The endolymphatic sac is required for normal development of the inner ear. Since it is inaccessible in situ for structural, physiological, and molecular studies, its dissection from animal models, especially mouse models, is essential. Dissection and preparation of the endolymphatic sac are extremely difficult due to its thin, delicate epithelial structure, and its proximity and adherence to adjacent tissues.
Our approach facilitates the visualization and identification of a sac and its separation from adjacent tissues. Diseases and disorders affecting the normal function of the endolymphatic sac can lead to loss of hearing and balance. Our technique is essential to elucidate the cellular, molecular, and physiological processes underlying endolymphatic sac functions.
Demonstrating the procedure will be Hyun Jae Lee, a postdoctoral visiting fellow from my laboratory. Start with arranging the inner ear preparation in a 35 millimeter tissue culture dish containing PBS with the root of the eighth cranial nerve oriented up. Using a No.4 forceps, hold the tissue at the cochlea and identify the vestibular aqueduct, the anterior and posterior semi-circular canals, the common crus, and the sigmoid sinus.
Then use a 27 gauge needle mounted on a one milliliter syringe to incise the dura mater and vestibular aqueduct, as well as the underlying connective tissues surrounding the endolymphatic sac. After making an incision into the connective tissue lateral to the endolymphatic sac, use forceps to hold the connective tissue and peel it by pulling it away from the temporal bone. Carefully remove any remaining debris and separate the endolymphatic sac epithelium from surrounding tissues.
For dissection of the opened endolymphatic sac, hold the stem part of the preparation to observe the cross-section of the lumen. Then insert a 27 gauge needle into the lumen and move it to cut the endolymphatic sac into two sheets. Hold the edge of each sheet-like tissue with forceps and separate them from each other.
In the representative analysis, a mid-sagittal section of the skull of these mice before and after half brain removal was visualized with tdTomato fluorescence. The endolymphatic sac was readily visible even without dissection. Inner ears from mice at embryonic day 16.6 as well as postnatal day 5 and 30 were isolated.
The endolymphatic sacs were observed under higher magnification. For P30 mice, the endolymphatic duct is difficult to isolate due to its encapsulation in bone. Immunohistochemistry of intact endolymphatic sacs from E16.5 and P5 was studied with anti-SLC26A4 antibody and phalloidin labeling.
Phalloidin was used to highlight the endolymphatic sac and the conjunctive tissue around it. High magnification images of opened endolymphatic sac epithelium at P5 show the SLC26A4 labeled in green is localized in the apical region of a subset of cells. The most important thing to remember when attempting this procedure is to be very gentle to avoid damaging the endolymphatic sac.
A fixed endolymphatic sac tissue isolated by this approach can be used for in situ hybridization, RNA scope, and immunohistochemistry. Live tissue can be used to prepare RNA for transcriptome analysis and for physiology. This approach has been used to study the transcriptome of the endolymphatic sac, either as a whole or in single-cell RNA-seq analysis to contribute to characterizing its different cell types.
