Name: social isolation model: a noninvasive rodent model of stress and anxiety
Uploaded: 2022-11-11T14:20:00
Description: Watch this Scientific Journal Video about Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety at JoVE.com

This work was funded by the National Institute of Health grant AA17991 (to J.L.), Carefree Biotechnology Foundation (to J.L.), University of Southern California (USC), USC Graduate School Travel/Research Award (to S.W.) Saudi Arabia Cultural Mission Scholarship (to A.A.O.), and Army Health Professions Scholarship Program (to A.S.S.).

All animal experiments are performed according to the protocols approved by the University of Southern California (USC) Institutional Animal Care and Use Committee (IACUC), and all methods are carried out in accordance with relevant guidelines, regulations, and recommendations.
1. Animals
<ol>
	<li>Obtain approval from appropriate animal care committees for the study.</li>
	<li>Set the vivarium to a dark-light 12 h cycle with controlled temperature and humidity between 24 &#...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+social+avoidance+and+social+fear.">Animal models of social avoidance and social fear.</a> Cell and Tissue Research. 354 (1), 107-118 (2013).</li><li>Wang, F., Kessels, H. 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S., Lumertz, F. S., Orso, R., Viola, T. W., de Almeida, R. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+social+isolation+on+the+oxytocinergic+system:+A+systematic+review+and+meta-analysis+of+rodent+data.">Impact of social isolation on the oxytocinergic system: A systematic review and meta-analysis of rodent data.</a> Neuroscience &amp; Biobehavioral Reviews. 134, 104549 (2022).</li><li>Mumtaz, F., Khan, M. I., Zubair, M., Dehpour, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+and+consequences+of+social+isolation+stress+in+animal+model-A+comprehensive+review.">Neurobiology and consequences of social isolation stress in animal model-A comprehensive review.</a> Biomedicine &amp; Pharmacotherapy. 105, 1205-1222 (2018).</li><li>Ranade, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comforting+in+mice.">Comforting in mice.</a> Nature Neuroscience. 24 (12), 1640 (2021).</li><li>Al Omran, A. J., 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=Social+isolation+induces+neuroinflammation+and+microglia+overactivation,+while+dihydromyricetin+prevents+and+improves+them.">Social isolation induces neuroinflammation and microglia overactivation, while dihydromyricetin prevents and improves them.</a> Journal of Neuroinflammation. 19 (1), 2 (2022).</li><li>Watanabe, S., 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=Dihydromyricetin+improves+social+isolation-induced+cognitive+impairments+and+astrocytic+changes+in+mice.">Dihydromyricetin improves social isolation-induced cognitive impairments and astrocytic changes in mice.</a> Scientific Reports. 12 (1), 5899 (2022).</li><li>Silva, J., 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=Modulation+of+hippocampal+GABAergic+neurotransmission+and+gephyrin+levels+by+dihydromyricetin+improves+anxiety.">Modulation of hippocampal GABAergic neurotransmission and gephyrin levels by dihydromyricetin improves anxiety.</a> Frontiers in Pharmacology. 11, 1008 (2020).</li><li>Porter, V. R., 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=Frequency+and+characteristics+of+anxiety+among+patients+with+Alzheimer's+disease+and+related+dementias.">Frequency and characteristics of anxiety among patients with Alzheimer's disease and related dementias.</a> Journal of Neuropsychiatry and Clinical Neuroscience. 15 (2), 180-186 (2003).</li><li>Hossain, M. M., 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=Prevalence+of+anxiety+and+depression+in+South+Asia+during+COVID-19:+A+systematic+review+and+meta-analysis.">Prevalence of anxiety and depression in South Asia during COVID-19: A systematic review and meta-analysis.</a> Heliyon. 7 (4), 06677 (2021).</li><li>. NHGRI. Knockout Mice Fact Sheet Available from: <a target="_blank" href="https://www.genome.gov/about-genomics/fact-sheets/Knockout-Mice-Fact-Sheet">https://www.genome.gov/about-genomics/fact-sheets/Knockout-Mice-Fact-Sheet</a> (2020)</li><li>Takahashi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+stress+and+aggression+in+murine+models.">Social stress and aggression in murine models.</a> Current Topics in Behavioral Neuroscience. 54, 181-208 (2022).</li><li>Lam, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+the+treatment+of+anxiety+disorders:+beyond+guidelines.">Challenges in the treatment of anxiety disorders: beyond guidelines.</a> International Journal of Psychiatry in Clinical Practice. 10, 18-24 (2006).</li><li>Sullens, D. 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Critical steps in the protocol include properly setting up the social isolation cages (i.e., wrapping of opaque bags and reducing the amount of bedding), minimizing the handling and disturbance of mice throughout the isolation period, and making sure the mice obtain and consume the agar with drug completely. It is critical that the vivarium or housing condition is maintained at a constant temperature and humidity, as well as minimized external interferences. Significant effort should be placed toward reducing as...

Anxiety disorders represent the largest class and burden of mental diseases in the United States (US), with related annual costs exceeding US$42 billion1,2,3. In recent years, anxiety and stress have heightened the prevalence of suicide and suicide ideation by over 16%4. Patients with chronic diseases are especially vulnerable to unintended secondary effects of mental distress or reduced cognitive function5. Current treatments for anxiety include psychotherapy, medications, or a combination of both6. However, despite this crisis, less than 50% of patients achieve full remission6,7. Anxiolytics such as benzodiazepines (BZs) and selective serotonin reuptake inhibitors (SSRIs) have significant drawbacks or produce little to no immediate effects8. Moreover, there is a relative scarcity of novel anxiolytics under development, challenged by the costly and time-consuming process of drug development9,10.
A critical step in the drug development process is the establishment and utilization of an animal model, such as mice, to study pathological changes and test drug safety and efficacy11. Current approaches to establishing anxiety animal models include 1) genetic manipulation, such as knocking out serotonin receptors (5-HT1A) or &#947;-aminobutyric acid A receptor (GABAAR) &#945; subunits12; 2) chronically administering anxiety-inducers such as corticosterone or lipopolysaccharides (LPS)13,14; or 3) administering environmental stress including social defeat and maternal separation15. These methods, however, may not realistically reflect anxiety induced throughout daily life and therefore may not be suitable for investigating the underlying mechanism or testing novel drugs.
Like humans, mice and rats are highly social creatures16,17,18. Social contact and social interactions are essential for optimal brain health and are critical for proper neurodevelopment during the rearing period19. Thus, maternal separation or social isolation during the rearing period results in mice that show more anxiety, depression, and changes in neurotransmission20. Moreover, social grooming or allogrooming is a common form of bonding or comforting behavior among mice and rats that live together21. Thus, socialization is an integral part of rodent life, and isolation negatively impacts their health.
In this context, the present protocol describes a novel anxiety model to mimic the intentional or unintentional patterns of social isolation in modern life. This social isolation (SI) model minimizes perceived distractions and invasiveness and utilizes adult wild type C57BL/6 mice and Sprague-Dawley (SD) rats. The protocol presented here focuses on the anxiety mice model based on our published evidence, which showed increased anxiety-like behavior, aggression, decreased cognition, and increased neuroinflammation as a result of social isolation22,23,24. Anxiety-like behavior is confirmed by the elevated plus maze (EPM) and open field (OF) tests, while cognitive function is measured by novel object recognition (NOR) and novel context recognition (NCR) tests. This model is useful for investigating anxiety and related disorders but can also be adapted or modified to study the natural progression and development of&#160;mild cognitive impairment as well as metabolic changes due to stress.

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	<tbody>
		<tr>
			<td>Black Plastic Bags</td>
			<td>Office Depot</td>
			<td>791932</td>
			<td>24&#34; x 32&#34;</td>
		</tr>
		<tr>
			<td>Elevated Plus Maze</td>
			<td>SD Instruments</td>
			<td>NA</td>
			<td>Black color</td>
		</tr>
		<tr>
			<td>Open Field enclosure</td>
			<td>SD Instruments</td>
			<td>NA</td>
			<td>White color</td>
		</tr>
		<tr>
			<td>Select Agar</td>
			<td>Invitrogen</td>
			<td>30391-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Square cotton for nesting (nestlet)</td>
			<td>Ancare Corporation</td>
			<td>NC9365966</td>
			<td>Divide a 2&#34; square piece into 4 pieces to create a 1&#34; square piece for isolation group</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh boat</td>
			<td>SIgma</td>
			<td>HS1420A</td>
			<td>Small, square white polystyrene</td>
		</tr>
	</tbody>
</table>

social isolation model: a noninvasive rodent model of stress and anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

All representative results and figures were modified from our recent publications22,23. To evaluate the effects of social isolation on anxiety and exploratory behavior, EPM and OF tests were performed 24 h following the end date of the 4 week social isolation period. Socially isolated mice spent significantly less time in the open arm (1.28 &#177; 0.17 min) compared to the control (2.31 &#177; 0.27 min), and a significantly longer time in the closed arm (3.31 &#1...

Watch this Scientific Journal Video about Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety at JoVE.com

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...

This model mimics human social, isolative life and it is a noninvasive and simple way to create a model of anxiety and mild cognitive impairment. This technique only requires the appropriate housing conditions and treatment auger, and it doesn't require tedious processes like injections or genetic manipulation. For social isolation of the animals, take a standard mouse cage with a 75 inch square floor space.Add half the amount of bedding and a one-inch square piece of cotton for nesting. Wrap the outside of the cage walls in opaque black plastic bags with no segment of the bag accessible from the inside. Secure the bag using tape and ensure that the animal's outside environment or surroundings are not visible.Leave the top and bottom of the cage unwrapped unless the mice can see neighboring animals through them. Keep the control mice in groups of two or three under normal caging conditions in a standard mouse cage with a full amount of bedding, a two-inch square piece of cotton or equivalent for nesting, with no wrapping of opaque bags. Ensure the group housed mice are compatible without conflict.If not, exclude the aggressor from the analysis. Change the cages only once a week during the dark cycle with the same plastic bag rewrapped to a new cage unless significant damage is present. For control mice, change the cages twice a week or more during the dark cycle.Continue isolating the mice for at least four weeks to observe the optimal results. To prepare the treatment, add 3%agar to deionized water and dissolve it at approximately 90 degrees Celsius using a heating plate or microwave. Prevent the bubbling solution from spillage or boiling over.Swirl the solution till it becomes homogenous. While the solution is still warm, add 5%sucrose. Swirl the homogenous solution before pouring the solution into a mold.Let it cool and solidify at room temperature or 4 degree Celsius. In the case of light sensitive treatment, protect the mold from light. Once solidified, cut the agar into cubes of 0.5 by 0.5 by 0.5 centimeters and store at 4 degrees celsius until administration.Administer the treatment at planned timing and frequency with as little handling as possible. Quietly and carefully place a single cube onto a small way boat or equivalent, such as the cap of a 50 milliliter chronicle tube during the dark phase of the light dark cycle without touching the mouse. Allow the mouse to consume the agar.After confirming the complete consumption of the agar, carefully removed the way boat from the cage and repeat as necessary. Socially isolated mice spend significantly less time in the open arm and a significantly longer time in the closed arm compared to the control group. In the open field test, socially isolated mice traveled less distance, reared less, and spent more time in the corners.Similarly, they spent less time in the central area than the control, indicating enhanced anxiety-like behavior. Socially isolated mice also displayed reduced novel object and novel context recognition compared to control, suggesting cognitive impairment. This technique allows us to study the development of anxiety and cognitive impairment rather than just the outcome of the disorders.

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