Xylazine hydrochloride

Three-week-old mice were anaesthetized with an intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Sigma-Aldrich, Burlington, MA, USA) and xylazine hydrochloride (10 mg/kg, Sigma-Aldrich, Burlington, MA, USA) and maintained at 37°C on a hot pad during the procedure. ABR recording was performed on both ears as described previously (Chen et al., 2003 (link)). Under anesthetic conditions, 25 μL 50 mg/mL gentamicin solution was injected through the tympanic membrane using a 30G needle into the right ear and the mice were kept lying on their left side until awake. Two weeks after gentamicin injection, recovery of the ear drum was confirmed under microscope after administering anesthesia, and ABR was performed on both injected ears and the contralateral control ears.

Six-week male CD-1 (ICR) mice were purchased from Shanghai Laboratory Animals Center. For HSV-1 infection, mice were anesthetized by intraperitoneal injection of 0.4 ml of a mixture consist of 500 ug/ml xylazine hydrochloride (Sigma, X1251) plus 4 mg/ml pentobarbital sodium (Solarbio) in sterile saline. Then 2 x 10⁵ pfu of virus was added onto each scarified cornea in 3 μl of PBS. For TG acquisition, mice were euthanized by cervical dislocation while under anesthesia with isoflurane and TG were collected into the lysis buffer and placed on dry ice before RNA extraction and placed into -80°C as described previously [59 (link)]. For western blot analysis, we homogenized TG in lysis buffer (Solarbio, R0020) and extracted protein according to the manufacturer’s protocol. For determination of viral titers in TG, TG were homogenized in cell culture media for titer determination. For eye swab collection, mice were anesthetized in an induction chamber with isoflurane (3% in oxygen 0.5 ml/min) using a V1 Table Top anesthesia machine (Colonial Medical Supply). Both eyes of each mouse were swabbed with cotton-tipped applicators, suspended in 1 ml of cell culture medium.

To obtain E15.5 embryos, Spic^GFP (CD45.2) homozygous male and C57BL/6 (CD45.2) female mice were mated in a cage overnight and separated on the next day. Pregnant mice were sacrificed 15 days later, the viable embryos were harvested, and the thymuses were isolated in ice-cold PBS. Congenic CD45.1 recipients were anesthetized by i.p. injection of ketamine hydrochloride (120 µg/g, Toronto Research Chemicals) and xylazine hydrochloride (12 µg/g, Sigma). The fur on the left flank was removed, and the left kidney was exposed by cutting the skin, muscle layer, and peritoneum. The kidney capsule was nicked with a G23 needle, and the fetal thymus was pushed into the pocket under the kidney capsule with a G23 needle equipped with a plunger from a spinal needle. After the kidney was re-positioned back into the peritoneal cavity, the peritoneum was sutured, and the skin was stapled with metal clips. Rymadil (Carprofen, 5 µg/g, Zoetis) was given subcutaneously to ease the wound pain, and Trimerin (Sulfadiazine at 0.5 mg/mL + Trimethoprim at 0.1 mg/mL) was given in the drinking water for the first 2 weeks after the surgery. The metal clips were removed from the skin after the first week, and the transplanted thymus and recipient’s endogenous thymus were harvested and analyzed 6 weeks after the kidney transplantation.

Ketamine hydrochloride, xylazine hydrochloride, A-438079 hydrochloride hydrate, BzATP [2′(3′)-O-(4-Benzoylbenzoyl) adenosine 5′-triphosphate triethylammonium] and memantine hydrochloride were purchased from Sigma Chemical Co., St. Louis, MO, United States and dissolved in sterile distilled water. After obtaining 180 min of baseline ECoG recordings, A-438079, at the doses of 20 and 40 μg, and BzATP, at the doses of 50 and 100 μg were administered into the lateral ventricle within a thin internal cannula (4.2 mm vertical to the bregma) in a volume of 2 μl. Memantine, at a dose of 5 mg/kg, was injected intraperitoneally in a volume of 0.5 ml. For the interaction groups, memantine was administered 10 min after the chosen doses of BzATP (100 μg) or A-438079 (20 μg) (Arslan et al., 2019 (link)). The sham group was given sterile distilled water (2 μl, i.c.v.).

WT (CD45.2) mice were anesthetized by i.p. injection of 120 µg/g body weight Ketamine hydrochloride (Toronto Research Chemicals) and 12 µg/g body weight Xylazine hydrochloride (Sigma). Anesthetized mice were taped to a 5-cm thick lead block so that the lead block covered the head and the chest down to the bottom of the rib cage. Then, they were irradiated with a lethal dose (1000 rad) from a ¹³⁷Cs source (Minishot II, AXR) so that only their abdomen and hind legs were exposed. After recovery from anesthesia, the mice were transfused i.v. with 10⁷ bone marrow cells from a congenic (CD45.1) donor. Then, they were given trimerin (0.5 mg/mL sulfadiazine + 0.1 mg/mL trimethoprim, China Chemical and Pharmaceutical Co., Tainan, Taiwan) in the drinking water for the first 2 weeks after the irradiation and analyzed after 6 weeks.

Isoprenaline, trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), ketamine, xylazine hydrochloride, and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma-Aldrich (St. Louis, MO, United States). 5,5′-Dithiobis-(2-nitrobenzoic) acid (DTNB) and pyrogallol were obtained from Cayman (Michigan, United States).