Name: a chronic sleep fragmentation model using vibrating orbital rotor to induce cognitive deficit and anxiety-like behavior in young wild-type mice
Uploaded: 2020-09-22T13:30:00
Description: Watch this Scientific Journal Video about A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice at JoVE.com

This work was supported by the National Natural Science Foundation of China (61327902-6 to W. Wang and 81801318 to F.F. Ding). We acknowledge Dr. Sigrid Veasy for establishing the SF experimental system and kindly providing technical details. We acknowledge Dr. Maiken Nedergaard for instructive comments for related experiments.

This protocol was approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.
1. Mice screening and preparation for the experiment
<ol>
	<li>Select wild-type adult (8&#8211;10 weeks old) male mice with weight of 20&#8211;28 g for the whole experiment. 
	NOTE: Wild-type C57BL/6 mice are obtained from the Hubei Research Center for Laboratory Animals, Hubei, China.</li>
	<li>Randomly assign all mice ...

Critical steps in the current protocol include setting up sleep fragmentation machines with the optimized parameters according to the study purpose and maintaining the mice in comfortable and quiet living environment throughout the entire modeling sessions. It is also crucial to decide the proper timing to interrupt or stop sleep fragmentation and arrange behavioral tests for those mice. Like other sleep manipulation models, it is important to perform the protocol in a dedicated room with controlled light cycles and void...

Sleep disturbance is increasingly common both in patients with sleep-disturbing conditions and healthy people with sleep-disturbing events. It has been observed that&#160;patients with neurodegenerative diseases, chronic pain, emotional stress, respiratory system diseases, urinary system diseases, etc., usually complain about unpleasant sleep experiences1,2,3,4,5. Obstructive sleep apnea (OSA), periodic limb movements in sleep (PLMS), sleep maintenance insomnia among other sleep disorders are the most common causes, which induce sleep fragmentation6,7. In developed countries, OSA has over 5% to 9% prevalence in adult population and 2% in child population8,9,10. Meanwhile, there is an increasing proportion of the healthy population experiencing sleep disturbance due to the overuse of smart phones, irregular sleep habits, annoying noises, and work duties, such as night shifts for caregivers. Sleep is acknowledged to be important for brain waste clearance11,12, memory consolidation13,14, metabolic balance15,16, among many other physiological processes. Yet, it still remains largely unknown whether long-term sleep disturbance gives rise to irreversible pathogenesis alterations in healthy human beings, and whether it is the etiology or a contributing factor of developing central nervous system diseases, such as neurodegenerative diseases in a couple of years down the road. Our goal is to report an experimental model that generates stable and evident cognitive deficit and anxiety-like behavior in young wild-type mice after a 2-month sleep fragmentation treatment. This model would be applied for answering the scientific questions listed above.
Sleep disturbance is listed as a potential risk factor for developing Alzheimer&#8217;s disease (AD) or dementia. Kang et al. first found and described the exacerbation of AD pathology by 6 h acute sleep deprivation17. Thereafter, many other studies reported that sleep deprivation or fragmentation could aggravate pathogenesis in transgenic AD mice models18,19,20. However, very few researchers have studied the consequence of sleep disturbance in young wild type mice; that is,&#160;whether sleep disturbance gives rise to AD-like behavior or pathological changes in young wild-type mice. In our recent publication, we reported that 2 months of sleep fragmentation induced evident spatial memory deficit and anxiety-like behavior, as well as increased intracellular Amyloid-&#946; (A&#946;) accumulation both in cortex and hippocampus in 2&#8211;3 month-old wild-type mice21. We also observed altered expression levels of endosome-autophagosome-lysosome pathway markers and microglia activation, which was similar to the pathological changes reported in APP/PS1 mice21,22.
This presented sleep fragmentation (SF) protocol was validated by Sinton et al.23 and modified by Li et al.24. In brief, an orbital rotor vibrating at 110 rpm interrupts sleep for 10 s every 2 min during light-ON phase (8:00 AM&#8211;8:00 PM). Sleep structure alteration in this model was previously characterized with electrophysiological sleep recordings and reported by Li et al.24, indicating a significant increase in the wake time and decrease in rapid eye movement (REM) sleep during the light-ON phase, with the total sleep and wake times (in 24 hour) unaffected after more than 4 weeks&#8217; modeling24. Currently, total sleep or partial sleep deprivation are the most commonly used sleep manipulation models. Total sleep deprivation is usually performed by sustained gentle handling or exposing the animal to novel objects, alternatively by continuously rotating a bar or a running treadmill25,26,27,28,29. Due to ethical reasons, total sleep deprivation is usually shorter than 24 h. The most commonly applied partial sleep deprivation model is the water platform method, which primarily ablating REM sleep30,31,32. Other approaches using either a treadmill or a bar that sweeps along the bottom of the cage, could induce sleep fragmentation when set on at fixed intervals33,34,35,36,37,38. It is noteworthy that SF interrupts sleep and intermittently causes arousals across all sleep stages24. One of the prominent advantages of this CSF model applying orbital rotor is that it can be performed continuously for months automatically controlled by machines, which avoids frequent processing labor daily except for regular monitoring. Furthermore, the apparatus would allow to simultaneously model multiple cages of mice under uniformed interventions. During entire modeling sessions, mice are housed in their home cages with usual bedding and nesting materials, while some other methods require exposure to diversified environments and inevitable stress.
Sleep fragmentation was previously characterized by the sleep manipulation method, which mimics frequent arousals during the sleep phase and substantial sleep rebound during the wake phase. In some literatures, CSF was regarded as the animal model for OSA39,40. In this study, the rationale of the chosen frequency of arousal to be 30 times per hour is based on the observation of arousal indices in patients with moderate-to-severe sleep apnea. It was observed that 4 weeks&#8217; sleep fragmentation significantly increased hypercapnic arousal latency and the tactile arousal threshold, which could at least last 2 weeks after recovery24. This phenotype was explained by revealing c-fos activation reduction in noradrenergic, orexinergic, histaminergic, and cholinergic wake-active neurons in response to hypercapnia, as well as reduced catecholaminergic and orexinergic projections into the cingulate cortex24. However, it is necessary to note that the most important feature in OSA is hypoxia caused by airway obstruction, which results in sleep disruption41,42. Sleep disturbance and repetitive hypoxia reciprocally interact with each other in OSA pathogenesis. Therefore, sleep fragmentation alone might not be able to fully demonstrate all key features of OSA in mice.
Herein, we present a standardized protocol to model chronic sleep fragmentation in young wild-type mice. Cognitive deficit and anxiety-like as well as depression-like behaviors after CSF treatment were evaluated by Morris water maze, Novel object recognition, Open field test, and Forced swimming test. It is important to note that this model should be taken as a whole that generates phenotypes of dysregulated sleep pattern, cognitive deficit, and anxiety-like behavior. The current model could potentially be applied, but not be limited, to the following purposes: 1) Further investigating the functional or molecular pathogenesis mechanisms induced by chronic sleep disturbance in young mice without genetic predisposition, 2) Identifying the direct pathway leading to neurodegeneration initiated by sleep disturbance, 3) Exploring the therapeutics for improving phenotypes induced by chronic sleep disturbance, 4) Studying the intrinsic protective/compensatory mechanisms in wild-type mice upon chronic sleep disturbance, 5) To be applied for studying sleep-wake regulation and state-transition mechanisms.

<table>
	<tbody>
		<tr>
			<td>Any-maze behavior tracking system</td>
			<td>Stoelting,Inc,USA</td>
			<td>-</td>
			<td>A video-tracking system which was used to record the behavior track of mice.</td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Hubei Research Center for Laboratory Animals, Hubei, China.</td>
			<td>-</td>
			<td>healthy male C57BL/6J mice aged 10-12 weeks were purchased from Hubei Research Center for Laboratory Animals</td>
		</tr>
		<tr>
			<td>Graphpad Prism 6.0 Software</td>
			<td>Graphpad Software,Inc.USA</td>
			<td>-</td>
			<td>Graphpad Prism 6.0 software was used to draw statistical graphs.</td>
		</tr>
		<tr>
			<td>Morris water maze system</td>
			<td>Shanghai XinRuan Information Technology Co.,Ltd,China</td>
			<td>XR-XM101</td>
			<td>The system was used to perform Morris water maze test</td>
		</tr>
		<tr>
			<td>Orbial rotor</td>
			<td>Shanghai ShiPing Laboratory Equipment Co.,Ltd,China</td>
			<td>SPH-331</td>
			<td>The orbital rotor was used to establish the chronic sleep fragmentation model</td>
		</tr>
		<tr>
			<td>Solid state timer</td>
			<td>OMRON Corporation, Kyoto, Japan</td>
			<td>H3CR-F8-300</td>
			<td>The solid state time was used to control the frequency and time of the rotor running</td>
		</tr>
		<tr>
			<td>Wooden Lusterless Tank</td>
			<td>-</td>
			<td>-</td>
			<td>length 30 cm, width 28 cm, height 35 cm The tank was used to perform open field test and novel object recognition test</td>
		</tr>
	</tbody>
</table>

a chronic sleep fragmentation model using vibrating orbital rotor to induce cognitive deficit and anxiety-like behavior in young wild-type mice

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer&#8217;s disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM&#8211;8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

All the representative results and figures were reproduced from our recent publication21. The reuse of the figures was permitted by the original journal.
The entire experimental design is illustrated in the order of time, which indicates the timing of CSF modeling, behavioral tests of MWM, NOR, OFT, and FST (Figure 1A). We obtained weights of mice every week from the CSF and the control groups, to monitor their general conditions during the...

Watch this Scientific Journal Video about A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice at JoVE.com

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely ...

Chronic sleep disturbance lead to neurodegenerative disease pathogenesis. We would like to provide a simple, stable, and long-term protocol to model frequent interruptions during human sleep to address this question. These mice minimizes handling labors and the artificial buyers.It can standardized interventions among mice within the same experimental group and between different experiments to produce repeatable behavioral and the pathological outcomes. This model can be applied to a variety of disease models such as depression and the Alzheimer's disease to study the impacts of chronic sleep disturbance in the presence of an underlying disease. Begin by randomly assigning three to five 20 to 28 gram 8 to 10-week-old adult wild-type male mice to CSF modeling and control cages to avoid social isolation stress.House both groups of cages in the same 21 to 23 degree Celsius and 35 to 60%humidity modeling room with a 12-hour light/dark cycle to maintain an identical surrounding environment. Then provide the mice with sufficient food and water using long nozzles with ball valve tips on the water bottles to prevent water leakage upon the platform movements and fastening the water bottles on top of each cage with a spring to avoid dislocation of the bottles during rotation. For chronic sleep fragmentation modeling, program an electrically controlled orbital rotor with a 67 by 110 centimeter platform to run during the 8:00 a.m.to 8:00 p.m. light phase and use a solid state timer to set the rotor speed to 110 revolutions per minute for a repetitive 10 seconds on, 110 seconds off cycle. Use thick springs to fasten the CSF cages on top of the rotor platform to prevent dislocation of the cages upon platform rotation and confirm that the food and water access are maintained during the orbital rotations.After setting up the model system, observe the cages for at least one hour to ensure that the orbital rotor is operating appropriately. During the modeling period, weigh the mice weekly at 8:00 a.m. when changing the bedding and remove any aggressors from the cages as necessary.At the end of the modeling period, continue to house and feed the mice in the modeling room. To perform a Morris Water Maze test, between 8:00 a.m. and 12:00 p.m.every day for five days, release one mouse at a time into a circular tank filled with 20 to 23 degree Celsius water in one of four quadrants for each trial. During the trial, use a video tracking system to record the escape latency of each mouse as it attempts to swim to the platform. If the mouse is unable to locate the platform within 60 seconds, guide the animal to the platform.Allow it to remain on the platform for 15 seconds before returning it to its cage. On the sixth day after training, remove the platform from the water tank and release the mouse from the northeast quadrant for one final 60-second swim period per animal. After CSF modeling, no differences in the weights of mice between the control and experimental groups are observed.The CSF group displays a reduced escapability in locating the platform over a period of five training days in the Morris Water Maze behavioral trial compared to the control animals. In the probe test, the CSF mice spent significantly less time proportionally in the targeted quadrant, crossing the previous platform location fewer times without an observable swimming speed difference. In the familiar phase of the novel object recognition test, there is no significant difference in the total exploration time between the CSF and control groups or in the exploration time between objects A1 and A2 in either group.In the test phase, the discrimination index of the CSF mice is significantly reduced compared to that of the control animals. In these representative open field test and forced swimming test evaluations, the CSF group spent less time in the central zone during the test than did the control group. CSF mice also exhibited a longer total distance of movement within the tank, suggesting increased spontaneous activity after modeling.Nevertheless, the CSF modeling did not induce depression-like behavior as verified by non-significant differences in the immobility time between the two groups subjected to forced swimming test. Selecting an appropriate vibrating cycle and modeling duration as well as a proper timeline for the CSF treatment and phenotype identification are important for the success of this experiment. Traditional sleep deprivation protocols require a great deal of work and the mostly for acute or short-term modeling.This protocol allows the study of a pathological mechanism of mild but long lasting sleep disturbance.

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