Ketamine

Electroretinogram (ERG) measurements were performed six weeks after immunization as previously described (53 (link), 54 (link)). Before carrying out the ERG under dim red light, mice were dark adapted overnight. The readings were done using a full-field flash electroretinograph (HMs ERG system, OcuScience, Henderson, NV, USA). After anesthesia with a ketamine (Ratiopharm, Ulm, Germany)/xylazine (Bayer healthcare, Berlin, Germany) cocktail (120/16 mg/kg), eyes were dilated and topically anesthetized. Reference electrodes were placed subcutaneously below the right and left ear and a ground electrode was placed in the base of the tail. Silver thread recording electrodes were placed in the center of the cornea. Scotopic flash ERGs were recorded at 0.1, 0.3, 1, 3, 10, and 25 cd*s/m² (n=6/group). Signals obtained from the corneal surface were then amplified, digitized, and averaged using ERG View 4.380R software (OcuScience). Briefly, the amplitude of the a-wave was measured from the pre-stimulus baseline (0 µV) to the trough of the a-wave. The amplitude of the b-wave was measured from the trough of the a-wave to the peak of the b-wave.

LV hemodynamic parameters were invasively measured. Rats (at 9 months of age) were anesthetized by intraperitoneal injection of a mixture of xylazine (4 mg/kg; Bayer, Germany) and ketamine (100 mg/kg; Dr E. Gräub AG, Switzerland), intubated and ventilated. The chest was opened and a microtip catheter (SPR-409, 2F, Millar Instruments, Houston, TX, USA) was gently inserted into the LV chamber. Hemodynamic parameters such as LV systolic (LVSP), LVEDP and heart rate were continuously recorded using LabChart (v7.3.2) and PowerLab System (8/30; both ADInstruments, Spechbach, Germany). Thereafter, the heart and lungs were removed and rinsed in ice-cold saline, before major blood vessels and connective tissue were removed, the heart blotted dry and weighed, and the heart or lung weight/body weight ratio calculated.

Mice were anesthetized with a 4:1 cocktail of ketamine and xylazine (Bayer) and perfused transcardially with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 m PBS. Brains were removed, postfixed for 6 h in 4% paraformaldehyde, and incubated overnight in 0.1 m PBS containing 30% sucrose. Cryosections (30 μm thick) were mounted on SuperFrost Plus glass slides for immunofluorescence analysis. Tissue sections were washed (10 min) in PBS; incubated in blocking solution containing 0.5% Triton X-100, 4% horse serum, and PBS (1 h, room temperature); and incubated overnight at 4°C in blocking solution containing the first primary antibody. Tissue was then washed in PBS (10 min), followed by incubation in secondary antibody for 1 h at room temperature. The primary antibodies used were as follows: anti-Tbr1 (1:500, chicken polyclonal; catalog #AB2261, Millipore); anti-CaMKIIa (1:500; mouse; catalog #SA-162, Biomol Research Laboratories); anti-Sim1 (1:1000; rabbit; catalog #ab4144, Millipore; RRID:AB_2187608); anti-Cux1 (1:100; mouse; catalog #sc-514008, Santa Cruz Biotechnology). The secondary antibodies used were as follows: Alexa Fluor 488 donkey anti-mouse (RRID:AB_141607); Alexa Fluor 647 donkey anti-chicken (RRID:AB_11194678); and Alexa Fluor 647 donkey anti-rabbit (RRID:AB_2536183; all diluted 1:1000).

To develop an ICA animal model, LPS (5 μg/5 μL) was injected directly into the mouse cerebellum as previously described but with some alterations^{25 (link)}. Briefly, male C57BL/6 mice (10 week old, Daehan Biolink Co., Ltd, Eumseong, Korea) were anesthetized deeply with intraperitoneal administration of 115 mg/kg of ketamine (Yuhan, Republic of Korea) and 23 mg/kg of Rompun (Bayer Korea Ltd., Republic of Korea) and placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). For injection into the cerebellum, a midline sagittal incision was made to expose the skull, and a burr hole was drilled. A total of 5 μL of LPS (1 mg/mL) or phosphate buffered saline (PBS) was injected once in the cerebellum (AP: − 2.5 mm; DV: − 2.5 mm, relative to the lambda) at a rate of 0.5 μL/min through a syringe pump connected to a Hamilton syringe (10 μL, 30G). The needle was left in place for an additional 5 min to limit reflux along the injection tract. Following injection, mice were moved into a cage with a warming pad to recover from surgery.