Rota rod apparatus

A rota-rod apparatus (Insight, Brazil) was used for the assessment of motor coordination. Initially, animals capable to stand on the rota-rod apparatus for 60 s (7 rpm) were selected 24 h before the test. The mice were divided into four groups of six animals each (n = 24) and were pretreated with H5 (20 and 40 mg/kg, p.o), saline, or diazepam (2.5 mg/kg). Each animal was individually evaluated on the rota-rod apparatus at 30, 60, 90, and 120 min after treatments, and the time (s) spent on top of the bar was recorded for up to 180 s [30 (link), 31 (link)].

In order to rule out antinociception false positive results, the effects of HEMh on spontaneous locomotor activity of male Swiss mice (n = 8 per group) was tested on the rotarod apparatus (Insight Ltd., Ribeirão Preto, Brazil). This method was used to eliminate any possible non-specific muscle relaxant effects of this extract [14 (link),17 (link)]. Twenty-four hours before the experiments, mice capable of remaining on the rotarod (4 cm in diameter, 6 rpm) for three periods of 60 s without falling were pre-selected. On the day of the experiment, the mice were treated with either vehicle (saline, 10 mL/kg, given by oral route), HEMh (62.5 mg/kg, given by oral route), or diazepam—a positive control (2 mg/kg, given by intraperitoneal route). One hour after oral treatment (saline or HEMh) and 30 min after diazepam injection, the mice were placed on the apparatus and the number of falls from the apparatus over 180 s was recorded with a stopwatch.

Experiments for evaluation of motor coordination balance of the animals were performed on the Rotarod apparatus (EFF412, Insight, Ribeirão Preto, SP, Brazil) as described in [64 (link)] and carried out with some modifications. The Rotarod is a rotating cylinder with a radius of 2.7 cm and a height of 40 cm. The device has four bays with 3 cm of space for each mouse. Four animals were evaluated in parallel. Animals that remain for a longer time in the rotating system of the device were considered better in motor coordination and equilibrium. The performance of Rotarod was continuously measured on a rotating rod since the mice should move forward to avoid falling. The animals were initially exposed to a pre-training for two consecutive days at four different speeds (16, 24, 28, and 32 rpm) for 3 min each, following the protocol of [65 (link)] modified. Animals that remained up 5 s were excluded according to [66 (link)]. On the 30th day, the animals were again placed on the rod three times in the accelerated mode when the residence time on the rotating rod in sec was recorded for up two minutes as [67 (link)].

To evaluate the possible non-specific muscle-relaxant or sedative effects of riparins, mice were submitted to the rota-rod test, as previously described [39 (link)]. The rota-rod apparatus (Insight, Ribeirão Preto, Brazil) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. The bar rotated at a constant speed of 8 revolutions per minute. The animals were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 120 s. Animals were treated with riparins (100 mg/Kg), diazepam (10 mg/Kg, reference drug) or vehicle (5% Tween 80 in distilled water; control group) and 40 min afterward were placed on a rotating rod. The resistance to falling was measured up to 120 s. The results are expressed as the average time (s) the animals remained on the rota-rod in each group.

To evaluate the motor performance, mice were submitted to the rotarod test, as previously described [32 (link)]. The rotarod apparatus (Insight, Ribeirão Preto, Brazil) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. The bar rotated at a constant speed of 8 revolutions per min. The animals were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 120 s [26 (link)]. At the test day, mice from different experimental groups were placed on a rotating rod and the resistance to falling was measured up to 120 s. Mice treated with diazepam (10 mg/kg; Cristália, Itapira, Brazil), the reference drug of the test, were placed on a rotating rod 1 h after treatment. The results are expressed as the average time (s) the animals remained on the rotarod in each group.

Since the behaviors evaluated in nociceptive tests depend on the integrity of the motor capacity of the mice, the motor performance was monitored throughout the experimental period using the rotarod test. We followed the methods of Santos et al. [49 (link)], and the method description partly reproduces their wording. The rotarod apparatus (Insight, Ribeirão Preto, Brazil) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. The bar rotated at a constant speed of 8 revolutions per min. The animals were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 120 s. On the test day, mice from different experimental groups were placed on a rotating rod, and the resistance to falling was measured up to 120 s. Mice treated with diazepam (10 mg/kg; Cristália, Itapira, Brazil), the reference drug of the test, were placed on a rotating rod one hour after treatment. The results are expressed as the average time (s) the animals remained on the rotarod in each group.

The rotarod apparatus (Insight^®, Brazil) consists of a grooved metal roller (8 cm in diameter), separated into 9 cm wide compartments elevated 16 cm. The rotarod is widely used to evaluate motor coordination of the front and hind limbs, as well as balance in rodents, which is the outcome most affected by ethanol. Ataxia resulted from cerebellar damage can be detected in the rotarod, mainly associated to beam walking test (Carter et al., 1999 (link); Oleas et al., 2013 (link)). The rotarod test is a useful tool to evaluate gait disturbance as well as motor coordination and at higher speeds, dysdiadochokinesia (Rozas et al., 2007 (link)). The animals were evaluated for their ability to remain on the rotating rod during five successive trials of 2 min at 8 rpm with an inter-trial interval of 60 s (Teixeira et al., 2014 (link)).