Fischer 344 rats

A total of 22 adult female Fischer 344 rats (160–180 g, Envigo Inc., Frederick, MD, USA) were housed according to the National Institutes of Health (NIH) and United States Department of Agriculture (USDA) guidelines. After receiving contusion injury, rats were randomly assigned to receive macrophage depletion via injection of liposomes filled with clodronate (SC/CLO group) or no depletion via injection of liposomes filled with phosphate buffered saline (PBS) (SC alone group). The institutional Animal Care and Use Committee (IACUC) of the University of Miami approved all animal procedures (IACUC protocol #15-079). A detailed timeline of the experimental procedures is shown in Figure 1.

Purified Schwann cell cultures were obtained from sciatic nerves of 2 adult female Fischer 344 rats (Envigo Inc., Frederick, MD, USA) following previously published protocols (Meijs et al., 2004). The resulting cultures were purified to greater than 95% (Takami et al., 2002). At passage 2 and at approximately 50% confluence, Schwann cells were transduced in D10/mitogen medium overnight with a lentiviral vector encoding enhanced green fluorescent protein (GFP), genes from Aequorea victoria, at a multiplicity of infection of 30. D10 medium consisted of Dulbecco's modified Eagle's medium (DMEM, ThermoFisher Scientific, Carlsbad CA, USA) and 10% fetal bovine serum (FBS, GE Healthcare Life Sciences, Logan UT, USA); the added mitogens were pituitary extract (20 µg/mL, Biomedical Technologies S.L., Madrid, Spain), forskolin (2 µM, Sigma-Aldrich, St. Louis MO, USA), and heregulin (2.5 nM, Genentech, San Francisco CA, USA). The production of the lentiviral vectors has been detailed elsewhere (Follenzi and Naldini, 2002; Blits et al., 2005). The transduction efficiency in the Schwann cell cultures was greater than 90%.

Liposomes filled with clodronate (50 mg/kg body weight; Encapsome, Brentwood TN, USA) were injected intraperitoneally at 1, 3, 6, 11, and 18 days following contusion injury (timeline in Figure 1) into the animals in the SC/CLO group. For animals in the SC alone group, the same volume of liposomes filled with PBS, based on the animal's body weight, was injected via the same route. The rats were anesthetized with 5% isoflurane and their abdomens were cleaned with 70% ethanol prior to each injection. The liposome vials were brought to room temperature and the contents mixed by gently inverting the containers before loading into the syringe. Macrophage depletion via this administration route was tested by giving the same dose of clodronate or PBS to 3 naïve animals (adult female Fischer 344 rats, 160–180 g, Envigo Inc.) in each group at days 1, 3, and 6, and then perfused at day 7.

A total of 42 adult female Fischer 344 rats (150-250 g, Envigo, CA) were used in this study. Animals had free access to food and water throughout the study. All surgery was done under anesthesia using a combination (2 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 g/ml), and acepromazine (0.25 mg/ml).