这项工作得到了中国国家自然科学基金 （82204088， 82273669） 和中国山东省自然科学基金 （ZR2021QH209） 的财政支持。

我们使用雄性 C57BL/6J 小鼠 （年龄：6-8 周;体重：20-22 g） 进行所有行为测试。小鼠经受稳定的条件（即温度，23 ± 1 °C;湿度，55% ± 5%，以及 7：00 开灯的 12/12 小时明暗循环）。所有行为测试均在 10：00 至 17：00 之间进行。所有动物实验均经青岛大学动物实验专业委员会伦理委员会批准。1 周的驯化期后，所有小鼠均暴露于可吸入环境污染物。
1. 接触污染物
<ol><li>滴注鼻内给药<ol><li>将可吸入的环境污染物溶解在 0.9% 盐水溶液中。使用 5% 硫酸锌溶液一次，已被证明会导致嗅觉功能障碍17。对于对照小鼠，施用 0.9% 生理盐水溶液。</li>
		<li>通过腹膜内注射 1% 戊巴比妥钠 18 麻醉小鼠，并使用脚趾捏反射评估麻醉深度。将兽医药膏涂抹在老鼠的眼睛上以防止干燥。将鼠标仰卧在倾斜的表面上，头部朝下。</li>
		<li>使用移液枪，在小鼠的一个鼻孔中注入 10 μL 溶液，让溶液自然吸入鼻腔。</li>
		<li>为确保鼠标的健康并防止任何潜在的不适，请在 10 分钟间隔后对另一个鼻孔重复吸气。</li>
		<li>在鼻内给药后 3 天，通过滴注在小鼠中，进行埋藏食物试验。</li>
	</ol></li>
</ol>2. 埋藏食物测试
<ol><li>在测试前 18-24 小时通过从家笼的食物料斗中取出所有食物颗粒来剥夺食物。更换老鼠的床上用品。不要取下水瓶。</li>
	<li>如下所述布置手术台。<ol><li>在测试开始前 1 小时，将装有小鼠的笼子带到手术室休息。</li>
		<li>在此期间安排手术室。使用透明 PVC 标准鼠标笼并将其标记为 A，因为这些笼子可以营造熟悉的环境。使用并将测试笼标记为 B，这是一种透明的 PVC 标准鼠笼。将实验后用于放置小鼠的公共笼子标记为笼子 C。</li>
		<li>用 3 厘米的垫料覆盖笼子 A 和 B，并用尺子测量它们。并排排列笼子，每个笼子之间的距离为 0.5 m（图 1）。</li>
		<li>将实验区域定义为半径为 2 m 的区域，中心是笼子的中心。将 2 m 范围之外的区域定义为观察区域。</li>
		<li>将笼子 C 和装有未经测试的小鼠的笼子尽可能远离实验区域。</li>
	</ol></li>
	<li>记录寻找食物的时间，如下所述。<ol><li>在笼子 B 中随机选择一个位置，将食物埋在被褥表面以下 1 厘米处，并将被褥表面抹平。</li>
		<li>将鼠标放入笼 A 中 4 分钟。定时结束时将小鼠转移到笼 B，打开视频设备，返回观察区域。</li>
		<li>当老鼠用前爪捡起食物块时，停止视频录制。在某些情况下，可以看到老鼠低头吃东西。这种行为也表明试验成功，即使鼠标没有用前爪夹住食物。</li>
		<li>记录数据。记录从接触笼 B 底部的垫子到为每只老鼠找到食物的时间。如果小鼠在 4 分钟后没有找到食物，则将找到时间记录为 240 秒的延迟时间。</li>
		<li>测试后将鼠标放入笼 C 中。</li>
		<li>从笼子 B 中取出饲料并将其放入密封袋中。更换笼子 B 中的食物，更换床上用品后测试下一只鼠标，如下所述。<ol><li>对于饲养在同一笼子中的小鼠，在笼子 B 中使用同一组垫料进行测试。对于不同笼子里的老鼠，用酒精清洗笼子，更换垫料。</li>
		</ol></li>
		<li>实验后向小鼠添加饲料和水。1 天后进行社会气味辨别实验。 注意：整个过程需要戴口罩，每只鼠标完成实验后需要更换透明手套，以尽可能避免气味交叉。保持运动的幅度尽可能小，以避免鼠标压力。</li>
	</ol></li>
</ol>3. 社会气味辨别实验
<ol><li>尿液采集<ol><li>分别收集性成熟雄性小鼠和雌性小鼠的尿液，并分装到等体积的试管中19。装在 2 mL 微量离心管中，每管含 300 μL 尿液。</li>
		<li>将样品储存在 -80°C 直至使用。解冻后摇动试管以均匀分布样品。不要反复解冻和冷冻样品。</li>
	</ol></li>
	<li>如下所述布置手术台。<ol><li>在测试开始前 1 小时，将装有小鼠的笼子带到手术室休息。</li>
		<li>在此期间，按照步骤 2.2.2-2.2.5 中的说明安排手术室。</li>
	</ol></li>
	<li>记录查找尿液的时间，如下所述。<ol><li>在笼 B 的两个宽边周围用胶带制作一个凹槽，其大小足以容纳含有 300 μL 男性和女性尿液的微量离心管。放置试管并暂时关闭两个试管盖。</li>
		<li>将鼠标放入笼 A 中并设置 4 分钟的倒计时。在计时结束时打开笼 B 的两管。</li>
		<li>将鼠标转移到笼 B 的中间位置，与两根管保持相同的距离，然后打开录像设备，轻轻缓慢地撤退到观察区域。</li>
		<li>当鼠标嗅到管壁/嘴巴甚至管内时，管子被成功闻到。此时按下秒表，将时间记录为闻到管子的时间（M s），然后继续记录停留时间（X s），直到鼠标离开管子。</li>
		<li>继续计时，嗅探另一根管子。当鼠标嗅到管壁/嘴巴甚至管内时，管子被成功闻到。按下秒表并记录闻到另一根管子所需的时间 （N s），然后继续记录停留时间 （Y s），直到鼠标离开管子。</li>
		<li>实验结束时将鼠标转移到笼 C。</li>
		<li>每次小鼠测试后更换笼 B 的尿管和床上用品。根据性别将用过的大鼠尿液放入干净的袋子中。</li>
		<li>如上所述更换手套并测试下一只鼠标。</li>
	</ol></li>
	<li>在无明显气味的实验室进行实验。避免使用散发强烈气味的个人产品。在整个手术过程中戴上手套和口罩，以尽可能避免异味传播。保持运动的幅度尽可能小，以避免鼠标压力。</li>
</ol>

评估吸入环境污染物导致小鼠嗅觉损害

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作者声明没有利益冲突。

本文介绍了两种旨在快速评估小鼠嗅觉障碍的基本方案。各种可吸入环境污染物导致小鼠不同程度的嗅觉功能障碍。埋藏食物测试用于评估检测挥发性气味的能力，而社会气味辨别实验用于评估动物辨别和区分各种社会气味的能力。这里的协议用于评估环境污染物对嗅觉功能的毒性影响。
吸入的环境污染物，包括臭氧、氨和柴油机尾气，具有可能影响小鼠在样品嗅探过程中?...

嗅觉障碍已成为一个值得注意的公共卫生问题，并且与神经退行性疾病的风险增加独立相关。这种情况会对整体健康状况产生不利影响，导致抑郁症状的发展，并导致生活质量下降。它的影响在对食物的感知改变、社交沟通的阻碍和负面情绪的加剧中得到了突出的体现1。包括鼻窦疾病、上呼吸道感染和创伤性脑损伤在内的各种因素被认为是导致人类嗅觉受损的原因2。值得注意的是，PM2.5 等可吸入环境污染物（估计在 2% 到 16% 之间）通过吸入的空气进入人体，穿过鼻腔，并到达特定的嗅觉专用区域，在那里它们被沉积 3,4,5,6,7。最近的研究结果表明，可吸入的环境污染物，包括 PM2.5 和氨，确实会伤害嗅觉感觉神经元 8,9,10。然而，需要进一步验证以确定这种损伤是否直接导致嗅觉功能障碍。因此，仔细评估可吸入环境污染物对嗅觉功能的影响尤为重要。
目前，许多研究实验室使用小鼠作为替代脊椎动物模型进行行为实验，旨在理解嗅觉功能的变化 11,12,13,14。小鼠被选为研究脊椎动物化学交流的首选模型系统，它表现出对觅食和社会交流至关重要的非凡嗅觉敏感性15。此外，用于观察和影响小鼠行为的一系列不断发展的工具使该物种对嗅觉功能研究极具吸引力16。
在这项研究中，我们采用埋藏食物测试和社会气味鉴别实验来评估暴露于可吸入环境污染物的小鼠模型的嗅觉损害。为了提高评估的精度，我们选择了最具代表性的方法来评估嗅觉功能。我们系统地改进了这种方法，以确保简单和清晰，使我们能够有效地衡量可吸入环境污染物引起的嗅觉功能障碍的程度。

<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

嗅觉障碍是一个重大的公共卫生问题，可独立预测神经退行性疾病的风险。吸入环境污染物暴露可能会损害嗅觉;因此，迫切需要评估吸入环境污染物暴露对嗅觉影响的方法。小鼠是嗅觉实验的理想模型，因为它们的高度发达的嗅觉系统和行为特征。为评估吸入环境污染物暴露对小鼠嗅觉功能的影响，提供了详细的埋藏食物检测和社会气味鉴别实验，包括实验准备、实验设施的选择和建设、检测过程和时间指标。同时，讨论了计时设备、操作细节和实验环境，以确保检测的成功。以硫酸锌为处理方法，证明了实验方法的可行性。该协议提供了一个简单明了的操作过程，用于评估吸入环境污染物对小鼠嗅觉功能的影响。

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.

作者聚焦：评估空气污染物的嗅觉影响 - 埋藏的食物和社会气味测试

研究吸入环境污染物对小鼠嗅觉功能的影响

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.

可吸入的环境污染物会损害小鼠的嗅觉功能。焚化炉和机动车辆排放的大气锌已被证明是一种吸入污染物，可导致过敏性肺部炎症20。硫酸锌被认为是导致嗅觉功能障碍的典型化合物之一21。因此，我们以硫酸锌为处理，通过鼻内滴注暴露小鼠，并使用埋藏食物测试和社会气味鉴别实验进行测试。在食物埋葬试验中，与对照组相比，暴露于硫酸锌的小鼠找到食物...

本文对埋藏食物试验和社会气味鉴别实验进行了详细说明，以评估吸入环境污染物暴露对小鼠嗅觉功能的影响。

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

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

283 次观看.  Qingdao University. 我们的研究方向是环境毒理学。它要求提供两种行为测试方法，用于启动嗅觉功能的影响或可遗传的环境污染物。我们通过某些食物测试和社会秩序判别实验提供了两种简单有效的方法来确定可遗传环境污染物对嗅觉功能的毒性。我们可以通过说服变量测试和社会秩序区分实验来综合评估可遗传环境污染物对嗅觉功能的影响。我们的协议使用视...