Behavioral Tests in Rodent Models of Stroke
At a glance
Introduction of Behavioral Tests in Rodent Models of Stroke
In a stroke, brain ischemia or bleeding damages central nervous system functions and results in a variety of neurological, motor, sensory, and cognitive dysfunctions. Given how damaging stroke can be, treatment is desperately needed. To facilitate stroke research and drug development, animal models of stroke have been established in several species. Among these, rodents are excellent models for exploring the understanding and effects of human stroke, as well as many other neurologic injuries, due to their high reproductive rate, low maintenance costs, availability of transgenic models, and similarity of vascular anatomy to humans.
In addition to examining histopathologic indices in rodents, it is important to assess functional outcomes after stroke. Due to the loss of limb function after stroke, many tests focus on motor and sensory testing. Since learning and memory deficits are also common after stroke, cognitive testing is also a key component in understanding the full scope of deficits. Therefore, it is important to have sensitive behavioral methods to detect the range of impairments that occur after a stroke. Behavioral assessment also provides the opportunity to monitor drug and cellular therapy by observing improvement in function over time.
Many methods are currently available for behavioral testing to assess post-stroke functional outcomes in rodent models. While there is no perfect test, there are a number of assessments that can be sensitive to detecting a range of impairments from overall to pattern-specific after stroke. Behavioral testing appears to be the most effective method for assessing whether an animal model mimics the clinical features of a specific disease.
Fig.1. Illustration of commonly used behavioral tests in rodent models of stroke. (Ruan, et al., 2020)
At Ace Therapeutics, we continue to innovate and provide behavioral testing services to assess behavioral changes in animal stroke models, such as motor dysfunction, sensory deficits, cognitive deficits, and mood disorders.
Related Services
Composite Scores in Rodent Models of Stroke
Bederson Scale and Neurological Scoring Scales
The Bederson scale is a global neurological assessment that measures neurological impairment after stroke. Tests include forelimb flexion, resistance to lateral push and circling behavior. A 0-3 scoring scale is used to assess behavioral deficits after stroke. This scoring scale is a simple way to reveal basic neurological deficits. Since the Bederson scale, many iterations of the scale have been developed and modified, all of which provide a simple way to detect impairment. Although easy to administer, the scale is limited in its neurological ratings due to its subjective nature. Additionally, in many common stroke models, deficits on the Bederson scale disappear quickly, making it less useful in detecting long-term deficits after stroke.
Modified Neurological Severity Scores (mNSS)
The modified neurological severity scores (mNSS) is a widely used tool for assessing neurological function in animal stroke studies, with scoring scales of 14 for mice and 18 for rats. It evaluates a range of functions, including motor skills, sensory perception, reflexes, and balance. Points are awarded for failures to perform specific tasks and deducted for absent reflexes, resulting in a composite score that indicates overall impairment. While the mNSS can effectively assess multiple deficits over 30-60 days, its simplicity may mask specific deficiencies, as some deficits may pertain to particular modalities. Additionally, the reflexes tested, such as the pinna and startle reflexes, may not be directly related to damage in the middle cerebral artery (MCA) territory.
Garcia Scale
The Garcia scale is a neurological assessment tool designed to evaluate motor and sensory functions in rats following middle cerebral artery occlusion (MCAO). It scores animals on various parameters, including spontaneous activity, limb movement symmetry, forelimb outstretching, climbing and grip strength, body proprioception, and vibrissae sensory function, with a total score indicating the severity of the stroke—lower scores reflect worse outcomes.
While the Garcia scale is easy to administer and covers both sensory and motor functions, it emphasizes forelimb performance, which may skew results in cases of severe hindlimb deficits. Therefore, it is recommended to use the Garcia scale alongside other assessments for a more comprehensive evaluation of stroke outcomes. Its effectiveness is particularly noted in ischemic stroke due to the controllable nature of lesion sites.
Motor and Sensorimotor Tests in Rodent Models of Stroke
Cylinder Test
The cylinder test is a behavioral assessment tool used to evaluate spontaneous forelimb use in rats, providing insights into the neural basis of spatial and motor behavior. In this test, a rat is placed in a transparent Plexiglas cylinder, where it explores vertical surfaces by rearing on its hindlimbs and using its forelimbs and vibrissae. Observers record the number of independent wall placements for each forelimb, allowing for the detection of asymmetry in limb use, particularly in animals with unilateral brain damage.
This test is recognized for its objectivity, ease of administration, and high inter-rater reliability. It is sensitive enough to identify chronic deficits that other assessments may overlook and can even detect mild neurological impairments. While no pre-training is necessary, obtaining baseline data is advisable to account for any pre-existing limb preferences. For optimal results, the test should be conducted during the animal's dark cycle and under red lighting, as rodents are more likely to explore in dim conditions.
Open Field Test
The open field test is a widely used assessment tool in rodent studies, designed to evaluate locomotor ability and exploratory behavior. Typically conducted in a wooden or plastic maze (50 cm x 50 cm x 38 cm), the test involves placing a mouse in the open field for 10 minutes while recording its movements via video. Key metrics include total travel distance, travel route, immobility time, and rear-up behavior, with automated tracking systems enhancing objectivity by minimizing human error.
In addition to assessing locomotion, the open field test can evaluate anxiety levels. Rodents often prefer the corners of the maze, so reduced time spent in the center indicates higher anxiety. However, using defecation and urination as emotional indicators is complicated by individual differences in food and water intake.
Pole Test
The pole test is a straightforward assessment designed to evaluate overall locomotor function in rodents, initially developed for mice to study bradykinesia and later adapted for stroke research. In this test, a mouse is placed on the tip of a vertical pole and must turn its head downward to descend. Key metrics recorded include the latency to turn (Tturn) and the total time to descend (TD). If the mouse falls or does not complete the task, specific protocols are followed to record the time appropriately. Overall, the pole test is easy to administer, requires minimal equipment, and provides an objective and sensitive measure of motor function, particularly useful for assessing long-term outcomes in ischemic stroke models.
Foot-Fault Test
The foot-fault test, also known as the grid walking test, is utilized to assess motor function and limb coordination in rodents. In this test, animals navigate an elevated grid with square openings, and a foot fault is recorded each time a paw slips or falls through the grid. The total number of steps taken and the number of foot faults per limb are quantified. Typically, sham controls exhibit few foot faults without side bias, while stroke-affected rodents show increased foot faults on the contralateral side.
Corner Test
The corner test is a behavioral assessment designed to evaluate sensorimotor dysfunction in rodents, initially developed for rats and later adapted for mice. In this test, animals are placed in a corner formed by two cardboards set at a 30° angle, where their vibrissae are stimulated. The direction in which the animal turns back is recorded; normally, there is no directional preference, but after a stroke, animals with contralateral limb deficits tend to turn towards their ipsilateral side. A higher frequency of ipsilateral turns indicates more severe stroke outcomes.
The corner test is advantageous due to its objectivity, quantitative nature, and ability to evaluate chronic sensorimotor deficits. However, it is not suitable for global stroke models, such as subarachnoid hemorrhage (SAH), where minimal sensorimotor asymmetry occurs. Additionally, the performance may be affected if animals are too sick or unmotivated, so long intervals between tests are recommended to mitigate this issue.
Adhesive Removal Test
The adhesive removal test is a widely utilized method for evaluating sensorimotor dysfunction and motor asymmetry in rodents. In this test, animals are placed in a transparent box with adhesive tapes attached to the hairless part of each forepaw. The time taken to contact and remove the adhesive is recorded. Typically, animals take longer to remove the tape from the contralateral forepaw, indicating sensorimotor deficits, while they perform normally with the ipsilateral forepaw.
The adhesive removal test is advantageous due to its objectivity and sensitivity for long-term assessments of sensorimotor function. However, it requires careful control of variables and multiple training sessions to ensure reliable data, as individual variability can affect results. Animals are typically trained for several days prior to stroke induction to minimize these variations.
Rotarod Test
The rotarod test is a widely used assessment for evaluating equilibrium and locomotor ability in rodents, particularly in disease models such as stroke. The test involves a rotating rod (3 cm diameter, 40 cm length) covered with a rough surface to prevent slipping. Animals are trained to remain on the rod, starting with no rotation and progressing to increasing speeds up to 20 rpm. The time taken before they fall off the rod is recorded, with faster falls indicating impaired locomotor function.
Wire Hanging Test
The wire hanging test is a functional assessment designed to evaluate various aspects of locomotor ability in rodents, including grip strength, endurance, and body coordination. In this test, animals are suspended on a wire 50 to 60 cm above the ground for up to four minutes, and the latency before falling is recorded as an indicator of muscle strength. Different hanging behaviors can be scored, ranging from simply falling off to successfully escaping by using all limbs and their tail. To enhance objectivity, modifications such as covering hind limbs with adhesive tape can be employed.
The wire hanging test is a straightforward, low-equipment method for assessing both short-term and long-term locomotor outcomes in stroke models. However, proper training is necessary to minimize individual variability and ensure consistent results.
Skilled Reaching Tasks
Skilled reaching tasks, particularly the single pellet reaching task, are designed to assess limb motor function in rodents, especially following neurological injuries like stroke. These tasks involve training animals to reach for food pellets through limited openings, which helps evaluate their functional recovery. In the single pellet task, a rodent is placed in a specially designed reaching box, where it must use its paw to retrieve pellets placed strategically to encourage reaching with the contralateral limb. The success and failure rates of these attempts provide insight into the animal's motor capabilities.
The single pellet reaching task is a sensitive and effective method for evaluating skilled motor deficits in both ischemic and hemorrhagic stroke, capable of assessing both short-term and long-term outcomes. However, it requires extensive training and is subject to individual variability in performance.
Cognitive Tests in Rodent Models of Stroke
Morris Water Maze
The Morris water maze is a widely used tool for assessing cognitive function, particularly spatial learning and memory in rodents. The setup consists of a round pool filled with opaque water, where animals must swim to find a submerged platform to escape. Key metrics analyzed include the initial heading angle, latency to reach the platform, and path length, which help evaluate swimming behavior and memory impairments.
Modified versions of the Morris water maze have been developed to assess spatial navigation memory and learning flexibility by changing the platform's location. These modifications allow for the detection of subtle deficits in search strategies as animals learn. A notable advantage of the water maze is that it produces highly quantifiable data without relying on olfactory cues, and it does not require food deprivation to motivate the animals.
Radial Arm Maze
The radial arm maze is a behavioral assessment used to evaluate learning and memory impairments in animal models of stroke, particularly those sensitive to hippocampal damage. The apparatus features a central platform with eight or twelve arms radiating outward, each containing food targets. To motivate the animals, they are typically food deprived.
The task can assess both reference memory, where the animal must remember the fixed locations of food targets across trials, and working memory, which requires the animal to recall which arms contained food in a single trial when the target locations change. Research indicates that ischemic animals tend to make more errors than non-ischemic ones, with some studies showing deficits in both reference and working memory. However, other findings suggest that ischemic rats experience greater impairments in working memory compared to reference memory.
Y-maze
The Y-maze is a widely used tool for assessing spontaneous alternation and spatial memory in rodents. Comprising three arms arranged at 120° angles, the Y-maze facilitates exploration behaviors and helps evaluate spatial memory by monitoring the tendency of animals to alternate their choices.
In a typical single-trial setup, an animal is placed in one arm and allowed to explore for a designated time (3 to 15 minutes). The sequence of visits and total entrances to each arm are recorded. Spontaneous alternation is calculated based on the animal's tendency to visit arms different from the previous two visits. To enhance the assessment and minimize habituation effects, a two-trial version of the task has been developed. In this setup, one arm is initially blocked, and when it is reopened during the second trial, the animal's preference for this novel arm is measured.
The Y-maze leverages natural exploration behavior to assess cognitive function, requiring no prior training and allowing for straightforward recording of results. It effectively evaluates both short-term and long-term cognitive outcomes in ischemic stroke models, while its sensitivity in hemorrhagic models appears limited. Researchers should consider inter-trial intervals to avoid habituation effects that may influence exploration behavior.
Novel Object Recognition/Location Tests
The novel object recognition and location tests are behavioral assessments used primarily in rats to evaluate memory and cognitive function. The one-trial object recognition test involves a series of training sessions where rats learn to distinguish between identical and novel objects in a Y-maze setup, followed by repeated trials to reinforce recognition of new items. A simplified version, the novel object recognition test, is conducted in an open field where the animal explores two identical objects and later one is replaced with a novel item, with the time spent exploring each recorded to measure recognition.
A similar approach is employed for assessing memory related to object location, where an object is moved to a different corner of the field. The animal's preference for the object in the new location indicates recognition of the change.
These tests are considered straightforward and sensitive for detecting long-term cognitive deficits, such as those resulting from stroke. However, they require prior training and their outcomes can be influenced by various factors like the exploration environment, object characteristics, time intervals between sessions, and the animal's state of anxiety or activity level. Notably, extended intervals between trials may significantly reduce the ability to recognize novel objects.
Elevated-plus Maze
The elevated-plus maze (EPM) is a widely utilized behavioral test for assessing anxiety levels in rodents, capitalizing on their natural tendency to seek safety in enclosed spaces. The maze consists of four arms: two open and two enclosed, elevated 50 cm above the ground. Rodents are placed in the center and allowed to explore for 10 minutes, with time spent and entries into each arm recorded. A higher percentage of time and entries in the open arms typically indicates lower anxiety levels.
Each of these behavioral tests has different principles, steps, and assessment criteria. An understanding of the preferred settings, strengths, and limitations of various behavioral assessment methods helps researchers to choose the best strategy based on their research objectives, which in turn helps to improve the reliability of their experimental results.
- Ruan, J., & Yao, Y. (2020). Behavioral tests in rodent models of stroke. Brain hemorrhages,1(4), 171-184.
