Hello, I'm David Dunton from Dr.Wung Lynn's lab at UMBC. In this video, we show an effective technique to prepare mouse nasal tissue with preserved anatomical organization. This technique allows one to obtain a single specimen containing nearly the entirety of nasal tissue, including respiratory and olfactory, epithelial and subepithelial tissue.
We show the step-by-step removal of various facial bones surrounding the nasal tissue that preserves even the most delicate of tissue, such as the terminates of the main olfactory epithelium. Our method has significant advantages over decalcification methods because it avoids harsh chemical treatment and bone removal can be accomplished in 20 to 30 minutes. Other advantages of this technique are that it is not limited by the age of the mouse and it can be performed using fixed or fresh tissue.
These advantages make our technique ideal for morphological, immunohistochemical, and physiological studies. Here we show a schematic diagram of the dorsal aspect of the bones of the skull and the nose. In this video, we will focus on the dissection of the bones of the nose.
This is a schematic diagram of the ventral aspect of the bones of the skull and the nose. This is a dorsal view with an un dissected skull and nose at the top and a dissected nose at the bottom. Shown now are the terminates of the main olfactory epithelium.
Here we show the bones of the nose starting from the dorsal aspect. This is the dorsal part of the zygomatic plate, followed by the zygomatic arch. This is the nasal bone, and next shown is the coddly located cribriform plate.
Far anterior is the location of the gruenberg ganglion. From the lateral viewpoint, we show the portion of the ethmoid bone that covers the turbo of the man olfactory epithelium, the grunberg ganglion, the maxilla, and the front incisors. Switching to a ventral viewpoint, we showed the molars, the front incisors, the palatine bone, and the vulner bone.
Begin the removal of the vulner bone by looking at the ventral aspect of the nose. To remove the vulner bone, use forceps to break the vulner bone along its length, and then remove the pieces of bone by flipping them away from the underlying tissue To avoid damage to the RO nasal organ. For this task, use jurors to break the ventral part of the front incisors.
Use a clamping motion and break the front incisors towards the location of the ulmer bone. Once the front incisors are broken, remove the remaining bone fragments that are connected to tissue by using forceps. To loosen the maxilla, start from the ventral aspect and break the maxilla just ventral to these zygomatic arch.
Flip the nose to the dorsal aspect and break the maxilla just anterior to the zygomatic arch. To loosen the maxilla to remove the right side of the nasal bone, clear away the remainder of the frontal bone coddle to the right side of the nasal bone. You'll use Ron jurors for this task.
It is necessary to exercise caution while using the Ron jurors to avoid damaging the thin cribriform plate because damage to the cribriform plate can cause the nose to split in half. Flip up and remove the broken bone. Next, flip the dorsal part of the zygomatic plate away from the underlying tissue.
Separate the nasal bone along its midline Using a pair of forceps or a eraser, grab this bone with forceps and gently move the bone from side to side while lifting up. This procedure should be done with some delicacy because it is easy to damage the underlying tissue while removing the bone. Once the bone is removed, you can see the underlying tissue separately, which is shown in this still image.
To remove the bone, simply cut it using forceps or scissors. Now go ahead and remove the anterior maxilla. If it has not already been removed at this point, the anterior maxilla is already loose, so remove it by breaking any remaining connections to tissue.
From a U dorsal viewpoint, remove the frontal bone that is just coddled to the left side of the nasal bone. Use the Ron jurors to break the frontal bone. You may use raw jaws or forceps to remove that piece of the frontal bone.
You may also loosen the maxilla at this time, although that is not necessary. To remove the left side of the nasal bone, use the forceps and gently separate the nasal bone for many of the surrounding bones. Then grab this bone with forceps and generally move the bone from side to side while lifting up to separate the nasal bone from the underlying tissue.
As the underlying tissue separates from the bone, you are sometimes able to use a lateral viewpoint to see the tissue connected to the bone. Use the forceps to gently separate the tissue connected to the bone, and then you'll remove the bone with scissors or forceps With the nasal bone removed. Remove the anterior maxilla on the left side of the nose using the same technique for removing the anterior maxilla on the right side of the nose.
The next step is to remove the zygomatic plate on the left side of the nose. There may still be some attachment of the zygomatic plate and underlying tissue, and if that is the case, then you may use the raw jurors to gently clamp and loosen the dorsal part of the zygomatic plate. Look at the ventral aspect of the nose.
To identify the molars and the palatine bone, the either the molars or the palatine bone may be removed. First, this is a view of the lateral aspect of the nose, and this view shows the removal of the molars on the right side of the nose. Using the urs with the right side of the maxilla removed, the palatine bone is removed.
Next, break the remaining maxilla and molars and remove any large bone fragments from the palatine bone or the maxilla. Break off and remove any remaining pieces of the ethmoid bone projecting coddle to the man manufacture epithelium. Place the fine forceps underneath the bone and flip up the bone to separate it from the underlying tissue.
Alternatively, use the Rogers as shown here to break the bone and then use the fine forceps to separate the bone from the underlying tissue. This is what the turbinate of the man olfactory epithelium look like. With the moid bone removed, this bone will be removed.
Next, it is coddle and medial to the nasal bone, and this bone will be removed using gers. Use caution during the step to avoid damaging the cribriform plate. Once the bone is broken, use forceps to remove the bone.
Next is a view of the nose. Once this bone has been removed, one last area to check is around the V nasal organ. Because there is a bone separating the paired tubes of the V nasal organ that I am just now removing in this dissection.
This is the dorsal viewpoint of a nose that has been dissected using our technique. Next, we've rotate a nose to show its various aspects. This is the dorsal view followed by the lateral view of the left side of the nose and the ventral view.
Now we show in detail the features of a nose fully dissected using our technique starting with the dorsal aspect of the nose. The first part we show is the turbinates of the main refactory epithelium. There are air bubbles in the tissue underlying the nasal bones, and these air bubbles are removed by vacuuming the sample from the lateral viewpoint.
We show the intact turbinate of the man olfactory epithelium. Notice that there are air bubbles present within the turbinates. Glandular tissue is anterior of the turbinate and respiratory epithelium is visible from this lateral viewpoint.
From the ventral viewpoint, the vulnerable nasal organ is visible. This picture shows an extension of our dissection technique, removing the brain olfactory bulbs and nose in one specimen. To prepare the tissue for sectioning, first take a plastic mold and fill it with optimum cutting temperature media.
Place the tissue of interest into the filled mold and place the mold inside a circle of silicone grease on a plastic way dish, a flask will be placed on the circle of silicone grease to create a seal for the vacuum. Our lab uses a vacuum created by running water. Once the vacuum is created by running, the water bubbles are pulled out of the tissue.
The vacuum is stopped by turning off the water and releasing the tubing. Next, retrieve the plastic mold. Here I am moving the tissue to its desired orientation before freezing the tissue.
Our lab uses dry ice to freeze down tissue. These are represented results of sectioning a nose using our dissection technique and staining with neutral red. This is a horizontal section showing the turbinates of the man effector epithelium, glandular tissue, and respiratory epithelium.
The second section is a coronal section that shows the convoluted folding pattern of the turbinates of the main olfactory epithelium. In this video, we show a technique to manually debone the nose and obtain a specimen ideal for morphological of immunohistochemical or physiological studies.
