Minocycline attenuates microglia activation and blocks the long-term epileptogenic effects of early-life seizures

Abstract

Innate immunity mediated by microglia appears to play a crucial role in initiating and propagating seizure-induced inflammatory responses. To address the role of activated microglia in the pathogenesis of childhood epilepsy, we first examined the time course of microglia activation following kainic acid-induced status epilepticus (KA-SE) in Cx3cr1(GFP/+) transgenic mice whose microglia are fluorescently labeled. We then determined whether this seizure-induced microglia activation primes the central immune response to overreact and to increase the susceptibility to a second seizure later in life. We used an inhibitor of microglia activation, minocycline, to block the seizure-induced inflammation to determine whether innate immunity plays a causal role in mediating the long-term epileptogenic effects of early-life seizure. First status epilepticus was induced at postnatal day (P) 25 and a second status at P39. KA-SE at P25 caused nearly a two-fold increase in microglia activation within 24h. Significant seizure-induced activation persisted for 7 days and returned to baseline by 14 days. P39 animals with prior exposure to KA-SE not only responded with greater microglial activation in response to "second hit" of KA, but shorter latency to express seizures. Inhibition of seizure-induced inflammation by 7 day minocycline post-treatment abrogated both the exaggerated microglia activation and the increased susceptibility to the second seizure later in life. The priming effect of early-life seizures is accompanied by modified and rapidly reactivated microglia. Our results suggest that anti-inflammatory therapy after SE may be useful to block the epileptogenic process and mitigate the long-term damaging effects of early-life seizures.

Figure 1. KA-SE induced acute and transient activation of microglia

P25 Cx3cr1^GFP/+ mice were injected i.p. with either KA (20mg/kg) or PBS and microglia activation within the hilus of the hippocampus assessed at 1(A–D), 7 (E–H) or 14 (I–L) days after administration of PBS or KA. Scale bar: 50μm. C & D 1 d PBS n=4; KA n=4; G & H, 7 d: PBS n=8, KA n=7; K & L 14 d: PBS n=7; KA n=5. (**p<0.01, ***p<0.001, Student’s t-test).

Figure 2. Early-life seizure increases microglia response and seizure susceptibility to later life seizures

Mice were injected with PBS or KA (20mg/kg) on P25 followed by an injection of KA on P39 (A. SK vs. B. KK). Scale bar: 50μm. C. Microglia activation in the hippocampal CA3 subregion was assessed 24h after last injection. SK n=5, KK n=5. (***p<0.001, Student’s t-test). D. Seizure susceptibility defined as the latency to the onset of grade III seizure was assessed. (SK n=6, KK n=8, ***p<0.001, Student’s t-test). 9/15 SK and 12/15 KK mice experienced status epilepticus (p = 0.42, Fisher’s exact test); 3/9 SK and 4/12 KK animals died after status.

Figure 3. Minocycline post-treatment reduces early life seizure-induced microglia activation

P25 mice were injected with PBS or KA (20mg/.kg) followed by either an i.p. injection of PBS (A. SS or B. KS) or minocycline (20 mg/kg, C. KM) for 7 consecutive days. Scale bar: 50μm. D. Microglia activation was assessed 1 d after last injection. SS n=6; KS n=5; KM n=7, p<0.001, One-Way ANOVA with post-hoc Tukey test (*p<0.05, **p<0.01).

Figure 4. Minocycline post-treatment prevents the priming effect of early-life seizures

P25 mice received an i.p. injection of PBS or minocycline (20 mg/kg) 3 hours after KA-SE induction and for the following six consecutive days. Following an additional 7 days, P39 mice received an i.p. injection of KA (A. SSK vs. B. KSK vs. C. KMK) and sacrificed 24 h later on P40. Scale bar: 50μm. D. Microglia activation was assessed 1 d after last injection. SSK n=4; KSK n=4; KM n=10, p<0.001, One-Way ANOVA with post-hoc Tukey test (***p<0.001). E. Seizure susceptibility was determined by latency (seconds) to grade III seizure. SSK n=6; KSK n=8; KM n=10, p<0.001, One-Way ANOVA with post-hoc Tukey test (***p<0.01). Comparable numbers of SSK, KSK and KMK experienced SE after KA injection at P39 (8/14, 8/9, 14/17, p>0.2, Fisher’s exact test). 6 mice from SSK, 1 from KSK and 3 from KMK had Grade II-III seizures while 2, 1 and 4 animals died, respectively.