Homeothermic blanket system

Male CD1 mice (n = 15) from Janvier Labs were included in this study at 18 days of age. The animals were maintained in temperature and humidity-controlled facilities. Ambient sound pressure levels inside the cages were below 40 dB SPL. For all hearing test experiments, mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.). Body temperature was maintained at 37°C (Microprobe Thermometer, BAT-12, WPI) with an isothermal pad (Homeothermic Blanket System, Harvard Apparatus). Prior to testing and to exclude middle ear damage, an otoscopic examination (using a binocular operating microscope) was performed on each mouse. Cochlear function was assessed via ABR and DPOAE at postnatal days 18, 21, 25, and 30 (i.e., P18, P21, P25, and P30). All the auditory tests were performed in a sound attenuated and electrically shielded recording chamber. After the final auditory test, animals were sacrificed for histological processing and scanning electron microscopy analyses of hair cell stereocilia. All procedures were approved by the Regional Ethics Committee for animal experiments in France (Comité d'Éthique pour l'Expérimentation Animale Auvergne; EC 92-12).

Animals were prepared for CCI as previously described^{36 (link)}. Male CD1 mice at postnatal (P) day 21 and P60-75 were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneal injection and positioned in a stereotaxic frame. Body temperature was monitored with a homeothermic blanket system (Harvard apparatus, Lewes, DE) containing a rectal probe and maintained at 37 °C with an autoregulated heating pad. A 4 mm craniotomy was made using a portable drill over the right parietal-temporal cortex (−2.5 mm A/P and 2.0 mm lateral from bregma). Injury was induced by moderate CCI using the eCCI- 6.3 device (Custom Design & Fabrication; 3 mm beveled steel impact tip) at a velocity of 5.0 +/− 0.3 m/s, 1.0 mm depth and 150 ms impact duration^{19 (link),37 (link)}. Sham controls received craniotomy only.

Adult CD1 mice were used for these experiments after approval was obtained by the IACUC of Weill Cornell Medical College. Mice were induced with isoflurane (2–4%) in 70% N₂:30% O₂ by facemask. After induction, the animal was maintained under isoflurane anesthesia using a small mask at ~1.5%. Animals were mounted in a stereotaxic frame. The heart rate, arterial blood oxygen saturation, and end-tidal CO₂ were monitored and maintained at normal values throughout the experiment. Temperature was monitored rectally and maintained at 37°C with a homeothermic blanket system (Harvard Apparatus, Holliston MA). A ~ 4 × 5 mm cranial window was opened over one hemisphere between Lambda and Bregma to expose the neocortex. The dura was carefully removed. Ruthenium-bipyridine-triphenylphosphine caged 4-aminopyridine (RuBi-4-AP) (200 μl, 10 mM) was topically applied or microinjected into neocortex at least 30 min before uncaging. For the focal illumination, 2 μl RuBi-4-AP solution was injected in neocortex at the depth of 300 μm using an UltraMicroPump (UMP-3, WPI, Sarasota, FL) via a glass electrode controlled at a speed of 100 nl/min. For control experiments, 4-AP ictal discharges were induced by injecting 4-AP (Sigma, 15 mM, 0.5 μl) at the same location using a Nanoject II injector as previously described (Schwartz and Bonhoeffer, 2001 (link); Zhao et al., 2009 (link)).