Behavioral Tests:

Irwin Test/Functional Observational Battery (FOB) is developed for rapid in vivo screening of new neurotrophic and neurotoxic substances. The battery of behavioral observations, assess six broad categories of CNS function including excitation, sedation, stereotypy, pain/anesthesia, reflexes, and autonomic balance.

The Beam walking test was designed for the investigation of ataxia-motor dysfunction in rodents. The test is used for detecting violations of the equilibrium and coordinating movement mediated by cerebellar dysfunction. This test is used to evaluate new drug candidates for their effect on coordinating movement.

The forced swim test is the main test to assess symptoms of depression using animal models. During the test when rodents are forced to swim, we are assessing the rodent’s active or passive behavior. Active behavior characterizes by active swimming, climbing, and looking for an escape route; passive behavior characterizes by stopping swimming, “giving up the search for escape,” floating on the water’s surface without struggling and showing the minimal movements necessary to keep an animal’s head above water. We measure and score swimming/escaping (active) or passive (floating/immobility) time in the tall cylinder with water. The forced swim test is commonly used to assess the antidepressant activity of compounds. The reduction of immobility time and increase of active swimming time is interpreted as an antidepressant-like effect of the pharmacological action.

The Morris/Radial Water Maze Test is a widely used method for studying spatial learning and memory. The method is based on the animal’s (rat or mouse) trying to find the hidden underwater platform and escape swimming in the pull of water by using visual cues. Morris Water Maze is an appropriate model for studies of such pathology as Alzheimer’s disease, depression, or amnesia, and age-related changes.

Method Advantages:

the aversive nature of the water motivates the animal to explore the territory
rapid learning
easy measuring of parameters
no requirement for food deprivation
less risk of potential confounding scent cues
both working and reference memory can be simultaneously tested in one experiment.

Method Limitations: The animal’s ability to swim and physical condition or endurance can impact the results. The act of being immersed in water and forced to swim can induce stress that may alter the outcomes of each repeated trial.

Time spent in the target quadrant of male C57Bl/6J aged and adult mice during the Morris water maze probe test. The mice were trained to achieve the hidden platform for 5 consecutive days, a probe test was performed 24 h after the last training with the absent platform.

Nociception Assays:

The Hot Plate Test allows for exploring the pain response caused by heat in rodents, as well as the influence of the centrally-acting analgesic on this process. Peripherally acting drugs are ineffective in this test.

The Hotplate test was carried out using IITC hot plate analgesia meter (Life Science, USA). The smooth metal surface was heated to 56±0,5 °C. The response was measured before and 15, 30, 45, and 60 min after IV injection of Vehicle (Saline) or test compound (10 mg/kg) (Mean ± SEM). Latency was measured until the first behavioral sign of nociception (limb fluttering, licking, withdrawal from the surface or animal jumping). The cut-off time for latency in the hotplate test was set at 20 s.

The tail-flick test is the most frequently used test for nociception in animals. Unlike the Hot plate test, the tail-flick test also allows local anesthetics to be tested. During the test, radiant heat applies to the rodent’s tail, and time is measured until the animal moves the tail away because of pain caused by the heat beam. The test set for the measurement of the nociceptive sensitivity of animals for the analgesic properties of substance assessment. Time increase before avoidance response to the thermal stimulus reveals an analgesic action.

Mice were treated with Vehicle (glycerol), or Benzocaine (12% benzocaine solution in glycerol) – 30 ul of solution were applied at the tail center (2 cm) for 5 min. The level of analgesia for each mouse was measured 5, 15, 30, 60, and 120 min after application of the test compound using an analgesia meter (Columbus Instruments, USA) with the intensity of thermal stimulus adjusted to a baseline tail-flick latency of 5.5±0.5 s. The tail exposure to infrared radiation was leveled at the site of application of test compounds (Mean ± SEM). Each measurement was performed at triplicates. The cut-off time for latency in the tail-flick test was set at 15 s.

Von Frey test The von Frey test involves applying a punctate stimulus to a given region of the rodent’s body, usually the plantar surface of the hind paw, and recording the stimulus intensity that evokes a withdrawal reflex. Stimuli are typically applied using calibrated fibers with a specific bending force. The purpose of this test is to measure mechanical nociception in order to evaluate the ability of an animal to detect a noxious stimulus. Von Frey remains the gold standard for determining mechanical thresholds in mice.

Von Frey tests for Balb/c male mice (8 w.o.) orally treated with paracetamol (300 mg/kg) 30 min after the treatment. Analgetic paracetamol depresses the mice’s nociceptive sensitivity, as indicated by the increased threshold for treated mice.

Physiological Tests:

Blood pressure (BP) in rodents is measured in the tail of the mouse or rat using volume pressure recording sensor technology with the CODA (Kent Scientific, USA) mouse/rat tail-cuff system.

Rodents’ blood pressure is measured by a tail-cuff method using the Coda Non-invasive Blood-Pressure System. Rodents are placed in plastic holders setting on the warming platform for 10 min to reduce stress and achieve a tail temperature of 32-34°C before the testing. Systolic (Sys) and diastolic (Dia) blood pressure is recorded. Mean blood pressure and pulse pressure (PP, difference between systolic and diastolic BP) values were then calculated. Typically measurement includes a set of 5 acclimations and 10 regular cycles, and the average blood pressure values are calculated taking into account accepted cycles only (M ± SEM).

The grip strength test is a non-invasive method for evaluating rodent muscle force in vivo. It allows studying the neuromuscular functions by determining the maximal peak force developed by a rodent when the operator pulls it out of a specially designed grid or bar available for both fore and hind limbs. The Grip Test is included in the Functional Observational Battery (FOB) to screen for neurobehavioral toxicity.

The grip strength test was performed in C57BL/6J male (M) and female (F) mice of different ages using a device 47200 Grip Strength Meter (Ugo Basil, Italy). The test is performed using a grating for two limbs according to the device instructions. The study of the grip strength parameter is carried out at least three times for each animal. The average value of the force obtained in gf units (gram force), which is then converted into Newton using the formula: A ∙ 0.00980665 = N, where A is the average value of the force in gf, N – average value of the force in Newton. The reproducibility of the method for mice is 16.5% (Mean ± SEM).

Rotarod test is a non-invasive method for evaluating rodent balance, physical condition, and motor coordination in vivo. It allows studying the neuromuscular functions by determining the maximal riding time (seconds) and speed at fall developed by a rodent, which naturally tries to stay on the rotating cylinder (rod) and avoid falling to the ground.