Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a 'two-hit' seizure model

Abstract

Early-life seizures result in increased susceptibility to seizures and greater neurologic injury with a second insult in adulthood. The mechanisms which link seizures in early-life to increased susceptibility to neurologic injury following a 'second hit' are not known. We examined the contribution of microglial activation and increased proinflammatory cytokine production to the subsequent increase in susceptibility to neurologic injury using a kainic acid (KA)-induced, established 'two-hit' seizure model in rats. Postnatal day (P)15 rats were administered intraperitoneal KA (early-life seizures) or saline, followed on P45 with either a 'second hit' of KA, a first exposure to KA (adult seizures), or saline. We measured the levels of proinflammatory cytokines (IL-1 beta, TNF-alpha, and S100B), the chemokine CCL2, microglial activation, seizure susceptibility and neuronal outcomes in adult rats 12 h and 10 days after the second hit on P45. The 'two-hit' group exposed to KA on both P15 and P45 had higher levels of cytokines, greater microglial activation, and increased susceptibility to seizures and neurologic injury compared to the adult seizures group. Treatment after early-life seizures with Minozac, a small molecule experimental therapeutic that targets upregulated proinflammatory cytokine production, attenuated the enhanced microglial and cytokine responses, the increased susceptibility to seizures, and the greater neuronal injury in the 'two-hit' group. These results implicate microglial activation as one mechanism by which early-life seizures contribute to increased vulnerability to neurologic insults in adulthood, and indicate the potential longer term benefits of early-life intervention with therapies that target up-regulation of proinflammatory cytokines.

Figure 1. Schematic of experimental design

Timeline of kainic acid (KA) injection, Minozac (Mzc) treatment, and Y-maze testing are shown. Following intraperitoneal (i.p.) injection of KA or saline (Sal) on postnatal day (P)15, rats were injected i.p. with Mzc (5 mg/kg) or Sal at 3 h and 9 h. Animals were allowed to recover for 30 days to P45. On P45, animals were administered KA or Sal. Y-maze testing was performed on alternating days from P46–P55. Animals were sacrificed at 30 d (P45 12 h) and 40 d (P55). Experimental groups at P45 and P55 include control (SSS), adult seizures (SSK), ‘two-hit’ (KSK), and Mzc-treated ‘two-hit’ (KMK).

Figure 2. Enhanced upregulation of proinflammatory cytokines in the ‘two-hit’ group, and prevention of this response by treatment with Mzc after early-life seizures

Effect of KA-induced early-life seizures on levels of the proinflammatory cytokines IL-1β (A), TNF-α (B), and S100B (C) in hippocampal homogenates after a second hit of KA on P45 and 12 h recovery. The cytokine levels were significantly increased in the ‘two-hit’ group (KSK) compared to controls (SSS) and adult seizures (SSK). Mzc treatment (KMK) on P15 prevented the enhanced increase in cytokine levels at P45. **, p < 0.01; ***, p < 0.001 vs. SSS.

Figure 3. Microglial activation is enhanced following a ‘second hit’ of seizures in adulthood, and this exaggerated response is prevented by administration of Mzc after early-life seizures

Quantification of microglial markers, Iba1 (A,B) and clusterin (C,D), in combined regions (CA1, CA2, CA3, DG, PoDG) of the hippocampus at 12 h (P45) and 10 d (P55) recovery following seizures at P45. (A and B) Increased numbers of Iba1-immunoreactive cells indicate increased microglial activation in adult seizures (SSK) and ‘two-hit’ (KSK) groups compared to controls (SSS) after exposure to KA on P45 at both time points. Prior treatment with Mzc on P15 (KMK) suppressed the enhanced increase in microglial activation in the ‘two-hit’ group at 12 h (A) and 10 d (B) recovery. (C and D) Quantification of clusterin-immunoreactive cells at the same time points. Clusterin-immunoreactive cells in controls, adult seizures, ‘two-hit’, and Mzc-treated groups show a pattern similar to Iba1 immunoreactivity. Mzc treatment suppressed the enhanced increase in clusterin immunoreactivity in the ‘two-hit’ group at 12 h (C) and 10 d (D). Representative photomicrographs of Iba1 (E–H) and clusterin (I–L) immunoreactive cells in the CA1 region of the hippocampus are shown. All photomicrographs are of P55 animals obtained at 10 d recovery after KA exposure on P45. Positively stained cells are brown. Sections were counterstained with hematoxylin (blue) for contrast. Scale bar, 100 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. SSS.

Figure 4. The chemokine CCL2 is upregulated following KA–induced seizures and this response is enhanced following a ‘second hit’ of seizures in adulthood

Levels of the chemokine, CCL2 were measured in hippocampal homogenates 12 hours after exposure to KA or Sal on P45. CCL2 levels were increased by KA (SSK), and this increase was greater in the ‘two-hit’ (KSK) group. Prior treatment with Mzc on P15 (KMK) prevented this enhanced response. **, p < 0.01 vs. SSS.

Figure 5. Mzc treatment after early-life seizures prevents the increased neuronal injury, susceptibility to seizures, and neurobehavioral impairment induced by a second hit in adulthood

(A) Representative photomicrographs of injured neurons (green) identified by FluoroJade-B (FJB), and DAPI (blue) fluorescent staining in the CA1 subfield of the hippocampus in a ‘two-hit’ animal (KSK) on P55 show the extent of neuronal injury in the ‘two-hit’ group and attenuation of this injury by Mzc treatment (KMK) on P15. (B) Quantification of FJB-fluorescent cells in the hippocampus of P55 animals. Neuronal injury is increased in the ‘two-hit’ (KSK) group compared to adult seizures (SSK), and this enhanced injury is prevented in the two-hit group (KMK) treated with Mzc on P15. (C) Qualitative assessment of neuronal nuclei (NeuN) staining shows increased neuronal loss in adult seizures and ‘two-hit’ groups, with greater neuronal loss evident in ‘two-hit‘ animals. Minimal or no neuronal loss was observed in controls and Mzc-treated animals. All photomicrographs are of P55 animals obtained at 10 d recovery after KA exposure on P45. Scale bar, 100 μm. (D) Reduced seizure severity in the Mzc-treated group (KMK) compared to the adult seizure (SSK) and the two-hit (KSK) groups. (E) Reduced seizure latency in the ‘two-hit’ (KSK) group after a second hit of KA on P45 compared to values for adult seizures (SSK). Prior treatment with Mzc on P15 in the ‘two-hit’ group (KMK) prevented this increased susceptibility, and was not different from the adult seizure group. (F) Hippocampal-dependent behavior measured by the Y-maze test from P46 to P55. Both the SSK and KSK groups showed behavioral impairment (reduced % alternation) compared to Sal-treated controls (SSS). Mzc treatment (KMK) after early-life seizures prevented this behavioral impairment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. SSS.