The authors wish to thank our funding agencies, which include NeuroDevNet (a National Centres of Excellence), the ALVA Foundation, the Women's and Children's Health Research Institute, and the University of Alberta.

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 inflammatio...

<ol>
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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...

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. Lubics13,14 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Breeding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Fisherbrand</td>
			<td>12-711-9AM</td>
			<td>Used for vaginal flushes.</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Hospira</td>
			<td>7983254</td>
			<td>The solution used to collect cells during vaginal flushes.</td>
		</tr>
		<tr>
			<td>400 &#181;l Microcentrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>05-408-120</td>
			<td>Used to hold the saline solution.</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica</td>
			<td>Leica ATC 2000</td>
			<td>For observation of the saline solution. Can be any light microscope used in the lab.</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Fisherbrand</td>
			<td>12-552-5</td>
			<td>The saline solution is placed on the slide. Can be any slides used in the lab.</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-F</td>
			<td>To coverslip the slides. Can use any coverslips used in the lab.</td>
		</tr>
		<tr>
			<td>Dietary&#160; Supplementation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Broccoli Sprouts seeds</td>
			<td>Mumm&#39;s Sprouting Seeds</td>
			<td></td>
			<td>Broccoli sprouts seeds are ordered and grown in the lab.</td>
		</tr>
		<tr>
			<td>Countertop Seed Sprouter Box</td>
			<td>Mumm&#39;s Sprouting Seeds</td>
			<td></td>
			<td>A box is used to germinate and grow the seeds prior to harvest.</td>
		</tr>
		<tr>
			<td>250 mL beaker</td>
			<td></td>
			<td></td>
			<td>The beaker is used to soak the seed. Any size beaker that would fit can be used.</td>
		</tr>
		<tr>
			<td>Maternal Inflammation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoplysaccharide (LPS)</td>
			<td>Sigma</td>
			<td>L3129</td>
			<td>The endotoxin used to mimic maternal inflammation.</td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD Syringe</td>
			<td>309659</td>
			<td>Used to inject the pregnant rat.</td>
		</tr>
		<tr>
			<td>Gauge (30G X 1/2)</td>
			<td>BD PrecisionGlide Needle</td>
			<td>305106</td>
			<td>Use the smallest needle to avoid pain and discomfort.</td>
		</tr>
		<tr>
			<td>Sterile Saline (0.9% Sodium Chloride, USP)</td>
			<td>Hospira</td>
			<td></td>
			<td>Saline is used to dissolve LPS.</td>
		</tr>
		<tr>
			<td>Weights</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Denver Instrument</td>
			<td></td>
			<td>For recording the weights. Can be any scale with 2 decimal places used in the lab.</td>
		</tr>
		<tr>
			<td>Neurodevelopmental Reflexes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin blunt rod</td>
			<td></td>
			<td></td>
			<td>Can be a paperclip or toothpick. This is for forelimb and hindlimb grasping.</td>
		</tr>
		<tr>
			<td>Round filter paper</td>
			<td>Whatman</td>
			<td>1001 150</td>
			<td>15 cm diameter paper used for gait analysis.</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher Scientific</td>
			<td>06-662-51</td>
			<td>For timing the time allocated to righting and gait.</td>
		</tr>
		<tr>
			<td>Blunt surface</td>
			<td></td>
			<td></td>
			<td>Can be an edge of a table. This is for hindlimb placing and cliff avoidance.</td>
		</tr>
		<tr>
			<td>Foam landing</td>
			<td></td>
			<td></td>
			<td>For when the pups perform accelerated righting.</td>
		</tr>
		<tr>
			<td>Video recorder</td>
			<td>Sony</td>
			<td>VCT-D580RM</td>
			<td>To record all reflexes tested. Must be able to record at 1/1000 fps</td>
		</tr>
		<tr>
			<td>Bell</td>
			<td></td>
			<td></td>
			<td>For auditory startle.&#160;</td>
		</tr>
		<tr>
			<td>Heat lamp or pad</td>
			<td></td>
			<td></td>
			<td>To maintain the body temperature of the pups underoing examination.</td>
		</tr>
	</tbody>
</table>

neurodevelopmental reflex testing in neonatal rat pups

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.

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...

Watch this Scientific Journal Video about Neurodevelopmental Reflex Testing in Neonatal Rat Pups at JoVE.com

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

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.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

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.

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Research

JoVE Journal

Behavior

19.4K visualizações.  University of Alberta. 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 thi...