Animal model of sepsis and vagotomy
Aged male Fisher rats (22-23 month-old) were obtained from Charles River Laboratories via the National Institute for Aging, National Institutes of Health (NIH). Animals were housed in a temperature-controlled room with a 12 h light-dark cycle and fed a standard Purina rat chow diet. Rats were allowed to acclimate to the environment for at least 5 days before being used for experiments. All experiments were performed in accordance with the NIH guidelines for the use of experimental animals, and the study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Feinstein Institutes for Medical Research.
Sepsis was induced in aged rats by cecal ligation and puncture (CLP) (22). Briefly, rats were anesthetized by 2% isoflurane inhalation. The abdomen was shaved and cleaned with iodine and alcohol solution, and a 2-cm midline incision was made. The cecum was then exposed and 70% of its length was ligated using 4-0 silk suture distal to the ileocecal valve. The cecum was punctured twice with an 18-G needle and a small amount of feces was squeezed out. The cecum then was returned to the abdominal cavity and abdominal incision was closed in layers and the animals were resuscitated with 30 ml/kg body weight normal saline subcutaneously. In sham rats, same surgical procedure was performed with the exception that their cecum was not ligated or punctured.
Vagotomy procedure was performed on a group of rats at the time of CLP as reported previously (23, 24). The trunks of subdiaphragmatic vagus nerve were transected. Briefly, the dorsal and ventral branches of the vagus nerve were dissected and each branch of the nerve was tied with surgical sutures at 2 points separated by about 1 cm, and then severed between the sutures. After surgery, animals were allowed to eat and drink food and water, respectively.
Administration of human ghrelin and human growth hormone into aged septic rats
Human ghrelin (Phoenix Pharmaceuticals, Belmont, CA) and human GH (ProSpec, Ness Ziona, Israel) were dissolved in normal saline. A 500 µl mixture of human ghrelin and GH (GG) was prepared and injected subcutaneously to CLP animals at 5 h after CLP. Each GG treated animal received 80 nmol/kg human ghrelin and 50 µg/kg human GH in a single bolus dose.
Splenocyte isolation and stimulation
Spleens were harvested at 20 h after CLP or sham-operation and homogenized by gentle grinding between frosted glass slides, followed by passage through a 70-µm cell strainer to obtain single cell suspension (BD Biosciences, San Jose, CA). The suspension was centrifuged at 1500 rpm for 5 min and the pellet was suspended into 44% percoll solution (Sigma, St. Louis, MO), then careflly overlay on top of 66% percoll solution, centrifuge 2000 rpm for 30 min at room temperature. After density gradient centrifuge, leukocytes formed a white fluffy ring at the interface between 44% percoll and 66% percoll and were collected into RPMI-1640 medium (Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin,10 mM HEPES and 0.5 µM 2-mercaptaethanol. Cell viability was greater than 90%. To evaluate immune responses of these cells, 2 × 106 cells were plated into a 24-well plate and stimulated with LPS (100 ng/ml, Sigma, St. Louis, MO) for 5 h. The released proinflammatory cytokines in the culture medium was measured.
Measurement of cytokines and analysis of lymphocytes, monocytes, and basophils number
Cytokine levels were quantified using an enzyme-linked immunosorbent assay (ELISA) kits specific for rat TNF-α, IL-6 (BD Biosciences) and TGF-β1 (eBioscience, San Diego, CA). All measurements were performed according to manufacturer’s instructions. Lymphocytes, monocytes, and basophils numbers in the blood were analyzed by using Cell-DYN 3700 analyzer (Abbott, Abbott Park, IL).
Western blot analysis
Spleen tissues were homogenized and lysed in RIPA buffer, 10 mM Tris buffer, pH 7.5 containing 0.1% Triton-X 100, 1 mM EDTA, 1 mM EGTA, protease inhibitor tablet (Thermo Fisher, Waltham, MA), phosphatase inhibitor tablet (Thermo Fisher). Protein concentration was determined by Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Tissue lysates were electrophoresed on 4-12% NuPAGE Bis-Tris Gel (Life Technologies) and transferred to nitrocellulose membranes. The membranes were then blocked with 0.1% casein in Tris buffer saline and incubated with anti-TGF-β (Proteintech, Chicago, IL), cleaved caspase-3 (Cell Signaling Technologies, Danvers, MA), and β-actin (Sigma) overnight at 4°C. After washing, membranes were incubated with infrared dye-labeled respective secondary Abs (LI-COR, Lincoln, NE). The Odyssey infrared image system (LI-COR) was used to analyze the target bands and intensities of bands was qualified using Image Studio Lite software (LI-COR).
Spleen tissues were fixed in 10% buffered formalin solution for 2 days and processed for paraffin sections using standard histology procedures. Paraffin sections were dewaxed and rehydrated in xylenes and series concentrations of alcohols, followed by antigen retrieval using antigen unmasking solution (Vector Labs, Burlingame, CA). Slides were then incubated with rabbit anti-rat PD-1 (1:50, Abcam, Cambridge, MA) and rabbit anti-rat HLA-DR (1:50 dilution, Proteintech, Chicago, IL) antibodies overnight at 4oC. Slides were washed with Tris-buffered saline containing 0.02% triton x-100 and further reacted with biotinylated anti-rabbit IgG (Vector Labs). Then, the slides were incubated with peroxidase conjugated avidin followed by reaction with substrate, DAB (Vector Labs). Slides were counterstained with hematoxylin and evaluated using a Nikon microscope.
All data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls (SNK) test and Student’s t-test. Differences in values were considered significant when P < 0.05.