This work was supported by Natural Science Foundation of Beijing (No.7162124), and Xin-ao Foundation for Beijing University of Chinese Medicine.

All the animals were maintained in accordance with the guidelines by the Chinese legislation on the ethical use and care of laboratory animals.
1. Pre-modeling Preparation
<ol>
	<li>Laboratory preparation
	<ol>
		<li>Run the UV lamp for at least 30 min before the experiment.</li>
		<li>Control the lab temperature at 25 &#177; 3 &#176;C, and relative humidity around 30%.</li>
		<li>Turn on the lab light at 6:00 and turn it off at 18:00 to establish a 12 h/12 h light/dark cycl...

<ol>
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C., Benedito-Silva, A. A., Tufik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+deprivation+induced+by+the+modified+multiple+platform+technique:+quantification+of+sleep+loss+and+recovery.">Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery.</a> Brain Research. 1004 (1-2), 45-51 (2004).</li><li>Alzoubi, K. H., Khabour, O. F., Tashtoush, N. H., AI-Azzam, S. I., Mhaidat, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Effect+of+Pentoxifylline+on+Sleep-Deprivation+Induced+Memory+Impairment.">Evaluation of the Effect of Pentoxifylline on Sleep-Deprivation Induced Memory Impairment.</a> Hippocampus. 23 (9), 812-819 (2013).</li><li>Pires, G. N., Tufik, S., Andersen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grooming+analysis+algorithm:+Use+in+the+relationship+between+sleep+deprivation+and+anxiety-like+behavior.">Grooming analysis algorithm: Use in the relationship between sleep deprivation and anxiety-like behavior.</a> Progress in Neuro-Psychopharmacology and Biological Psychiatry. 41, 6-10 (2013).</li><li>Yamashita, M., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+rat+model+of+central+fatigue+induced+by+chronic+sleep+disorder+and+excessive+brain+tryptophan.">Establishment of a rat model of central fatigue induced by chronic sleep disorder and excessive brain tryptophan.</a> Japanese Journal of Cognitive Neuroscience. 15, 67-74 (2013).</li><li>Arai, M., Yamazaki, M., Inoue, K., Fushiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+intracranial+injection+of+transforming+growth+factor-beta+relevant+to+central+fatigue+on+the+waking+electroencephalogram+of+rats+Comparison+with+effects+of+exercise.">Effects of intracranial injection of transforming growth factor-beta relevant to central fatigue on the waking electroencephalogram of rats Comparison with effects of exercise.</a> Progress in Neuro-Psychopharmacology and Biological Psychiatry. 26 (2), 307-312 (2002).</li><li>Han, C. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+behavioral+and+brain+changes+after+different+durations+of+the+modified+multiple+platform+method+on+rats:+An+animal+model+of+central+fatigue.">Distinct behavioral and brain changes after different durations of the modified multiple platform method on rats: An animal model of central fatigue.</a> PloS One. 12 (5), e0176850 (2017).</li><li>Tang, X., Yang, L., Sanford, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+in+sleep+and+motor+activity+in+rats.">Individual variation in sleep and motor activity in rats.</a> Behavioural Brain Research. 180 (1), 62-68 (2007).</li><li>Stanford, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Open+Field+Test:+reinventing+the+wheel.">The Open Field Test: reinventing the wheel.</a> Journal of Psychopharmacology. 21 (2), 134-135 (2007).</li><li>Ahn, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+anxiety+during+an+open+field+test+is+correlated+with+individual+differences+in+contextually+conditioned+fear+in+mice.">Basal anxiety during an open field test is correlated with individual differences in contextually conditioned fear in mice.</a> Animal Cells and Systems. 17 (3), 154 (2013).</li><li>Costa, A. A., Morato, S., Roque, A. C., Tin &#243;s, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+model+for+exploratory+activity+of+rats+with+different+anxiety+levels+in+elevated+plus-maze.">A computational model for exploratory activity of rats with different anxiety levels in elevated plus-maze.</a> Journal of Neuroscience Methods. 236, 44-50 (2014).</li><li>Liu, Z., Wu, Y., Liu, T., Li, R., Xie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+regulation+in+a+rat+model+of+exercise-induced+chronic+fatigue.">Serotonin regulation in a rat model of exercise-induced chronic fatigue.</a> Neuroscience. 349, 27-34 (2017).</li><li>Foley, T. E., Fleshner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroplasticity+of+dopamine+circuits+after+exercise:+implications+for+central+fatigue.">Neuroplasticity of dopamine circuits after exercise: implications for central fatigue.</a> NeuroMolecular Medicine. 10 (2), 67-80 (2008).</li><li>Leite, L. H., Rodrigues, A. G., Soares, D. D., Marubayashi, U., Coimbra, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+fatigue+induced+by+losartan+involves+brain+serotonin+and+dopamine+content.">Central fatigue induced by losartan involves brain serotonin and dopamine content.</a> Medicine &amp; Science in Sports &amp; Exercise. 42 (8), 1469-1476 (2010).</li></ol>

The MMPM is originally designed for sleep deprivation9. Rats are launched into a water tank with platforms fixed on the bottom. Driven by the instinctive fear of water, rats remain standing on the platforms and no sleep occurs. The study shows that different hours of sleep deprivation lead to various changes in rat behavior and mood, including recognition impairment10, negative emotions11, and central fatigue. Some researchers prove that chronic slee...

Fatigue is one of the main factors that threaten human health1. In the past decades, various researches have proved that fatigue is peripherally-triggered but centrally-driven, and always accompanied with emotional and cognitive disorders. Italian physiologist A. Mosso first proposed the word Central Fatigue2. It is generally defined as limited voluntary activity and cognition impairment due to dysfunction of impulse transmission in the central nervous system (CNS)3. Compared with peripheral muscle fatigue, central fatigue emphasizes changes in the CNS, as well as the consequent emotional/behavioral disturbances, including depression, anxiety, cognition impairment, and memory loss. One study shows that many factors can induce central fatigue, among which excessive physical activity and mental stress are quite indispensable4. As for the pathogenesis, theories like the tryptophan-kynurenine pathway hypothesis5 explain changes in certain pathways; however, more in-depth studies are still required&#160;to reveal the central-peripheral correlations of central fatigue.
As the underlying mechanism of central fatigue is still unclear, an effective animal model is quite important for further research. The existing fatigue models are mostly induced by excessive exercise, like treadmills6 and weight-loaded swimming7, with little concern on mental factors. To better simulate the development of central fatigue, our group developed a rat model with the MMPM. During the modeling process, rats remain standing on the narrow platforms in the Multiple Platform box for long hours including part of the sleep time. Different from excessive exercise models, the MMPM model uses partial sleep deprivation as a mental factor in consideration of the complex pathogenesis of central fatigue.
For model evaluation, we use the OFT and EPM tests to determine anxiety mood and voluntary activity. The ES test is performed to measure the peripheral muscle performance. In addition, we take the rat&#39;s brain and detect dopamine (DA)/serotonin (5-HT) content in both hypothalamuses to observe the central neurotransmitter differences.
The protocol presented below is designed to model central fatigue induced by repeated physical activity and lack of sleep, mimicking a common condition in human life. However, by adjusting the model duration, it can be used in many other fields, like in sleep observation and stress studies. In future research, we hope that this model will help discover more CNS changes and their connection with the peripheral system, to reveal the pathogenesis mechanism of central fatigue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>multiple platform sleep deprivation water tank</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>110cm x 60cm x 40cm. There are 15 plastic small platforms at the bottom. The small platform is 6.5cm in diameter and 8cm high</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Beijing Weitong Lihua Experimental Animal Technology Company</td>
			<td>license number SYXK (Beijing) 2016-0011</td>
			<td>Use 32 Wistar healthy male rats ,8 week old (200-210 g)</td>
		</tr>
		<tr>
			<td>Agilent 1100LC high performance liquid chromatograph&#160;</td>
			<td>Agilent</td>
			<td>&#160;G1379A, G1311A, G1313A , G1316A&#160;</td>
			<td>&#160;G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box</td>
		</tr>
		<tr>
			<td>DECADE II SDC electrochemical detector</td>
			<td>Dutch ANTEC company</td>
			<td></td>
			<td>glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)</td>
		</tr>
		<tr>
			<td>Biofuge Stratos high-speed refrigeration centrifuge</td>
			<td>HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VCX130 ultrasonic fracturing instrument</td>
			<td>SONICS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACS-ZEAS electronic scale</td>
			<td>Phos technology development, Beijing.</td>
			<td></td>
			<td>The weight of the weighing rats can be accurate to 0.1g.</td>
		</tr>
		<tr>
			<td>Open Field Box</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>wooden box of open field&#160; 100 cm by 100 cm x 40 cm, inside wall and bottom as the gray.The bottom is divided into 25 equal area squares, each of which is 20cm x 20cm, and the 16 grids along the outer wall are the external ones, and the other 9 grids are central.The camera is mounted above the median.</td>
		</tr>
		<tr>
			<td>Elevated Plus-maze</td>
			<td>Beijing zhongshi dechuang technology development co. LTD.</td>
			<td></td>
			<td>The open arms and close&#160; arms of the cross are composed of 30cm x 5cm x 15cm, and the central area is 5cm x 5cm, with a camera mounted above the center and 45cm high.</td>
		</tr>
		<tr>
			<td>rat swimming bucket.</td>
			<td>Zhenhua biological instrument equipment co., LTD. Anhui,China.</td>
			<td></td>
			<td>The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Shiya instrument co., LTD., changzhou,China.</td>
			<td></td>
			<td>Control water temperature</td>
		</tr>
		<tr>
			<td>Small water pump</td>
			<td>Xincheng technology co., LTD., chengdu,China.</td>
			<td></td>
			<td>Used for water tank and swimming behavior.</td>
		</tr>
		<tr>
			<td>Ethovition3.0 behavioral software.</td>
			<td>Nuldus,Netherlands</td>
			<td></td>
			<td>Measurement analysis of rat behavior videos.</td>
		</tr>
	</tbody>
</table>

a rat model of central fatigue using a modified multiple platform method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

We describe a rat model of central fatigue using MMPM. 24 Wistar rats are randomly divided into the control group and the model group, with 12 rats in each group. The model apparatus is designed as a water tank with narrow platforms on the bottom (Figure 1). Model Rats stand on the platforms for 14 h per day, including partial sleep time, for 21 days (Figure 2).
Behavio...

Watch this Scientific Journal Video about A Rat Model of Central Fatigue Using a Modified Multiple Platform Method at JoVE.com

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

This method can help answer key questions in the model construction of central fatigue. For example, how to produce fatigue from both physical and psychological aspects and how to assess it. The main advantage of this technique is to compound mental factors with modified multiple platform method to better simulate the onset of central fatigue.We first came up with the idea when we noticed the close association between intermittent sleep deprivation and central fatigue. We tried different hours and days of deprivation to induce central fatigue, and finally, found that 14 hours over 21 days was the right pattern. The model shows good validity in central fatigue, and it can be used in future studies to help review the underlying mechanism of the disease.Begin by constructing an opaque plastic tank without a cover. Fix 15 circular platforms on the bottom of the tank, which orderly distribute in three rows and five columns. Leave enough space between each platform, roughly 13 centimeters between rows and 10 centimeters between columns.Install a faucet in the water outlet on the lateral side of the tank. Finally, make an iron-wire netting cover for the tank, with a food box hanging on it. Begin by numbering the rats'tail roots with a marker.Weigh the rats, excluding the extremely light or heavy ones, and divide the rest randomly into the model and control groups. Put the rats gently into the clean cages, and allow them to acclimate to the lab for at least three days. Provide sufficient water and food supply.Begin by placing the tank on a flat service, then fill with seven centimeters of warm water, approximately one centimeter below the platform flat. Then put one day's worth of fodder and water into the food box, and hang it on the cover. Next, take the model group of rats out of the cage by the tail, and put them gently into the tank.Launch all the rats in the water instead of the platforms to motivate their fear of water. Make sure that every rat gets a platform to stand on, while rats of the control group stay in their original cages with sufficient food and water. Next, cover the tank and monitor the rats.After 14 hours, take the model rats out of the tank, and dry their hair with a dryer. Remark the rats'tails if it fades. Then, return the rat to the original cage with food and water.Finally, flush out the tank and open the faucet to out-flow the sewage. Then, sterilize the tank with a 75%ethanol spray. Open-field test results indicate that, compared with the control group, there is a significant decrease in both rearing movement and average velocity of voluntary activity in model rats.The elevated-plus maze test shows that 21 days of modeling decreased both entries and duration in the open arm significantly compared to the control group, while there was an increase in those in the close arm. The exhaustive swimming test indicated that the swimming duration of the model group is significantly shorter than the control group. Lastly, these results indicate that dopamine in the hypothalamus, as well as the ratio of dopamine to serotonin, significantly decreases in the model group compared to the control group, while serotonin content increases significantly.After watching this video, you should have a good understanding of how to build a multiple platform tank and apply it in your central fatigue model. This is a non-invasive model, which is easy to master. While trying this model, it is critical to keep an eye on the rest, as they are quite aggressive in the first two weeks, and the injury may occur.Though this model aims to provide insight into central fatigue, it can also be applied to other diseases by compounding factors or adjusting model durations, such as navigation fatigue and depression.

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Research

JoVE Journal

Neuroscience

8.8K 조회수.  Beijing University of Chinese Medicine. This method can help answer key questions in the model construction of central fatigue. For example, how to produce fatigue from both physical and psychological aspects and how to assess it. The main advantage of this technique is to compound mental factors with modified multiple platform method to better simulate the onset of central fatigue.We first came up with the idea when we noticed the close association between intermittent sleep deprivation and central fatigue. We tried differe...