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 neurode....

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 housi....

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.