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

Summary

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.

Abstract

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.

Introduction

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 deposited^3,4,5,6,7. Recent findings indicate that inhalable environmental pollutants, including PM2.5 and ammonia, can indeed harm olfactory sensory neurons^8,9,10. 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 function^11,12,13,14. 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.

Protocol

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 ± 1 °C; humidity, 55% ± 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

1. Intranasal administration by instillation
   1. 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.
   2. 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's eyes to prevent dryness. Position the mouse on its back on an inclined surface, with the head facing downward.
   3. Using a pipetting gun, administer 10 µL of the solution in one nostril of the mouse. Suspended the pipette tip above the nose of the mouse, so that the liquid drips onto the nose, allowing the mouse to naturally inhale the solution into the nasal cavity.
   4. 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.
   5. At 3 days after intranasal administration by instillation in mice, perform the buried food test.

2. Buried food test

1. 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.
2. Arrange the operating table as described below.
   1. At 1 h before the start of the test, take the cage containing the mice to the operating room to rest.
   2. 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.
   3. 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).
   4. 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.
   5. Keep cage C and cages containing untested mice as far away from the experimental area as possible.
3. Record the time to find food as described below.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. Put the mouse in cage C after the test.
   6. 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.
      1. 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.
   7. 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.

3. Social odor discrimination experiment

1. Urine collection
   1. 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 µL of urine in each tube.
   2. Store the samples at -80 ° C until use. Shake tubes to evenly distribute the sample after thawing. Do not thaw and freeze the sample repeatedly.
2. Arrange the operating table as described below.
   1. At 1 h before the start of the test, take the cage containing the mice to the operating room to rest. Allow the urine sample to reach room temperature.
   2. Arrange the operating room during this period as described in steps 2.2.2-2.2.5.
3. Record the time to find urine as described below.
   1. Make a groove with tape around each of the two wide sides of cage B large enough to hold the microcentrifuge tube containing 300 µL of male and female urine. Place the tubes and keep the two tube covers closed for now.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. Transfer the mouse to cage C at the end of the experiment.
   7. 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.
   8. Change gloves and test the next mouse as described above.
4. 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.

Results

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...

Discussion

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's capacity to discern and differentiate various social odors. The protocol here serves to evaluate the toxic effects of environmental pollutants on ol...

Materials

Name	Company	Catalog Number	Comments
0.5-10 μL  adjustable micropipette	Eppendorf, Germany	3123000225	Intranasal instillation
0.9% saline solution	Solarbio	7647-14-5	Dissolve pollutants
Anhydrous zinc sulfate	Macklin	7733-02-0	Expose mice
Centrifuge tube (2 mL)	Biosharp Incorporated	BS-20-M	Place urine
Electronic balance	Changzhou Ohaus Co.	EX125DZH	Weight anesthetics and pollutants
GraphPad Prism	GraphPad Software	8.0.1	statistic analysis
Handheld Dust detector	TSI Incorporated	DuatTrak 8532	Inhalation-exposed mice
Video recording equipment	Apple Inc.	iPhone 6s Plus	The activity time of mice was recorded
Vortex mixer	Haimen Kylin-Bell Lab Instruments Co.	Vortex-5	Mix solution