Evaluating Rodent Aerobics For Preclinical Ischemic Stroke Intervention Assessments

Stuart Little

One might probably associate rodents, particularly mice and rats, with sewers, cargoes, ships, or perhaps with an exceptional “French chef” or a friendly “Stuart”. Besides, tens and millions of rodents have played a central role in science for nearly 200 years, and as a matter of fact, the term “lab-rat” has popularly made its way into the urban lexicon. Out of roughly 100 million vertebrates used in research, 95% are either rats or mice. They closely replicate human response with ⁓90% genome similarity and form an obligatory fragment of scientific research to model human diseases.

Moreover, rodents serve as excellent experimental models for neurological disorders to screen newly-developed therapeutics and to study the resultant neurobehavioral outcome, as a prelude to their testing in well-controlled human clinical trials. In the article “Rodent Gymnastics: Neurobehavioral Assays in Ischemic Stroke” (published in Molecular Neurobiology), Dr. Rajanikant’s laboratory at the National Institute of Technology Calicut, India consolidated neurobehavioral tests performed on laboratory animals (in vivo) that are pertinent to neurological disorders, chiefly ischemic stroke. Ischemic stroke is one of the leading causes of death and disability worldwide. The major focus of this article is to categorize and highlight widely used in vivo sensorimotor, cognitive, anxiety-related and nociceptive neurobehavioral assays in ischemic stroke.

Sensorimotor tests

More than half of the stroke survivors are left with sensorimotor deficits that impair their physical mobility and leave them paralyzed. It is, therefore, imperative to study these deficits and neurobehavioral outcome to evaluate the degree of functional recovery following the administration of therapeutic interventions. The following tests are used in rodents to assess the post-stroke sensorimotor function and the corresponding functional recovery.

Adhesive removal test

Adhesive removal test is one of the rare tests sensitive enough to measure a sensory or motor deficit after an apparent full recovery. The test is performed by applying adhesive tapes to different parts of the animal body. The test varies depending on the body part that is involved in the tactical stimulation and the size of the tape used. In this method, values of time for contact and removal of adhesive for each paw is collected.

Tapered Beam Test

The tapered beam test is used to measure the limb impairments and to assess the motor coordination and balance in rodents following ischemia. The task involves animal walking across an elevated balance beam that tapers at one end and has an under-hanging ledge. Foot faults made with hind limbs and forelimbs are recorded as deficits in limb function. The foot faults increase in ischemic animals and the difficulty of the task increases.

Cylinder Test

Ischemia pertaining to basal ganglia may cause impaired forelimb and other motor functions. The cylinder test is used to check for the asymmetry in forelimb movement. When the animal is placed in the cylindrical enclosure, its exploratory instincts are set in by rearing up the hind limbs and probing with its forelimbs. The use of forelimb is noted when the whole paw is placed on the glass, indicating total body support. The use of both the left and right paws is calculated with reference to the total number of contacts.

Rotarod Test

The rotarod test is used to test motor coordination and balance. The animal is placed in the rotarod cylinder and the time it remains on the rotarod is measured. The speed of the cylinder slowly increases with time. Motor coordination between the experimental groups is evaluated by comparing the latency to fall on the very first trial. Motor learning can also be assessed by comparing the first trial with subsequent trials and is evident as an increased latency to fall over time.

Open field test

The open field test is a common measure of exploratory and general activity in rodents. It highlights the following behavior – line crossing, center square entries, center square duration, rearing, stretch attend postures, grooming, freezing, urination and defecation.

Grip Strength

Grip strength test is extensively used to evaluate the sensorimotor function following cerebral ischemia, affecting the basal ganglia region of the brain. It measures the muscle and limb strength of the animal. An automated grip strength meter with force sensor is used.

Staircase Test

The staircase test is used to find the effect on basal ganglia and subsequent motor functions. It is used to analyze the skilled paw reaching in rodents. The performances of both the left and right limbs are considered individually not requiring any contra-lateral restriction. The apparatus consists of a Plexiglas box with a removable double staircase on one end. Each step on this staircase contains a small trough in which food pellets are to be placed. The animals are trained prior and are made to go through a food deprivation regime. They are also made to familiarize with the food pellets. Test parameters are introduced during the training period, which includes variations in session time, reward diet, and starvation or restriction period.

Corner test

The corner test is used for identifying and quantifying sensorimotor and postural asymmetries. It has also been shown to provide a simple way for detecting contralateral deficits and ipsilateral turning biases. The test apparatus consists of two cardboards along the joint of which a small opening is left for encouraging the mice to enter all the way to the corner. As the animal reaches deep into the corner, facial whiskers touch the boards from both sides and get stimulated. The mice then rear up on their hind limbs and turn back to face the open end of the two boards. Naïve mice do not show a side preference, but animals subjected to ischemia turn toward the non-impaired side.

Cognitive tests

Besides sensorimotor deficits, cognitive decline deleteriously affects 20-80% of stroke survivors and occurs within first to three months of stroke onset causing mild cognitive impairment to dementia. However, the risk is associated with the demographic factors such as age, ethnicity, and occupation, as well as cerebrovascular factors. Rodents display cognitive impairment after ischemic stroke, and their neurobehavioral outcome could be studied using specifically designed cognitive tests.

Morris Water Maze Test

The Morris water maze test is used to measure the effect of neurocognitive disorders on spatial learning and possible neural treatments. Further, it is employed to test the effect of lesions on the brain in areas focused on memory. Behavioural testing is conducted in a circular pool of water and is made opaque by adding powdered milk. The initial heading angle, the latency to reach the platform and the path length are analyzed by their swimming pattern. A primary advantage of using the water maze over other common behavioral mazes is that there are no olfactory trails for animals to use scent tracking to find the target.

Passive Avoidance

Passive avoidance test is used to assess the cognitive memory of the animal pertaining to the hippocampus and amygdala. This test is a one-trial, fear motivated test with two chambers inside a shock cage. One chamber is lighted while other is dark. During the training period, the subject is introduced into the lit side of the cage and allowed to get acclimated for about 30 seconds. The door separating the two chambers is opened. Once the animal enters the dark chamber with all four paws in, the door is shut. A shock is administered through the floor following which the subject is removed. The next day the same protocol is followed without administering the shock. The latency or uncertainty of the animal to enter the dark chamber is measured. Following ischemia, rodents usually show less cognitive remembrance and perform worse than their sham counterparts in this test.

Fear Conditioning Test

Fear conditioning test is used as a paradigm to study the learning of animals to prevent aversive events. This test mainly relies on contextual and cued fear conditioning, which pertains to the hippocampus and amygdala, respectively. Stimuli such as foot shock, air puff, etc are used. A mouse is first placed in the shock cage equipped with sound system and made to acclimate to the surroundings. It is then subjected to a conditional auditory stimulus followed by a shock stimulus. The response along with the length of time the mouse spends frozen is video recorded. In the second run of the experiment, only the auditory stimulus is provided. The response of the mouse is again recorded.

Anxiety- related and nociceptive tests

Anxiety and depressive symptoms in stroke survivors are often overlooked, despite reports suggesting 55% of stroke patients suffering from post-stroke depression. Certain neurobehavioral assays have been developed to test anxiety-related symptoms in rodents after ischemic stroke. Pre-pulse inhibition test, tail suspension test, and forced swim test assess depression-like behavior. Pre-pulse inhibition test utilizes an auditory stimulus to monitor the acoustic startle response in animal models of focal ischemia. Ischemia can cause schizophrenic and obsessive-compulsive behavior, which in turn is signified by reduced startle inhibition. In a forced swim test, the rodent is placed in a cylinder filled with water. Wild-type animals exhibit escape behavior (swimming). However, depressed animals tend to give up sooner and stop swimming or just float on the water surface. The tail suspension test uses immobility of the animal suspended from its tail as a measurement of depression. More the immobility period, more the animal is said to be depressed. In a Tail Flick Test, thermal radiation is applied to the tail of the animal and its response to spinal nociception is observed. The reaction time or the “tail flick latency” is measured as the time from the application of heat source to the withdrawal of tail. A paradoxical observation is seen when different sites of the tail are tested. The reaction time decreases when the stimulus is applied to distant part of the tail.

Conclusion

The choice of an appropriate behavioral test is of cardinal importance in order to meet the specific research objective of the scientist. The rodent models satisfy fundamental requirements to model diseases that affect humans and bridge the gap between the evaluation criteria used in animal studies and clinical trials. However, there is no ‘one-size-fits-all’ approach, and some of the tests discussed above might not be instrumental in carrying out basic research. Moreover, further research is indispensable to develop more sensitive tests for the successful translation of experimental stroke research to clinical trials.

These findings are described in the article entitled Rodent Gymnastics: Neurobehavioral Assays in Ischemic Stroke, published in the journal Molecular Neurobiology. This work was led by Dr. Rajanikant G. K from the National Institute of Technology Calicut, India.

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