This work was supported by grants from the National Natural Science Foundation of China (82230014, 81930007, 82270342), the Shanghai Outstanding Academic Leaders Program (18XD1402400), the Science and Technology Commission of Shanghai Municipality (22QA1405400, 201409005200, 20YF1426100), Shanghai Pujiang Talent Program (2020PJD030), SHWSRS(2023-62), Shanghai Clinical Research Center for Aging and Medicine (19MC1910500), and Postgraduate Innovation Program of Bengbu Medical College (Byycxz21075).

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
<ol>
	<li>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).</li>
	<li>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).</li>
	<li>Place a steel rectangle platform (20 &#215; 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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Install a motor (see Table of Materials) on the motor mount with screws (Figure 1B-f).</li>
	<li>Secure a cooling fan (see Table of Materials) with self-locking bands on the T-shaped structure below the motor (Figure 1B-g).</li>
	<li>Fix the bearing end of a connecting rod to a platform corner facing the motor using screws (Figure 1B-h).</li>
	<li>Fix another end of the connecting rod to the shaft of the motor using screws (Figure 1B-i).</li>
	<li>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).</li>
	<li>Secure the cage on the rectangle platform with self-locking bands through the corner holes (Figure 1B-k).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Connect the input electrical wires of the power brick adapter to the time contactor (Figure 1B-m).</li>
</ol>
2. Induction of sleep deprivation
<ol>
	<li>Press the rightmost plus sign buttons on the left and right halves of the time contactor (see Table of Materials), respectively, until &#34;M&#34; appears on the mechanical counters on both sides (Figure 1C-a).</li>
	<li>Press the middle plus sign buttons on the left and right halves of the time contactor until &#34;5M&#34; appears on the mechanical counters on both sides (Figure 1C-b).</li>
	<li>Press the leftmost plus sign button on the left half of the time contactor until &#34;15M&#34; appears on the left mechanical counter (Figure 1C-c). 
	&#8203;NOTE: The time contactor will then be on for 15 min and off for 5 min in a cyclic mode.</li>
	<li>Put mice in the cage with water and food ad libitum.</li>
	<li>Supply the time contactor and the cooling fan with power. 
	NOTE: The platform will now be rocking at 10 rpm.</li>
	<li>Weigh each mouse at Zeitgeber time 0 (ZT0) every day. 
	&#8203;NOTE: The light is on from 8 AM (ZT0) to 8 PM (ZT12).</li>
</ol>
3. Oral glucose tolerance test
<ol>
	<li>Measure the fasting glucose levels in fasted mice by sampling blood from tail veins.</li>
	<li>Inject glucose solution into each mouse (2 g/kg body weight) intraperitoneally using 1 mL syringes.</li>
	<li>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.</li>
	<li>Put the mice back into the cage with the food and water ad libitum after the test.</li>
</ol>
4. Harvesting the brain tissues
<ol>
	<li>Decapitate the mice after adequate anesthesia by exposing them to isoflurane (2%) for 3-5 min.</li>
	<li>Expose the skull and make a 1 cm vertical cut at the skull using surgical scissors.</li>
	<li>Remove the skull using mosquito hemostats (see Table of Materials) to expose the brain tissue.</li>
	<li>Gently move the whole brain out of the cranial cavity using curved tweezers. 
	NOTE: Brain tissue should be removed according to local policies.</li>
	<li>Wash the brain tissue using cold phosphate-buffered saline (1x PBS, 4 &#176;C).</li>
	<li>Snap freeze the intact brain tissue in liquid nitrogen and transfer the tissue to -80 &#176;C for long-term storage . 
	&#8203;NOTE: When stored at -80&#176;C, the flash-frozen brain tissue is stable for at least 6 months.</li>
</ol>
5. Detection of gene expression by polymerase chain reaction (PCR)
<ol>
	<li>Thaw the brain tissues at 4 &#176;C or on ice.</li>
	<li>Transfer the tissue to a 1.5 mL microcentrifuge tube, and extract the total RNA using the TRIzol-based method13.</li>
	<li>Measure the concentration of RNA using a spectrophotometer (see Table of Materials) after RNA extraction.</li>
	<li>Perform reverse transcription of the total RNA (1 &#181;g) into complementary DNA (cDNA) using a commercial kit14.</li>
	<li>Measure gene expression levels by real-time reverse transcription polymerase chain reaction15.</li>
</ol>

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The authors have nothing to disclose.

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 used5,7,12. These physical approaches include the use of water platforms4, gentle handling5, sliding bar chambers6,24, rotating drums7, or orbital shakers8,9,25,26.
To induce sleep deprivation in mice effectively, ideal methods should awaken the mice with a non-stressful stimulus. The chosen device should also be automated and easily controllable to adjust the deprivation-rest cycles. Among the mentioned methods, the sliding bar chamber meets most of these requirements. However, it is expensive and may occasionally harm the mice. Another effective and minimally stressful method is the orbital shaker-based protocol7,8,9,25, where a cage is placed on a standard laboratory orbital shaker connected to a time controller, leading to repetitive sleep interruption. However, when specific study designs require multiple orbital shakers to run in parallel, the cost may become prohibitive for some research groups.
Inspired by the orbital shaker methods, the current study presents a detailed step-by-step protocol for establishing a rocker platform-based sleep deprivation equipment. Its cost is approximately one-tenth of laboratory orbital shakers, making it more accessible. The introduced device was validated to be effective in sleep deprivation in mice, as indicated by EEG/EMG monitoring data that showed significantly shortened sleep duration and increased sleep deprivation markers. Additionally, this rocker platform-based device did not significantly alter serum corticosterone levels in mice. Overall, we have introduced a new automated sleep deprivation device that is inexpensive, effective, minimally stressful, and controllable.
Despite its advantages, the current protocol has some limitations. Firstly, unlike ready-to-use commercially available devices, the sleep deprivation device introduced here requires assembly by the experimenters. However, detailed step-by-step protocols and illustrations have been provided to simplify the process. Secondly, not all the materials required for device establishment are commercially available, and some customization of materials may be necessary based on the specifications provided in this work. Thirdly, conventional water bottles used with the rocker platform may experience leakage, necessitating the use of customized water bottles to prevent this issue.
In conclusion, this study presents a cost-effective and efficient method for establishing an alternative device to induce sleep deprivation in group-housed mice. This protocol can aid researchers in investigating the effects and underlying mechanisms of sleep deprivation on a wide range of health conditions.

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 protocols5,8,9. 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 cycles10,11, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>NEST</td>
			<td>602002</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine ELISA kit</td>
			<td>Ruifan technology</td>
			<td>RF8885</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal cage</td>
			<td>ZeYa tech</td>
			<td>MJ2</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood glucose meter</td>
			<td>YuYue</td>
			<td>580</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>JieSiJie Laboratory Animal</td>
			<td>N/A</td>
			<td>Age: 8-10 weeks</td>
		</tr>
		<tr>
			<td>Connecting rod</td>
			<td>ShengXiang Tech</td>
			<td>N/A</td>
			<td>Length:&#160; 20 cm</td>
		</tr>
		<tr>
			<td>Cooling fan</td>
			<td>LiMing</td>
			<td>EFB0805VH</td>
			<td>Supply voltage: 5 V; Power consumption: 1.2 W; Air flow: 26.92 cfm; Dimensions: 40 mm * 40 mm * 56 mm</td>
		</tr>
		<tr>
			<td>Corticosterone ELISA kit</td>
			<td>Elabscience</td>
			<td>E-OSEL-M0001</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG/EMG recording and analysis system</td>
			<td>Pinnacle Technology</td>
			<td>8200-K1-iSE3</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>20071302</td>
			<td></td>
		</tr>
		<tr>
			<td>mosquito hemostats</td>
			<td>FST</td>
			<td>13011-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Motor and motor mount</td>
			<td>MingYang</td>
			<td>MY36GP-555</td>
			<td>Supply voltage: 24 V dc; Shaft diameter: 8 mm; Maximum output torque: 100 Kgf.cm; Maximum output speed: 10 rpm</td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 2000c</td>
			<td></td>
		</tr>
		<tr>
			<td>Power brick adapter</td>
			<td>MingYang</td>
			<td>QiYe-0243</td>
			<td>Input voltage: 110-220V ac; Output voltage: 24 V dc; Outputcurrent: 2 A; Cable length: 2 m</td>
		</tr>
		<tr>
			<td>qPCR commercial kit</td>
			<td>Vazyme</td>
			<td>Q711-02</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR measurement equipment</td>
			<td>Roche</td>
			<td>480</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangle platform attached with a screw-compatible steel cylinder</td>
			<td>Customized</td>
			<td>N/A</td>
			<td>Width: 20 cm; length: 25 cm; length of the cylinder: 30 cm, thickness: 2 mm</td>
		</tr>
		<tr>
			<td>Reverse RNA to cDNA commercial kit</td>
			<td>Vazyme</td>
			<td>R323-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw and nut</td>
			<td>Guwanji</td>
			<td>N/A</td>
			<td>Inner diameter: 6 mm, 12 mm</td>
		</tr>
		<tr>
			<td>Screw-compatible steel cylinder</td>
			<td>Customized</td>
			<td>N/A</td>
			<td>Length: 300 mm</td>
		</tr>
		<tr>
			<td>Slotted steel channels</td>
			<td>Customized</td>
			<td>N/A</td>
			<td>Length: 400 mm or 500 mm, thickness: 2 mm</td>
		</tr>
		<tr>
			<td>Time contactor</td>
			<td>LiXiang</td>
			<td>DH48S-S</td>
			<td>Supply voltage: 110-220 V ac; Units measured: hours, minutes, seconds; Contact configuration: DPDT</td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Vazyme</td>
			<td>R401-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a device for sleep deprivation in mice

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.

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 Homer1a in the brain (Figure 2E,F), which are markers of successful sleep deprivation17. Using an ELISA kit18, we observed that serum corticosterone levels were not significantly changed by the current sleep deprivation protocol (Figure 2G). Following sleep deprivation for 7 days, body and thymus weight significantly decreased (Figure 3A-D), consistent with previous reports19. Moreover, glucose tolerance was significantly impaired in mice after sleep deprivation (Figure 3E,F). To investigate the changes in clock gene expression, brain tissues were collected every 4 h throughout a day. We observed that the expression patterns of the clock genes in the brain were significantly changed after sleep deprivation (Figure 3G and Table 1), suggesting disruption of the molecular clock20.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65157/65157fig01v2.jpg" /> 
Figure 1: Establishment of the rocker platform-based device. (A) Illustrations depicting the major parts required for assembling the rocker platform-based device. (B) Detailed steps demonstrating the assembly of the sleep deprivation device. (C) Images displaying parameter settings in the time contactor. (D) Photograph of the fully assembled shaking chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65157/65157fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65157/65157fig02.jpg" /> 
Figure 2: Evaluation of sleep disturbance and serum corticosterone levels in mice. (A) Schematic diagram illustrating EEG/EMG recording in mice during sleep deprivation. (B) Representative EEG/EMG recordings traced in mice. (C) Representative EEG/EMG waveforms in mice during wakefulness, NREM, and REM. (D) Percentage of wakefulness duration, NREM duration, and REM duration recorded in sleep-disrupted mice and control mice (n = 4 mice/group). *P &#60; 0.05, ***P &#60; 0.001. Statistical analysis was performed using an unpaired t-test. Abbreviations: CTR, control group; NREM, Non-rapid eye movement; REM, Rapid eye movement; SD, sleep deprivation group. (E) mRNA levels of Homer1a in brain tissues measured in sleep-disrupted mice and control mice (n=4 mice/group). *P &#60; 0.05, Statistical analysis was performed using an unpaired t-test. Abbreviations: CTR, control group; Homer1a, Homer scaffolding protein 1a; SD, sleep deprivation group. (F) Adenosine content in brain tissue measured in sleep-disrupted mice and control mice (n=4 mice/group) using an ELISA kit. *P &#60; 0.05, Statistical analysis was performed using unpaired t-test at each time point. Abbreviations: CTR, control group; SD, sleep deprivation group. (G) Serum corticosterone concentration measured in sleep-disrupted mice and control mice (n = 4 mice per time point/group). Statistical analysis was performed using two-way analysis of variance. Abbreviations: CTR, control group; SD, sleep deprivation group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65157/65157fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65157/65157fig03.jpg" /> 
Figure 3: Pathophysiological changes after sleep deprivation in mice. (A) Representative images showing the sizes of mice in the indicated groups. Abbreviations: CTR, control group; SD, sleep deprivation group. (B) Changes in body weight after the initiation of sleep deprivation in the indicated groups (n = 24 mice per group). **P &#60; 0.001, statistical analysis was performed using an unpaired t-test. Abbreviations: CTR, control group; SD, sleep deprivation group. (C) Representative images showing the sizes of the thymus in the indicated groups. Abbreviations: CTR, control group; SD, sleep deprivation group. (D)&#160;Comparison of the ratios of thymus weight to body weight between the two groups (n = 24 mice per group). **P &#60; 0.001, statistical analysis was performed using an unpaired t-test. Abbreviations: CTR, control group; SD, sleep deprivation group. (E)&#160;Intraperitoneal glucose tolerance test results in the indicated groups (n = 5 per group). *P &#60; 0.01; **P &#60; 0.001, statistical analysis was performed using an unpaired t-test. Abbreviations: CTR, control group; SD, sleep deprivation group. (F) Comparison of the area under the curve (AUC) of the intraperitoneal glucose tolerance test between the sleep deprivation group and the control group (n = 5 per group). **P &#60; 0.001, statistical analysis was performed using an unpaired t-test. Abbreviations: CTR, control group; SD, sleep deprivation group. (G) Circadian expression patterns of the clock genes (Bmal1, Dbp, Cry1, Cry2, Nr1d1, Nr1d2, Per1, and Per2) in brain tissues were measured in the sleep deprivation group and control group (n = 4 mice per time point/group). Data were compared using non-linear cosinor regression, and the expression curves of the clock genes were fitted using the R package CircaCompare. P-values are provided as indicated. Abbreviations: A, amplitude; Bmal1, brain and muscle Arnt-like 1; Cry1, cryptochrome circadian regulator 1; Cry2, cryptochrome circadian regulator 2; CTR, control group; Dbp, D-site binding protein; M, mesor; Nr1d1, nuclear receptor subfamily 1 group D member 1; Nr1d2, nuclear receptor subfamily 1 group D member 2; P, phase; Per1, period circadian regulator 1; Per2, period circadian regulator 2; SD, sleep deprivation group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65157/65157fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Clock genes</td>
			<td>Bmal</td>
			<td>Dbp</td>
			<td>Cry1</td>
			<td>Cry2</td>
			<td>Nr1d1</td>
			<td>Nr1d2</td>
			<td>Per1</td>
			<td>Per2</td>
		</tr>
		<tr>
			<td rowspan="2">Rhythmicity</td>
			<td>Control</td>
			<td>P &#60; 0.05</td>
			<td>P &#60; 0.01</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.05</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.01</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
		</tr>
		<tr>
			<td>Sleep deprivation</td>
			<td>P &#60; 0.05</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
			<td>P &#60; 0.001</td>
		</tr>
		<tr>
			<td rowspan="2">Acrophase (Zeitgeber time)</td>
			<td>Control</td>
			<td>22</td>
			<td>12</td>
			<td>17</td>
			<td>15</td>
			<td>11</td>
			<td>14</td>
			<td>14</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Sleep deprivation</td>
			<td>10</td>
			<td>24</td>
			<td>4</td>
			<td>3</td>
			<td>22</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td rowspan="2">Mesor estimate</td>
			<td>Control</td>
			<td>0.933</td>
			<td>2.242</td>
			<td>1.136</td>
			<td>1.171</td>
			<td>1.799</td>
			<td>1.41</td>
			<td>1.289</td>
			<td>1.033</td>
		</tr>
		<tr>
			<td>Sleep deprivation</td>
			<td>0.826</td>
			<td>2.101</td>
			<td>1.094</td>
			<td>1.155</td>
			<td>1.756</td>
			<td>1.399</td>
			<td>0.999</td>
			<td>0.888</td>
		</tr>
		<tr>
			<td rowspan="2">Amplitude estimate</td>
			<td>Control</td>
			<td>0.099</td>
			<td>0.746</td>
			<td>0.305</td>
			<td>0.131</td>
			<td>0.494</td>
			<td>0.314</td>
			<td>0.294</td>
			<td>0.341</td>
		</tr>
		<tr>
			<td>Sleep deprivation</td>
			<td>0.108</td>
			<td>0.866</td>
			<td>0.342</td>
			<td>0.168</td>
			<td>0.503</td>
			<td>0.323</td>
			<td>0.388</td>
			<td>0.305</td>
		</tr>
	</tbody>
</table>
Table 1: The presence of the rhythmicity, mesor estimate, amplitude estimate, and the acrophase for the tested clock genes in each group.

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Establishing a Device for Sleep Deprivation in Mice

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.

Circadian rhythm disruption refers to the desynchronization between the external environment or behavior and the endogenous molecular clock, which ...

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1.3K Views.  The First Affiliated Hospital of Bengbu Medical College. 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.

Video: Establishing a Device for Sleep Deprivation in Mice

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...