# Round Window Reinforcement-Induced Changes in Intracochlear Sound Pressure
## 2 Materials and Methods
### HTB Functionality and ICSP Measurements  
After the surgical preparation, the METF and the inner ear hydrodynamic condition of the cochlea were assessed to confirm proper sound transmission from the ear canal to the cochlea.  
The METF was assessed as the \(V_{STAP}\) in response to acoustic stimulation. It was calculated and compared with the standard reference values published by the American Society for Testing and Materials (ASTM) [18].  
To verify that no air bubbles that could modify the hydrodynamics of the ICSP were present in the cochlear fluid, the phase difference between the \(V_{STAP}\) and \(V_{RWM}\) was calculated. A phase difference of 180\({}^{\circ}\) (SD \(\pm\) 7.2\({}^{\circ}\)) between the \(V_{STAP}\) and \(V_{RWM}\) in the frequency range between 100 and 500 Hz is an indication that no air bubbles are present in the cochlear ducts [19].  
For sensor insertion, a cochleostomy was performed by an experienced surgeon (A.D., M.C.) in the exposed bony basal turn of the cochlea. The first cochleostomy was performed in the ST and the second in the SV, approximately 1-2 mm medial to the ventral edge of the RWM. A skeeter drill (Medtronic Xomed, Jacksonville, FL, USA) with a diameter (0.6 mm) slightly larger than that of the sensor head (0.5 mm) was used. The drilling process was carried out under saline water to prevent air infiltration to the cochlea through the cochleostomies. The surgeon confirmed that no damages occurred to the anatomical structures during the cochleostomy procedure.  
Figure 1: (**A**) Custom-made intracochlear acoustic receiver (ICAR) sensor with a zoomed view on the sound receptor and micro-electromechanical system (MEMS) condenser microphone. (**B**) Experimental setup with a human temporal bone (HTB) sample, artificial ear canal (AEC), and two ICARs for simultaneous pressure measurement in SV and ST. For a precise sensor insertion into the cochlea, both hydrophones are positioned using multi-axis micromanipulator units.  
The HTBs were fixed with the cochleostomies located approximately at the highest point of the cochlea, as depicted in Figure 1B. The sensor used for ST measurements was navigated using a micromanipulator with seven degrees of freedom and a 10 \(\upmu\)m adjustment precision. The second sensor (SV measurement) was mounted on an articulated arm with a 2-axis micromanipulator at the base and an additional micro-positioning stage at the front end to control sensor insertion. A surgical microscope was used to insert the sensors into the cochlea. The insertion depth of the sensors was adjusted between 0.5 and 1.0 mm for all samples. The liquid level of the cochlea was maintained constant during sensor insertion to avoid air being entrained in the cochlea fluid. The gap between the outer rim of the sound receptor tube and the bony cochlear surface was sealed with a thin layer of soft tissue topped with a layer of Jeltrate to reinforce the seal (cf. Figure 2).  
The first ICSP measurement after sensor fixation was used as the baseline measurement for comparison with the ICSP data reported in the literature. The noise floor and signal-to-noise ratio (SNR) were determined while the acoustic stimulation was switched off. Only data with an SNR higher 7 dB were considered as results in the present study.