Ethilon

Studies were conducted with 197 male and female homozygous nude rats (Han:rnu/rnu Rowett) bred and maintained in an isolation facility in a pathogen free environment on a standard 12/12 h day and night cycle. Animals were fed a standard sterilised pellet diet and provided sterile tap water ad libitum. The athymic nude rat is T-cell deficient, but has normal complement and B-cell function [11 (link)]. 10 tumour spheroids (250-350 μm in diameter) were selected under a light microscope. The animals were anaesthetized with Hypnorm-Dormicum (0.4 ml/kg) s.c., the head secured in a stereotactic frame (Benchmark; Neurolab, St Louis, MO) and a short longitudinal incision was made in the scalp exposing the calvarium. A burr-hole was made 1 mm posterior to the bregma and 3 mm to the right of the sagittal suture using a micro-drill with a bit diameter of 2,9 mm. A Hamilton syringe with inner diameter of 810 μm was introduced to a depth of 2, 5 mm below the brain surface, and the spheroids were slowly injected and the syringe left in place for 3 min before withdrawal. The skin was closed with an Ethilon 3-0 suture. The tumours were allowed to grow for 4-5 months, then harvested and passaged onto new animals after initiation of spheroids in vitro. Animals were sacrificed at the onset of symptoms using CO₂ and the brains were removed. All procedures and experiments involving animals in this study were approved by The National Animal Research Authority and conducted according to the European Convention for the Protection of Vertebrates Used for Scientific purposes.

Adipose tissues were dissected, and resistance arterioles were isolated and cleaned of excess fat and connective tissues. Arterioles were then washed and prepared for measuring changes in the internal diameter in response to flow and acetylcholine (Ach) as previously described [26 (link),28 (link),29 (link)]. In summary, isolated arterioles were cannulated using glass micropipettes in an organ perfusion chamber, and both ends were secured using a 10-0 nylon Ethilon monofilament suture. Cannulated arterioles within the organ chamber were then placed on the stage of an inverted microscope attached to a video camera, a video monitor, and a video measuring device (model VIA-100; Boeckeler, Madison, WI, USA). The organ chamber was perfused with heated physiological salt solution (Krebs buffer) that contained the following components (mM): 4.4 KCL, 123 NaCl, 2.5 CaCl₂, 20 NaHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, and 11 glucose. The pH of the solution was kept at 7.4 ± 0.05, and the temperature was maintained at 37 °C. The buffer was also supplied with air mixture of 21% O₂, 5% CO₂, and 74% N₂. Each end of the cannulated arteriole was connected via silicon tubes to a physiological buffer-containing reservoir, and the intraluminal pressure gradient (10–100 cm H₂O) was established by modifying the distance between both reservoirs in equal and opposite directions [30 (link)].

Independent of the intervention, all mice were anesthetized with isoflurane (~ 2 to 3%; provided in 100% oxygen; CP-Pharma, Burgdorf, Germany) before being weighed and moved onto a heating pad (37 °C). Anesthesia was maintained at ~ 2% via a nose cone. Eye ointment, physiological saline (0.5 ml, 0.9% NaCl), Clindamycin (45 mg/kg, Ratiopharm, Ulm, Germany) and a single s.c. injection of Buprenorphine were applied. Anesthesia was then upheld for 15 min for the first intervention (anesthesia and analgesia alone). For osteotomy, the left femur was shaved and disinfected with alcoholic iodine solution. The osteotomy was conducted as described previously^{22 (link),66 (link),67 (link)}. A longitudinal skin incision was made between knee and hip, and the musculus vastus lateralis and musculus biceps femoris were bluntly separated to expose the femur. Two standardized external fixators (rigid: 18.1 N/mm; flexible: 3.2 N/mm, both RISystem, Davos, Switzerland) were used for stabilization. The external bar of the fixator was positioned parallel to the femur and all pins were positioned accordingly. Afterwards, an approximately 0.5 mm osteotomy gap was created between the second and third pin using a Gigli wire saw (0.44 mm; RISystem, Davos, Switzerland) and the gap was flushed with saline. Muscle and skin were closed with two layers of sutures (muscle: 5-0 Vicryl, skin: Ethilon 5-0, both Ethicon, Raritan, USA). For recovery, the mice were returned to their home cages under an infrared lamp and were closely monitored.

Animals were anesthetized with 3% isoflurane and maintained under anesthesia with 2% isoflurane and oxygen. Analgesia was provided by subcutaneous buprenorphine simbadol (0.1 mg/kg) injection pre-operatively and 24 h after surgery. All surgical procedures were performed using sterile microsurgical technique with an operating microscope (Superlux 40, Carl Zeiss).
To create the CP-TIB nerve graft, a freshly axotomized common peroneal (CP) nerve branch was grafted to the acutely or chronically denervated tibial (TIB). To mimic a delayed graft, the TIB nerve branch was transected 2–5 mm distal to the sciatic trifurcation and sutured to the quadriceps muscle. After 8 weeks of delay^{7 (link)}, the proximal (CP) and distal (TIB) stumps were aligned and sutured into either end of a 5 mm silicone conduit (Tuzic, Siliclear tubing, 4.6 mm OD, 1.98 mm ID) with 10/0 ethilon sutures to produce the CP-TIB graft with an interposed silicone conduit for cell and protein isolation. Immediate repair was achieved similarly but with immediate CP-TIB cross-suture (graft).

We assessed axon extension after acute injury and the formation of neuromuscular junctions (NMJ) at 8 weeks in Thy1-GFP rats. Rats were anesthetized with 5% isoflurane, and maintained under anesthesia with 1–2% isoflurane. Analgesia was provided by Meloxicam (2 mg/kg) pre-operatively and 24 h post-operatively. The sciatic nerve was exposed and a 10-mm silicone conduit placed between the transected nerve ends and sutured with single 10/0 sutures to create an 8 mm defect. The conduits were either filled with 0.7% agarose hydrogel, or three different concentrations of rat-specific IL10 (1 µg/mL, 3 µg/mL, 7 µg/mL, recombinant rat IL10, Peprotech, part #400-19) with 0.7% agarose as a carrier. The muscle and skin were re-apposed using 6-0 Ethilon sutures. Body temperature was maintained by an electrical heating pad placed below rat within the surgical field; body temperature and heating pad temperature were monitored to maintain physiologic body temperature. All surgeons were blinded to treatment groups during surgical procedures and tissue harvests.
To determine axon extension after injury, five groups of animals (IL10 at 1, 3, or 7 µg/mL in 0.7% agarose, unloaded agarose or empty conduit, n = 6–8 animals/group, male and female) were allowed to recover for 21 days. Animals were then anesthetized and euthanized (pentobarbital (250 mg/mL, 4 mL/kg), and the regenerative nerve bridge was harvested. Bridges were placed in a well with PBS and imaged using confocal microscope (Zeiss LSM510 confocal). One spectral window for the emission profile of GFP (488 nm) and the entire extension of the regenerative bridge was imaged using tiled, Z stack images. Axonal outgrowth from the distal stump was quantified by evaluating axonal regenerative profiles extending through contiguous regions of interest (ROI) at 250-µm intervals from the distal end of the proximal stump using BitPlane (Imaris) and calculating GFP + volumes for each ROI. Differences between groups were determined by evaluating GFP + volume and the interaction between dose and contiguous ROI in a mixed-effect model, in the same group of animals.
To determine the effect of exogenous IL10 on neuromuscular junction formation, two groups of animals (n = 8/group) underwent sciatic transection and a silicone conduit repair (10 mm) filled with 3 µg/mL IL10 in 0.7% agarose or agarose only (negative control). Animals were euthanized at 8 weeks NMJ formation in the extensor digitorum longus (EDL) muscle was assessed for terminal axons (green) innervating postsynaptic acetylcholine receptors (AChRs), labeled with rhodamine-ɑ-bungarotoxin (red) conjugated to Alexa Fluor-594^{54 (link)}. Briefly, the EDL muscles were harvested bilaterally for whole-mount confocal microscopy. The muscles were dissected into four separate muscle bellies to create thinner samples. The muscles were placed on Pyrex resin-coated dishes and stained with rhodamine alpha-bungarotoxin conjugated to Alexa Fluor-594 (2.5 µg/mL; Thermo Fischer Scientific, CA) at room temperature for 30 min to label the acetylcholine receptors in the postsynaptic membrane. The muscles were rinsed with phosphate-buffered saline and mounted under a coverslip for confocal microscopy. The concentration of alpha-bungarotoxin conjugate was validated using a dose curve in untreated GFP rats. The muscles were imaged using a Zeiss LSM510 confocal microscope. Two spectral windows using the Zeiss Zen image software were used: one using the emission profile of Alexa Fluor-594 (590 nm) and the emission profile of GFP (488 nm). To evaluate the thicker muscle samples, four equidistant z-series (stack) were taken across the muscle sample to acquire images of a minimum of 100 motor end plates. The observer was blinded to the treatment group at the time of acquisition of imaging and during image analysis. The images were used to count the number of innervated and non-innervated motor end plates; innervation was determined by the extension of a GFP-expressing axon to a positively labeled motor end plate. The proportion of innervated NMJ was standardized to each contralateral control.