These studies utilized eighty-five CD-1 timed-pregnant mice purchased from Charles River Laboratories (Wilmington, MA). The animals shipped on day 9 after mating and acclimated in the University of Vermont’s animal care facility for several days before undergoing surgery on day 15 of gestation. All experiments accorded with National Institutes of Health guidelines for laboratory animals and had approval from the Institutional Animal Care and Utilization Committee (IACUC) at the University of Vermont. After induction of isoflurane anesthesia, a laparotomy was performed using sterile technique and the right uterine horn exposed. Lipopolysaccharide (LPS) dosed at 250 µg or an equal volume of sterile saline (i.e. 100 µL) was injected intrauterine between gestational sacs 2 and 3 on the right uterine horn. At the completion of surgery, mice received one dose of buprenorphine (50 µg/kg) analgesic subcutaneously and were allowed to recover. Subsequently, the mice were euthanized at 2, 6, 12, 18, and 24 hours after LPS injection using isoflurane anesthesia and a lethal sodium pentobarbital injection (200 mg/kg intraperitoneal). The time zero (0-hour) control mice were euthanized without undergoing surgery or intrauterine injection.
The uterus, liver, lung, kidney, serum, and other tissues were harvested from the pregnant mice. Samples for protein assays were rinsed in normal saline, then immediately frozen in liquid nitrogen and stored at −80° C. Uterine tissue from the right uterine horn was typically designated for protein analysis, while the left uterine horn provided tissue for RNA analysis. Samples for RNA assays were rinsed in normal saline, placed in RNALater (Ambion, Inc., Austin, TX) and stored at 4° C for approximately 1 week, then removed from RNALater and stored at −80° C. For immunohistochemical studies, samples from the middle of the pregnant uterine horn were obtained at each of the time-points (i.e. time 0, 2, 6, 12, and 18 hour), then embedded in TissueTek O.C.T. compound (Sakura Finetek U.S.A., Torrance, CA) and immediately frozen in liquid nitrogen.
For the protein array and enzyme-linked immunosorbant assays (ELISA) studies, tissue was homogenized in ice cold 1X Cell Lysis buffer containing protease inhibitors. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockville, IL). For cytokine/chemokine protein array studies, uterine protein homogenates from four animals per time-point were equally pooled (125 µg each) for a total concentration of 500 µg protein. Expression of 40 mouse inflammation-related proteins was determined using the RayBio Mouse Inflammation Antibody Array kit (RayBiotech, Norcross, GA) (see for description of array). Membranes containing target inflammatory protein antibodies were incubated with blocking buffer, and then pooled protein samples were incubated with the membranes for 1–2 hours. The membranes were washed and then incubated in the biotinylated antibody solution at room temperature for 1–2 hours. The washing step was repeated, and diluted HRP-conjugated streptavidin was added to each membrane. After a 2-hour incubation, samples were washed a third time and developed to allow chemiluminescence visualization and densitometry using the Bio-Rad ChemiDoc XRS detection system (Bio-Rad Inc., Hercules, CA).
Total RNA was extracted from maternal mouse uterus, liver, lung, and kidney tissue using TRIzol reagent (Invitrogen Corp., Carlsbad, CA). Subsequently, genomic DNA was removed from samples using TURBO DNA-free (Ambion, Austin, TX). The RNA concentrations were determined using a NanoDrop spectrophotometer (NanoDrop, Inc., Wilmington, DE). Intact total RNA was confirmed by analysis of the 18S and 28S band patterns after formaldehyde-agarose gel electrophoresis.
For qualitative analysis of MCP-1 mRNA expression using reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA was synthesized from 1 µg of RNA template using the iScript cDNA Synthesis Kit (Bio-Rad) with random primers. Subsequently, the PCR was performed using the iTaq DNA polymerase kit (Bio-Rad) and mouse-specific sense and antisense primers for MCP-1 and the constitutively expressed gene beta-2-microglobulin (B2m) (see for primer sequences). The MCP-1 primers were designed over exon-exon splice sites to eliminate the possibility of amplifying genomic DNA. Tris borate EDTA (TBE) gels were made with 1–1.2% agarose and stained with GelRed (Biotium, Inc., Hayward, CA). Densitometric analysis of gels was performed using the Bio-Rad ChemiDoc XRS chemiluminescence detection system. Toll-like receptor 4 (TLR4) mRNA expression was also determined in uterine tissue using mouse TLR4 sense and antisense primers.
Primers used in RT-PCR and qRT-PCR studies
Real-time quantitative RT-PCR (qRT-PCR) was performed on an ABI Prism 7000 Sequence Detection System using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). For each sample, the cDNA was generated as described previously and used to amplify MCP-1, TLR4, and three constitutively expressed genes: B2m, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz), and hypoxanthine guanine phosphoribosyl transferase (Hprt) (see for primer sequences). Reactions were performed in triplicate for each sample. Following the qRT-PCR runs, standard curves generated for each primer set were used to calculate the relative quantities of all samples. The geometric mean of the quantities of the three constitutively expressed genes was used to normalize the target gene quantities. Negative (water) controls were run for each primer set in the qRT-PCR reaction to ensure the reagents were not contaminated and that no secondary primer structures were amplified. Specific sense and antisense primers used in RT-PCR and qRT-PCR runs were designed to span at least one exon-exon junction. PCR amplicons for all genes were sequence-verified by the University of Vermont’s DNA Analysis Core using an ABI 3130x1 Genetic Analyzer (16 Capillary) (Applied Biosystems).
The MCP-1 protein concentrations in individual tissue and serum samples were determined by ELISA using the mouse MCP-1 Assay kit (Invitrogen Corp.). The tissue samples were homogenized in 1X Cell Lysis buffer, and the total protein concentration was determined using BCA assays as previously described. Samples were added to MCP-1-antibody-coated microtiter wells and incubated for 2 hours. After washes, biotinylated MCP-1 antibody solution was incubated in the wells for 45 minutes. Streptavidin-HRP solution was then incubated in the wells for 45 minutes, followed by additional washes and the addition of Stabilized Chromogen reagent. After 20 minutes, Stop Solution was added, and absorbance of each well was read at 450 nm using a Synergy HT Multi-well Plate Reader (BioTek Instruments, Winooski, VT). The range for the ELISA was 9–2500 pg/ml of MCP-1.
For the immunohistochemical studies, 25 µm sections of uterine tissue were fixed in 4% paraformaldehyde on glass slides, then washed twice in phosphate buffered saline (PBS), and the endogenous peroxidases were quenched by incubating slides in a solution of 0.3% hydrogen peroxide in methanol. The slides were washed, blocked with rabbit serum and incubated in goat anti-MCP-1 polyclonal primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, the slides were washed in PBS, then incubated in anti-goat biotinylated IgG polyclonal antibody (Vector Laboratories, Burlingame, CA). The slides were then developed using the Vectastain Elite ABC kit (Vector Laboratories) and counterstained with hematoxylin.
Statistical analyses were performed using the one-way analysis of variance (ANOVA) or the nonparametric Kruskal-Wallis ANOVA on ranks, followed by multiple comparisons tests including the Student-Newman-Keals test, Dunn’s test (for the ELISA studies), and Dunnett test (for the qRT-PCR and kidney ELISA studies). Statistical significance occurred when p<0.05.