RAT MODIFIED IRWIN TEST (FUNCTIONAL OBSERVATIONAL BATTERY)

Animals

Studies are conducted in adult male and female Sprague Dawley rats (Envigo) in an AAALAC-accredited facility with approval from an Institutional Animal Care and Use Committee, and implemented in accordance with the Guide for the Care and Use of Laboratory Animals 8th Edition (National Research Council, 2011) with standards set by the National Institutes of Health.

Experimental procedure

The modified Irwin functional observation battery is a multi-parameter (autonomic, neuromuscular, sensorimotor, and behavioral) assessment of nervous system function in rodents. This systematic observational method describes and quantifies the behavioral and physiological state of rodents in response to drug exposures and can be used for multiple investigational goals including the selection of appropriate doses and optimal therapeutic window for subsequent assays. The procedure initially described by Irwin (1968) in mice was later adapted to rats (Moser, 1991; Mathiasen and Moser, 2018).

The modified Irwin test is conducted in appropriate group sizes determined by power analysis. Male and female rats (n=4) are evaluated in separate cohorts. Dose-responses are investigated, and in addition to a vehicle control, an unblinded, untreated, naïve control is used to establish baseline activity and to account for environmental variables that may affect the behaviors of the healthy animal. For all other animals, experimenters are blinded to the treatments, and dosing is performed by an independent experimenter.

Before testing, animals are handled over two days to familiarize them with the experimenter. Animals are visually evaluated by two independent observers simultaneously. Baseline behavioral observations are first recorded. Rats are then dosed and observations are recorded again at designated timepoints post-treatment depending on the asset.

Scoring Procedure

The estimated observation time required is less than 5 minutes per animal.

At each timepoint, the recorded observations include

body weight
respiration
urine staining
body temperature
head twitches
vocalizations
body position
jumping
auditory reflex
locomotor activity
tremor
righting reflex
sedation / excitation
twitch
reactivity to touch
ataxia
seizures
abdominal tone
piloerection
lacrimation
limb tone
ptosis
chromodacryorrhea
aggressiveness toward handler
exophthalmos
salivation
arching
unusual behavior
diarrhea
pupil size
writhing
increased urination and defecation
visual placement
grooming
bloody urine
modified grip strength
tail suspension
pinnae
corneal reflexes

Experimenters note increases, decreases, and severity of the behavioral symptoms or physiological manifestations. The standard rating system consists of a score of 0= normal activity of each specific outcome. Scores of 1, 2, 3, or 4 grade the level of behaviors observed as slight, moderate, extreme, or severe.

References

Irwin S (1968). Comprehensive observation assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia, 13 (3): 222-257. PMID: 5679627 DOI: 10.1007/BF00401402

Mathiasen JR, Moser VC (2018). The Irwin test and functional observational battery (FOB) for assessing the effects of compounds on behavior, physiology, and safety pharmacology in rodents. Curr Protoc Pharmacol, 83 (1): e43. PMID: 30179315 DOI: 10.1002/cpph.43

Moser VC (1991). Application of a neurobehavioral screening battery. J Am Coll Toxicol, 10 (6): 661-669. DOI: 10.3109/10915819109078658

National Research Council (2011). Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC: The National Academies Press

This work was conducted by PsychoGenics Inc. (Paramus, NJ) in collaboration with PSPP, NINDS, NIH under contract # 75N95019D00026