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.

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:TLR4/NF-ÎºB signaling plays a central mediator in response to danger signals released in the muscle ischemia-reperfusion injury (IRI). This study was designed to profile TLR4/NF-ÎºB-responsive microRNAs (miRNAs) in the skeletal muscles following IRI. Following 2 h of ischemia and subsequent reperfusion for indicated times (0 h, 4 h, 1 d, and 7 d) of the isolated thigh skeletal muscles based on femoral artery perfusion of C57BL/6, Tlr4â/â, and NF-ÎºBâ/âmice, the muscle specimens were analyzed with an miRNA array to detect the TLR4/NF-ÎºB-responsive miRNAs. Male C57BL/6 mice (10â12 weeks of age, 22â35 g) were purchased from BioLasco (Taiwan). Tlr4â/â (C57BL/10ScNJ) and NF-ÎºBâ/â (B6.Cg-Nfkb1tm1Bal/J) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All housing conditions were established and surgical procedures, analgesia, and assessments were performed in an AAALAC-accredited, SPF facility following national and institutional guidelines. Animal protocols were approved by the IACUC of Chang Gung Memorial Hospital. Mice were anesthetized with an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine, given as an intraperitoneal injection (0.01 ml/g body weight). After the induction of anesthesia, the mice were restrained in a supine position on a heating pad to maintain body temperature at 37Â°C. The quadriceps muscle perfused based on the femoral artery of the mouse was carefully isolated away from the femoral bone and the underlying adductor muscle group. In the ischemic group, ischemia was induced by placing a microvascular clamp carefully across the proximal site of vascular pedicle for 2 h and then the microvascular clamp was removed. Good vascular inflow and outflow through the pedicle was verified under direct magnified vision. In the sham-operated control group, the muscle was isolated without microvascular clamp being applied. The incision wound was closed with interrupted sutures (4-0 nylon) and the animals were allowed to awaken in the remaining periods of reperfusion. The harvested muscles were frozen in isopentane chilled in liquid nitrogen and stored at -80Â°C. For the miRNA array experiments, the isolated skeletal muscles of C57BL/6 mice after 2 h of ischemia and 0 h, 4 h, 1 d, and 7 d of reperfusion as well as of Tlr4â/âand NF-ÎºBâ/â mice after 2 h of ischemia and 1 d of reperfusion were used in 3 replicate experiments.

Project description:To profile the expression of circulating miRNAs in a mouse model of diet-induced obesity (DIO) with subsequent weight-reduction with low-fat diet (LFD), eighteen C57BL/6 male mice were grouped into three subgroups as: (1) Control: the mice fed with the standard AIN-76A (fat: 11.5 kcal%) diet for 12 wks; (2) DIO: the mice fed with 58 kcal% high-fat diet for 12 wks; (3) DIO+LFD: the mice fed with high-fat diet for 8 wks to induce obesity, then changed to 10.5 kcal% low-fat diet for subsequent 4 wks. C57BL/6 mice were purchased from BioLasco (Taipei, Taiwan). All housing conditions were maintained, and surgical procedures, including analgesia, were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited SPF facility according to national and institutional guidelines. In this experiment, eighteen C57BL/6 wild type male mice were randomly grouped into three subgroups (n=6 in each group): (1) Control: the control mice were fed ad libitum a standard AIN-76A (fat: 11.5 kcal%) diet for 12 wks; (2) DIO: the mice were fed ad libitum a 58 kcal% HFD (D12331; Research Diets Inc., New Brunswick, NJ) for 12 wks to induce obesity; (3) DIO+LFD: the mice fed ad libitum a 58 kcal% HFD (D12331) for 8 wks to induce obesity, then continued the feeding of 10.5 kcal% LFD (D 12329; Research Diets Inc.) for additional 4 wks. Weight measurements were performed on a weekly basis to for these three groups of mice. Evaluation of blood glucose levels was performed at the beginning and in the end of the experiment to confirm that the HFD-fed mice developed an obese and insulin resistant phenotype. After the end of experiment at 12w, all mice were killed. The abdominal WAT of each mice was removed and weighted. Paraffin-embedded abdominal WAT was sectioned at 5 M-NM-<m and stained with hematoxylin and eosin to measure mean adipocyte area. A volume of 1 mL of whole blood was collected into a plain tube and allowed to clot for 1 hour. The sera samples were aliquoted after centrifugation at 3,000 M-CM-^W g for 10 minutes and stored at M-bM-^HM-^R80M-BM-0C until further analysis.

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Profiling circulating microRNA expression in a mouse model of nerve allotransplantation

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