Ketamine hcl

To assess the cochlear surface morphology, the Cdk5rap1-KO and CNT mice were fixed using 4% paraformaldehyde by cardiac perfusion under deep anesthesia induced by intraperitoneal injection of 4 mg/kg xylazine (Bayer, Shawnee Mission, KS, USA) and 120 mg/kg ketamine-HCl (Daiichi Sankyo, Tokyo, Japan) in 0.9% NaCl. Following fixation, the bony capsule and lateral wall of the cochlea were removed. Samples were incubated with Texas Red-X phalloidin (1:100; Molecular Probes) for 30 min to stain F-actin. The cochlear surface morphology was imaged using a BZ-9000 fluorescence microscope. Five randomly selected surface images of the OC were captured at every turn of the cochlea at 40 × magnification for each group. The nusmber of IHCs and OHCs in a 140-μm basal segment of the basilar membrane was counted. Only HCs with an intact stereociliary bundle and a cuticular plate were counted per cochlear turn as previously described [32 (link), 44 (link), 45 (link)]. A second researcher verified the accuracy of the results (n = 5 for each genotype).

For plasmid inoculation into the mouse otocyst, E 11.5 timed-pregnant mice were anesthetized with intraperitoneal administration of 10 mg/kg of xylazine (Bayer, KS) and 80 mg/kg of ketamine–HCl (Daiichisankyo, Tokyo, Japan) in saline solution. Procedure details were described previously^{11 (link)}. Briefly, the uteri were gently removed from the pregnant mice following low midline laparotomy, Egfp-plasmid or Slc26a4-Egfp plasmid vectors with 0.1% fast green (Sigma, Missouri) as a tracking dye were injected by oral pressure into one side of otocysts in one or two embryos per a dam using heat-pulled glass micropipettes whose tip diameters were from 10 to 15 µm. Subsequently, the plasmid-filled otocysts were pinched on both sides of the embryonic head using a tweezer-style electrode paddle, then electroporated.

Pregnant mice were anesthetized, on E11.5, with intraperitoneal administration of 10 mg/kg of xylazine (Bayer, KS, USA) and 90 mg/kg of ketamine-HCl (Daiichisankyo, Tokyo, Japan) in saline. Details of the intrauterine injection into mice otocysts were described previously^{2 (link),34 ,35 (link)}. Progenitors of OSCs, which were used for transplantation, were dissociated from 6-well dishes into single cells using a 0.25% trypsin/0.02% EDTA solution and pipetting procedure. Briefly, each uterus was illuminated with a fiber-optic beam to identify the otocyst (Fig. 1 and Supplementary Figure 2B) after laparotomy, and subsequently, the cell admixture with 1% fast green (Sigma, St. Louis, Missouri, USA) as a tracking dye was injected by oral pressure into the right-side otocysts in one or two embryos per dam through the uterine wall using heat-pulled 20- to 30-μm glass microcapillaries (Fig. 1 and Supplementary Figure 1B). After the injections, the embryos were gently returned into the abdominal cavity and the abdominal skin was sutured with 30 mg/kg of chloramphenicol. For postnatal assessments, the treated embryos on E18.5 were passed to surrogates for further fostering.