Animal model and preparation
Guinea pigs are widely used in researches on auditory and inner ear disease due to the sensitive audition [16, 23, 24]. In this study all experiments were operated in vivo with adult guinea pigs of either sex. The whole animal experiment operations were conducted in accordance with the Guidance for Care and Use of Laboratory Animals of Shandong University and with support from the otorhinolaryngology department of Shandong Provincial Hospital.
The animals were anesthetized by single intraperitoneal injection of ethyl carbamate solution (1 g/kg body weight in 20 % sterile saline) in first step. Additional dose of 0.2 ml could be needed to ensure deep anesthesia of guinea pigs during the experiment. Once the animals were under deep anesthesia condition, they were then positioned on an operation table with animal heads stabilized in a head holder, preparing for the otologic surgical procedure. Considering the stability of the experiment and animal care, animals were placed in the thermostatic chamber to maintain body temperature at 38 °C. Next, the surgery was carried out. The animal’s bulla was exposed by retro-auricular incisions to provide access to the cochlea. Then the muscle tissue and soft tissue were dissected and the bulla’s posterior-lateral part was opened to get access to the round window niche. Figure 1b shows the surgical incision location and the exposed round window of cochlea.
ABR measurement
In this study the record of ABR was used as the index of auditory response. The Nicolet evoked potentials system (Endeavor CR, Nicolet Biomedical, USA) was used to record and measure the ABR signals. Three subdermal needle electrodes (DSN-1248 type, 13 mm length, 0.4 mm diameter, SunSpots series, Axon System, Inc. USA) were placed under the skin to obtain the ABRs. The record electrode was placed at the vertex of animal head, and the reference electrode was placed in the ipsilateral mastoid, while the ground electrode was placed in the neck muscles. Each acquired ABR data was filtered by the digital filter between 100 to 3000 Hz and averaged from 1024 trials in the Nicolet instrument to remove the irrelative noise and interference. All the recording operations were conducted in an electromagnetic shielding and sound-proof room.
Acoustic stimulation
After the surgery, we firstly gave the animals acoustic stimuli, serving as a reference to the later SWIR laser stimulation. The whole experiment was performed in the sound-proof circumstance to keep the results reliable.
The inner trigger acoustic stimulator of the Nicolet Evoked Potentials system was used to stimulate the inner ear. The sound stimuli were delivered to the animal right ear canal with a polyurethane foam earplug, which was coupled to a transducer (TIP300 type, Endeavor CR, Nicolet Biomedical, USA) via a silicone tube. The transducer was attached to the acoustic stimulator of the potentials system and it was calibrated with a digital phonometer before each measurement. A series of 11 Hz click-sound stimuli with alternating polarity were used in the study, which was normally used in relevant studies [19, 20]. The acoustically induced ABR at several sound pressure level (SPL) from 40 to 100 dB SPL in 20-dB steps were recorded, respectively. During the acoustic stimulation, the vibration of periosteum was transmitted through the middle ear to the cochlea, and then perceived by the auditory neurons.
Deafening procedure
Given that photoacoustic-induced vibration may also induce the auditory neural response in normal hearing animals [11, 16–18], laser stimulation with deafened animal is indispensable in order to verify the direct activation of auditory neurons by the SWIR laser. Therefore after the acoustic stimulation operations in normal hearing animals, extra deafening procedure was taken to deafen the guinea pigs’ cochlea acutely. The osseous spiral lamina was exposed carefully with a medical electrical drill and the basilar membrane was destroyed subsequently with a dissecting needle. The invasive procedure blocked the hair cells’ sensory function and caused hearing loss. Extra acoustic stimulation tests were done after the deafening operation to verify the loss of hearing. Then the laser stimulation was performed immediately after the acoustic test.
Laser stimulation
The pulsed SWIR laser with the central wavelength of 980 nm was used for the optical neural stimulation in vivo. The laser light was coupled into an optical fiber (Flexi Ray series, 105-μm-diameter core, Art photonics, Germany) for delivery. The laser pulse parameters, including pulse energy and pulse width, can be regulated with the adjustable current source and the real-time data can be monitored through a human machine interface.
Here in our study, we chose the modiolus of the cochlea as the stimulation site. When the round window was exposed after the animal surgery, the optical fiber was carefully inserted into the round window membrane using a three-axis micromanipulator (TSD-40 XYZ, SIGMA KOKI, Japan). Fiber position was carefully adjusted to make it directly oriented to the modiolus, the distance from the fiber distal end to the stimulated spiral ganglion cells was approximately 0.3 mm. Figure 1a shows the orientation and placement of the optical fiber towards the round window of the cochlea. Optical fiber was cleaned and cleaved before each measurement. The modulated pulsed laser light was then transmitted by the optical fiber and guided into the basal turn of the cochlea to irradiate the spiral ganglion.
In the first step, the effect of SWIR laser pulse energy to its evoked auditory neural response was investigated. The laser parameters of repetition rate and pulse width were kept to 11 Hz and 200 μs. And the laser single pulse energy was ranged from 0.05 to 0.5 mJ by adjusting the laser output peak power from 0.25 to 2.5 W. The pulse energy was measured in air with an energy meter (Nova II meter, PE10BF-C probe, Ophir Photonics, Israel) at the tip of optical fiber. The OABRs evoked with different laser pulse energy levels were recorded accordingly and the ABR wave III peak amplitude and the wave III absolute latency time were measured for later analysis.
Furthermore, the SWIR laser pulse width variation and its effects on neural response were studied. The laser pulse repetition rate remained 11 Hz and the peak output power was kept constant at 1 W during the stimulation process. Then the pulse width was adjusted from 100 to 800 μs in 100-μs steps. The OABR wave III peak amplitude and the wave III absolute latency time at each laser pulse width were recorded and measured subsequently.
Data statistical method
Each ABR data was averaged from 1024 trials and synchronized at the stimuli start point, for both acoustic and optical stimulations. In this study, the ABR waveforms were plotted with the peak positive voltage input of the amplifier with the recording electrode in the vertex site. And the wave III peak amplitude was used as the representation of auditory neural response intensity. Meanwhile, the time interval between the start point and the peak of wave III was plotted as wave III absolute latency time. The Origin 8.6 software was used for data processing and analysis. The ABR data through all experimental animals were averaged, with the mean, standard deviation and standard error of data calculated under the different stimulation parameters. The one-way analysis-of-variance (ANOVA) statistical model was implemented to verify if the value differences between individual animals were significant in the OABR parameters.