This protocol reduces the time for temporal bone processing from 9 to 10 months to 10 to 14 days. The main advantage of this technique is it allows for the rapid assessment of otopathology, which in our case is relevant for the assessment of COVID-19-associated middle ear pathology. This technique may be particularly helpful for time-sensitive studies of the temporal bone.
In the future, it may be further optimized for use studying inner ear pathology. Begin the harvest during autopsy and divide the cranial nerves sharply to the internal auditory canal. Make the first bony incision with the osteotome parallel to and just medial to the squamous portion of the temporal bone.
Then, perform the second bony incision parallel to the first at the medial border of the temporal bone. Make the third incision at three to four centimeters anterior and parallel to the petrous ridge. Next, make the fourth incision roughly parallel to the third at two centimeters posterior to the petrous ridge.
Use sharp dissection to free the soft tissue attached along the inferior edge of the specimen and remove the temporal bone. After the harvest, immediately submerge the tissue in 200 to 300 milliliters of 10%buffered formalin. Then, set a precision microsaw with twin diamond blades to a distance of five millimeters.
For rapid tissue decalcification, cut each specimen into three sections such that the central section of the temporal bone is five millimeters thick and those on either end are three to five millimeters thick. Place the sections in 200 to 300 milliliters of 0.37-molar EDTA for seven to 10 days at room temperature for softening. Hematoxylin and eosin staining of the middle ear mucosa and eustachian tube showed that the tissue is preserved after processing using this protocol.
Immunohistochemical analyses showed the presence of ACE2, transmembrane protease serine, and furin proteins within these tissues, providing a possible root for SARS-CoV2 infection in the respiratory epithelium of the middle ear. It is important to appropriately position the temporal bone tissue within the diamond saw. Alternative techniques that can be combined with this method include immunohistochemistry for different proteins, different tissue stains, and transcriptomics.
This technique allowed us to rapidly assess for the presence of the proteins involved in SARS-CoV2 infection within the middle ear during the COVID-19 pandemic.
