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

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.

本研究采用单细胞RNA测序（single-cell RNA sequencing）技术，对年轻与老年小鼠损伤脊髓实质（spinal cord parenchyma）内的免疫细胞以及脊髓脑膜（spinal cord meninges）中的淋巴管内皮细胞（lymphatic endothelial cells）进行转录组分析，旨在解析损伤后免疫应答的异质性及其在衰老过程中的改变规律。此外，从脊髓脑膜获取的转录组数据，可为阐明脑膜在脊髓损伤修复过程中的作用机制提供全新的分子层面视角。
实验设计如下：
针对脊髓挫伤模型，采用氯胺酮（100 mg/kg）与赛拉嗪（10 mg/kg）混合溶液对小鼠进行麻醉。于小鼠背部正中做15 mm皮肤切口，钝性分离结缔组织与肌肉，暴露T6至T13节段脊柱。于T9节段行椎板切除术以暴露背侧脊髓，使用固定于T7与T12横突的成角夹具稳定脊柱。采用无限地平线撞击器（Infinite Horizon Impactor，型号IH-0400）以90达因的力度构建脊髓挫伤模型。造模完成后，分别缝合肌肉与皮肤切口。
术后通过腹腔注射致死剂量的Euthasol®（体积分数10%）对小鼠实施安乐死，随后经心脏灌注含5 U/mL肝素的磷酸盐缓冲液（PBS）。摘取脊柱，使用装有PBS的注射器从脊柱中冲洗出脊髓。将脊髓置于预热的RPMI培养基中进行酶解：该培养基含1 mg/mL VIII型胶原酶、0.5 mg/mL DNase与体积分数2%胎牛血清（FBS），于37℃孵育30分钟，每15分钟进行一次吹打混匀。使用含体积分数10% FBS的RPMI培养基终止酶解反应。将样本通过70 µm滤膜过滤以获得单细胞悬液，随后以380×g离心5分钟收集细胞。采用CD45+磁珠（Miltenyi Biotec）与LS分离柱（Miltenyi Biotec）的配套方案，从脊髓单细胞悬液中富集CD45+免疫细胞。每份样本合并2根脊髓的细胞悬液后上样至分离柱。将每份样本分离得到的细胞混合后再次上柱，以提升细胞纯度。将细胞重悬于添加了0.04%非乙酰化牛血清白蛋白（BSA，Thermo Fisher Scientific）的PBS中，调整细胞浓度至1000个/μL。将富集得到的脊髓CD45+细胞上样至10X Genomics Chromium平台，构建带有细胞特异性与转录本特异性条形码的GEM（凝胶珠乳液，Gel Bead Emulsion）与cDNA文库；测序文库制备采用Chromium单细胞3’文库与凝胶磁珠试剂盒v3。随后采用Illumina NovaSeq6000测序平台对文库进行测序，预设每个细胞的测序数据量为100,000条reads。