Animals
Adult male wild type (WT) and Mer−/− Sprague-Dawley (SD) rats (age: 2 months, weight range: 230–250 g) were purchased from Cyagen Co., Ltd (Guangzhou, China). According to the Animal Ethics and Use Committee of Sichuan Cancer Hospital, all rats received humane care. The rats were housed in a specific pathogen-free room under standardized conditions (25 ± 2°C, 60% humidity, and a 12 h light/dark cycle) for 1 week before the experiment. All rats had free access to food and water. All animal procedures were performed according to the Institute Animal Care and Use Committees of the Sichuan Cancer Hospital and followed the National Institutes of Health’s Guide for the Care and the Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines, as well as recommendations of reduction, refinement, and replacement (known as the 3 Rs).
Group Assignments And Establishment Of Sepsis Model In Rats By Clp
The WT and Mer−/− rats were divided into two groups: (1) the sepsis group and (2) the sham group. Each group was then divided into 2 subgroups: intrathecal injection of ProS or normal saline (NS). The sepsis model was established by cecal ligation and puncture (CLP). The sample size was calculated by our preliminary experiment and previous studies [15, 16]. We assigned 10 rats to each subgroup (Fig. 1).
The model of sepsis was induced by CLP, as previously described [17]. After anesthetized with an intraperitoneal (i.p.) injection of 0.5% sodium pentobarbital (65 mg/kg), the rats were used tail clamping to determine a sufficient depth of anesthesia. After adequate anesthesia was established, we shaved and cleaned the abdomens of the rats before performing a 2-cm-long incision through the skin and rectus abdominis on the left side of the midline to expose the cecum. We located and carefully exteriorized each rat’s cecum and then ligated it with a 3–0 silk suture halfway from the base of the ileocecal valves. The ligated cecum was punctured twice with a 24-gauge needle to ensure that the wounds were infected with a small amount of stool. At last, the cecum was carefully returned to the abdomen and closed with a 3–0 silk suture. The cecum was returned to the abdomen after gentle manipulation in the sham group. In all groups, 10 mL/kg of 0.9% normal saline was subcutaneously injected after the abdomen was closed to restore the animals’ fluid levels.
Intrathecal Catheter Implantation
In this study, according to our preliminary experiments and previous studies[18], the rats were treated with an intrathecal injection of recombinant ProS (0.2 mg/kg) (9489-PS, R&D Systems) or an equal volume of NS at the 8 h, 1 day, 2 days and 3 days after CLP.
The catheter implantation was performed as described with slight modifications[19]. In brief, each rat was anesthetized with an intraperitoneal injection of 0.5% sodium pentobarbital (65 mg/kg) and placed in a prone position. A midline skin incision was made in the lumbar region (L2–S1), and the intervertebral membrane between L3 and L4 was exposed. A needle was then used to puncture the arachnoid membrane, and the PE-10 catheter was gently pushed into the subarachnoid space. The incisions were then closed in layers using 4 − 0 silk. During the surgery, the animal’s core body temperature was maintained at 37 ± 0.5°C with a thermostatically controlled heating pad during surgery.
Evaluation Of Neuromuscular Function
On day 4 after CLP, all the rats were placed in the dorsal recumbent position after anesthetization with 0.5% sodium pentobarbital (65 mg/kg). The right sciatic nerve was exposed at the thigh with non-compliant silk. Stimulation electrodes (RM6240 Systems, Inc., Chengdu, China) were attached to measure the nerve-mediated contraction of the tibialis anterior muscle with the following parameters: intensity, 3 V; duration, 0.2 ms; and frequency, 1 Hz. Compound muscle action potential (CMAP) was recorded with a receiving electrode attached to the tibialis anterior muscle before and at different times after surgery. The electromyographical data (amplitude, duration, and latency period of CMAP) were analyzed with RM6240 USB2.0S (I) version 1.0.2 software (RM6240 Systems, Chengdu Instrument Company, Chengdu, China). The motor conduction velocity (MCV) was calculated as the distance of conduction/latency time. The temperature of each rat was kept at 36–37°C using a heating light. Neuromuscular dysfunction was defined as a decrease of ≥ 20% of the lower limit of the average CMAP amplitude [20].
Tissue Preparation
Tissue preparation was performed in our previous studies[21]. After completing the evaluation of neuromuscular function, the animals were euthanized, and muscle tissues (gastrocnemius and tibialis anterior), spinal cord (L3-4 segments), and sciatic nerve were harvested in ice-cold phosphate-buffered saline ([PBS], pH 7.40) and transferred to 4% paraformaldehyde. After a 2-day fixation, the specimens were then embedded in paraffin. The samples were cut into 4-mm-thick sections on a rotary microtome (RM2135, Leica Instruments, Wetzlar, Germany) and placed onto glass slides. The sections were deparaffinized in dimethylbenzene, rehydrated successively in a gradient of ethanol, and washed with distilled water before the subsequent experiments. The fresh frozen specimens were prepared for the other analysis.
Enzyme-linked Immunosorbent Assay (Elisa)
The mouse TNF-α and IL-6 Enzyme-Linked Immunosorbent Assay (ELISA) Kit (CUSABIO BIOTECH Co., Ltd., China) was used following the manufacturer’s instructions. In brief, the supernate of tissue was added to a 96-well plate pre-coated with hamster monoclonal anti-mouse TNF-α capture antibody. After incubation at room temperature (25℃) for 2 h, samples were washed, and the detection antibody was added for an additional hour. After further washing, avidin-horseradish peroxidase was used for detection, and optical density was measured at 450 nm and 570 nm (Multiskan GO; Thermo Fisher Scientific).
Nissl Staining
Nissl staining was performed according to the previous study [18]. The L3-4 spinal cord slides were dehydrated with different concentration of alcohol, rehydrated in distilled water, then stained in Nissl Staining Solution (C0117, Beyotime) for 5 min at 37°C. Subsequently, we used the 95% ethyl alcohol to immerse the slices for 5 minutes, then used 100% alcohol to dehydrated, and finally used xylene to clean slides for 5 minutes. Spinal cord slices were mounted and observed under Nikon Eclipse E800 light microscope. The average quantities of Nissl bodies (neurons with a diameter ≥ 25 µm) were counted by randomly selecting five Nissl-stained sections at the same site of each animal.
Immunofluorescence Staining
For staining, the sections were incubated in primary antibodies made in 1% bovine serum albumin (BSA) solution to appropriate dilutions at 4 ℃ overnight. Then, the sections were washed twice, and secondary antibodies in 1% BSA were added for 1 h at room temperature. Primary antibodies were anti-Iba1 (c-32725, dilution 1:500; Santa Cruz Biotechnology, USA,) and anti-TLR4 (ab22048, dilution 1:400; Abcam, USA]. Secondary antibodies were goat anti-mouse Alexa-Fluor 594 (for Iba-1) and goat anti-mouse Alexa-Fluor 488 (for TLR4), and the stain DAPI was used at 1:400 (Invitrogen Molecular Probes). Five randomly selected sections from each group were observed using an Olympus optical microscope (BX51, Olympus, Tokyo, Japan), and 5 fields from each section were imaged for TLR4 and Iba-1 co-expression. Immunostained sections were quantitatively characterized by digital image analysis using Image Pro-Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). All the results were recorded by researchers blinded to the experimental group. TLR4 and Iba-1 were quantified as the average number of positive cells per field. A negative (no antibody) control was included.
Western-blotting Analysis
The protein from the tissue was extracted in a lysis buffer (Beyotime Institute of Biotechnology) using sonication. The lysate was separated by centrifugation at 8, 000 × g at 4°C for 8 min, and the supernatant was collected. The total protein concentration was analyzed with the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica). The membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS) for 1 h at room temperature (26°C), followed by overnight incubation with the following primary antibodies at 4°C: anti-Mer (1:500, 365499, Santa Cruz Biotechnology, Inc), anti-TLR4 (1:500, AF7017; Affinity Biosciences), anti-phosphorylated-STAT1 (1:500, AF3293; Affinity Biosciences), anti-p65 NF-κB (1:1000, AF5006; Affinity Biosciences), anti-SOCS 1 (1: 500, AF5378; Affinity Biosciences), anti-SOCS 3 (1: 500, AF6133; Affinity Biosciences), β-actin (1:1, 000, 47778, Santa Cruz Biotechnology, Inc) and anti-GAPDH (1:1, 000, Abcam, ab8245). After washing, the membranes were incubated with a secondary antibody (goat anti-rabbit: 1:1, 000, ZB-2301, goat anti-mouse: 1: 1, 000, ZB-2305, ZSGB-BIO) for 1 h at 37°C; the bands were visualized using the enhanced chemiluminescence kit (Beyotime, Institute of Biotechnology, Jiangsu Province, China), and the band intensity was measured using Quantity One software. All results were compared to β-actin or GAPDH to normalize the protein levels.
Statistical Analyses
All statistical analyses were performed using SPSS (version 17.0, SPSS). Values are expressed as mean ± SD. The levels of cytokine plasma and proteins (TLR4, NF-κB, SOCS1/3, and STAT1) at 4 days after CLP were analyzed using 1-way analysis of variance (ANOVA) to compare the within-group differences, and the Student–Newman–Keuls (SNK)-q test to compare the between-group differences. The differences in all data were considered statistically significant at a P value less than 0.05.