Project description:This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation. Experimental design Male Balb/c and C57BL/6 mice (age, 10M-bM-^@M-^S12 weeks; weight, 30M-bM-^@M-^S35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The Balb/c mice served as sciatic nerve allograft donors. The C57BL/6 mice were randomized into the group of nerve allograft transplantation with FK506 treatment and the group without FK506 treatment. 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 [18]. The mice were anesthetized by intraperitoneal injection of an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine. The anesthetized mice were restrained in a supine position on a heated pad to maintain the body temperature at 37M-BM-0C. Under aseptic conditions, with sterile povidone/iodine preparation and 70% ethanol and sterile instruments and drapes, the skin over the proximal right hindlimb was incised, and the underlying biceps femoris muscle was bluntly dissected to expose the sciatic nerve. An established mouse sciatic nerve allotransplantation model was used. In brief, 1-cm Balb/c donor sciatic nerve grafts were transplanted in reverse orientation into 0.5-cm sciatic nerve gaps created in the recipient Balb/c mice. Tension-free repair was then performed under an operating microscope with three 11M-bM-^@M-^S0 nylon (Ethicon Inc., Somerville, NJ) interrupted epineurial sutures under 40X magnification. The muscle was closed with 5M-bM-^@M-^S0 vicryl sutures, and the skin with interrupted 5M-bM-^@M-^S0 nylon sutures. The animals were monitored to ensure appropriate feeding and diet after surgery. FK506 was administered subcutaneously at 1 mg/(kg-d) throughout the experimental course. At 3, 7, and 14 d after initial surgery (n = 3 animals per group at each time point), the whole blood samples were collected at the indicated times in tubes containing anticoagulant. (F)3d, (F)7d and (F)14d were used to indicate the serum samples of allotransplantation mice with FK506 immunosuppression for 3, 7, and 14 d, respectively. (N)3d, (N)7d and (N)14d were used to indicate the serum samples of allotransplantation mice without FK506 immunosuppression and sacrificed at postoperative 3, 7, and 14 d, respectively. After the whole blood samples were incubated at room temperature for 15 min, they were centrifuged at 3,000 M-CM-^Wg for 10 min, white blood cells were slowly removed from the corresponding layers, and the serum was extracted and stored at M-bM-^@M-^S80M-BM-0C before processing for RNA analyses. Small RNA libraries generated from the serum of mice of above groups were analyzed using an Illumina HiSeq2000 Sequencer.

Project description: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 M-bM-^FM-^R Balb/c), (2) untreated allograft (C57BL/6 M-bM-^FM-^R Balb/c), and (3) allograft (C57BL/6 M-bM-^FM-^R 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. Male Balb/c and C57BL/6 mice (age, 10M-bM-^@M-^S12 weeks; weight, 30M-bM-^@M-^S35 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/(kgM-bM-^@M-"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 M-bM-^@M-^S80M-BM-0C 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)M-bM-^@M-^Saccredited SPF facility was used. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Chang Gung Memorial Hospital.

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Identification of circulating miRNAs in a mouse model of nerve allograft transplantation under FK506 immunosuppression by Illumina small RNA deep sequencing

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