Domtor

Euploid and Ts65Dn young adult mice (2 to 2.5 month-old) were anesthetized with a combination of 1 mg/Kg medetomidine (Domtor, Pfizer) and 75 mg/Kg ketamine (Imalgene 500, Merial), and immobilized in a stereotaxic frame (Harvard Apparatus). Bilateral injections were performed at the level of the ventral hippocampus at the following coordinates relative to bregma (anterior-posterior = −3.3 mm, medial-lateral = +/− 3 mm, dorso-ventral = −3.3 mm and −2.3 mm) using a 5 μl Hamilton syringe. Up to 1008 transducing units (3 μl of viral suspensions of Lv-Control or Lv-miR155-802T) were injected into each hemisphere at a rate of 0.2 μl/min, under the precise control of an infusion pump (Ultramicropump, World Precision Instruments). The needle was left in place for 5 min after injection and then slowly retracted from the brain. Before complete withdrawal, the needle was allowed to dwell for an additional 5 min. After surgical intervention, the animals were injected subcutaneously with a dose of 2 mg/kg of atipamezole (Antisedan, Pfizer) for anaesthetic reversal. The analgesic, buprenorphine (Buprex, ScheringPlough) was also administered intraperitoneally at a dose of 0.05 mg/Kg twice a day for the following 72 h after intervention. Mice were euthanized and the hippocampus dissected at day 23 after infusion.

Two skin samples of 6 mm were obtained from each Ibizan hound under intravenous sedation. Dogs were sedated with a combination of 0.2 ml of medetomidine (Domtor^®, Pfizer, Madrid, Spain), 0.2 ml of butorphanol (Torbugesic Vet 10 mg/ml, Zoetis Spain, S.L., Madrid, Spain), and 0.3 ml of alphaxalone (Alfaxan 10 mg/ml®, Jurox Limited Microbial Developments, Malvern, UK). One skin sample was obtained from a LST-positive reaction at 72 hours [12 (link)] and another from normal-looking skin. Skin biopsies of LST-positive reactions were cut into two halves. One half was fixed in 10% formalin for the aforementioned immunohistological study [19 (link)] and the other half and the whole skin biopsy from normal-looking skin were submerged in RNA later (RNAlater^® Stabilization Solution, Ambion, Inc., Austin, Texas) stored at 4°C overnight and then kept at -80°C until used.
Moreover, skin biopsies from normal-looking skin from control dogs were used as control samples for immune gene expression. These samples were characterised by histologically normal skin and negative for Leishmania quantitative PCR.