Domitor

Adult C57BL/6J mice (2 females and 1 male, 11–17 weeks old) were subjected to the same Hargreaves protocol as described above for baseline measurements. After completed stimulation, 40 min were allowed to pass until the mouse was injected intraperitoneally with 0.7–0.8 ml ketamine (Ketalar, 10 mg/ml, Pfizer) and medetomidine (Domitor, 1 mg/ml, Orion Pharma; 1:1), followed by perfusion and tissue preparation for RNAscope, as described above. Same mice but separate sections have been used in a manuscript under revision. No mice were excluded from the analysis.

The mouse was either subjected to pinching (1 female and 2 males) or scratching (2 females and 1 male) of the skin on the right dorsolateral calf. The pinching was performed 5× for 5 s using tweezers, with 5 s resting periods in between each pinching episode. The scratching was conducted for 30 s with 2 Hz and ∼300 mN (30.6 g), using an artificial mouse claw in scratch position. Forty minutes after application of the stimulus, the mouse was injected intraperitoneally with 0.05 ml ketamine (Ketalar, 10 mg/ml, Pfizer) and 0.05 ml medetomidine (Domitor, 1 mg/ml, Orion Pharma), followed by perfusion and tissue preparation for RNAscope, as described above.

Mice were anesthetized with intraperitoneal injection of 60 mg/kg ketamine (Ketaminol^®, 50 mg/mL; Pfizer Oy Animal Health, Espoo, Finland) and 0.4 mg/kg medetomidine (Domitor^®, 1 mg/mL; Orion Pharma, Espoo, Finland). The mouse pupils were dilated with topically applied 0.5% tropicamide (Oftan^® Tropicamid, 5 mg/mL; Santen Pharmaceutical Co., Ltd., Tampere, Finland). Under full anaesthesia, volumes of 1 µL of Cy3-pullulan (5 mg/mL) or Cy3-pullulan-DEX (5 mg/mL) in PBS (pH 7.4) were injected intravitreally into mice using Hamilton microinjector (Hamilton Co., Reno, NV, USA). A topical eye drop (Viscotears^®, Alcon, Finland) was applied after intravitreal injections to prevent dryness of the cornea. Quality of intravitreal injections was confirmed by optical coherence tomography (OCT) and fundus camera (Phoenix MICRON^TM, Berkeley, CA, USA).
After 24 h the mice were sacrificed, the eyes were removed and incubated in a 4% PFA solution for 2 h. The eyes were stored in 1% PFA solution until further processing of organotypic retinal cultures. The following procedures were performed according to published protocols [38 (link),39 (link),40 (link),41 (link),42 (link)] and method described in SI-2.

All mice used in experiments were of the slc-ICR strain, and were purchased from SLC Japan, Inc. The mice were bred and maintained at the experimental animal facility of Osaka University Graduate School of Medicine and were killed with an overdose of a mixture of Vetorphale (0.5 mg/mL, Meiji), Dormicum (0.4 mg/mL, Roche) and Domitor (0.03 mg/mL, Orion Pharma) by peritoneal injection. This study was approved by the institutional committee of Osaka University and all experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Osaka University Medical School.

In view of their impaired fertility, Mfp2^–/– mice were generated from heterozygous breeding pairs and identified by genotyping, as described (Baes et al., 2000 (link)). Upon comparison of the retinal morphology, gene expression and lipid composition, we observed no differences between Mfp2^+/+ and Mfp2^+/– mice, and, therefore, both genotypes were used as control for the Mfp2^–/– mice. All mice were bred in a C57Bl6/J background. Animals were bred in the animal housing facility of the KU Leuven, had ad libitum access to water and standard rodent food and were kept on a 14/10 h light and dark cycle. All mice were sacrificed between 2 and 6 pm. Mice were anesthetized by an intraperitoneal injection of a mixture of medetomidine (1 mg/kg; Domitor^®, Orion Pharma) and ketamine (75 mg/kg; Nimatek^®, Dechra) and sacrificed via cervical dislocation, unless stated otherwise. All experiments were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of Animals in Ophthalmic and Visual Research, the Guidelines for Care and Use of Experimental Animals (NIH) and the European Directive 2010/63/EU, and were fully approved by the Research Ethical Committee of the KU Leuven (P166/2017).

Breeding, maintenance and genotyping of heterozygous VEGFR-3 mutant mice (Chy mice) were performed as described previously [10 (link)]. The mice were maintained on a C3H background and the breeding yielded either Chy mice (VEGFR3^+/Chy) or wildtype (wt) siblings (VEGFR-3^+/+). The mice were anesthetized with a mixture of ketamine (12.2 mg/ml; Ketalar, Pfizer) and metetomidine (24.3 μg/ml; Domitor, Orion Pharma) during micropuncture and wick-in-needle experiments, and with 1 % isofluorane in a combination with O₂ and N₂ during all other procedures. Animals were euthanized with CO₂. All animal experiments were conducted in accordance with the regulations of the Norwegian State Commission for Laboratory Animals, which are consistent with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and Council of Europe (ETS 123). Experiments were performed with the approval from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited Animal Care and Use Program at University of Bergen.