Transcriptomic differences in cochlear cells isolated from mice implanted with dexamethason-eluting electrode arrays or standard electrode arrays

The inflammatory foreign body response (FBR) following cochlear implantation (CI) can negatively impact CI outcomes, including increased electrode impedances. This study aims to investigate the long-term efficacy of dexamethasone eluting cochlear implant and locally delivered dexamethasone, a potent anti-inflammatory glucocorticoid, on the intracochlear FBR and electrical impedance post-implantation in a murine model. The left ears of CX3CR1+/GFP Thy1+/YFP (macrophage-neuron dual reporter) mice were implanted with dexamethasone-eluting cochlear implants (Dex-CI) or standard implant (Standard-CI) while the right ear served as unoperated control. Another group of dual reporter mice was implanted with a standard CI electrode array followed by injection of dexamethasone in the middle ear to mimic current clinical practice (Dex-local). Mouse implants were electrically stimulated with serial measurement of electrical impedance. Dex-CI reduced electrical impedance and inflammatory FBR in the murine model for an extended period. Dex-local in the murine model is ineffective for long-term reduction of FBR and electrode impedance. Our data suggest that dexamethasone eluting arrays are more effective than the current clinical practice of locally applied dexamethasone in reducing FBR and electrical impedance. Following cochlear implantation (CI), mice were euthanized at 33 days post-CI, implanted and contralateral cochlea were microdissected, tissues collected separately in DMEM F-12 media and lysed with accutase at 37°C for 30 minutes on a shaker. The media was replaced with 2 mL of DMEM F-12 containing 5% FBS to stop the lysis. The tissue was triturated for 2 minutes and filtered through a 20 µm filter (pluriSelect Life Science, El Cajon, CA, United States). The filtered cells were centrifuged at 300 x g for 3 minutes and resuspended in 90 µL MACS buffer (0.5% BSA in PBS). 10 µL of CD11b microbeads (71-097-142, Miltenyi Biotec, Auburn, Ca, USA) were added to the cell suspension and incubated for 15 minutes at 4°C, washed in MACS buffer followed by centrifugation 350 x g for 5 minutes resuspended in 500 µL MACS buffer. Cell suspension was then applied to a prewashed column in a magnetic holder to collect flow-through containing Cd11b negative cells. Column was then washed 3 times with 500 µL MACS buffer with negative flow-through captured each time. The CD11b microbead-bound cells were then eluted with 1 mL MACS buffer. Samples were centrifuged at 300 x g for 3 minutes and adjusted with MACS buffer to 3 x 106 cells/mL. Samples at this concentration were used for 10x cell capture. Single-cell captures were performed following the manufacturer’s recommendations on a 10x Genomics Controller. The targeted number of captured cells ranged from 3231 to 3572 per run. Library preparation was performed according to the instructions in the 10x Genomics Chromium Single Cell c’ Chip Kit V2. Libraries were sequenced on a Nextseq 500 instrument (Illumina, San Diego, CA) and reads were subsequently processed using 10x Genomics CellRanger analytical pipeline using default settings and 10x Genomics downloadable mm10 genome as previously described.

人工耳蜗植入（cochlear implantation, CI）后引发的炎症性异物反应（inflammatory foreign body response, FBR）会对人工耳蜗植入效果产生负面影响，其中包括电极阻抗升高。本研究旨在探究载地塞米松人工耳蜗与局部递送的地塞米松（一种强效抗炎糖皮质激素）对小鼠模型中植入术后耳蜗内炎症性异物反应（FBR）及电极阻抗的长期改善效果。本研究对CX3CR1+/GFP Thy1+/YFP（巨噬细胞-神经元双报告基因）小鼠的左耳植入载地塞米松人工耳蜗（Dex-CI）或标准人工耳蜗（Standard-CI），以右耳作为未手术对照；另一组双报告基因小鼠则植入标准人工耳蜗电极阵列，随后在中耳注射地塞米松以模拟当前临床操作，即局部递送地塞米松组（Dex-local）。研究对小鼠植入物进行电刺激，并连续测量电极阻抗。结果显示，载地塞米松人工耳蜗（Dex-CI）可在小鼠模型中长期降低电极阻抗与炎症性异物反应（FBR）；而局部递送地塞米松组（Dex-local）在小鼠模型中无法长期降低炎症性异物反应（FBR）与电极阻抗。本研究数据表明，载地塞米松电极阵列在降低炎症性异物反应（FBR）与电极阻抗方面，优于当前临床采用的局部应用地塞米松方案。本研究在人工耳蜗植入（CI）后第33天处死小鼠，对植入侧与对侧耳蜗进行显微解剖，将组织分别收集于DMEM/F12培养基中，随后使用Accutase在37℃摇床中裂解30分钟；更换为2mL含5%胎牛血清（FBS）的DMEM/F12培养基以终止裂解反应。将组织研磨2分钟，随后通过20μm滤膜（pluriSelect Life Science, 美国加利福尼亚州埃尔卡洪市）进行过滤。将过滤后的细胞以300×g离心3分钟，重悬于90μL磁激活细胞分选（MACS）缓冲液（含0.5%牛血清白蛋白（BSA）的磷酸盐缓冲液（PBS））。向细胞悬液中加入10μL CD11b磁珠（货号71-097-142, Miltenyi Biotec, 美国加利福尼亚州奥本市），于4℃孵育15分钟，用MACS缓冲液洗涤后以350×g离心5分钟，并重悬于500μL MACS缓冲液。将细胞悬液施加至置于磁架上的预洗过的分选柱中，收集含有CD11b阴性细胞的流出液；随后用500μL MACS缓冲液洗涤分选柱3次，每次收集对应的阴性流出液。将结合CD11b磁珠的细胞用1mL MACS缓冲液洗脱。将样本以300×g离心3分钟，并用MACS缓冲液调整细胞浓度至3×10^6个/mL。该浓度的样本用于10x单细胞捕获。单细胞捕获操作按照制造商的说明在10x Genomics控制器上完成，每次运行的目标捕获细胞数为3231至3572个。文库制备按照10x Genomics Chromium单细胞cDNA芯片试剂盒V2的说明书进行操作。文库在Nextseq 500测序仪（Illumina, 美国加利福尼亚州圣迭戈市）上进行测序，随后按照此前的方法，使用10x Genomics CellRanger分析流程（默认参数）及10x Genomics可下载的mm10小鼠参考基因组对测序读段进行处理。