We used male Sprague–Dawley rats (200–250 g). The animals stayed in groups under normal conditions with access to enough food and water ad libitum. The procedures of the study were approved by the Local Ethics Committee for Animal Experimentation (101/2013 September).
Experimental groups were assigned as five groups: control (n = 16), sham (n = 16), CLP + saline (n = 16), CLP + immunoglobulin G (IgG) (250 mg/kg, iv) (n = 16), and CLP + immunoglobulin enriched with immunoglobulin M (IgGAM) (250 mg/kg, iv) (n = 16). Blood and brain samples were taken in two sets of experiments after CLP to see the early (24 h) and late (10 days) effects of treatment.
To assess cerebral complement expression alterations, apoptosis, and gliosis induced by sepsis and to delineate the impact of IVIg treatment on these parameters, a CLP-based sepsis model was performed as described previously . After intraperitoneal ketamine administration (100 mg/kg), a longitudinal midline abdominal incision was made with a scalpel. A small scissor was used to extend the incision and gain entry into the peritoneal cavity. The cecum was isolated and exteriorized with blunt anatomical forceps; care was taken not to breach or damage the mesenteric blood vessels. To induce high-grade sepsis, the cecum was ligated and perforated by a single through-and-through puncture midway between the ligation using an 18-G needle. After removing the needle, a small amount of feces was extruded from both the mesenteric and antimesenteric penetration holes and the cecum was relocated into the abdominal cavity. Following the closure of the wound, the animals were resuscitated by injecting warmed saline (37 °C; 5 ml per 100 g body weight) subcutaneously. The sham-operated rats underwent the same procedure, except for the ligation and perforation of the cecum. The control rats received neither any surgical intervention nor IVIg treatment. At the early stage (24 h) of the sepsis model, the survival rate of the CLP + saline group was 6/8 (75%), whereas in the control, sham, CLP + IgG, and CLP + IgGAM group, the survival rates were 8/8 (100%). In the late stage (10 days), the survival rates were 5/8 (62.5%) in the CLP + saline group, 7/8 (87.5%) in the CLP + IgG and CLP + IgGAM groups, and 8/8 (100%) in the control and sham groups.
Administration of immunoglobulins
The animals were given human IgG, 250 mg/kg (Octapharma; Vienna, Austria), or IgGAM, 250 mg/kg (Pentaglobin; Biotest, Dreieich, Germany), intravenously via penile vein 5 min after the CLP procedure. After the IV injection, the animals were placed back in their cages for recovery.
ELISA for serum total complement activity and complement levels
To assess the effects of IVIg treatment on systemic complement activity and to validate the complement activity inhibiting effect of IVIg, serum total complement activity (CH50), C3, and soluble C5b-9 levels were measured by ELISA kits (Neoscientific, Cambridge, MA, USA), as per the manufacturer’s instructions. Optical density was measured at 450 nm, and concentrations were calculated by referring to a standard curve.
Expression levels of major complement factors, complement regulators, and apoptosis factors in septic encephalopathy model were evaluated by real-time PCR. Total RNA from each brain sample was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and RNA was quantified by OD 260. Four micrograms of total messenger RNA (mRNA) were reverse transcribed using superscript II reverse transcriptase and oligo dT primer (Invitrogen). Specific primers (Additional file 1: Table S1) were optimized using primer3 software and generated by Qiagen (Hilden, Germany). The specificity of the primers was verified with a Blast search through NCBI. The quantitative real-time PCR reactions were performed with the SYBR Green kit (Roche Diagnostics, Mannheim, Germany) using 2 μl of cDNA and 0.6 μl of each primer in a 20-μl final volume. Quantitative PCR was performed using Light Cycler (Roche Diagnostics) for 40 cycles at 95 °C for 15 s and at annealing temperatures of 60 °C for 20 s and 72 °C for 30 s. All samples were studied as duplicates, and three housekeeping genes were used as reference genes. Data were analyzed according to ΔΔCt method, and the results were expressed as relative mRNA levels.
To confirm the real-time PCR results of most crucially altered complement and apoptosis factors at the protein level, immunoblotting experiments were conducted. Twenty micrograms of each brain lysate was loaded and separated by 4–20% SDS–polyacrylamide gradient gel electrophoresis and then transferred to 0.45-μm polyvinylidene fluoride membranes (100 V, 80 min). After blocking for 1 h in Phosphate buffered saline with tween 20 (PBST) (10 mM sodium phosphate, 0.9% NaCl, and 0.1% Tween 20) containing 5% non-fat dry milk, blots were incubated overnight at 4 °C with the primary antibodies (Additional file 2: Table S2) in PBST containing 3% non-fat milk. The blots were washed four times with PBST (40 min) and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBST containing 3% non-fat dry milk. Immunoreactivity of the protein bands were detected by enhanced chemiluminescent autoradiography (ECL kit, Amersham Pharmacia Biotech, Piscataway, NJ). A molecular weight standard (Bio-Rad Laboratories, Hercules, CA) was loaded in the last lane of each gel to assess relative molecular mass of detected bands. The immune blot bands were quantified through measurement of band intensity with ImageJ software using the same pixel scale for all pictures. Band intensities were normalized by β-actin expression and expressed as arbitrary units.
TUNEL and immunohistochemistry
The presence and intensity of infiltrating immune cells, apoptosis, and gliosis were evaluated with immunohistochemical methods. Brain samples were first evaluated by standard hematoxylin and eosin (H&E) staining. To investigate the presence of potential infiltrating immune cells and reactive gliosis, immunohistochemistry studies were performed. Brain samples were first treated with 4% paraformaldehyde overnight at 4 °C, immersed in 40% sucrose overnight at 4 °C, and subsequently snap frozen in liquid nitrogen. Seven-micrometer-thick frozen sections were serially incubated with 0.3% H2O2 for 20 min, 10% goat serum for 1 h, and primary antibodies (Additional file 2: Table S2) overnight at 4 °C. The sections were then incubated in biotinylated goat anti-human IgG (1:2000, Vector Laboratories, Burlingame, CA), and the immunoreactivity was developed by serial incubation with avidin–biotin peroxidase (Vector Laboratories) for 1 h and diaminobenzidine . TUNEL staining was done with an apoptosis detection kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Rat spleen sections were used as a positive control in all experiments. The presence of immune cells, apoptosis, and gliosis were visualized by two independent blinded observers.
Serum complement factor and activity levels, brain mRNA expression levels measured by real-time PCR, and normalized brain protein expression levels measured by immunoblot experiments were compared among different treatment arms by using ANOVA followed with Tukey adjustment for multiple pairwise comparisons. p < 0.05 was considered statistically significant.