The cylinder test, also known as a paw reach test, is a simple and objective behavioral asymmetry test that is now widely used to predict localized ischemic damage to the forelimb motor cortex in rodents.

Ace Therapeutics offers the cylinder test to determine limb use asymmetry in rodent models of stroke. This test detects long-term stroke outcomes in ischemic stroke and cerebral hemorrhagic models and can be used to assess the neuroprotective effects of new therapies.

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Stroke Causes Sensory-Motor Asymmetry

One of the most striking symptoms of stroke is sensory-motor asymmetry. The cylinder test is commonly used to predict localized ischemic damage to the motor cortex of the forelimb in rodents. When rodents are placed in a cylinder, they explore by standing upright and touching the cylinder wall with their forelimb paws for postural support. Following an ischemic injury to the sensory-motor cortex of the forelimb, they rely more on the unaffected forelimb paw for postural support, resulting in a reduction in the number of affected paw touches, which is referred to as forelimb asymmetry.

What is the Cylinder Test?

The cylinder test was originally used to assess spontaneous locomotor activity in the forelimbs following CNS injury. The test has been modified to focus on the spontaneous motor behavior of the forelimb during gait and exploratory stance, and is the test recommended by experts for stroke recovery studies in rodents.

In the test, rodents are placed in a transparent cylinder (9 cm diameter x 15 cm height for mice and 20 cm diameter x 30 cm height for rats) and observed. Rodents actively explored vertical surfaces, standing on their hind limbs and exploring surfaces with their forelimbs and antennae. As they explore their environment, their forelimb movements are recorded by a video camera. A mirror is placed behind the column to record when the animal turns away from the camera. Testing in a dark environment encourages locomotion, especially since stroke reduces movement in rodents.

Fig.1. A rat's spontaneous forelimb use being assessed using the cylinder test. (Schaar, et al., 2010)

Readouts in the Cylinder Test

When assessing behavior in the cylinder, record the number of times the right forelimb, the left forelimb, and both forelimbs independently leaned against the wall at the same time. Animals with unilateral brain injury show asymmetry in forelimb use during vertical exploration.

A total of 20 movements were recorded for each test. The final score = (non-impaired forelimb movement - impaired forelimb movement)/(non-impaired forelimb movement + impaired forelimb movement + movement with both limbs).

Fig.2. Assessments of cylinder test after photothrombosis. (Li, et al., 2014)

Advantages and Disadvantages of the Cylinder Test

• The cylinder test is a simple and objective test of behavioral asymmetry that is sensitive to chronic dysfunction not detected by other tasks and has high inter-rater reliability.
• This test requires no special equipment and has minimal administration requirements.
• No pre-training is required, but it is desirable to obtain baseline data to test for preoperative bias, as sometimes some animals demonstrate the ability to use a limb independently.
• It is best to perform this assessment in the animal's dark cycle and under red light conditions, as rodents are more inclined to explore in dark environments.
• This test is capable of detecting minor neurological damage.
• The cylinder test is capable of detecting long-term stroke outcomes in ischemic stroke and intracerebral hemorrhage (ICH) models, but is not applicable to whole brain injury models such as subarachnoid haemorrhage (SAH).

Applications of the Cylinder Test in Rodent Models of Stroke

The cylinder test is a sensitive measure for evaluating limb asymmetry and the efficacy of neuroprotective treatments in stroke research.

Applications of the Cylinder Test in Rodent Models of Ischemic Stroke

The cylinder test effectively assesses limb use asymmetry in both ischemic and hemorrhagic stroke models. In ischemic stroke, studies show that mice use their ipsilateral forelimb more frequently than the contralateral limb following middle cerebral artery occlusion (MCAO), with asymmetry peaking at day 3 post-injury and remaining detectable for up to 15 days, but disappearing by day 40. Long-term limb asymmetry is also observed in hypoxic ischemia and focal ischemia models. Notably, bone marrow cell treatment improved recovery, eliminating asymmetry in ischemic rats by day 28. In a long-term study, significant asymmetry persisted even at day 77.

Applications of the Cylinder Test in Rodent Models of Hemorrhagic Stroke

For hemorrhagic stroke, the cylinder test reveals increased ipsilateral limb usage in rats after collagenase-induced ICH, with performance improving following rehabilitation treatment. Similar findings exist in autologous blood-induced ICH, where treatments can restore contralateral forelimb use. In contrast, SAH typically does not induce limb asymmetry in this test, likely due to its global impact on brain function.

1. Schaar, K. L., Brenneman, M. M., & Savitz, S. I. (2010). Functional assessments in the rodent stroke model. Experimental & translational stroke medicine, 2, 1-11.
2. Li, H., et al. (2014). Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice. BMC neuroscience, 15, 1-13.