Materials and Methods

Animals
WT male and female BALB/c mice were purchased from National Cancer Institute (NCI, Frederick, MD) and were allowed to habituate to the institutional animal facility for at least 1 week before experimental use (8–9 wks old). Breeding pairs for BALB/c CD154 KO mice were obtained from Dr. Abul Abbas at the University of California San Francisco and bred in the animal facility of University of New England (UNE). BALB/c CD4 KO mice were originally obtained from Dr. William T. Lee of the Wadsworth Center, New York State Department of Health and are currently maintained at the UNE animal facility by breeding homozygous KO mice. All mice were group-housed with food and water ad libitum and maintained on a 12-h light/dark cycle. Since no significant gender differences in all parameters measured were detected, both male and female mice (roughly 1:1 ratio) were used in all experiments. The Institutional Animal Care and Use Committee (IACUC) at UNE approved all experimental procedures. For the experiment conducted in the Dartmouth College (Figure 4A), The Institutional Animal Care and Use Committee (IACUC) at Dartmouth College approved the experimental procedures.

L5Tx and assessment of mechanical sensitivity
WT BALB/c and BALB/c CD154 KO mice were randomly assigned into sham and L5Tx groups. L5Tx and sham surgeries were performed as previously described [4]. The mechanical sensitivity of each mouse was measured with von Frey filaments (Stoelting, Wood Dale, IL) using the Up-Down paradigm (detailed in [20]) as previously described [4]. The fifty percent threshold force needed for paw withdrawal was calculated and used to represent the mechanical sensitivity. No significant differences in basal mechanical sensitivity were observed between WT and CD154 KO mice. The experimenter performing the behavioral tests was blinded to the experimental groups (including both the genotype and type of surgery).

Flow cytometry
As described previously [4], following transcardiac perfusion with phosphate buffered saline (PBS), mononuclear cells for flow cytometric analysis were prepared from an individual spleen, pooled lumbar lymph nodes (LNs) (5 mice), or pooled mouse lumbar spinal cord tissue (10 mice) by discontinuous Percoll (Amersham, Piscataway, NJ) gradients, and the total mononuclear cell number was determined using a hemacytometer with trypan blue (Sigma, St Louis, MO) prior to intracellular flow cytometric analysis. Due to the limited number of infiltrating cells available for analysis following the collection procedure (even with 10 mice pooled together) and the fact that the vast majority of infiltrating CD4+ T lymphocytes were observed in the ipsilateral side of dorsal horn area [4], lumbar spinal cord tissues were not separated as ipsilateral vs. contralateral in these experiments. In general, the intracellular staining was performed as follows. Briefly, following the final PBS wash, mononuclear cells were resuspended in staining buffer (2% fetal bovine serum (FBS)/0.09% NaN3/PBS) containing anti-mouse-CD16/CD32 monoclonal antibody (mAb) (clone 2.4G2; 1 µg/50µl/tube) and incubated on ice for 30 minutes. A combination of three fluorescent mAbs (Table 1) was added to each tube for surface staining (30 minutes on ice). After two PBS washes, the cells were fixed with 1% formaldehyde/PBS at room temperature for 20 minutes, washed with PBS again, and then permeabilized with a permeabilization buffer (0.5% saponin (Sigma)/2% FBS/0.09% NaN3/PBS) at room temperature for 10 minutes. Cells were then blocked with 10% isotype-matched serum (either mouse or rat serum) (in 0.5% saponin/PBS) on ice for 30 minutes. One selected fluorescent mAb (Table 1) was added to each tube for intracellular staining (30 minutes on ice). After being washed with permealization buffer, cells were resuspended in 1% formaldehyde/PBS and stored at 4°C until flow cytometric analysis. All samples were analyzed with an Accuri C6 flow cytometer (BD Biosciences-Accuri, Ann Arbor, MI) and FlowJo analysis software (Tree Star, Inc., Ashland, OR). For spleen and LN samples, a total of 20,000 events were collected and analyzed. For spinal cord mononuclear cells, due to the limited numbers of cells obtained, all events in each sample were analyzed. Non-stained cells, proper isotype controls, and surface-stained only controls were included as controls. For intracellular cytokine staining IFN-γ, IL-4, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF)), 0.1% brefeldin A (Sigma)/PBS was used in place of PBS from tissue harvesting up to the fixation step. All experiments were repeated several times in order to collect sufficient data for statistical analyses (i.e., when pooled samples were used, each pooled sample was considered as n=1).

Determination of tissue levels of IFN-γ
Spleens and lumbar spinal cords were collected following transcardiac perfusion and processed for the assessment of IFN-γ levels via enzyme-linked immunosorbent assay (ELISA). Tissue homogenates were prepared as described previously [13] and stored at −80°C until analysis. Levels of IFN-γ were determined with a Duo Set ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturers’ protocol. The IFN-γ level of each sample was normalized based on the protein concentration of the particular sample as determined by the BCA protein assay (Pierce-Thermo Scientific, Rockford, IL).

Immunohistochemistry (IHC)
Fluorescent IHC for CD4, CD154, and GFAP was performed on the L5 segment of lumbar spinal cord sections following a previously published procedure [13]. For CD4, a FITC-rat-anti-mouse CD4 mAb (clone RM4–5; 1:100) (BD Biosciences, San Diego, CA), was used; for CD154, a primary mAb, purified Armenian hamster-anti-CD154 (clone MR1; 1:100) (BD Biosciences), and a secondary antibody (Ab), TRITC-goat-anti-Armenian hamster IgG (1:5000) (Jackson Immuno Research Laboratories, West Grove, PA), were used. For GFAP, a primary Ab, polyclonal rabbit-anti-GFAP (1:10,000) (DAKO UK Ltd., UK) and a secondary Ab, Cy3-goat-anti-rabbit IgG (1:800) (Jackson Immuno Research Laboratories), were used. DAPI-containing mounting media (either VECTASHIELD® (Vector laboratories, Burlingame, CA) or Fluoromount-GT (VWR, Bristol, CT)) was used. “Non-stained” and “no primary Abs” controls were included when performing IHC, and no significant fluorescent signal was detected from these controls. For CD4 and CD 154 IHC, images were taken with an Olympus fluorescence microscope (U-ULH) (Olympus Optical Co.) and an Olympus Q-FIRE camera. For GFAP IHC, slices were examined with a Nikon Eclipse E800 fluorescence microscope (Nikon Instruments Inc., Melville, NY) and a SPOT RT Slider CCD microscope digital camera (Burlingame, CA). To determine GFAP expression in the spinal cord dorsal horn, black and white images were analyzed using Image J (NIH). Both the total dorsal horn area and GFAP immunostaining intensity were measured for each L5 dorsal horn image (represented images shown in Figure 5A). A ratio of total intensity/total dorsal horn area was then calculated and used as the GFAP immunoreactivity for each L5 dorsal horn slice. The average GFAP immunoreactivity of three slices from the same tissue sample were calculated and used as the GFAP expression for that particular sample in the final data analysis. The experimenter performing the analyses was blinded to the experimental groups.

Statistical analyses
All data were graphed with SigmaPlot 10.0 (Systat Software, Inc. San Jose, CA) and analyzed with SigmaStat 3.5 (Systat Software, Inc. San Jose, CA). Appropriate analyses of variance (ANOVA) were performed followed by the Student-Newman-Keuls (SNK) Post hoc test. All data are presented as mean ± SEM when applicable. p<0.05 was considered as statistically significant.