The acute phase response and soman-induced status epilepticus: temporal, regional and cellular changes in rat brain cytokine concentrations

Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA; CRL: CD[SD]-BR, 250 - 350 g) were treated with HI-6 dichloride (Walter Reed Army Institute of Research, Silver Spring, MD; 125 mg/kg, i.p.) 30 minutes prior to GD administration and with atropine methyl nitrate (AMN, Sigma-Aldrich, St. Louis, MO; 2.0 mg/kg, i.m.) 1 minute after GD administration. Vehicle control animals received HI-6, AMN and saline, while naïve animals received no injections. GD (GD-U-2323-CTF-N, purity 98.8 wt%) was diluted in saline at USAMRICD. GD (1.6 LD₅₀ = 180 μg/kg) was administered subcutaneously in the scruff of the neck and the rat was observed for convulsive activity. This dose of GD produces within minutes [24] a 100% generalized convulsive seizure rate that is maintained up to 24 hours [3]. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), and the Animal Welfare Act of 1966 (P.L. 89-544), as amended. The animal care program at this institute is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Experimental, vehicle control and naïve animals were deeply anesthetized with a sodium pentobarbital solution (70 mg/kg, i.p.) then euthanized by decapitation at 0.5, 1, 3, 6, 12, 24, 48 or 72 hours after onset of convulsions. Following euthanasia, piriform cortex, hippocampus and thalamus tissue was extracted and processed into lysate as previously described [25]. Briefly, the brain regions were excised, rinsed with cold PBS and snap frozen in liquid nitrogen. The tissues were weighed and homogenized in ice-cold triple detergent lysis buffer containing a Complete™ protease inhibitor cocktail (Roche Biochemicals, Indianapolis, IN) at a ratio of 1 ml buffer to 50 mg tissue. Samples were allowed to stand at 4°C for at least 30 minutes before centrifugation at 8000 G for 5 minutes and removal of the lysate for assaying. Cytokine concentrations were quantified using a rat cytokine multiplex bead immunoassay kit containing IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IL-17, and TNF-α (LINCO Research, St. Charles, MO). The bead immunoassay procedure used 25 μl of sample (94 ± 8 μg protein) per well and was conducted according to the manufacturer's instructions with each individual cytokine standard curve and sample assayed in duplicate. The plate was read on a Luminex™ 100 instrument (Bio-Rad Laboratories, Hercules, CA) and analyzed with either BioRad or STaRStation software (Applied Cytometry, Sacramento, CA). The number of replicates for the experimental samples are as follows: piriform cortex, n = 6 for each time point and naïve; hippocampus, n = 6 for each time point except for naïve (n = 5), 6 hr (n = 5) and 24 hr (n = 7); thalamus, n = 5 for each time point and naïve except for 0.5 hr (n = 6), 6 hr (n = 4), 12 hr (n = 3), 24 hr (n = 6) and 48 hr (n = 6). Time matched vehicle controls (n = 3 per time point) were analyzed individually and condensed into a single vehicle control comparison group when no significant difference was found between these samples over time by analyte or brain region.

Separate from the animals used in the multiplex bead array immunoassay, experimental, vehicle control and naïve animals were deeply anesthetized, euthanized by decapitation at 12 hours after seizure onset and perfused with isotonic saline followed by 4% paraformaldehyde via cardiac puncture. Brains were immediately frozen at -20°C, cut on a Leica CM3050 S cryostat (Thermo Shandon, Inc.; Pittsburgh, PA) at 40 microns and stored in cryobuffer (30% each of glycerol, ethylene glycol and water, 10% 2 × phosphate buffer) until use. Free float fluorescent IHC labeling was conducted as previously described [25]. Experimental and vehicle control samples had an n = 3 for each cytokine/cell type combination. The antibodies used were as follows: rabbit anti-IL-1α (1:1000; ab9875, Abcam, Cambridge, MA), rabbit anti-IL-1β (1:1000; ab9787, Abcam), rabbit anti-IL-6 (1:500; ab6672, Abcam), mouse anti-NeuN to label neurons (1:1000; MAB377, Chemicon, Temecula, CA), mouse anti-GFAP to label astrocytes (1:1000; MS-280-P, NeoMarkers, Fremont, CA), and mouse anti-cd11b to label microglia and macrophages (1:1000; CBL1512, Chemicon). Alexafluor™ fluorescent-tagged secondary and tertiary antibodies (Molecular Probes, Eugene, OR) were used for visualization. Tissue sections labeled with only secondary and tertiary antibodies were used as secondary controls. Sections were viewed and digitally captured with an Olympus BX51 microscope equipped with an Olympus DP-70 high-resolution color CCD digital camera (Opelco, Dulles, VA). An Olympus BX61 equipped with a DSU spinning disk confocal system and DP-70 CCD camera was used to confirm same cell co-localization (Opelco). Images of 40 μm tissues were acquired using a z step interval of 1 μm and analyzed using Slidebook™ software (Opelco). Publication images were compiled using Adobe Photoshop CS digital image software. Color levels and background labeling were reduced and evened using the levels tool. All input levels (0-255) were normalized in the RGB channel as follows: highlight input levels were set at the peak of the image histogram, midtone levels were set at 0.8 and shadow levels were set either at the edge of the histogram closest to 255 or at 180, whichever was greater.

Immunoassay data were evaluated by ANOVA with a post-hoc Dunnett's analysis and expressed in pg/ml. Data points calculated below the minimum detectible concentrations (MinDC) for the assays were conservatively set at -0.01 pg/ml of the MinDC for statistical analyses. Values are expressed as mean ± SEM. Differences were considered significant at the level of p ≤ 0.05.

Of the ten inflammatory cytokines investigated, concentrations of four significantly increased: IL-1α, IL-1β, IL-6, and TNF-α as shown in Figures 1-4. No significant changes were observed for IL-2, IL-4, IL-10, IL-12p70, IL-13 or IL-17 during the time course (data not shown). Concentrations of IL-1α significantly increased at 12 hours after SE onset in the piriform cortex (205 ± 99 pg/ml v. 14 ± 7 pg/ml vehicle control), hippocampus (234 ± 29 pg/ml v. 34 ± 29 pg/ml vehicle control) and thalamus (303 ± 213 pg/ml v. 45 ± 13 pg/ml vehicle control)(Figure 1). IL-1β significantly increased in the piriform cortex at 12 hours (41 ± 10 pg/ml v. 16 ± 10 pg/ml vehicle control) and in the thalamus at 12 hours (48 ± 28 pg/ml) and 24 hours (24 ± 12 pg/ml v. 5 ± 1 pg/ml vehicle control) (Figure 2). No significant changes in IL-1β concentration were observed in the hippocampus though a definite trend was observed, with an approximate 3-fold increase over vehicle control values. Significant IL-6 concentration increases were observed in all brain regions at 12 and 24 hours (Figure 3) though peak expression varied by region. In the piriform cortex, IL-6 concentrations significantly increased at 12 hours (2399 ± 1238 pg/ml) and peaked at 24 hours (3200 ± 894 pg/ml) compared to vehicle control (38 ± 8 pg/ml). Peak levels in the hippocampus were observed at 12 hours (2462 ± 1489 pg/ml) with a small decrease at 24 hours (2386 ± 814 pg/ml v. 99 ± 48 pg/ml vehicle control) with the true peak likely occurring between these time points. In the thalamus, IL-6 peaks at 12 hours (2706 ± 913 pg/ml) though concentration increases are still significant at 24 hours (1711 ± 909 pg/ml) compared to vehicle control (13 ± 13 pg/ml). Lastly, TNF-α significantly increased in the piriform cortex at 6 (20 ± 7 pg/ml) and 12 hours (32 ± 15 pg/ml v. 4.43 ± 0.0 pg/ml [below MinDC]), in the hippocampus at 6 (27 ± 7 pg/ml) and 12 hours (37 ± 10 pg/ml v. 13 ± 3 pg/ml vehicle control) and in the thalamus at 12 hours (61 ± 31 pg/ml v. 6 ± 3 pg/ml vehicle control) (Figure 4). Naïve and vehicle controls were not significantly different from each other for any factor measured in any region. To further identify the source of these cytokines, neurons, astrocytes and microglia expressing IL-1α, IL-1β and IL-6 were identified in each brain region using IHC at the 12-hour time point, the peak of cytokine expression for the majority of these brain regions. Localization of TNF-α was not pursued due to a lack of effective IHC antibodies.

The cytokines IL-1α (Figure 5) and β (Figure 6) (collectively IL-1) are functionally similar and likewise have similar labeling patterns in the piriform cortex (Figure 5A and 6A, left), hippocampus (dentate gyrus shown; Figure 5B and 6B, left) and thalamus (lateral posterior region shown; Figure 5C and 6C, left). Labeling was absent in vehicle (Figure 5 and 6A, B, & 6C right) and secondary controls (not shown). In the piriform cortex, IL-1 positive cells were located in all 3 layers with IL-1α found predominantly in layer I and IL-1β found in layer II. In the hippocampus, IL-1 positive cells were found primarily in the polymorphic layer of the dentate gyrus (PoDG) and the CA3 pyramidal layer closest to the dentate gyrus. IL-1 positive cells were also found in the laterodorsal and lateral posterior nuclei of the thalamus. To identify these cells, sections were co-labeled with antibodies specific for neurons, astrocytes or microglia and for IL-1α or β. Positive immunoreactivity for IL-1 was absent in both neurons (Figure 5 and 6D) and astrocytes (Figure 5 and 6E). However, strong and abundant expression of IL-1α and β was observed in many activated microglia (Figure 5F). IL-1β, but not IL-1α, also appeared in morphologically identified dystrophic microglia (Figure 6F, white arrow). Cellular expression of IL-1 was the same regardless of the brain region investigated.

IL-6 immunolabeling was present in the piriform cortex (Figure 7A, left), hippocampus (dentate gyrus shown; Figure 7B, left) and thalamus (Figure 7C, left). IL-6 labeling was mostly absent in vehicle controls (Figure 7A, B, & 7C right), though light diffuse immunoreactivity in a limited number of piriform cortex and thalamus neurons was occasionally observed. Cellular expression of IL-6 was the same regardless of the brain region investigated. IL-6 labeling was moderate to strong, with both a diffuse and punctate distribution in neuronal cell bodies (Figure 7D). IL-6 positive neurons were localized primarily in layers II and III of the piriform cortex, the pyramidal and extrapyramidal regions of the hippocampus and many of the thalamic nuclei. Diffuse labeling of IL-6 was also present in activated astrocytes (Figure 7E) but often sparse and less intense than in neurons. Though co-localization was infrequent, IL-6 was often found coincident with astrocytes around the vasculature in all studied brain regions. No co-localization was observed between IL-6 and microglia (Figure 7F). To confirm that the cellular origin of IL-6 did not change with time, 24 hour tissues were also labeled with IL-6. No differences in cellular expression of this cytokine were observed between 12 and 24 hours (data not shown).