Profiling circulating microRNA expression in a mouse model of nerve allotransplantation (part 2). Mus musculus

Background. The lack of noninvasive biomarkers of rejection remains a challenge in the accurate monitoring of deeply buried nerve allografts and precludes optimization of therapeutic intervention. This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation with or without immunosuppression. Methods. Balb/c mice were randomized into 3 experimental groups, that is, (1) untreated isograft (Balb/c → Balb/c), (2) untreated allograft (C57BL/6 → Balb/c), and (3) allograft (C57BL/6 → Balb/c) with FK506 immunosuppression. A 1-cm Balb/c or C57BL/6 donor sciatic nerve graft was transplanted into sciatic nerve gaps created in recipient mice. At 1, 3, 7, 10, and 14 d after nerve transplantation, nerve grafts, whole blood, and sera were obtained for miRNA expression analysis with an miRNA array and subsequent validation with quantitative PCR. Overall design: Male Balb/c and C57BL/6 mice (age, 10–12 weeks; weight, 30–35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The Balb/c mice were randomized into 3 experimental groups: (1) untreated isograft, (2) untreated allograft, and (3) allograft with FK506 treatment. Additional Balb/c and C57BL/6 mice served as sciatic nerve isograft and allograft donors, respectively. These species of mice were selected on the basis of disparity at the MHC locus and prior experience with reciprocal rejection of grafts between these murine strains. FK506 was administered subcutaneously at 1 mg/(kg•d) throughout the experimental course, unless indicated otherwise. At 1, 3, 7, 10, and 14 d after initial surgery (n = 6 animals per group at each time point), sciatic nerve grafts were harvested, and whole blood samples were obtained. Additional sham-operated mice were subjected to the same procedure, including opening of the skin and muscle layers and exposing the sciatic nerve, but without nerve transection or nerve grafting, to measure the effect of FK506 injection on the expression of circulating miRNA. To test the effect of the discontinuation of FK506 treatment on the expression of circulating miRNA, whole blood was drawn at 0, 2, 4, and 6 d after discontinuation of FK506 injection in an additional group of mice with allografts and immunosuppression for 7 d. The whole blood samples (1 mL per mouse) were collected at the indicated times in tubes containing anticoagulant. After the whole blood samples were incubated at room temperature for 15 min, they were centrifuged at 3000 rpm for 10 min, white blood cells were slowly removed from the corresponding layers, and the serum was extracted and stored at –80°C before processing for RNA analyses. All the housing conditions and the surgical procedures, analgesia, and assessments were in accordance with national and institutional guidelines, and an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)–accredited SPF facility was used. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Chang Gung Memorial Hospital.

背景
目前，移植排斥反应的无创生物标志物仍存在缺失，这给深埋式神经异体移植物的精准监测带来了挑战，同时也阻碍了治疗干预方案的优化。本研究旨在明确伴或不伴免疫抑制的神经异体移植过程中，循环微RNA（microRNAs）的表达谱。
方法
将Balb/c小鼠随机分为3个实验组：(1) 未处理的同基因移植组（Balb/c→Balb/c），(2) 未处理的异体移植组（C57BL/6→Balb/c），以及(3) 经FK506免疫抑制的异体移植组（C57BL/6→Balb/c）。取长度为1 cm的Balb/c或C57BL/6供体坐骨神经移植物，移植至受体小鼠的坐骨神经缺损部位。于神经移植后1、3、7、10和14天，采集移植物、全血及血清样本，采用miRNA芯片进行miRNA表达分析，并通过定量聚合酶链反应（quantitative PCR）进行后续验证。
整体实验设计
雄性Balb/c与C57BL/6小鼠（周龄10~12周，体重30~35 g）购自中国台湾宜兰的BioLasco公司。将Balb/c小鼠随机分为3个实验组：(1) 未处理同基因移植组，(2) 未处理异体移植组，以及(3) 经FK506处理的异体移植组。额外选取Balb/c及C57BL/6小鼠分别作为坐骨神经同基因移植供体与异体移植供体。选择这两种小鼠品系的依据为二者的主要组织相容性复合体（MHC，major histocompatibility complex）基因座存在差异，且既往研究已证实这两种品系小鼠间会发生移植物相互排斥反应。FK506以1 mg/(kg·d)的剂量经皮下注射给药，贯穿整个实验周期，除非另有说明。于初次手术后1、3、7、10和14天采集样本（每个时间点每组6只动物），收获坐骨神经移植物并获取全血样本。额外设置假手术组小鼠，仅实施相同的手术操作（切开皮肤与肌肉层并暴露坐骨神经，但不进行神经切断或移植物移植），以评估FK506注射对循环miRNA表达的影响。为评估FK506给药中断对循环miRNA表达的影响，另选取一组已实施异体移植并接受7天免疫抑制治疗的小鼠，在停止FK506注射后的0、2、4和6天采集全血样本。在指定时间点采集全血样本（每只小鼠1 mL），采集时使用含抗凝剂的采血管。全血样本于室温孵育15分钟后，以3000转/分钟离心10分钟，缓慢吸取对应分层中的白细胞，提取血清并置于-80℃冰箱保存，直至进行RNA分析。所有饲养条件、手术操作、镇痛处理及实验评估均符合国家及机构指南要求，实验在经国际实验动物护理评估与认证协会（Association for Assessment and Accreditation of Laboratory Animal Care, AAALAC）认证的SPF级动物设施中开展。本实验的动物实验方案经高雄长庚纪念医院实验动物管理与使用委员会（Institutional Animal Care and Use Committee, IACUC）批准。