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Rodent Behavioral Tests Sensitive to Functional Recovery and Compensation
Introduction
Research scientists rely upon rodent models to help us better understand and test potential treatments for stroke survivors. Although there are limitations in the use of rodents to study human neurological disorders, it is possible to identify and focus on the functional overlaps and similarities between species’ brains and behaviors when designing studies investigating stroke prevention, acute neuroprotection, and chronic poststroke interventions. There is overwhelming evidence that the fundamental organization of the rat brain, for instance, is not that different from humans in that there is much overlap in sensory and motor systems, and cognitive, emotion, and attention processes. Researchers can utilize both species-typical and cross-species behavioral patterns to sensitively probe the impact of stroke and determine the efficacy of possible clinically relevant treatments through careful selection and analysis of tests of rodents’ behaviors and by making careful comparisons to similar human behavior or cognitive processes.
Common rodent models of focal ischemic or hemorrhagic stroke cause damage to the sensorimotor cortex and/or striatum, producing sensory and motor impairments primarily in forelimb or hind limb function, and in more severe models, experimental stroke can damage the hippocampus and related brain structures, producing impairments in cognitive domains. The severity of injury and the resolution of impairments through interventions or treatments can be assessed utilizing well-established rodent behavioral tests in these stroke models (see Refs. ). However, one major criticism of using rodent models for stroke research has been that often researchers report that rodents quickly “recover” from stroke-induced impairments, whereas humans do not. In most cases, this recovery is not true brain repair or functional recovery, but actually is a demonstration of the ability of the animal to learn new compensatory behaviors that mask ongoing impairments. Rodents, as is also true with humans, are really good at compensating for impairments, and thus many studies have reported “recovery” on tasks over time when in fact with careful methods, ongoing impairments are unmasked and compensatory behaviors can be revealed . Through careful selection of behavioral tests, usually using both quantitative and qualitative methods of analysis, it is possible to more sensitively distinguish between behavioral patterns used to compensate for impairments versus true brain repair or remodeling. There is no agreed-upon battery of neurological tests that are routinely used to screen for clinically effective treatments. However, there are a number of well-established behavioral tests commonly used that are sensitive to lesion severity or lesion location. Some of these tests are more suited for acute studies, whereas others can reveal chronic ongoing impairments in function. There are also varying degrees to which these tests can be used to distinguish between recovery of function and compensatory behaviors.
In this brief review, we will describe in more detail a few commonly used tests of rodent sensory, motor, and cognitive function. These specific behavioral tests have been chosen for further discussion because they can provide both quantitative and qualitative data or methods to distinguish compensatory behaviors from true recovery of function. Additionally, we have compiled a more extensive list, although not exhaustive, of common poststroke rodent behavioral assays and indicate some of their limitations and strengths and whether they are best for rats, mice or both ( Table 75.1 ; adapted and expanded from Ref. ).
Table 75.1
Tests of Experimental Stroke Impairments
Adapted from Zarruk JG, Garcia-Yebenes I, Romera VG, et al. Neurological tests for functional outcome assessment in rodent models of ischaemic stroke. Rev Neurol 2011;53(10):607–18.
| Tests | Symptoms Evaluated | Sensitive Acutely/Chronically | Species | Disadvantages | Advantages |
|---|---|---|---|---|---|
| Sensorimotor tests | |||||
| Adhesive removal test | Sensory | +/+ | R & M | Training needed; less sensitive in models without a cortical injury | High sensitivity long after ischemic damage, including MCAO models with small cortical damage |
| Beam walking tests | Motor coordination | +/+ | R & M | Training needed; compensatory bias in beams without a ledge | Good sensitivity in rats and mice long after ischemic damage; addition of a ledge increases sensitivity |
| Corner test | Whiskers sensitivity | +/+ | M | Less sensitive in models with cortical lesions; low evidence in rats | Simple and fast; no training needed; evaluates long-term dysfunctions in striatal infarcts |
| Rotarod test | Motor coordination and balance | +/+ | R & M | Low sensitivity in mice 72 h after MCAO; confounding factors; training needed | Good sensitivity and reliability in rats |
| Bilateral tactile stimulation test | Tactile discrimination | +/− | R & M | Task can be time consuming | Magnitude of sensory asymmetry is sensitive to minor impairments, long-lasting deficits in MCAO, and focal ischemia |
| Open field test | Locomotor activity | +/− | R & M | Low sensitivity with small infarcts; only useful in short-term protocols | Assesses anhedonia and stress |
| Cylinder test | Motor coordination | +/− | R & M | Decreased sensitivity in mice with small infarcts | Easy and fast to apply; it is sensitive 1 month after ischemia |
| Forelimb placing | Vibrissa evoked limb placement | −/+ | R | It is subjective (double blind); sensitivity decreases due to a high spontaneous recovery | Simple and fast; detects neurological impairments in models of striatal and/or cortical lesions |
| Swim task | Forelimb inhibition | +/+ | R | Little training needed; resistant to recovery | |
| Staircase/Montoya test | Fine motor coordination and sensory neglect | +/+ | R & M | Long training needed; Large number of excluded animals | Good sensitivity and reliability in MCAO models with small infarcts, and long after the surgery |
| Single-pellet | Fine motor coordination, sensory neglect, forelimb movement abnormalities | +/+ | R & M | Long training times; time-consuming video analysis | Great sensitivity and reliability up to at least 11 months after focal damage |
| Pasta handling | Manual dexterity; oral motor deficits | +/+ | R & M | Must acclimate animals to being filmed while eating; can be difficult to quantify | Simple to administer; no shaping or training |
| Foot fault | Motor coordination | +/− | R & M | Poor sensitivity in mild ischemia | Very sensitive in severe ischemia models |
| Ladder task | Forelimb and hind limb function; motor coordination and paw grip | +/+ | R & M | Animals learn to compensate unless ladder rung spacing is varied | Minimal training; qualitative and quantitative data; sensitive chronically |
| Elevated body swing test | Muscle strength | +/+ | R & M | Less experience in mice | Simple and fast; no training needed; sensitive even 30 days after MCAO |
| Cognitive tests | |||||
| Morris water maze | Spatial learning and memory | +/+ | R & M | Less sensitive in mice 72 h after MCAO; training needed | Very good sensitivity in rats long after ischemia |
| Passive avoidance tests | Avoidance learning | +/+ | R & M | Expensive equipment; training needed | Very high sensitivity long after ischemia in rats and mice; easy to perform |
| Neurological scales | |||||
| Modified neurological severity score | General neurological assessment | +/− | R & M | Low sensitivity at late times after ischemia; subjective | Gives an overall degree of ischemic injury; easy and fast |
Tests of experimental stroke impairments. Summary of several commonly used behavioral tests used to assess neurological damage following experimental stroke in rats (R) and mice (M), including tests that have shown sensitivity (+) during the acute and chronic periods after stroke.
MCAO , Middle cerebral artery occlusion.
Forelimb Function Tasks
Hemiparesis of the hand and arm are the most prevalent, enduring, and disabling impairments following stroke in humans and there are many well-established tests developed to assess upper-extremity impairments in rodents following different unilateral stroke models ( ; Table 75.1 ). One highly reliable task is the single-pellet reaching task. Although it is more time consuming and requires prior training, the test is sensitive to chronic upper-extremity impairments, including digit function, and can be used to reveal compensatory behaviors and recovery of normalized movement patterns.
Single-Pellet Reaching Task
Although there are several variations, most single-pellet reaching tasks for mice and rats are fairly similar in their general methods for administering the task, collecting data, and analyzing the data (e.g., Refs. ). Because this is a food-motivated task, animals are first food restricted (∼85–90% initial starting weight), acclimated to a clear-reaching chamber and then are trained to reach through a narrow window to retrieve food pellets (or seeds for mice). Pellets are placed in a shallow well on a shelf placed outside the window at a distance between ∼1 and 2 cm. Once animals show a limb preference, researchers can encourage the use of just that one limb by inserting a wall in the cage ipsilateral to the reaching limb or placing a bracelet or cast on the nonreaching limb to inhibit that limb’s ability to reach the pellet. Over several days to weeks, animals are trained to reach through the window, grasp the pellet, withdraw their limb and place the pellet in their mouths. This is defined as a “Success.” Animals are trained to an asymptotic level of ∼40–60% success rate, maintained over two to three consecutive days. A success rate is calculated as the percentage of successful reaches of the total number of reach attempts [(total successes/total reach attempts) ×100]. Quantitative data can also be collected which includes the number of “Failures” and “Drops”. A Failure is when an animal either reaches the upper limit of times without grasping the pellet or reaches for the pellet and knocks it out of the well. A Drop is when an animal grasps the pellet, retracts the arm into the cage but fails to place the pellet into its mouth. Drops may also denote a sensory impairment because the animal fails to recognize that a pellet is in its paw.
When assessing limb impairments and recovery, a minimum of 10–30 trials should be used. The specific number of trials depends upon the frequency of test administration and study design. When testing the efficacy of an intervention or treatment, the assignment of animals to groups should be counterbalanced to ensure reaching performance is matched at baseline (before stroke induction), poststroke, and prior to treatment (when possible). This task is highly sensitive to distal forelimb impairments for up to 11 months poststroke, even following fairly small unilateral strokes in rats . There are several variations of skilled reaching tasks, and other reach-to-grasp tasks, that can also be used to evaluate forelimb dexterity .
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