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

Summary

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

Abstract

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 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.

Introduction

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 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'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.

Protocol

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

1. Laboratory preparation
   1. Run the UV lamp for at least 30 min before the experiment.
   2. Control the lab temperature at 25 ± 3 °C, and relative humidity around 30%.
   3. Turn on the lab light at 6:00 and turn it off at 18:00 to establish a 12 h/12 h light/dark cycle.
2. Multiple Platform box construction
   1. Construct an opaque plastic tank without a cover of 110 × 60 × 40 cm³.
   2. Fix fifteen circular platforms (h = 8 cm, d = 6.5 cm) on the bottom of the tank, which orderly distribute in three rows and five columns. Leave enough space between each platform, roughly 10 cm between columns and 13 cm between rows.
   3. Set a water outlet on the lateral side of the tank and install a faucet.
   4. Make an iron-wire netting cover for the tank with a food box hanging on it.
3. Grouping and housing rats
   NOTE: Wistar male rats of 8 weeks old, weighing approximately 200 - 210 g, are used in the experiment. The rats live in groups during the modeling process.
   1. Number the rats' tail roots with a marker pen.
   2. Weigh the rats, exclude the extremely light or heavy ones, and divide the rest randomly into the model and control groups.
   3. Put the rats gently into the clean cages and allow them to acclimate to the lab for at least 3 days. Provide sufficient water and food supply.

2. Modeling with MMPM

NOTE: The process starts at 18:00 and ends at 8:00 the next day, for a total of 14 h per day, over 21 days. To avoid interference factors, the same person is required to conduct the entire experiment, while wearing the same lab coat. 10 Wistar rats are used in the experiment.

1. Place the tank on a flat surface, e.g., the floor. Then fill the tank with roughly 7 cm of warm (25 ± 3 °C) water, approximately 1 cm below the platform flat.
2. Prepare enough food and drink for all the rats in the tank for 1 day. Put fodder and water into the food box and hang it on the cover.
   NOTE: Some smart rats learn to rest on the food box. If so, drive them back into the tank.
3. Take the model group rats out of the cage, grab them 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.
4. Cover the tank. Monitor the rats to avoid accidental injury. If a rat stays in water for more than 1 h without climbing onto the platform, pick it out of the tank and remove it from the test.
5. After 14 h, take the model rats out of the tank and dry their hair with a dryer. Re-mark the rats' tails if it fades. Return the rats to their original cages and provide them with sufficient food and water.
6. Flush every corner of the tank. Elevate one side of the tank and open the faucet to outflow the sewage.
7. Sterilize the tank with a 75% ethanol spray and expose it to the UV light.

3. Model Assessment: Behavioral Test

NOTE: All the tests are performed in the behavioral lab. Noise and extra light are not allowed during the test to avoid disturbance. If possible, use the same person(s) to conduct each test. A dark coat and gloves are required for grey scale recognition in image processing. Perform the OFT first as it has the least effect on rat behavior.

1. OFT
   1. Check the recorder over the Open Field Box to make sure it is properly connected to the workstation and covers every corner of the box. Adjust the lighting to eliminate shadows in the box.
   2. Move the rats into the behavioral lab in their original cages. Allow them to acclimate for at least 1 h before the test.
   3. Clean and sanitize the box with 75% ethanol to ensure that there is no excrement or smell left from previous experiment.
   4. Remove a rat from the cage by its back and put it gently into the central area of the box. Quickly retreat arms from the box so as not to block the shot.
   5. Input the rat's number and start recording. Count and record the frequency of the rat's vertical activities, including rearing and climbing.
   6. After 5 min, stop recording, take the rat out of the box, and return it to the cage.
   7. Repeat steps 3.1.3 - 3.1.6 until all the rats have finished the test.
2. EPM
   1. Perform the pre-check and acclimation steps as for the OFT (steps 3.1.1 - 3.1.2).
   2. Remove a rat from the cage by its back and put it gently onto the junction part of the two arms. Land the rat towards the left open arm and leave quickly so as not to block the shot.
   3. Input the rat's number and start recording. Count and record the frequency of different arm entrances. If the rat drops off the maze in the test, pick it up and send it back to the maze. Record detailed information for data analysis.
   4. After 5 min, stop recording, take the rat out, and return it to the cage.
   5. Remove the excrement and wipe the maze with 75% ethanol to eliminate the former rat's smell.
   6. Repeat steps 3.2.2 - 3.2.5 until all the rats have finished the test.
3. ES test
   1. Fill the swimming tank (70 × 30 × 110 cm³) with 80 cm of warm (25 ± 3 °C) water.
      NOTE: If there is a thermostat in the tank, the water temperature should be set around 37 °C, which is similar to the rat's body temperature. If not, set it to room temperature to keep it constant.
   2. Make a load for each rat with pin bunches and tie it gently on its tail root. The load weighs 10% of the rat's weight.
   3. Grab a rat by the tail and throw it into the swimming tank. If the rats huddle or cling to the wall, set them apart and drive them back into water.
   4. Start timing at the moment when the rat is put into water and stop timing when it's exhausted, which is demonstrated as the failure to struggle out of water with the mouth and nose beneath the water for more than 10 s.
      NOTE: Sometimes, exhaustion and drowning occur suddenly. Be sure to have enough experimenters to record and save the animal at the same time.
   5. Remove the exhausted rats out of the water without interrupting others. Dry their hair, re-mark their numbers, and send them back to cage.
   6. Change the water in the tank after one group finishes. After all the rats are done, empty the swimming tank, and clean and sterilize it with ethanol and UV light.

4. Model Assessment: Central Neurotransmitter Detection

1. Anesthetize the rat with intra-peritoneal injection of 10% chloral hydrate (3 mL/kg) until it is unconscious.
2. Decapitate the rat.
3. Make a longitudinal incision along the post-medial line, open the cranium to both sides, and expose the brain. Turn the cranium over, remove the brain, and put the brain on an ice bag.
4. Separate and remove the hypothalamus, which is the diamond-shaped area in the central part of the base of the brain that has a clear boundary with surrounding tissues. Place it in a sterile tube and freeze it with liquid nitrogen. Store all the samples in a -80 °C refrigerator.
5. Detect the content of DA and 5-HT in the hypothalamus using high-performance liquid chromatography (HPLC)⁸.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
multiple platform sleep deprivation water tank	Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine		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
Wistar rats	Beijing Weitong Lihua Experimental Animal Technology Company	license number SYXK (Beijing) 2016-0011	Use 32 Wistar healthy male rats ,8 week old (200-210 g)
Agilent 1100LC high performance liquid chromatograph	Agilent	G1379A, G1311A, G1313A , G1316A	G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box
DECADE II SDC electrochemical detector	Dutch ANTEC company		glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)
Biofuge Stratos high-speed refrigeration centrifuge	HERAEUS
VCX130 ultrasonic fracturing instrument	SONICS
ACS-ZEAS electronic scale	Phos technology development, Beijing.		The weight of the weighing rats can be accurate to 0.1g.
Open Field Box	Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine		wooden box of open field  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.
Elevated Plus-maze	Beijing zhongshi dechuang technology development co. LTD.		The open arms and close  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.
rat swimming bucket.	Zhenhua biological instrument equipment co., LTD. Anhui,China.		The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.
Thermometer	Shiya instrument co., LTD., changzhou,China.		Control water temperature
Small water pump	Xincheng technology co., LTD., chengdu,China.		Used for water tank and swimming behavior.
Ethovition3.0 behavioral software.	Nuldus,Netherlands		Measurement analysis of rat behavior videos.