Surgifoam

Human tonsils were collected within several hours of routine tonsillectomies performed by B. Tweel at the Mount Sinai Health System in New York City under an Institutional Review Board-approved protocol #20-00930. Human tonsil explants were dissected into 1-mm tissue blocks that were plated on top of a collagen sponge (Surgifoam; Ethicon) and maintained in RPMI 1640 medium (Life Technologies) containing 15% fetal bovine serum (FBS; Gibco), 2mM GlutaMax (Life Technologies), 1 mM sodium pyruvate (Corning), 1% minimal essential medium (MEM) nonessential amino acids (Corning), 2.5 μg/ml amphotericin B (HyClone), 50 mg/ml gentamicin sulfate (Corning), and 0.3 mg/ml Timentin (bioWORLD) as previously described (50 (link), 120 (link)). The human tonsil explant tissue blocks were left uninfected or individually inoculated with 5 μL of HIV-1_NL-CI (equivalent to 3.24 ng of p24). Complete media changes were performed post-infection on days 2, 5, and 8. Suspension cells for flow cytometry and scRNA -seq were harvested by centrifugation (2000 x g, 5 min) of tonsil explant supernatants at indicated time points.

This experiment was approved by the McGill University Animal Care Committee (Protocol # 2019-8098) and performed according to the guidelines of the Canadian Council on Animal Care. For hemostatic sealing of the volumetric hepatic injury, the rats were anesthetized using isoflurane (4% isoflurane in oxygen) in an induction chamber. Anesthesia was maintained at 2% isoflurane using a nose cone during the surgery. A volume of 1 mL of saline was injected subcutaneously. Abdominal hair was removed, and the rats were placed over a heating pad for the duration of the surgery. The liver was exposed via a laparotomy. A volumetric injury of 4 mm diameter and 3 mm depth was made to the liver using a biopsy punch. Cylindrical-shaped SURGIFOAM (Ethicon) or LIMB of 5 mm diameter and 4 mm depth was inserted into the wound immediately. The amount of blood loss until hemostasis was reached and the time to hemostasis was recorded for each group. After the hemostatic sealing was confirmed, the incision was closed using sutures. Two weeks after the implantation, the rats were euthanized by 5% isoflurane induction followed by CO₂ inhalation. Livers with the implants were excised and fixed in 4% paraformaldehyde solution for 48 h for histological analysis.

Mice were anesthetized with isoflurane inhalation. Surgery was performed under an Omano surgical microscope (OM2300S-V7, 7-40X). A 1–1.5 cm midline neck incision was used. The superficial tissues were bluntly dissected and lateralized with a mini retractor. The neck muscles were gently dissected to locate the mouse’s right auditory bulla and the auditory capsule on the right side of the head, which are the landmarks to locate the foramen lacerum. A prepared piece of Surgifoam (Ethicon) at approximately 1–1.5 mg was gently delivered into the foramen lacerum using curved forceps. The Surgifoam was positioned between the trigeminal nerve root and the cochlea bulla. After removing the retractor and replacing the tissues, the skin was closed with 6-0 nylon monofilament (Ethicon) sutures. Mice in the sham group underwent the same surgical procedure including neck shaving, skin incision, muscle dissection, and foramen lacerum exposure without nerve root compression. The duration of the surgery ranged from 8 to 12 minutes per mouse. Tamoxifen was intraperitoneally administrated to Fos-iCre-ERT2 (Fos^2A-iCreER-knockin) mice immediately after the FLIT procedure.

Pre-anesthetic medication varied for each specific patient, but as a basic protocol, intravenous midazolam (0.25 mg/kg) and ketamine hydrochloride (1 mg/kg) were used followed by an intravenous bolus of propofol (5 mg/kg). Anesthesia was maintained using isoflurane as well as a continuous infusion of fentanyl (10 μg/kg/h). The same surgeon (FB), with substantial neurosurgical experience, performed all surgeries, using the technique described in the literature (20 (link)). The patient was positioned in dorsal recumbency, and the neck gently extended under a folded towel, which allows widening of the intervertebral disc spaces. The ventral cervical area was prepared for surgery by clipping the hair and local application of povidone-iodine. A sterile patch (Opsite Incise; Smith and Nephew, London, United Kingdom) was placed over the surgical field. Surgical material used for surgery was standard, including Surgairtome drill, Kerrison forceps, special curettes, burs and absorbable gelatin sponge (Surgifoam, Ethicon, USA). In a few dogs, additional disc fenestrations were performed at disc spaces where severe disc mineralization/dehydration or very minimal disc protrusion were noted on MRI, as a preventative measure to future clinical disc herniation.

Under 2% isoflurane anesthesia, we performed a T13 laminectomy using micro scissors (FST, #15010–09) or a crescent blade (FST, #10317–14). If required, we used a high-speed bone drill (Foredom, K.1070) to clear cement over the T13 laminectomy area. After laminectomy, we immediately covered the exposed spinal cord with a gelatin sponge (Ethicon, Surgifoam^®) soaked in sterile saline. Using dual fine forceps, we pulled any remaining dura laterally to expose the leptomeninges. We inhibit fibrosis with GORE^® PRECLUDE^® Pericardial Membrane, which we purchased from a third-party medical vendor (dotmed.com). Next, we cut the GORE^® PRECLUDE^® Pericardial membrane to the size of the laminectomy opening using micro scissors and placed it directly over the exposed spinal cord. To limit ingrowth, we placed small pieces of Surgifoam around the membrane. We blotted these small pieces dry with an absorbent spear (Beaver-Visitec International, Weck-Cel^®). Next, to protect and hold the PRECLUDE^® membrane in place, we spread Kwik-Sil over the wider working area. We removed animals from anesthesia and monitored them closely post-op until they ambulated. We administered one dose of carprofen (5.0 mg/kg, sc) for postoperative analgesia. Mice with implants recovered from laminectomy for at least one week before proceeding to the window placement.