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 environ...

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. Chapter 8, Unit 8.24 (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, 11.18.1-11.18.21 (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 (2 and 3-Spec Issue), 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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5-10 &#956;L&#160; 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&#160;Incorporated</td>
			<td>DuatTrak <img alt="Equation 1" src="/files/ftp_upload/66818/66818eq01.jpg" style="margin: auto;" />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&#160;</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.

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

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

Our research is environmental toxicology. It asks to provide two behavioral testing methods for starting effects or inheritable environmental pollutants on olfactory function. We provide two simple and effective methods by certain food tests and the social order discrimination experiment for determining the toxicity of inheritable environmental pollutants on olfactory function.We can comprehensively evaluate the impact of inheritable environmental pollutants on olfactory function by convincing the variable test and social order discrimination experiment matters. Our protocol uses a video device to time and record mouse behavior, addressing the efficiency of observes not being able to observe the behavior of mice in the observation area.

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...

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.

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

Research

JoVE Journal

Environment

Performing Buried Food Test to Assess the Effects of Inhaled Pollutants on Olfactory Function in Mice

To begin, dissolve inhalable environmental pollutants in a 0.9%saline solution. Use a 5%zinc sulfate solution once as it has been shown to cause loss of smell. Using a pipetting gun, administer 10 microliters of the solution into one nostril of the mouse, allowing it to naturally inhale the solution into the nasal cavity.Remove all chow pellets from the food hopper of the home cage, to carry out food deprivation, 18 to 24 hours before the test. Change the bedding materials for the mice. Take the cage containing the mice to the operating room to rest one hour before the start of the test.Arrange the operating room during this period. Use transparent PVC standard mice cages marked as A and squirrel cages marked as B for testing. Mark the common cage used to place the mice after the experiment as cage C.Cover cages A and B with three centimeters of bedding material, and measure them with a ruler.Then arrange the cages side by side, with 0.5 meters between each cage. Next, define the experimental area as a two-meter radius space centered on the cages, with the area beyond two meters designated as the observation area. Keep cage C and cages containing untested mice as far away from the experimental area as possible.Now, select a position at random in cage B.Bury the food one centimeter below the surface of the bedding and smooth the surface of the bedding. Then place the mouse in cage A for four minutes. At the end of the time, transfer the mouse to cage B and turn on the video device before returning to the observation area.Stop the video recording when the mouse picks up the food lock with its forepaw. Record the time from contact with the mat at the bottom of cage B until the food is found for each mouse. After the test, put the mouse in cage C and add feed and water.In the food burial test, mice exposed to zinc sulfate were unable to find food for 240 seconds compared to the control group.

186 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.

Assessing Olfactory Impairment in Mice Due to Inhaled Environmental Pollutants

概要