Journal of Neuroscience Methods 251 (2015) 138–142
Contents lists available at ScienceDirect
Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth
Basic Neuroscience
An experimental evaluation of a new designed apparatus (NDA) for the rapid measurement of impaired motor function in rats M. Jarrahi ∗ , B. Sedighi Moghadam, H. Torkmandi Laboratory of Behavioral Neuroscience, Research Center and Department of Physiology, Semnan University of Medical Sciences, Semnan 15131-38111, Iran
h i g h l i g h t s • • • • •
We evaluate a new apparatus for the rapid measurement of impaired motor function in rats. We examine experimentally the capability of the new apparatus for impaired motor function in rats. We compare experimentally the capability of the new apparatus with rotarod for impaired motor function in rats. The new apparatus is more sensitive than rotarod for evaluating of impaired motor system function. The sensitivity of the new apparatus increases by faster rotation speeds.
a r t i c l e
i n f o
Article history: Received 26 March 2015 Received in revised form 17 May 2015 Accepted 28 May 2015 Available online 5 June 2015 Keywords: Rat Motor coordination Rotarod Beam walking New designed apparatus Ethanol
a b s t r a c t Background: Assessment of the ability of rat to balance by rotarod apparatus (ROTA) is frequently used as a measure of impaired motor system function. Most of these methods have some disadvantages, such as failing to sense motor coordination rather than endurance and as the sensitivity of the method is low, more animals are needed to obtain statistically significant results. New method: We have designed and tested a new designed apparatus (NDA) to measure motor system function in rats. Our system consists of a glass box containing 4 beams which placed with 1 cm distance between them, two electrical motors for rotating the beams, and a camera to record the movements of the rats. The RPM of the beams is adjustable digitally between 0 and 50 rounds per minute. Results: We evaluated experimentally the capability of the NDA for the rapid measurement of impaired motor function in rats. Also we demonstrated that the sensitivity of the NDA increases by faster rotation speeds and may be more sensitive than ROTA for evaluating of impaired motor system function. Comparison with existing methods: Compared to a previous version of this task, our NDA provides a more efficient method to test rodents for studies of motor system function after impaired motor nervous system. Conclusions: In summary, our NDA will allow high efficient monitoring of rat motor system function and may be more sensitive than ROTA for evaluating of impaired motor system function in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The balance and motor coordination maintaining is a complex function in which several portions of nervous system are involved (Agid, 1990; Johansson and Cole, 1992; Zelenin et al., 2010). Assessment of balance and motor coordination can be used to measure the effects of experimental manipulations or other test compounds
Abbreviations: NDA, new designed apparatus; ETH, ethanol; ROTA, rotarod apparatus; VEH, vehicle; RPM, rotations per minute. ∗ Corresponding author. Tel.: +98 9122312187; fax: +98 2333654209. E-mail address: [email protected] (M. Jarrahi). http://dx.doi.org/10.1016/j.jneumeth.2015.05.023 0165-0270/© 2015 Elsevier B.V. All rights reserved.
on rats or mice nervous system (Hamm et al., 1994; Carter et al., 2001; Fujimoto et al., 2004; Schaar et al., 2010). Widely used protocols and three well-established used methods for assessing balance and motor coordination in rats and mice are ROTA test, footprint analysis and beam walking (Goldstein and Davis, 1990; Rozas et al., 1997; Bervar, 2000). Currently, ROTA is the most prevalent tool utilized extensively to measure and assesses the balance and motor coordination (Jansen and Low, 1996; Luong et al., 2011; Mandillo et al., 2014). Although, there are several studies regarding the utility of ROTA for motor impairment in laboratory applications (Hamm et al., 1994; Boix et al., 2010), the primary disadvantage of this method is that a number of animals with reduced motor coordination will drop at
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the beginning, while for those that do continue, the assessment will soon start to measure endurance rather than motor coordination per se. Also there are numerous commercial versions of this apparatus on the market, but a few have disadvantages, such as failing to accelerate at an adequate speed to detect motor coordination rather than endurance (Stanley et al., 2005; Deacon, 2013) and as the sensitivity of the procedure specially with constant speed ROTAs is low, more animals are required to obtain statistically significant results (Jones and Roberts, 1968). In this research we report the development and testing of an inexpensive apparatus to investigate the balance and motor coordination with more accuracy which we think that can overcome the limitations of ROTA. Therefore, we aim to compare NDA and ROTA on a rat model by conducting a series of motor coordination tests after administration of different doses of ETH and see its effects on different rotation speeds and compare the two apparatuses on rats. 2. Materials and methods 2.1. Animals Adult male Wistar rats (200 ± 10 g) were individually housed in cages (50 cm × 26 cm × 25 cm) and kept on a 12 h-light/dark cycle (lights on at 06:00 h). Food and water were accessible ad libitum. The ambient environment was maintained at a constant temperature (22 ± 2 ◦ C) and relative humidity (50–60%). All studies were conducted between 10:00 and 14:00 h. The experimental protocol was approved by the Ethical Review Board of Semnan University of Medical Sciences (Iran) and all of the experimental trials were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Care was taken to decrease the number of animals that were used in each experiment. The rats were kept in their testing room for about 2 h each day, three days before any behavioral testing. During this time, they were alternatively handled by the investigator. Both subjects and researchers were kept blind about all experiments. 2.2. Drugs It is well known that ETH produces dramatic impairments in balance and motor coordination in rats and also it is one of the most powerful first-line drugs that could examine in all studies which deals with balance and motor in coordination. This was the reason for the choice of ETH for our work. Based on a preliminary, pilot study, the lowest dose of ETH (1.5 g/kg) was chosen, which significantly disturb balance and motor coordination of rats in ROTA test. Also in this study we found that the lowest dose of ETH (0.15 g/kg) could significantly disturb balance and motor coordination of rats in NDA test. ETH solutions were made by diluting ETH (Sigma–Aldrich, 99.5%) with 0.9% saline and doses of 0.15 and 1.5 g/kg were prepared and injected intraperitoneally at a volume of 2 ml/kg. Control groups were given equivalent injections of 0.9% saline as a VEH. 2.3. ROTA ROTA is a test used to assess motor coordination and motor learning in rodent models of CNS disorders. The Rotarod (Harvard Apparatus Ltd., Fircroft Way, Edenbridge, Kent) was used for measurement of impaired motor function (Jones and Roberts, 1968). The rats were placed on the rotating rod with constant rotations of 4, 8, 16 or 20 RPM, the latency to fall is recorded, where the rats fall safely below the rotating rod.
Fig. 1. Schematic illustration of the new designed apparatus. Rat was placed on the two middle beams. The Box A contains electrical motors. The Box B is the lateral camera. The anterior camera (not shown) was located parallel to the horizontal level of beams and to some extent lower to record the errors of forelimb movements more exactly. For further details see the text.
2.4. NDA The NDA is illustrated in Fig. 1 was made to the design of the author by the engineering sections of the neuroscience department, University of Semnan. The apparatus consists of two electrical motors for rotating the beams (Box A in Fig. 1), a camera to record the movements of the rats (Box B in Fig. 1), 4 beams with 1 m length which placed with 1 cm distance between them and a glass box containing the three above-mentioned components of the system (Fig. 1). Beams surfaces were covered with a soft rubber. The beams are 1.2 cm in diameter, supported 30 cm higher than the bottom of the apparatus. The two middle beams are rotating in different ways. The beams number 1 and 4 are rotating inwardly to force the rats to stay on the two middle beams. The system is 10 cm in width and 50 cm height from the floor of the box. The RPM of the beams is adjustable digitally between 0 and 50 rounds per minute. In all NDA tests, the rotation speeds of lateral beams were 10 RPM and the rotation speeds of middle beams were the same as the rotation speeds of rod in ROTA defined in our protocol.
2.5. Experimental groups 192 male rats were randomly allocated into 24 groups and were examined by the ROTA or the NDA at 4, 8, 16 or 20 RPM. The rats were treated by VEH or either ETH (0.15 or 1.5 g/kg) and the rotation testing in all experiments was done by administration of VEH or ETH and using of the ROTA or NDA. Based on this protocol we had 24 groups and each group contained eight rats. All groups includes: (ROTA4 + VEH), (ROTA8 + VEH), (ROTA16 + VEH), (ROTA20 + VEH), (NDA4 + VEH), (NDA8 + VEH), (NDA16 + VEH), (NDA20 + VEH), (ROTA4 + ETH0.15 g/kg), (ROTA4 + ETH1.5 g/kg), (ROTA8 + ETH0.15 g/kg), (ROTA8 + ETH1.5 g/kg), (ROTA16 + ETH0.15 g/kg), (ROTA16 + ETH1.5 g/kg), (ROTA20 + ETH0.15 g/kg), (ROTA20 + ETH1.5 g/kg), (NDA4 + ETH0.15 g/kg), (NDA4 + ETH1.5 g/kg), (NDA8 + ETH0.15 g/kg), (NDA8 + ETH1.5 g/kg), (NDA16 + ETH0.15 g/kg), (NDA16 + ETH1.5 g/kg), (NDA20 + ETH0.15 g/kg) and (NDA20 + ETH1.5 g/kg). Behavioral tests on the ROTA or the NDA were done immediately 5 min after VEH or ETH injections.
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2.6. Experimental procedures in the ROTA and the NDA tests The rats were placed on a rotating rod in the ROTA 5 min after injections and the rotation speeds of 4, 8, 16 or 20 RPM was selected in each group based on our protocol. At the same time the duration of remaining rat on the rod for each rotation speed was measured. All trials repeated three times for each rat in group. Results were expressed as the duration of animals that succeeded in remaining on the rod until the cut-off time (60 s) was reached. The total time that the rats stayed on the ROTA and the time to falling from it were recorded. The trials with NDA were also setup with speeds of 4, 8, 16 or 20 RPM for middle beams, and the rats were placed on the beams 5 min after injections. Each rat of related groups was placed on middle beams, 3 times with 5 min time intervals. The anterior camera (not shown in Fig. 1) was placed in front of system parallel to the horizontal level of the beams and to some extent lower to record the errors of movements more exactly. After reviewing the films, we measured the times in which the forelimb of the rats dropped between beams of the new system (which we named it hand error) within 40 s. The first 10 s of the trials was ignored. In ROTA, we measured the time in which the rats were able to stay over the rotating rod maximizing 60 s. The criteria for measuring the hand errors was dropping the forelimb between the beams of the NDA and seeing the forelimb under the level of beams in camera film. 2.7. Statistics A blind data analysis was done to prevent bias. All values were presented as mean ± SEM using the SPSS software program for windows version 19 (IBM Corp). All data were subjected to the Shapiro–Wilk test for normality. The data followed a normal distribution were analyzed using One-Way ANOVA. When the assumptions of a normal distribution and homogeneity of variance were provided, homogeneity of variances was tested by Levens test. In our study One-Way ANOVA followed by the Games–Howel test post hoc analysis was used for comparing between the VEH and ETH groups in each speed rotation. Also The VEH or ETH groups in each rotation speeds of 4, 8, 16 and 20 RPM of the ROTA or the NDA were analyzed and compared by One-Way ANOVA followed by the Tukey’s test post hoc analysis. The data were analyzed by a difference were considered significant if the P value was less than 0.05. 3. Results In this study, information of the mean fall latencies or hand errors related to VEH or ETH (0.15 and 1.5 g/kg) groups in each rotation speeds (4, 8, 16 or 20 RPM) of the ROTA or the NDA were compared and analyzed. The results did not show noteworthy differences in mean fall latencies or hand errors between VEH and ETH (0.15 g/kg) groups in each rotation speed of 4, 8, 16 or 20 RPM in ROTA tests, but all comparisons in the NDA showed significant differences between VEH and ETH (0.15 g/kg) groups in all rotation speeds as were seen in 4 (F5,54 = 37.3, P = 0.043), 8 (F5,54 = 37.3, P = 0.013),16 (F5,54 = 37.3, P = 0.005) and 20 (F5,54 = 37.3, P = 0.05) RPM. Also the results showed significant differences between VEH and ETH (1.5 g/kg) groups in rotation speeds of 4 (F5,54 = 37.3, P = 0.029), 8 (F5,54 = 37.3, P = 0.024), 16 (F5,54 = 37.3, P = 0.006) and 20 (F5,54 = 37.3, P = 0.001) RPM in ROTA tests, and all comparisons in the NDA showed significant differences between VEH and ETH (1.5 g/kg) groups in all rotation speeds as were seen in 4 (F5,54 = 37.3, P = 0.0001), 8 (F5,54 = 37.3, P = 0.0001), 16 (F5,54 = 37.3,P = 0.0001) and 20 (F5,54 = 37.3, P = 0.001) RPM. Furthermore the VEH or ETH (0.15 and 1.5 g/kg) groups between different rotation speeds of 4, 8, 16 or 20 RPM of the ROTA or the
NDA were compared and the results showed significant differences (F3,36 = 0.034, P = 0.023) between VEH groups in rotation speeds of 4 and 20 RPM in the NDA and also showed significant differences (F3,36 = 0.027, P = 0.020) between ETH (1.5 g/kg) groups in rotation speeds of 4 and 20 RPM in the NDA. There was also significant differences (F3,36 = 4.13, P = 0.033) between VEH groups in rotation speeds of 4 and 20 RPM in the ROTA tests. The difference was also significant (F3,36 = 4.13, P = 0.006) between ETH (1.5 g/kg) groups in rotation speeds of 4 and 20 RPM in the ROTA tests. We also saw significant differences between ETH (0.15 g/kg) groups in rotation speeds of 4 and 20 RPM (F3,36 = 4.13, P = 0.015) and rotation speeds of 8 and 20 RPM (F3,36 = 4.13, P = 0.037) in the NDA.
4. Discussion The results of our experiment indicate that the NDA may consider as an alternative to the ROTA. As expected, faster speed rates of rods or beams led to decrease latencies to fall in ROTA tests and it increased the rate of error movements in the NDA (Fig. 2). As the results show, the mean duration of the staying of rats on the ROTA that received 0.15 g/kg ETH compare to the rats that were injected only VEH do not show significant differences, consistent with prior studies (Bogo et al., 1981; Bettica et al., 2012). While in the NDA the mean number of hand errors in rats that received 0.15 g/kg ETH compare to the rats were injected only VEH show significant differences in all speed rotations (P < 0.05). Overall, results show that the NDA may have more sensitivity compare to the ROTA. Also the result shows that both in the ROTA and the NDA, the sensitivity of the apparatus in detecting difference between the two groups increase by raising the speed of rotation. In statistical analysis, it was determined that the highest sensitivity for detecting difference between the two groups of VEH or ETH was seen at rotation speed 20 RPM both in the ROTA (P < 0.01) and the NDA (P < 0.05), that is consistent with previous studies (Hamm et al., 1994; Bettica et al., 2012). Although we needs more research to find the disadvantages of the NDA, it seems that this system lacks some of the disadvantages of motor coordination measuring in the ROTA. Some of the disadvantages of it includes: It is poor in detecting minor deficits or improvements in coordination (Carter et al., 2001), usually needs expensive equipment (Metten et al., 2004) and more animals are required to obtain statistically significant results (Jones and Roberts, 1968). In addition to the mentioned items, we believe that it is likely there are some genotypes of animals that may not be testable on the ROTA due to their tendency to jump from the rod instead of running on top (Rusty et al., 2003). So applying the NDA may be useful for comparing certain genotypes of rats. Although the lowest dose of ETH in literature used to create imbalance in rat was 0.5 and 0.3 g/kg (Bogo et al., 1981; Bettica et al., 2012), we preferred to use 0.15 g/kg ETH to compare the ROTA and the NDA groups in our studies based on our research protocol and the result of a pilot study (Data not shown). As the previous findings suggest that compared to the beam-walking and beambalance tasks, the ROTA task is a more sensitive and efficient index for assessing motor impairment produced by brain injury (Hamm et al., 1994), therefore we preferred to compare the ROTA and the NDA. To the best of our knowledge, there is no report of a similar device to our NDA so far. Therefore we compare it with the ROTA. In addition to lacking of some disadvantages, in the NDA the beams design is horizontal and the axis of beams rotation is perpendicular to the longitudinal axis of the animal. So this design structure prevents extreme fatigue in animals moving on the beams which in turn may create imbalance. Also it appears that the NDA is more sensitive to measure lateral imbalance relation to anterior
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Fig. 2. Comparison between VEH (saline as a VEH) and ethanol (ETH) (0.15 or 1.5 g/kg) in the rotarod apparatus (ROTA) or in the new designed apparatus (NDA) in different rotation speeds of 4, 8, 16 or 20 RPM. The comparisons included: (A); (ROTA + VEH) vs (ROTA + ETH0.15 g/kg), (B); (NDA + VEH) vs (NDA + ETH0.15 g/kg), (C); (ROTA + VEH) vs (ROTA + ETH1.5 g/kg) and (D); (NDA + VEH) vs (NDA + ETH1.5 g/kg). All tests were done 5 min after injections (VEH or ETH). The results are expressed as mean ± SEM.
posterior imbalance in animal. We suggest that this advantage of the new apparatus may open a new window to assessing further aspects of nervous system elements supposed to be involved in balance and motor coordination such as impairment in gravity sensing, inability to move, and impairment of weight distribution. Also, we agree that, our work has several limitations such as lacking the repetition of the main experiments, comparison the NDA with the beam walking test and testing other drugs (many depressants such as barbiturates and benzodiazepines) when comparing the NDA and ROTA. So further research is needed to determine this
by assessing these effects on deteriorations of motor system using the NDA.
5. Conclusion Taken together, our results indicates that the NDA has more sensitivity compare to the ROTA. So the new apparatus may be able to detect some of the subtle deterioration of motor coordination which is not possible by the ROTA.
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Conflict of interest statement The authors certify that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments This research was founded and supported by a research grant (grant number: 393) from Semnan University of Medical Sciences (Semnan, Iran). Their contribution is gratefully acknowledged. Also we would like to have a special thank to Dr. Amir Hushang Bakhtiary for his advice and Dr. Nasrolah Moradi for proofreading. References Agid Y. From posture to initiation of movement. Rev Neurol 1990;146:536–42. Bervar M. Video analysis of standing – an alternative footprint analysis to assess functional loss following injury to the rat sciatic nerve. J Neurosci Methods 2000;102:109–16. Bettica P, Squassante L, Groeger JA, Gennery B, Winsky-Sommerer R, Dijk DJ. Differential effects of a dual orexin receptor antagonist (SB-649868) and zolpidem on sleep initiation and consolidation, SWS, REM sleep, and EEG power spectra in a model of situational insomnia. Neuropsychopharmacology 2012;37:1224–33. Bogo V, Hill TA, Young RW. Comparison of accelerod and ROTA sensitivity in detecting ethanol- and acrylamide-induced performance decrement in rats: review of experimental considerations of rotating rod systems. Neurotoxicology 1981;2:765–87. Boix JO, Cauli O, Felipo V. Developmental exposure to polychlorinated biphenyls 52, 138 or 180 affects differentially learning or motor coordination in adult rats. Mechanisms involved. Neuroscience 2010;167:994–1003. Carter RJ, Morton J, Dunnett SB. Motor coordination and balance in rodents. Curr Protoc Neurosci 2001 [Chapter 8: Unit 8.12]. Deacon RM. Measuring motor coordination in mice. J Vis Exp 2013;75:e2609.
Fujimoto ST, Longhi L, Saatman KE, Conte V, Stocchetti N, et al. Motor and cognitive function evaluation following experimental traumatic brain injury. Neurosci Biobehav Rev 2004;28:365–78. Goldstein LB, Davis JN. Beam-walking in rats: studies towards developing an animal model of functional recovery after brain injury. J Neurosci Methods 1990;31:101–7. Hamm RJ, Pike BR, O’Dell DM, Lyeth BG, Jenkins LW. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. J Neurotrauma 1994;11:187–96. Jansen EM, Low WC. Long-term effects of neonatal ischemic–hypoxic brain injury on sensorimotor and locomotor tasks in rats. Behav Brain Res 1996;78: 189–94. Johansson RS, Cole KJ. Sensory-motor coordination during grasping and manipulative actions. Curr Opin Neurobiol 1992;2:815–23. Jones BJ, Roberts DJ. The quantitative measurement of motor in co-ordination in naive mice using an accelerating rotarod. J Pharm Pharmacol 1968;20:302–4. Luong TN, Carlisle HJ, Southwell A, Patterson PH. Assessment of motor balance and coordination in mice using the balance beam. J Vis Exp 2011;49:2376. Mandillo SI, Heise L, Garbugino GP, Tocchini-Valentini A, Giuliani S, Wells S, et al. Early motor deficits in mouse disease models are reliably uncovered using an automated home-cage wheel-running system: a cross-laboratory validation. Dis Model Mech 2014;7:397–407. Metten PK, Best L, Cameron AJ, Saultz AB, Zuraw JM, Yu CH, et al. Observer-rated ataxia: rating scales for assessment of genetic differences in ethanol-induced intoxication in mice. J Appl Physiol 2004;97:360–8. Rozas G, Guerra MJ, Labandeira-Garcia JL. An automated rotarod method for quantitative drug-free evaluation of overall motor deficits in rat models of parkinsonism. Brain Res Brain Res Protoc 1997;2:75–84. Rusty NR, Wahlsten D, Crabbe JC. Influence of task parameters on rotarod performance and sensitivity to ethanol in mice. Behav Brain Res 2003;141:237–49. Schaar KL, Bernneman MM, Savits SI. Functional assessments in the rodent stroke model. Exp Transl Stroke Med 2010;2:13. Stanley JL, Lincoln RJ, Brown TA, McDonald LM, Dawson GR, Reynolds DS. The mouse beam walking assay offers improved sensitivity over the mouse rotarod in determining motor coordination deficits induced by benzodiazepines. J Psychopharmacol 2005;19:221–7. Zelenin PV, Beloozerova IN, Sirota MG, Orlovsky GN, Deliagina TG. Activity of red nucleus neurons in the cat during postural corrections. J Neurosci 2010;30:14533–42.
Contents lists available at ScienceDirect
Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth
Basic Neuroscience
An experimental evaluation of a new designed apparatus (NDA) for the rapid measurement of impaired motor function in rats M. Jarrahi ∗ , B. Sedighi Moghadam, H. Torkmandi Laboratory of Behavioral Neuroscience, Research Center and Department of Physiology, Semnan University of Medical Sciences, Semnan 15131-38111, Iran
h i g h l i g h t s • • • • •
We evaluate a new apparatus for the rapid measurement of impaired motor function in rats. We examine experimentally the capability of the new apparatus for impaired motor function in rats. We compare experimentally the capability of the new apparatus with rotarod for impaired motor function in rats. The new apparatus is more sensitive than rotarod for evaluating of impaired motor system function. The sensitivity of the new apparatus increases by faster rotation speeds.
a r t i c l e
i n f o
Article history: Received 26 March 2015 Received in revised form 17 May 2015 Accepted 28 May 2015 Available online 5 June 2015 Keywords: Rat Motor coordination Rotarod Beam walking New designed apparatus Ethanol
a b s t r a c t Background: Assessment of the ability of rat to balance by rotarod apparatus (ROTA) is frequently used as a measure of impaired motor system function. Most of these methods have some disadvantages, such as failing to sense motor coordination rather than endurance and as the sensitivity of the method is low, more animals are needed to obtain statistically significant results. New method: We have designed and tested a new designed apparatus (NDA) to measure motor system function in rats. Our system consists of a glass box containing 4 beams which placed with 1 cm distance between them, two electrical motors for rotating the beams, and a camera to record the movements of the rats. The RPM of the beams is adjustable digitally between 0 and 50 rounds per minute. Results: We evaluated experimentally the capability of the NDA for the rapid measurement of impaired motor function in rats. Also we demonstrated that the sensitivity of the NDA increases by faster rotation speeds and may be more sensitive than ROTA for evaluating of impaired motor system function. Comparison with existing methods: Compared to a previous version of this task, our NDA provides a more efficient method to test rodents for studies of motor system function after impaired motor nervous system. Conclusions: In summary, our NDA will allow high efficient monitoring of rat motor system function and may be more sensitive than ROTA for evaluating of impaired motor system function in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The balance and motor coordination maintaining is a complex function in which several portions of nervous system are involved (Agid, 1990; Johansson and Cole, 1992; Zelenin et al., 2010). Assessment of balance and motor coordination can be used to measure the effects of experimental manipulations or other test compounds
Abbreviations: NDA, new designed apparatus; ETH, ethanol; ROTA, rotarod apparatus; VEH, vehicle; RPM, rotations per minute. ∗ Corresponding author. Tel.: +98 9122312187; fax: +98 2333654209. E-mail address: [email protected] (M. Jarrahi). http://dx.doi.org/10.1016/j.jneumeth.2015.05.023 0165-0270/© 2015 Elsevier B.V. All rights reserved.
on rats or mice nervous system (Hamm et al., 1994; Carter et al., 2001; Fujimoto et al., 2004; Schaar et al., 2010). Widely used protocols and three well-established used methods for assessing balance and motor coordination in rats and mice are ROTA test, footprint analysis and beam walking (Goldstein and Davis, 1990; Rozas et al., 1997; Bervar, 2000). Currently, ROTA is the most prevalent tool utilized extensively to measure and assesses the balance and motor coordination (Jansen and Low, 1996; Luong et al., 2011; Mandillo et al., 2014). Although, there are several studies regarding the utility of ROTA for motor impairment in laboratory applications (Hamm et al., 1994; Boix et al., 2010), the primary disadvantage of this method is that a number of animals with reduced motor coordination will drop at
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the beginning, while for those that do continue, the assessment will soon start to measure endurance rather than motor coordination per se. Also there are numerous commercial versions of this apparatus on the market, but a few have disadvantages, such as failing to accelerate at an adequate speed to detect motor coordination rather than endurance (Stanley et al., 2005; Deacon, 2013) and as the sensitivity of the procedure specially with constant speed ROTAs is low, more animals are required to obtain statistically significant results (Jones and Roberts, 1968). In this research we report the development and testing of an inexpensive apparatus to investigate the balance and motor coordination with more accuracy which we think that can overcome the limitations of ROTA. Therefore, we aim to compare NDA and ROTA on a rat model by conducting a series of motor coordination tests after administration of different doses of ETH and see its effects on different rotation speeds and compare the two apparatuses on rats. 2. Materials and methods 2.1. Animals Adult male Wistar rats (200 ± 10 g) were individually housed in cages (50 cm × 26 cm × 25 cm) and kept on a 12 h-light/dark cycle (lights on at 06:00 h). Food and water were accessible ad libitum. The ambient environment was maintained at a constant temperature (22 ± 2 ◦ C) and relative humidity (50–60%). All studies were conducted between 10:00 and 14:00 h. The experimental protocol was approved by the Ethical Review Board of Semnan University of Medical Sciences (Iran) and all of the experimental trials were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Care was taken to decrease the number of animals that were used in each experiment. The rats were kept in their testing room for about 2 h each day, three days before any behavioral testing. During this time, they were alternatively handled by the investigator. Both subjects and researchers were kept blind about all experiments. 2.2. Drugs It is well known that ETH produces dramatic impairments in balance and motor coordination in rats and also it is one of the most powerful first-line drugs that could examine in all studies which deals with balance and motor in coordination. This was the reason for the choice of ETH for our work. Based on a preliminary, pilot study, the lowest dose of ETH (1.5 g/kg) was chosen, which significantly disturb balance and motor coordination of rats in ROTA test. Also in this study we found that the lowest dose of ETH (0.15 g/kg) could significantly disturb balance and motor coordination of rats in NDA test. ETH solutions were made by diluting ETH (Sigma–Aldrich, 99.5%) with 0.9% saline and doses of 0.15 and 1.5 g/kg were prepared and injected intraperitoneally at a volume of 2 ml/kg. Control groups were given equivalent injections of 0.9% saline as a VEH. 2.3. ROTA ROTA is a test used to assess motor coordination and motor learning in rodent models of CNS disorders. The Rotarod (Harvard Apparatus Ltd., Fircroft Way, Edenbridge, Kent) was used for measurement of impaired motor function (Jones and Roberts, 1968). The rats were placed on the rotating rod with constant rotations of 4, 8, 16 or 20 RPM, the latency to fall is recorded, where the rats fall safely below the rotating rod.
Fig. 1. Schematic illustration of the new designed apparatus. Rat was placed on the two middle beams. The Box A contains electrical motors. The Box B is the lateral camera. The anterior camera (not shown) was located parallel to the horizontal level of beams and to some extent lower to record the errors of forelimb movements more exactly. For further details see the text.
2.4. NDA The NDA is illustrated in Fig. 1 was made to the design of the author by the engineering sections of the neuroscience department, University of Semnan. The apparatus consists of two electrical motors for rotating the beams (Box A in Fig. 1), a camera to record the movements of the rats (Box B in Fig. 1), 4 beams with 1 m length which placed with 1 cm distance between them and a glass box containing the three above-mentioned components of the system (Fig. 1). Beams surfaces were covered with a soft rubber. The beams are 1.2 cm in diameter, supported 30 cm higher than the bottom of the apparatus. The two middle beams are rotating in different ways. The beams number 1 and 4 are rotating inwardly to force the rats to stay on the two middle beams. The system is 10 cm in width and 50 cm height from the floor of the box. The RPM of the beams is adjustable digitally between 0 and 50 rounds per minute. In all NDA tests, the rotation speeds of lateral beams were 10 RPM and the rotation speeds of middle beams were the same as the rotation speeds of rod in ROTA defined in our protocol.
2.5. Experimental groups 192 male rats were randomly allocated into 24 groups and were examined by the ROTA or the NDA at 4, 8, 16 or 20 RPM. The rats were treated by VEH or either ETH (0.15 or 1.5 g/kg) and the rotation testing in all experiments was done by administration of VEH or ETH and using of the ROTA or NDA. Based on this protocol we had 24 groups and each group contained eight rats. All groups includes: (ROTA4 + VEH), (ROTA8 + VEH), (ROTA16 + VEH), (ROTA20 + VEH), (NDA4 + VEH), (NDA8 + VEH), (NDA16 + VEH), (NDA20 + VEH), (ROTA4 + ETH0.15 g/kg), (ROTA4 + ETH1.5 g/kg), (ROTA8 + ETH0.15 g/kg), (ROTA8 + ETH1.5 g/kg), (ROTA16 + ETH0.15 g/kg), (ROTA16 + ETH1.5 g/kg), (ROTA20 + ETH0.15 g/kg), (ROTA20 + ETH1.5 g/kg), (NDA4 + ETH0.15 g/kg), (NDA4 + ETH1.5 g/kg), (NDA8 + ETH0.15 g/kg), (NDA8 + ETH1.5 g/kg), (NDA16 + ETH0.15 g/kg), (NDA16 + ETH1.5 g/kg), (NDA20 + ETH0.15 g/kg) and (NDA20 + ETH1.5 g/kg). Behavioral tests on the ROTA or the NDA were done immediately 5 min after VEH or ETH injections.
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2.6. Experimental procedures in the ROTA and the NDA tests The rats were placed on a rotating rod in the ROTA 5 min after injections and the rotation speeds of 4, 8, 16 or 20 RPM was selected in each group based on our protocol. At the same time the duration of remaining rat on the rod for each rotation speed was measured. All trials repeated three times for each rat in group. Results were expressed as the duration of animals that succeeded in remaining on the rod until the cut-off time (60 s) was reached. The total time that the rats stayed on the ROTA and the time to falling from it were recorded. The trials with NDA were also setup with speeds of 4, 8, 16 or 20 RPM for middle beams, and the rats were placed on the beams 5 min after injections. Each rat of related groups was placed on middle beams, 3 times with 5 min time intervals. The anterior camera (not shown in Fig. 1) was placed in front of system parallel to the horizontal level of the beams and to some extent lower to record the errors of movements more exactly. After reviewing the films, we measured the times in which the forelimb of the rats dropped between beams of the new system (which we named it hand error) within 40 s. The first 10 s of the trials was ignored. In ROTA, we measured the time in which the rats were able to stay over the rotating rod maximizing 60 s. The criteria for measuring the hand errors was dropping the forelimb between the beams of the NDA and seeing the forelimb under the level of beams in camera film. 2.7. Statistics A blind data analysis was done to prevent bias. All values were presented as mean ± SEM using the SPSS software program for windows version 19 (IBM Corp). All data were subjected to the Shapiro–Wilk test for normality. The data followed a normal distribution were analyzed using One-Way ANOVA. When the assumptions of a normal distribution and homogeneity of variance were provided, homogeneity of variances was tested by Levens test. In our study One-Way ANOVA followed by the Games–Howel test post hoc analysis was used for comparing between the VEH and ETH groups in each speed rotation. Also The VEH or ETH groups in each rotation speeds of 4, 8, 16 and 20 RPM of the ROTA or the NDA were analyzed and compared by One-Way ANOVA followed by the Tukey’s test post hoc analysis. The data were analyzed by a difference were considered significant if the P value was less than 0.05. 3. Results In this study, information of the mean fall latencies or hand errors related to VEH or ETH (0.15 and 1.5 g/kg) groups in each rotation speeds (4, 8, 16 or 20 RPM) of the ROTA or the NDA were compared and analyzed. The results did not show noteworthy differences in mean fall latencies or hand errors between VEH and ETH (0.15 g/kg) groups in each rotation speed of 4, 8, 16 or 20 RPM in ROTA tests, but all comparisons in the NDA showed significant differences between VEH and ETH (0.15 g/kg) groups in all rotation speeds as were seen in 4 (F5,54 = 37.3, P = 0.043), 8 (F5,54 = 37.3, P = 0.013),16 (F5,54 = 37.3, P = 0.005) and 20 (F5,54 = 37.3, P = 0.05) RPM. Also the results showed significant differences between VEH and ETH (1.5 g/kg) groups in rotation speeds of 4 (F5,54 = 37.3, P = 0.029), 8 (F5,54 = 37.3, P = 0.024), 16 (F5,54 = 37.3, P = 0.006) and 20 (F5,54 = 37.3, P = 0.001) RPM in ROTA tests, and all comparisons in the NDA showed significant differences between VEH and ETH (1.5 g/kg) groups in all rotation speeds as were seen in 4 (F5,54 = 37.3, P = 0.0001), 8 (F5,54 = 37.3, P = 0.0001), 16 (F5,54 = 37.3,P = 0.0001) and 20 (F5,54 = 37.3, P = 0.001) RPM. Furthermore the VEH or ETH (0.15 and 1.5 g/kg) groups between different rotation speeds of 4, 8, 16 or 20 RPM of the ROTA or the
NDA were compared and the results showed significant differences (F3,36 = 0.034, P = 0.023) between VEH groups in rotation speeds of 4 and 20 RPM in the NDA and also showed significant differences (F3,36 = 0.027, P = 0.020) between ETH (1.5 g/kg) groups in rotation speeds of 4 and 20 RPM in the NDA. There was also significant differences (F3,36 = 4.13, P = 0.033) between VEH groups in rotation speeds of 4 and 20 RPM in the ROTA tests. The difference was also significant (F3,36 = 4.13, P = 0.006) between ETH (1.5 g/kg) groups in rotation speeds of 4 and 20 RPM in the ROTA tests. We also saw significant differences between ETH (0.15 g/kg) groups in rotation speeds of 4 and 20 RPM (F3,36 = 4.13, P = 0.015) and rotation speeds of 8 and 20 RPM (F3,36 = 4.13, P = 0.037) in the NDA.
4. Discussion The results of our experiment indicate that the NDA may consider as an alternative to the ROTA. As expected, faster speed rates of rods or beams led to decrease latencies to fall in ROTA tests and it increased the rate of error movements in the NDA (Fig. 2). As the results show, the mean duration of the staying of rats on the ROTA that received 0.15 g/kg ETH compare to the rats that were injected only VEH do not show significant differences, consistent with prior studies (Bogo et al., 1981; Bettica et al., 2012). While in the NDA the mean number of hand errors in rats that received 0.15 g/kg ETH compare to the rats were injected only VEH show significant differences in all speed rotations (P < 0.05). Overall, results show that the NDA may have more sensitivity compare to the ROTA. Also the result shows that both in the ROTA and the NDA, the sensitivity of the apparatus in detecting difference between the two groups increase by raising the speed of rotation. In statistical analysis, it was determined that the highest sensitivity for detecting difference between the two groups of VEH or ETH was seen at rotation speed 20 RPM both in the ROTA (P < 0.01) and the NDA (P < 0.05), that is consistent with previous studies (Hamm et al., 1994; Bettica et al., 2012). Although we needs more research to find the disadvantages of the NDA, it seems that this system lacks some of the disadvantages of motor coordination measuring in the ROTA. Some of the disadvantages of it includes: It is poor in detecting minor deficits or improvements in coordination (Carter et al., 2001), usually needs expensive equipment (Metten et al., 2004) and more animals are required to obtain statistically significant results (Jones and Roberts, 1968). In addition to the mentioned items, we believe that it is likely there are some genotypes of animals that may not be testable on the ROTA due to their tendency to jump from the rod instead of running on top (Rusty et al., 2003). So applying the NDA may be useful for comparing certain genotypes of rats. Although the lowest dose of ETH in literature used to create imbalance in rat was 0.5 and 0.3 g/kg (Bogo et al., 1981; Bettica et al., 2012), we preferred to use 0.15 g/kg ETH to compare the ROTA and the NDA groups in our studies based on our research protocol and the result of a pilot study (Data not shown). As the previous findings suggest that compared to the beam-walking and beambalance tasks, the ROTA task is a more sensitive and efficient index for assessing motor impairment produced by brain injury (Hamm et al., 1994), therefore we preferred to compare the ROTA and the NDA. To the best of our knowledge, there is no report of a similar device to our NDA so far. Therefore we compare it with the ROTA. In addition to lacking of some disadvantages, in the NDA the beams design is horizontal and the axis of beams rotation is perpendicular to the longitudinal axis of the animal. So this design structure prevents extreme fatigue in animals moving on the beams which in turn may create imbalance. Also it appears that the NDA is more sensitive to measure lateral imbalance relation to anterior
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Fig. 2. Comparison between VEH (saline as a VEH) and ethanol (ETH) (0.15 or 1.5 g/kg) in the rotarod apparatus (ROTA) or in the new designed apparatus (NDA) in different rotation speeds of 4, 8, 16 or 20 RPM. The comparisons included: (A); (ROTA + VEH) vs (ROTA + ETH0.15 g/kg), (B); (NDA + VEH) vs (NDA + ETH0.15 g/kg), (C); (ROTA + VEH) vs (ROTA + ETH1.5 g/kg) and (D); (NDA + VEH) vs (NDA + ETH1.5 g/kg). All tests were done 5 min after injections (VEH or ETH). The results are expressed as mean ± SEM.
posterior imbalance in animal. We suggest that this advantage of the new apparatus may open a new window to assessing further aspects of nervous system elements supposed to be involved in balance and motor coordination such as impairment in gravity sensing, inability to move, and impairment of weight distribution. Also, we agree that, our work has several limitations such as lacking the repetition of the main experiments, comparison the NDA with the beam walking test and testing other drugs (many depressants such as barbiturates and benzodiazepines) when comparing the NDA and ROTA. So further research is needed to determine this
by assessing these effects on deteriorations of motor system using the NDA.
5. Conclusion Taken together, our results indicates that the NDA has more sensitivity compare to the ROTA. So the new apparatus may be able to detect some of the subtle deterioration of motor coordination which is not possible by the ROTA.
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Conflict of interest statement The authors certify that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments This research was founded and supported by a research grant (grant number: 393) from Semnan University of Medical Sciences (Semnan, Iran). Their contribution is gratefully acknowledged. Also we would like to have a special thank to Dr. Amir Hushang Bakhtiary for his advice and Dr. Nasrolah Moradi for proofreading. References Agid Y. From posture to initiation of movement. Rev Neurol 1990;146:536–42. Bervar M. Video analysis of standing – an alternative footprint analysis to assess functional loss following injury to the rat sciatic nerve. J Neurosci Methods 2000;102:109–16. Bettica P, Squassante L, Groeger JA, Gennery B, Winsky-Sommerer R, Dijk DJ. Differential effects of a dual orexin receptor antagonist (SB-649868) and zolpidem on sleep initiation and consolidation, SWS, REM sleep, and EEG power spectra in a model of situational insomnia. Neuropsychopharmacology 2012;37:1224–33. Bogo V, Hill TA, Young RW. Comparison of accelerod and ROTA sensitivity in detecting ethanol- and acrylamide-induced performance decrement in rats: review of experimental considerations of rotating rod systems. Neurotoxicology 1981;2:765–87. Boix JO, Cauli O, Felipo V. Developmental exposure to polychlorinated biphenyls 52, 138 or 180 affects differentially learning or motor coordination in adult rats. Mechanisms involved. Neuroscience 2010;167:994–1003. Carter RJ, Morton J, Dunnett SB. Motor coordination and balance in rodents. Curr Protoc Neurosci 2001 [Chapter 8: Unit 8.12]. Deacon RM. Measuring motor coordination in mice. J Vis Exp 2013;75:e2609.
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