Establishing a Device for Sleep Deprivation in Mice

Summary

The present protocol outlines a method for setting up a cost-effective rocker platform-based device used for inducing sleep deprivation in mice. This device has proven to be effective in causing disruptions in electroencephalogram (EEG)-evidenced sleep patterns, as well as inducing metabolic and molecular changes associated with sleep deprivation.

Abstract

Circadian rhythm disruption refers to the desynchronization between the external environment or behavior and the endogenous molecular clock, which significantly impairs health. Sleep deprivation is one of the most common causes of circadian rhythm disruption. Various modalities (e.g., platforms on the water, gentle handling, sliding bar chambers, rotating drums, orbital shakers, etc.) have been reported for inducing sleep deprivation in mice to investigate its effects on health. The current study introduces an alternative method for sleep deprivation in mice. An automated rocker platform-based device was designed that is cost-effective and efficiently disrupts sleep in group-housed mice at adjustable time intervals. This device induces characteristic changes of sleep deprivation with minimal stress response. Consequently, this method may prove useful for investigators interested in studying the effects and underlying mechanisms of sleep deprivation on the pathogenesis of multiple diseases. Moreover, it offers a cost-effective solution, particularly when multiple sleep deprivation devices are required to run in parallel.

Introduction

Circadian rhythm disruption refers to the desynchronization between the external environment or behavior and the endogenous biological clock. One of the most common causes of circadian rhythm disruption is sleep deprivation¹. Sleep deprivation not only negatively affects human health but also significantly increases the risk of many diseases, including cancer² and cardiovascular diseases³. However, the mechanisms underlying the detrimental effects of sleep deprivation remain largely unknown, and establishing sleep deprivation models is essential to enhance our understanding in this regard.

Various methods for sleep deprivation in mice have been reported, such as the use of water platforms⁴, gentle handling⁵, sliding bar chambers⁶, rotating drums⁷, and cage agitation protocols^5,8,9. Sliding bar chambers automatically sweep bars across the bottom of the cage, forcing the mice to walk over them and stay awake. Cage agitation protocols involve placing cages on laboratory orbital shakers, resulting in efficient sleep disruption. While these methods are automatic and effective, they can be expensive when multiple devices are required to run in parallel, especially for specific study designs that involve a large number of sleep-deprived mice needed for circadian gene profiling. On the other hand, water platforms and gentle handling protocols are cheaper and simpler methods commonly used to induce sleep deprivation. However, the water platform does not allow automatic control of prespecified deprivation-rest cycles^10,11, and gentle handling requires continuous vigilance from the researchers to disturb sleep. Additionally, other modalities, like rotating drums, can be confounded by social isolation or stress¹².

Inspired by the orbital shaker-based method, we aim to introduce a protocol for establishing a rocker platform-based device for sleep deprivation in mice. This method is cheap, effective, minimally stressful, controllable, and automated. The current protocol allows us to create a rocker platform-based device at a cost approximately ten times cheaper than that of orbital shakers, based on our accessibility. This device effectively disrupted sleep in group-housed mice and induced characteristic changes of sleep deprivation with minimal stress response. It will be especially useful for researchers interested in investigating the effects and underlying mechanisms of sleep deprivation on the pathogenesis of multiple diseases, particularly when the study involves multiple-group sleep deprivation in parallel.

Protocol

All animal experimental protocols in this study were approved by the Laboratory Animal Welfare Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Male C57BL/6J mice, aged between 8 to 10 weeks, were used in the study. The animals were obtained from a commercial source (see Table of Materials). The major parts required for establishing the device are listed in Figure 1A.

1. Preparation of the sleep deprivation device

1. Secure one end of a 50 cm slotted steel channel at the middle of a 40 cm slotted steel channel with screws (see Table of Materials) to make a T-shaped structure; repeat the process and make two such T-shaped structures (Figure 1B-a).
2. Make the two T-shaped structures stand upwards parallelly 30 cm apart, and connect the bottoms of the two T-shaped structures with a 30 cm screw-compatible steel cylinder (see Table of Materials) using screws (Figure 1B-b).
3. Place a steel rectangle platform (20 × 25 cm) (see Table of Materials) in between the two T-shaped structures (Figure 1B-c).
   NOTE: If ready-to-use steel rectangle platforms of the specified size are not available, one can make one by welding 2 mm thick flat steels.
4. Secure each end of a 30 cm screw-compatible steel cylinder attached in the platform to two bearings fixed on each of the T-shaped structures at 10 cm down from the top (Figure 1B-d).
5. Secure a motor mount (see Table of Materials) on one of the T-shaped structures at 25 cm down from the top using screws (Figure 1B-e).
   NOTE: Alternatively, construction adhesive can be used to secure the motor mount on the T-shaped structure instead of crews.
6. Install a motor (see Table of Materials) on the motor mount with screws (Figure 1B-f).
7. Secure a cooling fan (see Table of Materials) with self-locking bands on the T-shaped structure below the motor (Figure 1B-g).
8. Fix the bearing end of a connecting rod to a platform corner facing the motor using screws (Figure 1B-h).
9. Fix another end of the connecting rod to the shaft of the motor using screws (Figure 1B-i).
10. Drill two 4 mm holes at each of the four corners of a plastic container or standard animal cage (see Table of Materials) using an electric drill, and drill two 4 mm holes and a lower 6 mm hole on the left side of the cage (Figure 1B-j).
11. Secure the cage on the rectangle platform with self-locking bands through the corner holes (Figure 1B-k).
12. Drill a 5 mm hole in the cap of a 50 mL centrifuge tube with an electric drill, and plug in the hole with a long nozzle equipped with a ball valve to prevent water leakage.
    NOTE: Hydrogel would be an alternative option for water supply if customizing water bottles is difficult.
13. Secure the customized water bottle on the left side of the cage using self-locking bands through the two 4 mm holes, with the nozzle going through the 6 mm hole (Figure 1B-l).
14. Connect the output electrical wires of the power brick adapter to the two terminals of the motor (Figure 1B-l).
    NOTE: There is no specific polarity requirement for connecting the wires to the motor terminals.
15. Connect the input electrical wires of the power brick adapter to the time contactor (Figure 1B-m).

2. Induction of sleep deprivation

1. Press the rightmost plus sign buttons on the left and right halves of the time contactor (see Table of Materials), respectively, until "M" appears on the mechanical counters on both sides (Figure 1C-a).
2. Press the middle plus sign buttons on the left and right halves of the time contactor until "5M" appears on the mechanical counters on both sides (Figure 1C-b).
3. Press the leftmost plus sign button on the left half of the time contactor until "15M" appears on the left mechanical counter (Figure 1C-c).
   ​NOTE: The time contactor will then be on for 15 min and off for 5 min in a cyclic mode.
4. Put mice in the cage with water and food ad libitum.
5. Supply the time contactor and the cooling fan with power.
   NOTE: The platform will now be rocking at 10 rpm.
6. Weigh each mouse at Zeitgeber time 0 (ZT0) every day.
   ​NOTE: The light is on from 8 AM (ZT0) to 8 PM (ZT12).

3. Oral glucose tolerance test

1. Measure the fasting glucose levels in fasted mice by sampling blood from tail veins.
2. Inject glucose solution into each mouse (2 g/kg body weight) intraperitoneally using 1 mL syringes.
3. Collect blood samples through the tail vein, and test the blood glucose at 15 min, 30 min, 60 min, and 120 min after glucose injection, respectively.
4. Put the mice back into the cage with the food and water ad libitum after the test.

4. Harvesting the brain tissues

1. Decapitate the mice after adequate anesthesia by exposing them to isoflurane (2%) for 3-5 min.
2. Expose the skull and make a 1 cm vertical cut at the skull using surgical scissors.
3. Remove the skull using mosquito hemostats (see Table of Materials) to expose the brain tissue.
4. Gently move the whole brain out of the cranial cavity using curved tweezers.
   NOTE: Brain tissue should be removed according to local policies.
5. Wash the brain tissue using cold phosphate-buffered saline (1x PBS, 4 °C).
6. Snap freeze the intact brain tissue in liquid nitrogen and transfer the tissue to -80 °C for long-term storage .
   ​NOTE: When stored at -80°C, the flash-frozen brain tissue is stable for at least 6 months.

5. Detection of gene expression by polymerase chain reaction (PCR)

1. Thaw the brain tissues at 4 °C or on ice.
2. Transfer the tissue to a 1.5 mL microcentrifuge tube, and extract the total RNA using the TRIzol-based method¹³.
3. Measure the concentration of RNA using a spectrophotometer (see Table of Materials) after RNA extraction.
4. Perform reverse transcription of the total RNA (1 µg) into complementary DNA (cDNA) using a commercial kit¹⁴.
5. Measure gene expression levels by real-time reverse transcription polymerase chain reaction¹⁵.

Results

The established device for sleep deprivation in mice is shown in Figure 1D. At day 7 after sleep deprivation commencement, electroencephalogram (EEG) and electromyography (EMG) monitoring¹⁶ indicated that the device significantly reduced sleep duration and increased wakefulness duration in mice (Figure 2A-D). Meanwhile, the current protocol significantly increased adenosine build-up and mRNA levels of

Discussion

Mouse models of sleep deprivation are essential for studying the effects of sleep disruption on various diseases, including cardiovascular disease²¹, psychiatric conditions²², and neurological disorders²³. Among the existing sleep deprivation strategies in mice, physical approaches that involve repetitive short-term interruption of sleep are the most commonly used^5,7,

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Axygen	MCT-150-C-S
50 mL centrifuge tube	NEST	602002
Adenosine ELISA kit	Ruifan technology	RF8885
Animal cage	ZeYa tech	MJ2
Blood glucose meter	YuYue	580
C57BL/6J Mice	JieSiJie Laboratory Animal	N/A	Age: 8-10 weeks
Connecting rod	ShengXiang Tech	N/A	Length:  20 cm
Cooling fan	LiMing	EFB0805VH	Supply voltage: 5 V; Power consumption: 1.2 W; Air flow: 26.92 cfm; Dimensions: 40 mm * 40 mm * 56 mm
Corticosterone ELISA kit	Elabscience	E-OSEL-M0001
EEG/EMG recording and analysis system	Pinnacle Technology	8200-K1-iSE3
Isoflurane	RWD	20071302
mosquito hemostats	FST	13011-12	Surgical instrument
Motor and motor mount	MingYang	MY36GP-555	Supply voltage: 24 V dc; Shaft diameter: 8 mm; Maximum output torque: 100 Kgf.cm; Maximum output speed: 10 rpm
NanoDrop 2000c	Thermo Scientific	NanoDrop 2000c
Power brick adapter	MingYang	QiYe-0243	Input voltage: 110-220V ac; Output voltage: 24 V dc; Outputcurrent: 2 A; Cable length: 2 m
qPCR commercial kit	Vazyme	Q711-02
qPCR measurement equipment	Roche	480
Rectangle platform attached with a screw-compatible steel cylinder	Customized	N/A	Width: 20 cm; length: 25 cm; length of the cylinder: 30 cm, thickness: 2 mm
Reverse RNA to cDNA commercial kit	Vazyme	R323-01
Screw and nut	Guwanji	N/A	Inner diameter: 6 mm, 12 mm
Screw-compatible steel cylinder	Customized	N/A	Length: 300 mm
Slotted steel channels	Customized	N/A	Length: 400 mm or 500 mm, thickness: 2 mm
Time contactor	LiXiang	DH48S-S	Supply voltage: 110-220 V ac; Units measured: hours, minutes, seconds; Contact configuration: DPDT
TRIzol	Vazyme	R401-01