Rapid, Coordinate Inflammatory Responses after Experimental Febrile Status Epilepticus: Implications for Epileptogenesis

Activated microglia after eFSE. IBA1-IR cells are visible 24 h after eFSE in dorsal hippocampus in both eFSE rats (A, B, C) and control rats (A', B', C'). D, While the number of IBA1-IR cells is similar between the eFSE group (n = 4) and control group (n = 4), eFSE rats have a larger percentage of amoeboid microglia. Ramified (or nonactivated) microglia have slender, long processes with small soma, while amoeboid (or activated) microglia have short, thick processes with a distinct large soma. Solid arrows point to amoeboid microglia, while empty arrows point to ramified microglia. E, There are no differences in mRNA levels for CD11b (1 h, n = 5; 3 h, n = 6; 24 h, n = 6; 96 h, n = 4), which can increase as microglia become active. Data are presented as the mean ± SEM. Scale bars: A–C', 200 μm; insets, 5 μm. *Statistically significant at p < 0.05. CTL, Control. SO, stratum oriens. SP, stratum pyramidale. SR, stratum radiatum. SLM, lacunosum- moleculare. ML, molecular layer. GCL, granule cell layer. SVZ, subventricular zone.

Activated astrocytes after eFSE. GFAP-IR cells are visible in dorsal hippocampus regions CA1, CA3, and the hilus of the dentate gyrus 24 h after eFSE (A, B, C; n = 4) but not in control rats (A', B', C'; n = 4). Solid arrows depict GFAP⁺ cells. D, Quantification reveals a significant increase in the number of GFAP-IR cells compared with controls. E, mRNA levels of GFAP are significantly increased 3 and 24 h after eFSE, but not at 1 or 96 h (1 h, n = 5; 3 h, n = 6; 24 h, n = 6; 96 h, n = 4). Data are presented as the mean ± SEM. Scale bars: A–C', 200 μm; insets, 5 μm. *Statistically significant at p < 0.05. CTL, Control. SO, stratum oriens. SP, stratum pyramidale. SR, stratum radiatum. SLM, lacunosum- moleculare. ML, molecular layer. GCL, granule cell layer. SVZ, subventricular zone.

HMGB1 translocation in hippocampal neurons after eFSE. A, In control animals (n = 3), HMGB1 is confined to the nucleus of cells (open arrows) in area CA1 of dorsal hippocampus. B, One hour after the end of eFSE, HMGB1-IR is seen in the processes of neurons (closed arrows; n = 4), indicating translocation of HMGB1 from the nucleus. C, Translocation is still evident 3 h after the end of eFSE (n = 4). D, By 24 h after eFSE, mostly nuclear HMGB1 is observed (n = 4). E, Quantification of HMGB1 immunocytochemistry as the percentage of HMGB1-IR cells with translocation over total HMGB1-IR cells shows a significant increase in the percentage of HMGB1 translocation 1 and 3 h after eFSE with a return to control conditions by 24 h. F–M, Double labeling of HMGB1 and NeuN (both in soma and processes) demonstrates that HMGB1 translocation occurs from neurons (F–H) and not from astrocytes, as shown with GFAP (I–K), or microglia, as shown with CD11b (L, M; CTL n = 3; 1 h, n = 4; 3 h, n = 4). O, There is no increase in the levels of HMGB1 mRNA, indicating that translocation is achieved using already available protein (1 h, n = 5; 3 h, n = 6; 24 h, n = 6; 96 h, n = 4). P, TLR4, one receptor through which proinflammatory HMGB1 acts, mRNA is upregulated 24 h after eFSE with a return to baseline values by 96 h (1 h, n = 5; 3 h, n = 6; 24 h, n = 6; 96 h, n = 4). Data are presented as the mean ± SEM. Scale bars, 100 μm. *Statistically significant at p < 0.05. CTL, Control. SO, stratum oriens. SP, stratum pyramidale. SR, stratum radiatum.

Transient cytokine upregulation after eFSE. A, IL-1β mRNA levels are significantly upregulated 1 and 3 h after eFSE (CTL = 23; 1 h, n = 10; 3 h, n = 10; 24 h, n = 12; 96 h, n = 4). B, Variability of IL-1β mRNA expression between rats is apparent when data are presented as individual animals where some rats have high levels of IL-1β expression and others are indistinguishable from controls. C, IL-1r1 mRNA levels are significantly upregulated 3 and 24 h after eFSE, with obvious interanimal variability (CTL, n = 20l; 1 h, n = 10; 3 h, n = 10; 24 h, n = 13; 96 h, n = 4). D, While never statistically significant, IL-6 levels trend upward for some animals at 24 h with a return to baseline values by 96 h (CTL, n = 9; 1 h, n = 4; 3 h, n = 4; 24 h, n = 10; 96 h, n = 4). E, TNF-α mRNA levels are significantly augmented by 3 h and return to baseline by 24 h (CTL, n = 25; 1 h, n = 10; 3 h, n = 10; 24 h, n = 12; 96 h, n = 4). F, COX2 mRNA levels are significantly upregulated at every tested time point (CTL, n = 11; 1 h, n = 6; 3 h, n = 6; 24 h, n = 6; 96 h, n = 4). G, GFAP, COX2, and IL-6 protein levels are augmented with interanimal variation 24 h after eFSE (n = 4) compared with littermate controls (n = 4). Densitometric analyses of protein levels normalized and compared with controls are provided in H. Data are presented as the mean ± SEM. *Statistically significant at p < 0.05.

Interanimal variation of inflammatory mediator expression after eFSE. A, Three hours after eFSE, COX2, GFAP, TNF-α, and IL-1r1 levels were significantly increased. When the expression profile of an individual rat is examined across these mediators, trends in expression appear (n = 4). For example, rat 1 has the highest expression across all explored mediators, while rat 4 has virtually no inflammatory response after eFSE. Rats 2 and 3 both fall in between rats 1 and 4, with moderate expression profiles. B, A similar pattern is found 24 h after eFSE as well (n = 6). COX2, IL-1r1, TLR4, and GFAP were all significantly upregulated at this time point. As seen with the 3 h time point, rats with high expression in one mediator have high expression in all others, and vice versa. Rat 6 has the highest expression of all examined mediators, while Rat 5 is a nonresponder.