Ketavet

For laser photocoagulation mice were anesthetized with a mixture of ketamine (100 mg/kg body weight, Ketavet; Pfizer Animal Health) and xylazine (5 mg/kg body weight, 2% Rompun; Bayer HealthCare) diluted in 0.9% sodium chloride by intraperitoneal (i.p.) injection and their pupils dilated with a topical drop of phenylephrine 2.5%–tropicamide 0.5%. A slit-lamp-mounted diode laser system (Quantel Medical Vitra, 532 nm green laser, power 100 mW, duration 100 ms, and spot size 100 µm) was used to generate three equal laser burns around the optic nerve in each eye with a cover glass as a contact lens³⁹ . For gene expression and protein analysis, 20 laser burns were applied to both eyes. To validate rupture of Bruch’s membrane, infrared images were recorded using Spectralis™ HRA/OCT device (Heidelberg Engineering) to analyze post-laser retinal structure and laser lesion size in vivo. In case of cataract and corneal epithelial edema before laser photocoagulation, unsuccessful laser burn without Bruch’s membrane rupture, or severe choroidal hemorrhages, eyes were excluded from further analysis.

Animal studies were performed in accordance with the guidelines of the European Parliament for the use of living animals in scientific studies and in accordance with the German law for protection of animals. The protocol was approved by Heinrich-Heine-University Animal Care Committee and by the district government of North-Rhine-Westphalia (LANUV, NRW, AZ 84-02.04.2013.A486).
Gene-targeted mice lacking either PLD1 or PLD2 were described before (Dall’Armi et al., 2010 (link)). Both mutant mouse lines were intercrossed to create constitutive Pld1^-/-/Pld2^-/- mice and the corresponding wild-type littermates were bred from breeder pairs. For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed. Experiments were done with male mice aged 10–12 weeks. Mice were anesthetized with Ketamin (100 mg/kg Ketavet^®, Pfizer, berlin, Germany) and Xylacin (5 mg/kg, Xylazin 2% Bernburg, Medistar, Ascheberg, Germany) by intraperitoneal (i.p.) injection before opening the thorax for removal of the heart or euthanasia was performed by cervical dislocation.

We primed micro-osmotic Alzet pumps (model 1007D) under sterile conditions one day before implantation and left them in an incubator at 37.0°C overnight. We anesthetized the animals with a ketamine (Ketavet, Pfizer, Germany, 150 mg/kg) and xylazine (Rompun, Bayer, Germany, 15 mg/kg) mixture dissolved in 8.5 ml of sterile 0.9% saline. Head skin incision was performed exposing the skull.
We implanted the cannula 0.2 mm to the right from Bregma corresponding to the location of the right ventricle. A drop of cyanoacrylate clay (Weicon, Germany) was introduced between the cannula cap and the skull. We slowly moved the cannula down until the cap touched the skull. We left the animals for 5 min to ensure drying of the clay. Then, we carefully sutured the skin above the cannula with a 5–0 polypropilen thread (Ethicon, USA).
Animals received lidocaine gel (Xylocain, AstraZeneca, Germany) locally and 0.5 ml of ringer lactate solution (B Braun Melsungen, Germany) intraperitoneally to substitute the liquid loss. We placed the cage with operated animals onto the 37°C warm bed for 2 hours.
The animals received a total amount of 84±16.8 µl of PEDF (20 µg/ml in CSF) or CSF at a pumping rate of 0.5±0.1 µl/hour.

For acoustic trauma (AT), animals were anesthetized (75 mg/kg ketamine hydrochloride, Ketavet, Pharmacia, Pfizer, Karlsruhe, Germany; 5 mg/kg xylazine hydrochloride, Rompun 2 %, Bayer Leverkusen, Germany; in injection water to give an application volume of 5 ml/kg body weight) and exposed to intense pure tone noise (10 kHz, 116 dB sound pressure level (SPL) for 40 min) in a reverberating chamber, binaurally in an open field [25 (link)]. Control sham-exposed animals underwent the same procedure but with no traumatic sound presented (i.e., the speaker remained turned off).

Eight female Dutch Belted rabbits weighing 2–2.5 kg were obtained from Kitayama Labes Co. Ltd. (Arai, Ina, Nagano Prefecture, Japan). Prior to the experiments, the animals were kept in colonies of a maximum of five animals with access to forage and water ad libitum. Experiments were conducted according to institutional guidelines and following approval of the experimental plan by the local government committee for the protection of animals (Regierungspräsidium von Mittelfranken, Ansbach, Germany; permit number 54–2532.1-16/11). For laser treatment and for lens- and ERG-measurements, the animals were anaesthetized with 50 mg/kg Ketamine (KetaVet, Pfizer Pharma GmbH, Berlin, Germany) and 10.5 mg/kg Xylazine (Rompun 2%, Bayer AG, Leverkusen, Germany). For additional local anesthesia of the cornea, oxybuprocaine hydrochloride eye drops (Conjuncain EDO; Bausch&Lomb, Berlin, Germany) were given. After surgical interventions the rabbits were kept in individual boxes with access to forage and water ad libitum. Animals were euthanized 6 months after surgery by intravenous injection of 300 mg pentobarbital (Narcoren; Merial GmbH, Sanofi, Paris, France) and the eyes were removed for histological analyses.

Animals were deeply anesthetized with an intraperitoneal injection of Ketavet (Pfizer; 100 mg/kg) and Rompun (Bayer Healthcare; 4 mg/kg), then perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4; 10 min) followed by 4% paraformaldehyde in 0.1 M PBS (30 min). After perfusion, brains were immediately dissected out of the skulls, post-fixed for an additional hour and washed in 0.1 M PBS.