neonatal rat reflex methodology is to learn how to perform these tasks quickly, accurately, and comparably. This method can answer key questions in the field of nerve developmental disabilities such as cerebral palsy. The main advantage of this technique is that it offers a way to test early developmental reflexes in newborn rat pups.

Like humans, newborn rodents have early developmental reflexes that offer a window into later development. Individuals new to this method will struggle as the reflexes occur quickly and can be difficult to score. It is important to be as consistent as possible.

The implications of this technique extend toward the therapy and diagnosis of cerebral palsy and developmental disabilities. Determination of reflex behaviors is the gold standard for the ascertainment and the efficacy of therapeutic interventions. Visual demonstration of this method is critical as the steps are difficult to learn through written words.

To begin, acclimate the rats to the environment and to human handling as described in the accompanying text protocol. Once acclimated, mate the rats to produce a litter of pups for testing. Beginning on post-natal day three, transfer the pups to a quiet room at a consistent time each day.

Let them acclimate for a minimum of one hour prior to performing an array of neurodevelopmental reflex tests. In the room, set up a heating lamp or heat pad to maintain the pup's body temperature at 36.5 degrees Celsius. Also, record the pup's weight each day.

Next, set up a video camera with a shutter speed of at least 1/1000th of a second to analyze the reflex tests frame by frame. Position the camera so that it will be directly above or beside the rat pup and the testing materials during each test. For each of the following tests, perform the test each day until the pup is able to perform the task on two consecutive days.

The date of the first occurrence is the date which should be recorded for data quantification. Beginning on postnatal day three, perform the forelimb grasp reflex test. For this test, place a blunt rod against the palm of each of the pup's forepaws, one at a time, and manually apply light pressure.

The 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.

Next, place a blunt rod against the sole of each hindpaw, and apply light pressure manually to conduct the hindlimb grasp reflex test. 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.

Acquisition of this reflex occurs when both hindpaws grasp the rod for two days in a row. Also beginning on postnatal day three, conduct the righting reflex test. For this test, firmly hold the pup in a supine position, so that all four paws are upright.

Let go of the pup and immediately start the timer. Righting is achieved when the pup is able to flip or roll over on to all four paws and each paw is perpendicular to the body. Give each pup a maximum of 15 seconds to achieve this goal.

Beginning on postnatal day four, perform the hindlimb placing reflex test. For this 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. Also beginning on postnatal day four, conduct the cliff avoidance test. For this test, 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. Begin the evaluation of gait on postnatal day six. For this test, place pups in the center of a 15 centimeter diameter circle and allow the pup 30 seconds to move off the circle.

A successful gait is performed when the rat pup is able to move both forepaws outside the circle in less than 30 seconds. Begin the analysis of auditory startle on postnatal day 10. Here, something, such as a bell, that is capable of producing a loud noise, is required.

Position the bell directly over the pup. Ring the bell, and then 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.

Starting on postnatal day 12, place the 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.

Also starting on postnatal day 12, begin observing the pup's eyelids. Record the day both eyelids open. On postnatal day 14, begin the accelerated righting testing.

For this test, place the pup in a supine position 30.48 centimeters above a foam landing pad. Drop the pup and observe its ability to right itself. Proper righting refers to when the pup is able to turn over to the prone position and land on its paws.

Newborn pups exposed to in utero inflammation were born significantly smaller than controls. Dietary supplementation with broccoli sprouts was able to increase the birth weights of these pups. Along with birth weight, 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.

Following birth, examination of the neonatal reflex hindlimb placing in these groups showed a significant delay in the acquisition of this reflex for pups exposed to in utero inflammation compared to controls. Dietary supplementation with broccoli sprouts prevented the delay. The results of the neonatal reflex cliff avoidance test revealed that pups exposed to in utero inflammation are significantly delayed in acquiring a protective response.

Again, broccoli sprouts consumption prevented significant delay. This same result was seen following examination of the neonatal reflex gait. During this procedure, other methods like daily weight recordings can be performed in order to answer additional questions like daily growth rate, catch-up growth, the severity of injury, and the effect of growth restriction.

After its development, this technique paved the way for researchers in the field of neuroscience to explore developmental reflex testing in neonatal rodents. After watching this video, you should have a good understanding of how to perform early developmental reflex testing in newborn rat pups. Moreover, you should understand the significance of early behavioral reflexes as they provide an early biomarker of injury and the effect of therapeutic intervention.