Experimental animals
Female Sprague-Dawley rats 11 weeks were purchased from Razi Institute (Tehran, Iran). After arrival, all rats were housed in standard control caging at the standard condition of temperature (21±1°C) and controlled humidity. All rats were given ad libitum access to food and water over the study period. The rats were housed one per wire cage. Animals were randomly divided into three groups: Spinal cord injured animals (SCI) (n=6), Sham control (laminectomy alone without spinal cord injury, n=6); and intact control group (n=6). All surgical and postoperative care procedures were performed in accordance with the Animal Ethics Committee of Tarbiat Modares University, Tehran, Iran.
Animal surgical procedures
Rats were anesthetized with a mixture of xylazine (100-150 mg/kg) and ketamine (60-90 mg/kg) by intraperitoneal (IP) injection, and laminectomy performed at the T9-T10 thoracic level to expose the spinal cord. The vertebral column was stabilized using clamps on T8 and T11 vertebrae, and rats received low-level thoracic injury by dropping a 10-g weight (a 2-mm metal rod) from 12.5 mm height centered above the spinal cord. Afterward, the impact rod was immediately detached following the injury, and overlying muscle layers and skin were sutured. During recovery, animals were placed on heating pads and monitored. The animals were housed individually in cages and bladders of rats were expressed manually daily until they recovered their function. Antibiotic (Cefazolin, Jaber Ibn Hayan Co., Tehran) was given with a dose of 50 mg/kg of body weight once a day for the first 7 days post-surgery to prevent infection. Moreover, 6 ml of sterile saline was injected subcutaneously for three days. Rats always had ad libitum access to food and water, with some food pellets placed at the bottom of each cage to have easy access to the pellets. The sham groups underwent laminectomy similar to that performed in the SCI group, but without impact injury.
Lumbar lymph node isolation
Rats were deeply anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (10 mg/kg) one week after thoracic spinal cord injury. For lumbar lymph nodes isolation, each rat was put in its prone position, a median incision was made into the skin and peritoneum of the lower abdomen to open the abdominal cavity, and the intestines were then retracted to have access to the rat lumbar lymph nodes, which could be observed singly or in pairs located along each side of the distal abdominal aorta at the bifurcation into the iliac arteries [10]. All the Lumbar lymph nodes were isolated.
Lumbar lymph node size and weight estimation
Lumbar lymph nodes were harvested, any additional connective tissue and fat covering the lymph node were removed, and all lumbar lymph nodes weighed. Lymph node size was calculated using a two-dimensional area of lymph nodes.
Total cell number, cell viability, and cell death rate measurement
Single-cell suspensions were isolated from excised lumbar lymph nodes by forcing the tissues through a fine wire mesh. Cells were washed and resuspended in PBS containing 10% FCS. Total cell number was enumerated with Cellometer (Nexcelom), an automated cell counter. The cell viability and cell death rate were also determined using acridine orange/propidium iodide (AO/PI) assay. Cell counts were performed in duplicates.
CFSE assay
Single-cell suspensions were generated from excised lumbar lymph nodes within seven days after SCI from all groups. The cells in RPMI-1640 medium (GIBCO, New York) supplemented with 10% fetal bovine serum (Gibco, New York) were cultured in flat-bottomed wells on a 96-well microtiter plate. Cells (2×105 cells per well) were cultured for 72 hours in an antigen-free medium (unstimulated control) or together with PHA (10 µg/ml) at 37°C in 5% CO2. After rinsing with RPMI-1640, the cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) (CellTrace TM CFSE Cell Proliferation Kit, Invitrogen, Molecular Probes, USA). The cells were stained at a final concentration of 1 µM, then incubated for 72 h at 37°C and unstained cells were used to distinguish the background autofluorescence. The unstimulated cells from lumbar lymph nodes were used as a reference to fix the zero point of the peak for the undivided population in FlowJo software (FlowJo, Ashland, OR, USA). The area of lymphocytes was gated according to light scattering characteristics (size/granularity). Ten thousand events were acquired for each sample on FACS CanII instrument (BD Company, USA) and data were analyzed by FlowJo software.
Cytokine analysis
ELISA kits (DuoSet; R&D System, Oxon, UK) were used to evaluate the concentrations of either IL-4 (lower and upper quantitation limits were 15.625 and 1000 pg/ml, respectively), and IFN-γ (lower and upper quantitation limits were 39.1 and 2500 pg/ml, respectively; samples were diluted 1:30). Mononuclear cells collected from lumbar lymph nodes of animal groups 7 days following thoracic spinal cord injury were stimulated with PHA (10µg/ml) in 96-well plates for 72h and then levels in the culture supernatants were determined using ELISA kits as described by the manufacturer.
Statistical analysis
All Statistical analysis was performed with GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA). Data distribution was assessed by the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for the analysis of data. Data were presented as mean ± SD. P values less than 0.05 were considered statistically significant.