The outer hair cell can be divided into three domains: the apex, the base, and the lateral wall. With the use of filipin, a polyene fluorescent antibi- otic that binds to cholesterol, we found under fluo- rescence microscopy that the lateral wall mem- branes were less intensely stained than the apical and basal membranes. This difference in filipin fluo- rescence between the lateral walls and the ends diminished when cells were incubated in water-sol- uble cholesterol before staining, suggesting that exogenous cholesterol enters the lateral wall. Under confocal microscopy, we studied the incor- poration pattern of a fluorescent cholesterol ana- logue, NBD-cholesterol. NBD-cholesterol did not stain the apical membranes whereas it intensely labeled the lateral wall. The micropipette aspiration technique was used to assess the effect of choles- terol on lateral wall stiffness. The lateral wall stiffness parameter of cells treated with water-soluble cho- lesterol (n = 23) was significantly higher than that of controls (n = 27): 0.76 ± 0.24 (mean ± SD) versus 0.46 ± 0.10 (Student’s t test, p < 0.001). In conclusion, cholesterol has different distributions among outer hair cell membranes, and when water-soluble cho- lesterol is incorporated into the cells, the outer hair cell lateral wall stiffness parameter increases. (Otolaryngol Head Neck Surg 1998;119:14-20.) 14 L ittle is understood about how an elevated blood cholesterol level increases the risk of hearing loss in human beings 1-5 and animals. 6-9 The cochlear outer hair cell is a highly polarized, cylindrical, mechano- sensory cell of epithelial origin with three functional domains: the apex, with its stereocilia, which is involved with mechanoelectrical transduction; the base, with synaptic transmission; and the lateral wall, with its unique electromechanical transduction. The third process, electromotility, results in length changes of the cell, 10 which can occur at acoustic fre- quencies (>20 kHz). 11 Electromotility is thought to be responsible for the remarkable sensitivity of mam- malian hearing. 12 The functional differences among the domains place constraints on the lipid composition of their mem- branes. Cholesterol, by promoting close packing of the acyl chains of phospholipids, 13 reduces not only the lipid bilayer permeability to small molecules 14 but also membrane fluidity. The outer hair cell thus faces the dilemma of requiring cholesterol for intracellular integrity and, at the same time, of minimizing the pos- sible impact of cholesterol on membrane mechanics. Freeze-fracture images of the cell’s membranes have suggested that cholesterol distribution to the lateral wall differs from that to the apex and base. 15 Here we show the location and relative concentration of cholesterol in cochlear outer hair cell membranes and the effect of water-soluble cholesterol incorporation on the cell’s mechanical properties. The results are in three parts: (1) localization of cholesterol within the cell, (2) uptake of exogenous cholesterol by the cell, and (3) measurement of lateral wall stiffness. METHODS AND MATERIAL Outer Hair Cell Isolation Guinea pigs of either sex weighing 200 to 300 gm were killed by decapitation. Outer hair cells were isolated from the inner ear by conventional methods. 10 Morphologic criteria of cells for inclusion in the study included a characteristic bire- fringence and crisp cylindrical shape without regional swelling. The cytoplasm in the axial core immediately apical First Place—Resident Basic Science Award 1997 Contribution of membrane cholesterol to outer hair cell lateral wall stiffness THANH-VAN N. NGUYEN, MD, and WILLIAM E. BROWNELL, PhD, Houston, Texas From the Bobby R. Alford Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine. Supported by the American Academy of Otolaryngology–Head and Neck Surgery Foundation Resident Research Grant and the National Institute on Deafness and Other Communication Disorders DC-00354 grant. Presented at the Annual Meeting of the American Academy of Otolaryngology–Head and Neck Surgery, San Francisco, Calif., Sept. 7–10, 1997. Reprint requests: W. E. Brownell, PhD, Bobby R. Alford Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine, NA505, One Baylor Plaza, Houston, TX 77030. Copyright © 1998 by the American Academy of Otolaryngology– Head and Neck Surgery Foundation, Inc. 0194-5998/98/$5.00 + 0 23/10/88638