Ketamine

All chemicals were purchased from EMD Chemicals Inc. (Darmstadt, Germany) unless otherwise stated. Dopamine hydrochloride, Tris base, Tris hydrochloride, 3,4-ethylenedioxythiophene (EDOT), and GBR-12909 were purchased from Sigma-Aldrich (St. Louis, MO). Eticlopride hydrochloride was purchased from Tocris Bioscience (Avonmouth, Bristol, U.K.). Nafion solution (LQ-1105) was purchased from Ion Power Inc. (New Castle, DE) and used as provided. Ketamine was purchased from Phoenix Pharmaceuticals Inc. (Ketaject, St. Joseph, MO). Artificial cerebral spinal fluid (aCSF) was made by dissolving 15 mM Tris, 126 mM NaCl, 2.5 mM KCl, 20 mM NaHCO₃, 2.0 mM NaH₂PO₄, 1.2 mM Na₂SO₄, 1.2 mM CaCl₂, and 2.0 mM MgCl₂ in 18.2 MΩ water (EMD Millipore) and the pH was adjusted to 7.40 using HCl. To produce an aCSF gelatin, porcine gelatin (0.5% by mass) was added and the solution was heated until it dissolved. The mixture was allowed to set at room temperature.

Ketamine and xylazine were purchased from Phoenix Pharmaceuticals Inc., (St Joseph, MI). Buprenorphine was purchased from Rickitt & Colman (Richmond, VA). Dura-Pen was from Vedco Inc. (Overland Park, KS). 17β-estradiol sulfate, Propylpyrazole triol (PPT) and 2,3-bis(4-hydroxyphenyl)-proprionitrile (DPN) were purchased from Sigma Aldrich (St. Louis, MO). G1 was purchased from Tocris Biosciences (Ellisville, MO). The ER agonists were dissolved in DMSO for stock solution and the working concentration was a 1:50 dilution in Sterile saline. Sterile saline was purchased from B. Braun Medical (Irvine, CA).

Immunocompetent mice were infected intratracheal with 10⁶ fungal spores according to Andrianaki et al. [6 (link)]. The mice were anesthetized by intraperitoneal injection of 0.2 ml mixture of ketamine (82.5 mg/kg, Phoenix, St. Joseph, MO) and xylazine (6 mg/kg, Lloyd Laboratories, Shenandoah, IA). The intratracheal injection of the fungal spores was performed according to Luo et al. [27 (link)]. The mice were then euthanized by cervical dislocation at different time points including 4, 24, and 72 h, the lungs were homogenized, and fungal CFU counts were assessed. All animal studies have been taken place at the Lundquist Institute for Biomedical Innovations, Torrance, CA, U.S.A. Animal studies were approved by the IACUC of the Lundquist Institute for Biomedical Innovations at Harbor-UCLA Medical Center and according to the NIH guidelines for animal housing and care.

This study was carried out in strict accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research, set out by the Association of Research in Vision and Ophthalmology (ARVO) and was approved by Tufts University Institutional Animal Care and Use Committee (IACUC) protocol B2011-150 and Tufts University Institutional Biosafety Committee registration 2011-BRIA68. For experiments 6 weeks old C57Bl/6J mice (Jackson laboratory, Bar Harbor, ME) were used. Animals were anesthetized by intraperitoneal injections of a mixture containing 0.1mg/g body weight Ketamine (Phoenix™, St Joseph, MO) and of 0.01mg/g body weight Xylazine (Lloyed, Shenandoah, IA). Mice were kept warm during anesthesia. Mice were sacrificed by CO₂ inhalation followed by cervical dislocation.