Project description:We profiled the expression of circulating microRNAs (miRNAs) in mice exposed to gram-positive and gram-negative bacteria using Illumina small RNA deep sequencing. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. Male C57BL/6 mice (age, 10–12 weeks; weight, 30–35 g) were purchased from BioLasco (Yi-Lan, Taiwan). 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 body temperature at 37°C. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) purchased from Caliper (Caliper, USA) were used to induce bacterial infection in the mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). To create mixed gram-negative and gram-positive bacterial infection, 1 × 108 Xen14 bacteria and 1 × 108 Xen29 bacteria/100 μL of PBS were used for wound contamination. Three animal models were used to create bacterial infection routes: subcutaneous injection (hereafter referred to as (I)), cut wound (hereafter referred to as (C)), and skin grafting (hereafter referred to as (S)). In the (I) model, E. coli and/or S. aureus suspensions were injected subcutaneously into the backs of the mice using an Fr. 25 needle. In the (C) model, a 1 cm incision wound was created in the midline of the back, smeared with E. coli and/or S. aureus suspension, and the wound was closed directly with a 4-0 nylon suture. In the (S) model, a 1×1 cm rectangular full- thickness skin graft was lifted from the backs of the mice, E. coli and/or S. aureus suspensions were spread over the wound bed, and the skin graft was reattached and closed with a 4-0 nylon suture. An additional group of animals in each of these three models was inoculated with PBS to serve as a negative control. Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) 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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Circulating miRNA expression after subcutaneous injection of Gram negative and positive bacteria in the mice

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