Histoacryl glue

Animal use was approved by the Comité Régional d'Ethique du Languedoc –Roussillon, France and the veterinary office of the Canton of Zurich, Switzerland. Injection of viral constructs in pup and adult mice were conducted as described previously15 (link)49 (link). Briefly, postnatal day 3–4 mice (as indicated) or postnatal day 30 CB17/SCID mice were anaesthetized with isoflurane inhalation and placed under a Zeiss stereomicroscope (Stemi 2000, Carl Zeiss Microscopy GmbH). The gluteus superficialis and biceps femoris muscles were separated to reveal the cavity traversed by the sciatic nerve. A thin glass needle filled with coloured viral solution (3 μl for pups or 8 μl for adult injections) was introduced into the nerve with a micromanipulator (IM—3C, Narishige Japan Group). This solution was injected over 10 min with short pressure pulses using a microinjector (Pneumatic Picopump PV820, World Precision Instruments) coupled to a 3 MHz function pulse generator (GFG8215, Langlois), so that regions distant of the injection site could be filled. The nerve was replaced in the cavity, the muscles were readjusted, and the wound was closed with Histoacryl glue (BBraun, Germany). When fully awake, pups were put back to the mother and the litter treated with antibiotics to prevent infection. Sacrifices were done by CO₂ inhalation.

Volume overload HF was induced by the surgical creation of an arteriovenous fistula (A-V) using the needle technique as previously described) [15 (link),37 (link),41 (link),42 (link),65 (link),83 (link)]. Briefly, under anesthesia (Isoflurane: 5% for induction; 2% for maintenance balanced with O₂), the inferior vena cava and the abdominal aorta were exposed using a midline incision. Both vessels were clamped caudal to the renal artery and to the aortic bifurcation respectively. The aorta was punctured using an 18-gauge needle and advanced until it perforated the adjacent vena cava. Immediately afterward, a drop of histoacryl® glue (B. Braun, Germany) was used to seal the aorta at the puncture point. Sham-operated rats underwent the same anesthesia and surgical procedures but without performing the A-V fistula.

The proximal end of the catheter was led subcutaneously from the neck to a midline incision in the abdomen and inserted in the abdominal cavity through a puncture hole. Intraperitoneal catheters ended in the peritoneal cavity and were anchored on the left side of the abdominal wall with Histoacryl^® glue (B. Braun Medical).

For recordings from BWMs animals were immobilized with Histoacryl glue (B. Braun Surgical, Spain) and a lateral incision was made to access neuromuscular junctions (NMJs) along the ventral nerve cord. The basement membrane overlying muscles was enzymatically removed by incubation in 0.5 mg/ml collagenase for 10 s (C5138, Sigma-Aldrich, Germany). Muscles were patch-clamped in whole-cell mode at 22°C using an EPC10 amplifier with head stage connected to a standard HEKA pipette holder for fire-polished borosilicate pipettes (1B100F-4, Worcester Polytechnic Institute, Worcester, MA, USA) of 4–7 MΩ resistance. The bath solution contained: NaCl 150 mM; KCl 5 mM; CaCl₂ 5 mM; MgCl₂ 1 mM; glucose 10 mM; sucrose 5 mM; HEPES 15 mM, pH 7.3 with NaOH, ~330 mOsm. The pipette solution contained potassium-gluconate 115 mM; KCl 25 mM; CaCl₂ 0.1 mM; MgCl₂ 5 mM; BAPTA 1 mM; Na₂ATP 5 mM; Na₂GTP 0.5 mM; cAMP 0.5 mM; cGMP 0.5 mM; HEPES 10 mM, pH 7.2 with 1 M KOH, ~320 mOsm. Recordings were conducted at a holding potential of −60 mV. Light activation was performed using a LED lamp at 470 nm (KSL-70, Rapp OptoElectronic, Germany; 8 mW/mm²) and controlled by Patchmaster software (HEKA, Germany). Data were analyzed by Patchmaster (HEKA, Germany) and MiniAnalysis (Synaptosoft, USA) software.