This work was supported by research grants (NIH/NIDCD 009269, 012831 and ARRA administrative supplement NIH grants) to Weihong Lin. We especially thank Mr. Tim Ford at UMBC for his technical assistance in videotaping and processing. We also wish to thank Dr. Daphne Blumberg, Ms. Chere Petty at UMBC and Mr. Nicholas McCollum from Olympus America Inc. for their equipment assistance in videotaping.

1. Mouse Nose Preparation
We used adult C57BL/6 background mice in this study. All animal care and procedures are approved by the Animal Care and Use Committees (IACUC) of University of Maryland, Baltimore County.

 1.1 Acquiring the nose from paraformaldahyde-fixed mice
<img src="/files/ftp_upload/50538/50538fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50538/50538fig1highres.jpg"/> 
Figure 1. Bones ...

<ol>
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Here, we demonstrated a step-by-step procedure for isolating intact olfactory and respiratory tissue from the mouse nose by sequentially removing the surrounding bones while sparing the tissue below. We show that careful bone removal can preserve even the most delicate tissues in their entirety. We also share insight into possible modifications of this technique, in which we isolate both the brain and nose tissue together to preserve the nerve connection. This new method provides a means for isolating whole olfactory ...

The mammalian nasal cavity contains various types of tissues and organs that serve distinct functions. The nasal cavity makes up the entry portion of the upper respiratory tract, which allows air travel into and out of the lungs. Inhaled air passes through the nasal cavity where it undergoes temperature and humidity conditioning 1 as well as cleaning or filtering to remove irritating and toxic substances and infectious microorganisms 2. Both treatments are carried out by nasal epithelia and subepithelial tissues, including glands and vessels and are critical for protecting the lower airways and the lungs. In addition to its role in respiration and epithelial defense, the nasal tissue also contains peripheral sensory apparatuses of the olfactory and trigeminal systems, which detect a wide range of chemical substances in the passing air. Depending on which system is activated, sensory detection of chemicals in the nose can elicit either a sense of smell, irritation, or pain 3,4. 
The peripheral olfactory system is complex and made up of several anatomically separated olfactory sensory organs within the nasal cavity. Among them, the main olfactory epithelium (MOE) is the largest, which makes up approximately 45-52% of the nasal epithelia in rodents 5 and is located in the posterior region. In the anteroventral region, there is a pair of tubular structures known as the vomeronasal organ 6, which sit along each side of the nasal septum. Two additional small groupings of olfactory sensory neurons, known as the septal organ of Masera 7,8 and the Gruneberg ganglion 9, reside along the ventral septum and the dorsal entry region of the nasal cavity, respectively. These peripheral organs contain neuro-epithelia with distinctive features in morphology, cell marker expression, and physiological function. Together they detect thousands of odor molecules with exquisite sensitivity 10-12. 
In addition to the olfactory sensory organs, the nasal cavity also houses other sensory systems. It is known that peptidergic trigeminal nerve fibers are present in the nasal epithelium, especially the respiratory epithelium 13,14. Some of these fibers detect irritating and toxic chemicals and are responsible for initiating protective reflexes such as coughing and sneezing 4,15. Irritating odorous and bitter compounds can also be detected by a recently discovered population of solitary chemosensory cells (SCCs), many of which are innervated by trigeminal nerve fibers 16-19. These SCCs are located in higher density in the entry region of the nasal cavity and vomeronasal entry ducts, hinting that they may also serve a protective function 16-18. Thus, nasal epithelia can differ substantially in function, morphology, and cell composition depending on their anatomical locations.
Even within a single and specialized epithelium, there are regional differences. The MOE is one such example. The MOE lines various turbinates, which are complicated and curled structures. Because of them, different regions of the MOE experience different air flow rates, and thus, different diffusion and clearance rates of airborne odor molecules 20. Also, it is known that olfactory sensory neurons (OSNs) expressing a given odor receptor are located in one of four circumvented zones of the MOE 21,22. How this location difference affects an OSN&#x2019;s response to odorants is largely not known. In addition, some OSN populations exhibit regional preference. Guanylyl cyclase-D (GC-D)-expressing OSNs have zonal distributions favoring the cul-de-sac regions of the ectoturbinates 23,24. More recently, we found a subpopulation of canonical OSNs that expresses transient receptor potential channel M5 (trpM5) and is preferentially located in the lateral and ventral regions 25. These results indicate that MOE is not uniform. However, how these regional differences affect olfactory coding is not understood. This is in part because thorough physiological investigation of the MOE and the nose has been limited by the difficulty of obtaining intact nasal epithelia with preserved anatomical organization using current methods.
The nasal epithelia are predominantly surrounded by the anterior bones of the skull, including the nasal, maxilla, palatine, zygomatic, and ethmoid bones. In adult mice and other rodent models, these bones are hard and difficult to remove without damaging the closely associated nasal tissue, particularly the delicate turbinates. Often, chemical-based decalcification is used to soften bones to allow cryosectioning of nasal tissues for morphological, immunohistochemical, and in situ hybridization studies; however, depending on the age of the animal, the decalcification process can last overnight up to 7 days 24,26-28. This treatment is also limited because it requires tissue be fixative-preserved. Additionally, chemical decalcification can be harsh and affect the immunolabeling of some sensitive antibodies 29,30. For physiological studies, live tissue is required, and thus, these experiments are often conducted on isolated OSNs or MOE slices obtained from neonates whose skull bones are thin and soft 17,31,32. Physiological studies can also utilize whole mount preparations by splitting the head 25,33,34, but usually only the medial surface of the nose is easily accessible, limiting physiological recordings on other areas. 
Here, we describe an effective, manual deboning method to prepare intact nasal tissues with preserved original anatomical organization and morphology. We sequentially remove the major bones of the anterior skull under a dissection microscope to expose an almost entirely intact nasal epithelium while keeping the thin turbinate bones intact unless the mice are very old and cryosectioning is needed. We also extend the method to preserve the connection between the nasal tissues and olfactory bulbs, as well as the rest of the brain, thus facilitating simultaneous examination of both peripheral and central circuits. Our method can be used to prepare paraformaldehyde-fixed, as well as fresh, live nasal tissue. Thus, our method is expected to facilitate morphological, immunohistochemical and physiological studies of respiration, olfaction, and nasal damage and illness.

<table border="1">
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Dissection</td>
 </tr>
 <tr>
 <td>Rongeur, 1.0 mm Jaw width</td>
 <td>World Precision Instruments (WPI)</td>
 <td>501333</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps, Dumont 3 </td>
 <td>WPI</td>
 <td>503235 </td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps, Dumont 55</td>
 <td>WPI</td>
 <td>14099</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps, Dumont AA</td>
 <td>Fine Science Tools (FST)</td>
 <td>11210-20</td>
 <td></td>
 </tr>
 <tr>
 <td>Specimen forceps, Serrated</td>
 <td>VWR</td>
 <td>82027-440</td>
 <td></td>
 </tr>
 <tr>
 <td>Operating scissors</td>
 <td>WPI</td>
 <td>501753</td>
 <td></td>
 </tr>
 <tr>
 <td>Iris scissors, Straight</td>
 <td>Miltex</td>
 <td>V95-304</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection microscope</td>
 <td>Olympus</td>
 <td>SZ40</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Tissue embedding</td>
 </tr>
 <tr>
 <td>Optimum cutting temperature (OCT) compound</td>
 <td>Sakura Finetek</td>
 <td>4583</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic embedding mold</td>
 <td>VWR</td>
 <td>15160-215 </td>
 <td></td>
 </tr>
 <tr>
 <td>Aspirator vacuum pump</td>
 <td>Fisher Scientific</td>
 <td>09-960-2</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 	 <td></td>
 <td></td>
 <td></td>
 <td>Section staining </td>
 </tr>
 <tr>
 <td>Neutral red</td>
 <td>ACROS Organic</td>
 <td>CAS 553-24-2</td>
 <td>Nuclei staining</td>
 </tr>
</table>

an effective manual deboning method to prepare intact mouse nasal tissue with preserved anatomical organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

Using this method, we can reliably obtain almost entirely intact nasal tissue. Figure 2A shows an image of adult nasal specimen from a paraformaldehyde-fixed head. In this specimen, all four sub-olfactory sensory organs, including the MOE, septal organ, the Gruneberg ganglion, and VNO, are intact. Also, the respiratory epithelia and subepithelial tissues, such as glands and vessels, are preserved. We have successfully used this method in a number of studies in which we investigated morphology, distri...

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An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

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JoVE Journal

Biology

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