Name: studying the effects of inhaled environmental pollutants on olfactory function in mice
Uploaded: 2024-09-13T13:40:00.000+00:00
Duration: 4 min
Description: Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental ...

This work was financially supported by the National Natural Science Foundation of China (82204088, 82273669) and the Natural Science Foundation of Shandong Province, China (ZR2021QH209).

We used male C57BL/6J mice (age: 6-8 weeks; weight: 20-22 g) for all behavioral tests. The mice were subjected to steady conditions (i.e., temperature, 23 &#177; 1 &#176;C; humidity, 55% &#177; 5%, and 12/12 h light-dark cycle with lights on at 7:00). All behavioral tests were performed between 10:00 and 17:00. All animal experiments were approved by the Ethics Committee of the Professional Committee of Animal Experiments of Qingdao University. After a 1-week acclimation period, all mice were exposed to inhalable environmental pollutants.
1. Exposure to pollutants
<ol>
	<li>Intranasal administration by instillation
	<ol>
		<li>Dissolve inhalable environmental pollutants in a 0.9% saline solution. Use 5% zinc sulfate solution once, which has been demonstrated to cause olfactory dysfunction17. For control mice, administer 0.9% normal saline solution.</li>
		<li>Anesthetize the mouse by intraperitoneal injection of 1% sodium pentobarbital (57 mg/kg)18 and assess the depth of anesthesia using the toe-pinch reflex. Apply vet ointment to mouse&#39;s eyes to prevent dryness. Position the mouse on its back on an inclined surface, with the head facing downward.</li>
		<li>Using a pipetting gun, administer 10 &#181;L of the solution in one nostril of the mouse.&#160;Suspended the pipette tip above the nose of the mouse, so that the liquid drips onto the nose,&#160;allowing the mouse to naturally inhale the solution into the nasal cavity.</li>
		<li>To ensure the well-being of the mouse and prevent any potential discomfort, repeat the inhalation for the other nostril after a 10 min interval.</li>
		<li>At 3 days after intranasal administration by instillation in mice, perform the buried food test.</li>
	</ol>
	</li>
</ol>
2.&#160;Buried food test
<ol>
	<li>Carry out food deprivation at 18-24 h before the test by removing all chow pellets from the food hopper of the home cage. Change the bedding materials for mice. Do not remove the water bottle.</li>
	<li>Arrange the operating table as described below.
	<ol>
		<li>At 1 h before the start of the test, take the cage containing the mice to the operating room to rest.</li>
		<li>Arrange the operating room during this period. Use and mark transparent PVC standard mice cages as A as these make a familiar environment. Use and mark the test cage as B, which is a transparent PVC standard squirrel cage. Mark the common cage used to place the mice after the experiment as cage C.</li>
		<li>Cover cages A and B with 3 cm of bedding material and measure them with a ruler. Arrange the cages side by side, with a distance of 0.5 m between each cage (Figure 1).</li>
		<li>Define the experimental area as an area with a radius of 2 m, with the center being the center of the cages. Define the area outside the range of 2 m as the observation area.</li>
		<li>Keep cage C and cages containing untested mice as far away from the experimental area as possible.</li>
	</ol>
	</li>
	<li>Record the time to find food as described below.
	<ol>
		<li>Select a position at random in cage B, bury the food 1 cm below the surface of the bedding, and smooth the surface of the bedding.</li>
		<li>Put the mouse into cage A for 4 min. Transfer the mice to cage B at the end of the timing, turn on the video device, and return to the observation area.</li>
		<li>When the mouse picks up the food block with its forepaw, stop the video recording. In some cases, mice can be seen eating with their heads bent over the food. This behavior also indicates trial success, even if the mouse is not holding the food with its forepaw.</li>
		<li>Record data. Record the time from contact with the mat at the bottom of cage B until food was found for each mouse. If the mice do not find food after 4 min, record the find time as a delay time of 240 s.</li>
		<li>Put the mouse in cage C after the test.</li>
		<li>Remove the feed from cage B and put it in a sealed bag. Replace the food in cage B and test the next mouse after changing the bedding material, as described below.
		<ol>
			<li>For mice housed in the same cage, test with the same set of bedding materials in cage B. For mice in different cages, clean the cage with alcohol and replace the bedding materials.</li>
		</ol>
		</li>
		<li>Add feed and water to mice after the experiment. Perform the social odor discrimination experiment 1 day later. 
		NOTE: Masks need to be worn throughout the process, and transparent gloves need to be changed after each mouse finishes the experiment to avoid odor cross as much as possible. Keep the amplitude of the movements as small as possible to avoid mouse stress.</li>
	</ol>
	</li>
</ol>
3. Social odor discrimination experiment
<ol>
	<li>Urine collection
	<ol>
		<li>Separately collect the urine of sexually mature male mice and female mice and aliquot into tubes in equal volumes19. Pack in 2 mL microcentrifuge tubes with 300 &#181;L of urine in each tube.</li>
		<li>Store the samples at -80 &#176; C until use. Shake tubes to evenly distribute the sample after thawing. Do not thaw and freeze the sample repeatedly.</li>
	</ol>
	</li>
	<li>Arrange the operating table as described below.
	<ol>
		<li>At 1 h before the start of the test, take the cage containing the mice to the operating room to rest. Allow the urine&#160;sample to reach room temperature.</li>
		<li>Arrange the operating room during this period as described in steps 2.2.2-2.2.5.</li>
	</ol>
	</li>
	<li>Record the time to find urine as described below.
	<ol>
		<li>Make a groove with tape around each of the two wide sides of cage B large enough to hold the microcentrifuge tube containing 300 &#181;L of male and female urine. Place the tubes and keep the two tube covers closed for now.</li>
		<li>Put the mouse in cage A and set a countdown for 4 min. Open the two tubes of cage B at the end of the timing.</li>
		<li>Transfer the mouse to the middle position of cage B, at the same distance from the two tubes, then turn on the video recording equipment and gently and slowly retreat to the observation area.</li>
		<li>When the mouse sniffs the tube wall/mouth or even inside the tube, the tube is successfully smelled. At this time, press the stopwatch and record the time as the time of smelling the tube (M s), then continue to record the residence time (X s) until the mouse moves away from the tube.</li>
		<li>Continue timing the time sniffing the other tube. When the mouse sniffs the tube wall/mouth or even inside the tube, the tube is successfully smelled. Press the stopwatch and record the time it took to smell the other tube (N s), then continue to record the residence time (Y s) until the mouse moves away from the tube.</li>
		<li>Transfer the mouse to cage C at the end of the experiment.</li>
		<li>Change the urine tubes and bedding material of cage B after each mouse test. Put the used mouse urine in a clean bag according to sex.</li>
		<li>Change gloves and test the next mouse as described above.</li>
	</ol>
	</li>
	<li>Perform the experiment in a laboratory without special odor, such as strong fragrances or the smell of food, if there are, thoroughly ventilate before experimenting. Avoid personal products that emit strong odors. Wear gloves and masks throughout the procedure to avoid odor crossing as much as possible. Keep the amplitude of the movements as small as possible to avoid mouse stress.</li>
</ol>

Assessing Olfactory Impairment in Mice Due to Inhaled Environmental Pollutants

<ol>
	<li>Sch&#228;fer, L., Schriever, V. A., Croy, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+olfactory+dysfunction:+causes+and+consequences.">Human olfactory dysfunction: causes and consequences.</a> Cell Tissue Res. 383 (1), 569-579 (2021).</li><li>Keller, A., Malaspina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+consequences+of+olfactory+dysfunction:+a+patient+report+series.">Hidden consequences of olfactory dysfunction: a patient report series.</a> BMC Ear Nose Throat Disord. 13 (1), 8 (2013).</li><li>Schroeter, J. D., 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=Application+of+physiological+computational+fluid+dynamics+models+to+predict+interspecies+nasal+dosimetry+of+inhaled+acrolein.">Application of physiological computational fluid dynamics models to predict interspecies nasal dosimetry of inhaled acrolein.</a> Inhal Toxicol. 20 (3), 227-243 (2008).</li><li>Schroeter, J. D., Garcia, G. J., Kimbell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+fluid+dynamics+approach+to+assess+interhuman+variability+in+hydrogen+sulfide+nasal+dosimetry.">A computational fluid dynamics approach to assess interhuman variability in hydrogen sulfide nasal dosimetry.</a> Inhal Toxicol. 22 (4), 277-286 (2010).</li><li>Keyhani, K., Scherer, P. W., Mozell, 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=Numerical+simulation+of+airflow+in+the+human+nasal+cavity.">Numerical simulation of airflow in the human nasal cavity.</a> J Biomech Eng. 117 (4), 429-441 (1995).</li><li>Hahn, I., Scherer, P. W., Mozell, 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=Velocity+profiles+measured+for+airflow+through+a+large-scale+model+of+the+human+nasal+cavity.">Velocity profiles measured for airflow through a large-scale model of the human nasal cavity.</a> J Appl Physiol. 75 (5), 2273-2287 (1993).</li><li>Zhang, Z., 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=Exposure+to+particulate+matter+air+pollution+and+Anosmia.">Exposure to particulate matter air pollution and Anosmia.</a> JAMA Netw Open. 4 (5), e2111606 (2021).</li><li>Ekstr&#246;m, I. A., 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=Environmental+air+pollution+and+olfactory+decline+in+aging.">Environmental air pollution and olfactory decline in aging.</a> Environ Health Perspect. 130 (2), 27005 (2022).</li><li>Adams, D. 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=Nitrogen+dioxide+pollution+exposure+is+associated+with+olfactory+dysfunction+in+older+U.S.+adults.">Nitrogen dioxide pollution exposure is associated with olfactory dysfunction in older U.S. adults.</a> Int Forum Allergy Rhinol. 6 (12), 1245-1252 (2016).</li><li>Prah, J. D., Benignus, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decrements+in+olfactory+sensitivity+due+to+ozone+exposure.">Decrements in olfactory sensitivity due to ozone exposure.</a> Percept Mot Skills. 48 (1), 317-318 (1979).</li><li>Shi, Z., 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=Chronic+exposure+to+environmental+pollutant+ammonia+causes+damage+to+the+olfactory+system+and+behavioral+abnormalities+in+mice.">Chronic exposure to environmental pollutant ammonia causes damage to the olfactory system and behavioral abnormalities in mice.</a> Environ Sci Technol. 57 (41), 15412-15421 (2023).</li><li>Hern&#225;ndez-Soto, 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=Chronic+intermittent+hypoxia+alters+main+olfactory+bulb+activity+and+olfaction.">Chronic intermittent hypoxia alters main olfactory bulb activity and olfaction.</a> Exp Neurol. 340, 113653 (2021).</li><li>Islam, 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=Odor+preference+and+olfactory+memory+are+impaired+in+Olfaxin-deficient+mice.">Odor preference and olfactory memory are impaired in Olfaxin-deficient mice.</a> Brain Res. 1688, 81-90 (2018).</li><li>Wang, H., 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=Inducible+and+conditional+activation+of+ERK5+MAP+kinase+rescues+mice+from+cadmium-induced+olfactory+memory+deficits.">Inducible and conditional activation of ERK5 MAP kinase rescues mice from cadmium-induced olfactory memory deficits.</a> Neurotoxicology. 81, 127-136 (2020).</li><li>Chamero, P., Leinders-Zufall, T., Zufall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+genes+to+social+communication:+molecular+sensing+by+the+vomeronasal+organ.">From genes to social communication: molecular sensing by the vomeronasal organ.</a> Trends Neurosci. 35 (10), 597-606 (2012).</li><li>Mohrhardt, 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=Signal+detection+and+coding+in+the+accessory+olfactory+system.">Signal detection and coding in the accessory olfactory system.</a> Chem Senses. 43 (9), 667-695 (2018).</li><li>Liu, X., 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=Type+3+adenylyl+cyclase+in+the+MOE+is+involved+in+learning+and+memory+in+mice.">Type 3 adenylyl cyclase in the MOE is involved in learning and memory in mice.</a> Behav Brain Res. 383, 112533 (2020).</li><li>Li, X., 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=Polyhexamethylene+guanidine+aerosol+triggers+pulmonary+fibrosis+concomitant+with+elevated+surface+tension+via+inhibiting+pulmonary+surfactant.">Polyhexamethylene guanidine aerosol triggers pulmonary fibrosis concomitant with elevated surface tension via inhibiting pulmonary surfactant.</a> J Hazard Mater. 420, 126642 (2021).</li><li>Yang, M., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+behavioral+assessment+of+mouse+olfaction.">Simple behavioral assessment of mouse olfaction.</a> Curr Protoc Neurosci. , (2009).</li><li>Huang, K. L., 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=Zinc+oxide+nanoparticles+induce+eosinophilic+airway+inflammation+in+mice.">Zinc oxide nanoparticles induce eosinophilic airway inflammation in mice.</a> J Hazard Mater. 297, 304-312 (2015).</li><li>Burd, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+study+of+the+effects+of+intranasal+zinc+sulfate+irrigation+on+the+mouse+olfactory+epithelium+and+olfactory+bulb.">Morphological study of the effects of intranasal zinc sulfate irrigation on the mouse olfactory epithelium and olfactory bulb.</a> Microsc Res Tech. 24 (3), 195-213 (1993).</li><li>Zou, 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=Methods+to+measure+olfactory+behavior+in+mice.">Methods to measure olfactory behavior in mice.</a> Curr Protoc Toxicol. 63, 1-21 (2015).</li><li>Ryalls, J. M. W., 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=Anthropogenic+air+pollutants+reduce+insect-mediated+pollination+services.">Anthropogenic air pollutants reduce insect-mediated pollination services.</a> Environ Pollut. 297, 118847 (2022).</li><li>Schiffman, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livestock+odors:+implications+for+human+health+and+well-being.">Livestock odors: implications for human health and well-being.</a> J Anim Sci. 76 (5), 1343-1355 (1998).</li><li>Albrechet-Souza, L., Gilpin, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+predator+odor+avoidance+model+of+post-traumatic+stress+disorder+in+rats.">The predator odor avoidance model of post-traumatic stress disorder in rats.</a> Behav Pharmacol. 30, 105-114 (2019).</li><li>Davies, D. A., 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=Inactivation+of+medial+prefrontal+cortex+or+acute+stress+impairs+odor+span+in+rats.">Inactivation of medial prefrontal cortex or acute stress impairs odor span in rats.</a> Learn Mem. 20 (12), 665-669 (2013).</li><li>Landers, M. S., Sullivan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+neurobiology+of+infant+attachment+and+fear.">The development and neurobiology of infant attachment and fear.</a> Dev Neurosci. 34 (2-3), 101-114 (2012).</li><li>Drobyshevsky, A., 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=Antenatal+insults+modify+newborn+olfactory+function+by+nitric+oxide+produced+from+neuronal+nitric+oxide+synthase.">Antenatal insults modify newborn olfactory function by nitric oxide produced from neuronal nitric oxide synthase.</a> Exp Neurol. 237 (2), 427-434 (2012).</li><li>Arbuckle, E. P., 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=Testing+for+odor+discrimination+and+habituation+in+mice.">Testing for odor discrimination and habituation in mice.</a> J Vis Exp. (99), e52615 (2015).</li></ol>

The authors declare no conflicts of interest.

This article introduces two fundamental protocols designed for the swift assessment of olfactory impairment in mice. Varied inhalable environmental pollutants result in distinct levels of olfactory dysfunction in mice. The buried food test is employed to assess the capability to detect volatile odors, while the social odor discrimination experiment evaluates the animal&#39;s capacity to discern and differentiate various social odors. The protocol here serves to evaluate the toxic effects of environmental pollutants on ol...

Olfactory impairment has emerged as a noteworthy public health concern and is independently associated with an increased risk of neurodegenerative diseases. This condition can adversely affect overall well-being, contribute to the development of depressive symptoms, and result in a diminished quality of life. Its impact is prominently observed in the altered perception of food, hindrance in social communication, and heightened negative feelings1. Various factors, including sinonasal disease, upper respiratory tract infection, and traumatic brain injury, have been considered contributors to olfactory impairment in humans2. Notably, inhalable environmental pollutants such as PM2.5, estimated to range from 2% to 16%, enter the body through inspired air, traverse the nasal cavity, and reach specific olfaction-dedicated regions where they are deposited3,4,5,6,7. Recent findings indicate that inhalable environmental pollutants, including PM2.5 and ammonia, can indeed harm olfactory sensory neurons8,9,10. However, further validation is required to ascertain whether such damage directly leads to olfactory dysfunction. Hence, a meticulous evaluation of the effects of inhalable environmental pollutants on olfactory function is of particular importance.
Currently, numerous research laboratories employ mice as an alternative vertebrate model for behavioral experiments aimed at comprehending changes in olfactory function11,12,13,14. Mice were chosen as the preferred model system for investigating vertebrate chemical communication, and it exhibits a remarkable olfactory sensitivity crucial for foraging and social communication15. Furthermore, the continually evolving array of tools for observing and influencing mouse behavior has rendered this species exceptionally appealing for research on olfactory function16.
In this study, we employed the buried food test and the social odor discrimination experiment to assess olfactory impairment in a mouse model exposed to inhalable environmental pollutants. To enhance the precision of evaluation, we opted for the most representative method for assessing olfactory function. We systematically refined this method to ensure simplicity and clarity, allowing us to gauge the extent of olfactory dysfunction induced by inhalable environmental pollutants effectively.

<table><tbody><tr><td>0.5-10 &amp;mu;L&amp;nbsp; adjustable micropipette</td><td>Eppendorf, Germany</td><td>3123000225</td><td>Intranasal instillation</td></tr><tr><td>0.9% saline solution</td><td>Solarbio</td><td>7647-14-5</td><td>Dissolve pollutants</td></tr><tr><td>Anhydrous zinc sulfate</td><td>Macklin</td><td>7733-02-0</td><td>Expose mice</td></tr><tr><td>Centrifuge tube (2 mL)</td><td>Biosharp Incorporated</td><td>BS-20-M</td><td>Place urine</td></tr><tr><td>Electronic balance</td><td>Changzhou Ohaus Co.</td><td>EX125DZH</td><td>Weight anesthetics and pollutants</td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>8.0.1</td><td>statistic analysis</td></tr><tr><td>Handheld Dust detector</td><td>TSI&amp;nbsp;Incorporated</td><td>DuatTrak &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66818/66818eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;8532</td><td>Inhalation-exposed mice</td></tr><tr><td>Video recording equipment</td><td>Apple Inc.</td><td>iPhone 6s Plus</td><td>The activity time of mice was recorded</td></tr><tr><td>Vortex mixer</td><td>Haimen Kylin-Bell Lab Instruments Co.</td><td>Vortex-5&amp;nbsp;</td><td>Mix solution</td></tr></tbody></table>

studying the effects of inhaled environmental pollutants on olfactory function in mice

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental pollutants exposure may impair olfaction; thereby, there is an urgent need for methods to evaluate the effects of inhaled environmental pollutants exposure on olfaction. Mice are ideal models for olfactory experiments because of their highly developed olfactory system and behavioral characteristics. To assess the effects of inhaled environmental pollutants exposure on olfactory function in mice, a detailed buried food test and social odor discrimination experiment is provided, including the experiment preparation, the selection and construction of experimental facilities, the testing process, and indexes of time. Meanwhile, timekeeping equipment, operational details, and the experimental environment are discussed to ensure the success of the assay. Zinc sulfate is used as the treatment to demonstrate the feasibility of the experimental approach. The protocol provides a simple and clear operational process for assessing the effects of inhaled environmental pollutants on olfactory function in mice.

Inhalable environmental pollutants impair olfactory function in mice. Atmospheric zinc emitted from incinerators and motor vehicles has been demonstrated to be an inhaled pollutant that can result in allergic lung inflammation20. Zinc sulfate is considered one of the typical compounds to cause olfactory dysfunction21. Therefore, we use zinc sulfate as the treatment to expose mice by intranasal instillation and test using the buried food test and social odor discrimination e...

Author Spotlight: Assessing the Olfactory Effects of Airborne Pollutants &#8212; Buried Food and Social Odor Tests

This paper provides a detailed description of the buried food test and social odor discrimination experiment to assess the effects of inhaled environmental pollutants exposure on olfactory function in mice.

Studying the Effects of Inhaled Environmental Pollutants on Olfactory Function in Mice

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental ...

studying-effects-inhaled-environmental-pollutants-on-olfactory

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

680 Views.  Qingdao University. This paper provides a detailed description of the buried food test and social odor discrimination experiment to assess the effects of inhaled environmental pollutants exposure on olfactory function in mice.

Video: Author Spotlight: Assessing the Olfactory Effects of Airborne Pollutants — Buried Food and Social Odor Tests

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Behavior

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Biology

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Neuroscience

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Medicine

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...