Alzet

Intrathecal cannula catheterization was performed on the same day of SNI surgery. Under anesthesia a small laminectomy was made over the thoracic spinal cord^{40 (link)}. A polyethylene catheter (Alzet, Charles River Ltd, UK) was inserted under the dura mater in the lumbar enlargement and attached to a subcutaneous osmotic pump (Alzet 1007D, Charles River Ltd). For subcutaneous oligomer administration pumps were not connected to the cannula. An LNA-based miR-21-5p inhibitor and scrambled control oligomer were custom-made as fluorescein amidite (FAM)-labeled compounds by Exiqon (Denmark). Sequences are reported in Supplementary Table 1. The oligomers were mixed (1:5 v/v) with i-Fect™ in vivo transfection reagent (Neuromics, 2B Scientific, UK) and delivered for 7 days at 12 pmol/day. At the end of treatments, catheter and pump were checked to ascertain efficient delivery.

Luciferin (40 mg ml⁻¹) was intraperitoneally administered into Per1-luc mice at a controlled flow rate (1 μl h⁻¹ by an osmotic pump (Alzet, Charles River, Saint Aubin Les Elbeuf, France); 10, 15 or 30 μl h⁻¹ by iPRECIO pump (Primtec, Tokyo, Japan)). The osmotic pump was implanted into the intraperitoneal cavity. The iPRECIO pump was connected to a ‘modified free moving animal system' (complete tethering system for mice, Eicom, Shimotoba, Kyoto, Japan) which consisted of a liquid swivel (TCS1-20, 11-μl single-channel swivel, 0.65-mm connections), balance arm, crisscross mouse harness and a tube that was guided into the intraperitoneal cavity through a subcutaneous tunnel from an incision in the dorsal neck to a ventral abdomen incision (Supplementary Fig. 1d,e). After the operation, mice were placed in a cage until the start of the experiment.

Animal surgery was performed as previously described [15 (link)]. Briefly, rats were anesthetized and mounted in a stereotaxic frame. Two hundred μl of purified IgG (NMO-IgG₁, 2 mg/ml) or NaCl (Control-IgG) were infused into the CSF during seven days at a rate of 1 μL/hour using a sterile mini-osmotic pump implanted subcutaneously (Osmotic pump ALZET, Charles Rivers, France). After seven days, rats were anesthetised and received an intracardiac perfusion of 100 mL phosphate-buffered saline (PBS; 0.1 M, pH 7.4). Then, rats were sacrificed using pentobarbital, brain was then removed and frozen in isopentane at -30°C, and further stored at -80°C.
Brain tissue was embedded in Tissue-Tek OCT and subsequently cut on a cryomicrotome [15 (link)]. Brain slices were used for immunohistochemical (IHC) study of tight junction proteins.
NMO-rats were defined as those receiving NMO-IgG and control-rats as those receiving NaCl. Animal care and procedures were carried out in accordance with the European Directive 2010/63/UE and followed the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines. The study was approved by the local Lyon 1 University Animal Care Committee (B2012-80 project).

Rats and mice anesthetized under isoflurane (Iso-vet®, Piramal, UK) (1–3%) were implanted subcutaneously with an osmotic minipump (Alzet, Charles River) containing either isoproterenol (1.5 mg/kg/day for rat or 30 mg/kg/day for mouse) (iso-pump) or vehicle for 14 or 28 days to develop either EACH or HF, respectively, or as otherwise stated.

All animal experiments were conducted in strict accordance with the Home Office Animals (Scientific Procedures) Act 1986. Female C57BL/6 mice, aged 10–12 weeks and weighing at least 25 g prior to use in experiments, were housed and maintained in SPF conditions at the University of East Anglia, Norwich, UK in accordance with HO regulations, and all procedures were performed by fully-trained and licenced researchers. Experimental animals were closely monitored and were killed by rising CO₂ and cervical dislocation, at the time points described in the text, prior to subsequent tissue collection. All animals were regularly monitored for clinical signs; any displaying signs beyond those expected within the moderate limits of the procedures would be immediately sacrificed by the above methods and were not included in experimental data. Osmotic minipumps (Alzet, model 2002, Charles River, Margate, UK) were inserted subcutaneously to anaesthetised animals.

Intrathecal cannula catheterization was performed on the same day of SNI surgery. After anesthesia, a small laminectomy was made over the thoracic spinal cord. A polyethylene catheter (Alzet, Charles River Ltd, UK) was inserted under the dura mater in the lumbar enlargement and attached to a subcutaneous osmotic pump (Alzet 1007D, Charles River Ltd). The locked nucleic acid (LNA)-based miR-23a inhibitor and interfered control oligomer were custom-made as fluorescein amidite (FAM)-labeled compounds by Exiqon (Copenhagen, Hovedstaden, Denmark). The oligomers were mixed (1:5 v/v) with i-Fect™ in vivo transfection reagent (Neuromics, 2B Scientific, UK) and delivered for 7 days via an osmotic pump at a rate of 12 pmol/day.