Ketamine

All mice were anesthetized with a 100 mg/kg ketamine (Hospira Inc., Lake Forest, Illinois, United States) and 10 mg/kg xylazine (AnaSed, Shenandoah, Indiana, United States) solution injected intraperitoneally. The toe pinch method was utilized to ensure the mice were fully anesthetized prior to tumor implantation. The lower peritoneal region of each mouse was prepared in a sterile field using povidine-iodine (Purdue Products, Stamford, Connecticut, United States). For LNCaP and PC-3 xenografts, $200\mu \mathrm{L}$ of cell suspension in fresh media ( $1\times {10}^6\mathrm{cells}$ ) was injected into both hind flanks of each mouse subcutaneously. The mice were monitored weekly following implantation for tumor growth and general well-being.

For intracranial (i.c.) injections, age-matched (5–7 weeks) C57BL/6 miR-155^+/+ mice (wildtype (WT)) or miR-155^−/− mice were anesthetized with an intraperitoneal (i.p.) injection of 200 μl of a mixture of ketamine (Hospira, Lake Forest, IL, USA) and xylazine (Phoenix Pharmaceutical, Saint Joseph, MO, USA) in Hank’s balanced salt solution (HBSS). Mice were injected intracranially (i.c.) with 200 plaque-forming units (PFU) of JHMV (strain V34) suspended in 30 μl HBSS [39 (link)]. Clinical severity was assessed using a previously described four-point scoring scale [40 (link)]. For analysis of viral titers, mice were sacrificed at indicated time points. One half of each brain was homogenized and used in a plaque assay performed using the DBT mouse astrocytoma cell line [41 (link)]. The DM-JHMV (2.5 × 10⁵ PFU) strain [31 (link), 42 (link)] was used to immunize experimental mice via i.p. injection to generate virus-specific T cells. This is an established and reliable method to accurately measure T cell responses following JHMV infection [42 (link), 43 (link)]. RAG1^−/− mice were purchased from Jackson Laboratories. All animal studies were reviewed and approved by the University of Utah Animal Care and Use Committee.

All reagents were purchased and used as received unless otherwise indicated. MCC950 sodium salt was acquired from Selleckchem.com (Houston, TX; Catalog No. S7809) and formulated to 4.4 mg/mL in a vehicle consisting of 10% dimethyl sulfoxide (DMSO, Sigma, Burlington, MA; Catalog No. D8418) and 90% Dulbecco’s phosphate buffered saline (DPBS, Gibco, Waltham, MA; Catalog No. 14190–144). Ketamine (NDC 0409–2051-15) was obtained from Hospira, Inc. (Lake Forest, IL). Xylazine (NDC 593990110–20), proparacaine hydrochloride (NDC 17478–263-12), and fluorescein (NDC 17478–253-10) were acquired from Akorn, Inc. (Lake Forest, IL). Tropicamide (NDC 61314–354-01) was purchased from Sandoz (Basel, Switzerland) and phenylephrine (NDC 42707–102-15) was obtained from Paragon BioTeck, Inc. (Portland, OR). GenTeal Severe gel (0065–8064-01) was purchased from Alcon laboratories, Inc (Fort Worth, Texas).

For surgeries, mice were anesthetized with intraperitoneal ketamine (80 mg/kg; Hospira, Inc., Lake Forest, IL, USA) and xylazine (20 mg/kg; Akorn, Decatur, IL, USA). Retinal ischemia-reperfusion was performed as described previously [18 (link)]. Briefly, pupils were dilated with 1% atropine sulfate (Akorn, Inc., Lake Forest, IL, USA). The anterior chamber was cannulated with a 32-gauge needle attached to a line from a saline reservoir at a height calibrated to yield 120 mmHg. The intraocular pressure (IOP) was elevated to 120 mm Hg for 45–60 min; I/R injury and choroidal non-perfusion was evident by the whitening of the anterior segment of the globe and blanching of the episcleral veins [47 (link)]. During infusion, topical anesthesia (0.5% tetracaine HCl) was applied to the cornea. After ischemia, the needle was immediately withdrawn, allowing for rapid reperfusion; IOP was normalized, and reflow of the retinal vasculature was confirmed by observation of the episcleral veins. Topical antibiotic was applied to the cornea to minimize infection. IR injury was performed in one eye, with the other undergoing sham surgery, in which the needle was inserted into the anterior chamber without elevating the IOP. Mice were killed 1, 3 or 14 days post-IR and eyes were processed.

The animals were anesthetized with xylazine (10 mg/kg, i.m., IVX; Animal Health Inc., Greeley, CO) and ketamine (40 mg/kg, i.m.; Hospira, Inc., Lake Forest, IL), and placed on a heating pad in a sound-isolated chamber. To ensure the ear canal was free of wax and that there was no canal deformity, inflammation of the tympanic membrane, or effusion in the middle ear, the external ear canal and tympanic membrane were examined using an operating microscope. Needle electrodes were placed subcutaneously near the test ear, both at the vertex and at the shoulder of the test ear side. Each ear was stimulated separately with a closed-tube sound-delivery system sealed into the ear canal. To measure the auditory brainstem response, tone bursts with a 1 ms rise time were applied at 4, 8, 12, 16, 24, and 32 kHz and thresholds obtained for each ear; the tone-burst stimulus intensity was increased in steps of 5 dB. The threshold was defined as an evoked response of 0.2 μV from the electrodes. ABR measurements were taken before noise exposure, 1 hour after noise exposure (temporary threshold shift), and 2 weeks after noise exposure (permanent threshold shift).