Atipamezole

Mice were anesthetized by intraperitoneal injection of a combination of 0.05 mg/kg fentanyl, 0.5 mg/kg medetomidine (Pfizer, Berlin, Germany), and 5 mg/kg midazolam (Ratiopharm, Ulm, Germany) and placed on a heated surgical pad to keep the body temperature constant. The right kidney was exposed through median abdominal incision, and mice were subjected to ischemia by clamping the renal pedicle with a nontraumatic microaneurysm clamp (Braun, Melsungen, Germany), which was removed after 45 min. The incision was closed with a 5–0 suture (Ethicon, Livingston, Scotland, UK) and surgical staples (Hugo Sachs GmbH, March, Germany). Postoperatively, anesthesia was antagonized by subcutaneous injection of a combination of 2.5 mg/kg atipamezole (Pfizer), 0.5 mg/kg flumazenil (Delta Select, Pfullingen, Germany), and 1.2 mg/kg naloxone (Inresa, Bartenheim, France). Mice were sacrificed by combined cervical dislocation and exsanguination. Tissue specimens of the ischemic right and the contralateral nonischemic kidney were collected on day 18 for histological analysis and embedded in paraffin according to standard protocols.

To study the effect of eugenol on LPS-induced lung injury, mice were randomly designated into 4 groups, with 8–10 mice in each group. Group (1) [Saline] was control, received saline (NaCl 0.9%) by intraperitoneal (IP) and intra-tracheal (IT) routes. Group (2) [Eug] received 200 mg/kg of eugenol intraperitoneally (IP) and saline by IT route. The third group (3) [LPS] received saline by IP route and LPS (5 µg/mouse) by intra-tracheal instillation. Group (4) [Eug+LPS] was treated intraperitoneally with 200 mg/kg of eugenol and instilled by LPS. Mice received saline (groups 1 and 3) or eugenol (group 2 and 4) intraperitoneally (IP) once per day for two consecutive days. For each group, three hours after the second injection, mice were anesthetized with a cocktail of anesthetics (75 mg/kg ketamine (Virbac Sante Animale, Carros, France) plus 1 mg/kg medetomidine (Pfizer, Paris, France)). Subsequently, they were instilled, intra-tracheally, with 5 µg of LPS (groups 3 and 4) or physiological saline (groups 1 and 2). The mice were aroused by an IP injection of 1 mg/kg atipamezole (Pfizer), a medetomidine antagonist.

The drug response trial was separated into two portions. Heifer B was used in the first portion and Heifer A was used in the second portion (Table 1). Infrared images were taken from the right flank, the coronary band, the foot, and the hind area (Fig. 1; ak, g, d, co). In the first portion, where only xylazine (Rompun, Bayer) was utilized, baseline images were taken every 7.5 min for three sets with the FLIR SC2000 camera at a distance of 0.8 m. The animal was then injected with 0.02 mg/kg of xylazine (Rompun, Bayer) intravenously to obtain mild sedation while maintained in the calorimetric chamber, and a subsequent set of images were taken after this injection every 7.5 min for a total of 12 sets. The second portion of the trial involved xylazine and atipamezole (Antisedan, Pfizer), with xylazine first administered to the animal (0.02 mg/kg; IV), followed by atipamezole (0.10 mg/kg; IV) 40 min after the xylazine. A set of baseline images was taken every 20 min for three sets, followed by the xylazine injection. A set of images was then taken every 20 min following the xylazine injection for two more sets, and after injection of atipamezole, two additional sets of images were taken every 20 min. Calorimetry measures were taken for the animals throughout the trial, with information gathered for heat production.

All experiments were performed on adult C56Bl/6J and CD-1 mice (10–12 weeks old at the start of the experiment), housed under a 12/12 h light/dark cycle with ad libitum access to food and water. The experiments were approved by the KU Leuven Animal Ethics Committee and followed the guidelines of the ARVO statement for the use of animals in ophthalmic and Vision Res.
Surgical procedures to induce experimental glaucoma and for viral vector or recombinant BDNF administration were performed under general anaesthesia, induced by intraperitoneal (i.p.) administration of ketamine (75 mg/kg body weight, Anesketin, Eurovet, Belgium) and medetomidin (1 mg/kg body weight, Domitor, Janssen Pharmaceutics, Belgium). Additionally, for glaucoma induction and intravitreal (ivt) injections, a droplet of local analgesic, oxybuprocaïne hydrochloride (0.4%, Unicaïne, Théa, Belgium) was applied to the eye. For intracollicular vector injections, the head region was topically analgesized by applying lidocaine hydrochloride (5%, Xylocaine, AstraZeneca, UK) to the scalp. After any surgical procedure, waking was encouraged by i.p. administration of atipamezole (1 mg/kg body weight, Antisedan, Pfizer, Belgium). During recovery, tobramycin eye ointment (Tobrex, Alcon, TX, USA) was applied to the cornea of both eyes to prevent desiccation and mice were placed on a heat pad to maintain body temperature.

During the 3^rd or 4^th week of voluntary wheel running or sedentary conditions rats underwent stereotaxic surgery for microdialysis guide cannulae implantation. Cannulae implantation was performed during the 3^rd and 4^th week of running because previous work from our lab indicates exercising rats that undergo surgery during this period are still protected from behavioral deficits dependent that follow unpredictable and inescapable tail shock [25 (link)]. Rats were anesthetized with a cocktail of ketamine (0.75 mg/kg i.p.; Vedco, St. Joseph, MO, USA) and medetomidine (0.5 mg/kg i.p.; Pfizer, New York, NY, USA). Guide cannulae were implanted unilaterally in the right or left DMS (-0.2 A/P, ± 2 M/L, -3.8 D/V from bregma) or DLS (-0.2 A/P, ± 4.3 M/L, -4.5 D/V from bregma), based on the Paxinos and Watson atlas. Rats were immunized with a single subcutaneous injection of penicillin (0.25mL/Kg; of Combi-Pen, Agrilbas, St. Joseph, Missouri, USA). Atipamezole (0.5 mg/kg i.p.; Pfizer, New York, NY, USA) was administered following surgery to reverse the effects of medetomidine.