Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

Summary

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

Abstract

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed “paw-dragging”. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia – two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Introduction

The goal of neural regeneration strategies is to demonstrate both tissue repair and functional recovery. Functional recovery is typically evaluated with behavioural tests that measure functional deficits, in this case involving motor skills that are associated with damage to the specific brain regions. Traumatic brain injury or ischemic damage to the sensorimotor forelimb area of the cortex can be demonstrated by a number of behavioural tests. One such test, the cylinder test is used extensively in rats to assess functional deficits in forelimb activity¹. The test has a low set-up cost requiring only a cylinder, camera and table with a transparent top. It i....

Protocol

Ethics statement: All experiments were approved by Memorial University of Newfoundland's Animal Care Ethics Committee according to the guidelines of the Canadian Council on Animal Care.

1. Mice

1. Use adult mice. In this study, adult male FVBN mice (n=10) between 2-4 months were used. House mice on a 12:12 hr reverse light-dark cycle and provide standard rodent chow and water ad libitum.

2. Materials Required for the Cylinder Test

1. Obtain a table with a transparent top to film the cylinder test. The dimensions of the table are irrelevant, the top must be plexiglass or glass, and there must be e....

Results

We have previously demonstrated that paw-dragging behaviour appears following a focal ischemic injury to the forelimb sensorimotor cortex and is a positive indicator of damage⁴. Intra-cortical injections of ET-1 into the forelimb sensorimotor cortex were used to induce an ischemic lesion (Figure 8A,B). This study examined whether paw-dragging behaviour extended for longer than 14 days post-injury for its potential use to assess functional recovery. Mice were tested in the cylinder test on the .......

Discussion

The key points to establish when quantifying paw-dragging behaviour in the cylinder test are the following: i) quantify the number of paw-drags versus total paw touches for each paw before brain injury to establish a baseline; ii) quantify the number of paw-drags versus total paw touches for each paw following the ischemic injury; and iii) discriminate between a paw-drag and the lateral motion of the paw along the cylinder wall during lateral rotation of the mouse’s torso.

Paw-dragging i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plexi-glass cylinder			17.5 cm high, 9.5 cm outer diameter, 8.8 cm inner diameter, wall thickness 0.35 cm (or 3.5 mm)
viewing table			54x56x66.5 cm (width x length x height), top of table is a 51x51 cm sheet of plexiglass.
mirror			34x58 cm mirror
video camera	Sony	DCR-SR42	Video camera with onboard storage, SD functionality, 40x optical zoom
computer	Dell	Optiplex 760	Processor: Intel, 3.0 GHz, Memory 4.00GB (RAM)
computer monitor	Samsung	S22C350H
Excel (Microsoft Office Professional Plus)	Microsoft	v14.0.7106.5003
VLC Media Player	Video LAN	v2.1.2	Media player with playback speed modulation and video support
External Hard Drive	Western Digital	WDBAAU0020HBK-01	2 TB