Age-dependent immune and lymphatic responses after spinal cord injury [RNA-seq, aged]

We used single-cell RNA sequencing to profile immune cells in the injured spinal cord parenchyma and lymphatic endothelial cells in the spinal cord meninges from young and aged mice. This help us understand the heterogeneity of the immune response after injury and how it is altered in aging. Moreover, the data obtained from the spinal cord meninges provides novel molecular insights into how the meninges may contribute to the repair process. Overall design: For the contusion injury, mice were anesthetized with ketamine (100 mg/kg)-xylazine (10 mg/kg) mixture. A 15-mm midline skin incision was made, and the connective tissue and muscles were bluntly dissected to expose T6-T13. Laminectomy was performed at T9 to expose the dorsal spinal cord, and the vertebral column was stabilized with angled clamps attached to the T7 and T12 transverse processes. The contusion injury was delivered using an Infinite Horizon Impactor (IH-0400 Impactor) with a force of 90 dynes. After injury, the muscles and skin were sutured separately. Mice were humanely euthanized with an intraperitoneal injection of a lethal dose of Euthasol® (10% v/v), followed by transcardiac perfusion of PBS with 5 U/mL of Heparin. Vertebral columns were obtained, and spinal cords were flushed from the vertebral column using a PBS-filled syringe. Spinal cords were enzymatically digested with prewarmed RPMI containing 1 mg/mL collagenase VIII, 0.5 mg/mL DNase, and 2% FBS for 30 minutes at 37°C with trituration every 15 minutes. The enzymes were neutralized using RPMI with 10% FBS. Samples were passed through a 70 µm filter to obtain single-cell suspensions and pelleted 380 xg for 5 minutes. CD45+ cells were enriched from the spinal cord single-cell suspensions following the protocol for CD45+ microbeads (Miltenyi Biotec) and LS columns (Miltenyi Biotec). Two spinal cords per sample were pooled and applied to each column. Isolated cells per sample were pooled and were passed through the column again to increase purity. Cells were resuspended to a concentration of 1000 cells/uL in PBS supplemented with 0.04% non-acetylated BSA (Thermo Fisher Scientific). The CD45+ cells enriched from the spinal cord were loaded onto a 10X Genomics Chromium platform for GEM and cDNA generation with cell- and transcript-specific barcodes and sequencing libraries made using the Chromium Single Cell 3' Library and Gel Bead Kit v3. Libraries were then sequenced using the Illumina NovaSeq6000 targeting 100,000 reads per cell.