Autosamdri 931

The rats with the 4VO were sacrificed seven days after the procedure under anesthesia using inhalation of 2% isoflurane. Rats in the sham group were sacrificed without any procedures at the same weeks in 4VO model, after exposing the heart, we made a small incision through the apex of the left ventricle to insert the perfusion catheter and another small incision in the right auricle to let the perfusate escape. Subsequent to transcardiac perfusion with 200 mL of normal saline for exsanguination, the rats were perfused with 300 mL of 4% formaldehyde. The temporal bones containing the inner ear were dissected out and then immersed in the Karnoysky’s fixative (2% glutaraldehyde–2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for two hours at 4℃. The otolithic organs were dissected under the stereo microscope (Stemi-305, ZEISS; Oberkochen, Germany) and again immersed in the Karnoysky’s fixative for six hours at 4℃. And then the tissues were washed with 0.1 M phosphate buffer and postfixed with 1% OsO₄ for 1.5 hours. Then, we performed dehydration by gradually increasing the concentrations of ethanol, drying with the critical point dryer (Autosamdri-931, tousimis; Rockville, MD, USA), and coating with platinum by ion sputter (EM-ACE600, Leica; Wetzlar, Germany). The specimens were then subjected to scanning electron microscope (SEM; MERLIN, ZEISS).

Hydrogels were produced by directly mixing 150 μL of 1 M NaCl into 850 μL of the cellulose dispersion (0.072 wt%) in a mould (d = 1.6 cm). The mould was sealed with parafilm and left to gel overnight. Alternatively, 940 μL of the dispersion (0.065 wt%) was mixed with 60 μL 0.1 M HCl.
An alternative way for hydrogel formation was by diffusion. A mould filled with 1 mL of the fibril dispersion (0.061 wt%) was dipped into a NaCl (150 mM) or an HCl (6 mM) solution. A dialysis membrane (Spectrum Laboratories Standard RC Tubing, 6–8 kDa cut off) and a Teflon membrane (Sigma-Aldrich, 1.0 μm × 47 mm), which prevented the gel from sticking to the cellulose-based dialysis membrane, were used to cover the mould to allow the diffusion of the salt and protons into the dispersion.
Hydrogels were carried over into a customized metal mesh cage and placed in 100 mL of 50% EtOH at 5 °C for 24 h for solvent exchange. The second and third exchanges took place in 97% EtOH (100 mL), and in 99% EtOH (50 mL), respectively, followed by supercritical CO₂ drying (Tousimis, Autosamdri 931).