Targeting Seizure-Induced Neurogenesis in a Clinically Relevant Time Period Leads to Transient But Not Persistent Seizure Reduction

Abstract

Mesial temporal lobe epilepsy (mTLE), the most common form of medically refractory epilepsy in adults, is usually associated with hippocampal pathophysiology. Using rodent models of mTLE, many studies including work from our laboratory have shown that new neurons born around the onset of severe acute seizures known as status epilepticus (SE) are crucial for the process of epileptogenesis and targeting seizure-induced neurogenesis either genetically or pharmacologically can impact the frequency of chronic seizures. However, these studies are limited in their clinical relevance as none of them determines the potential of blocking new neurons generated after the epileptogenic insult to alleviate the development of chronic seizures. Therefore, using a pilocarpine-induced SE model of mTLE in mice of either sex, we show that >4 weeks of continuous and concurrent ablation of seizure-induced neurogenesis after SE can reduce the formation of spontaneous recurrent seizures by 65%. We also found that blocking post-SE neurogenesis does not lead to long-term seizure reduction as the effect was observed only transiently for 10 d with >4 weeks of continuous and concurrent ablation of seizure-induced neurogenesis. Thus, these findings provide evidence that seizure-induced neurogenesis when adequately reduced in a clinically relevant time period has the potential to transiently suppress recurrent seizures, but additional mechanisms need to be targeted to permanently prevent epilepsy development.SIGNIFICANCE STATEMENT Consistent with morphological and electrophysiological studies suggesting aberrant adult-generated neurons contribute to epilepsy development, ablation of seizure-induced new neurons at the time of the initial insult reduces the frequency of recurrent seizures. In this study, we show that continuous targeting of post-insult new neurons in a therapeutically relevant time period reduces chronic seizures; however, this effect does not persist suggesting possible additional mechanisms.

Keywords: adult neurogenesis; epilepsy; hippocampus; mTLE; neural stem cell; newborn neurons.

Figure 1.

Four weeks of blocking post-seizure neurogenesis does not suppress chronic seizures. A, Experimental design. B, Graph showing frequency of SRS between Pilo/Veh and Pilo/GCV groups. Student's t test, p = 0.5772. C, Graph showing SRS duration between Pilo/Veh and Pilo/GCV groups. Student's t test, p = 0.0808. D, Graph showing time course analysis of seizure frequency of Pilo/Veh and Pilo/GCV groups over the period of 14 d. E, Representative microscopic images showing DCX immunoreactivity between Pilo/Veh and Pilo/GCV groups. Inset, Typical DCX+ cell. F, Graph showing the number of DCX-expressing cells in the SGZ between the Pilo/Veh and Pilo/GCV groups. Student's t test, p < 0.0001. G, Representative microscopic images showing Prox1 immunoreactivity of the mature granule neuron between Pilo/Veh and Pilo/GCV groups. Inset, Typical Prox1+ EGCs. H, Graph showing Prox1+ EGCs in the Pilo/Veh and Pilo/GCV groups. Student's t test, p < 0.0001. Scale bar, 100 μm; inset, 25 μm. N = 6–7 animals per group. ns=not significant. pilo, Pilocarpine. *p < 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 2.

Greater than 4 weeks of blocking post-seizure neurogenesis suppresses chronic seizures. A, Experimental design. B, Graph showing frequency of SRS between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0147. C, Graph showing SRS duration between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.4351. D, Graph showing time course analysis of seizure frequency of Pilo/Veh and Pilo/GCV groups over the period of 14 d. E, Representative microscopic images showing DCX immunoreactivity between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Inset, Typical DCX+ cell. F, Graph showing the number of DCX-expressing cells in the SGZ between the Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0055. G, Representative microscopic images showing Prox1 immunoreactivity of the mature granule neuron between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Inset, Typical Prox1+ EGC. H, Graph showing Prox1+ EGCs in the Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0025. Scale bar, 100 μm; inset, 25 μm. N = 8–9 animals per group. ns = not significant. *p < 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

Figure 3.

Reduced seizures from 8 weeks of post-seizure ablation does not persist. A, Experimental design. B, Graph showing frequency of SRS between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.7819. C, Graph showing SRS duration between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.5698. D, Graph showing time course analysis of seizure frequency of Pilo/Veh and Pilo/GCV groups over the period of 14 d. E, Representative microscopic images showing DCX immunoreactivity between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Inset, Typical DCX+ cell. F, Graph showing the number of DCX-expressing cells in the SGZ between the Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0657. G, Representative microscopic images showing Prox1 immunoreactivity of the mature granule neuron. Inset, Typical Prox1+ EGC. H, Graph showing Prox1+ EGCs in the Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0004. Scale bar, 100 μm; inset, 25 μm. N = 5–7 animals per group. ns = not significant. *p < 0.05, ***p ≤ 0.001.

Figure 4.

Effect of ablation of seizure-induced neurogenesis on survival of new neurons. A, Experimental design for 4 weeks of ablation of post-seizure neurogenesis. B, Graph showing the number of BrdU/Prox1+ cells between Pilo/Veh and Pilo/GCV groups. Student's t test, p = ns. C, Experimental design for 8 weeks of ablation of post-seizure neurogenesis. D, Graph showing the number of BrdU/Prox1+ cells between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0066. E, Experimental design for 8 weeks of ablation of post-seizure neurogenesis and recording at 20-WPSE. F, Graph showing the number of BrdU/Prox1+ cells between Pilo/Veh/Veh and Pilo/GCV/GCV groups. Student's t test, p = 0.0025. G, Representative microscopic images showing BrdU/Prox1 immunoreactivity of the ectopic granule neurons. Arrowheads show typical BrdU/Prox1+ EGCs. H, Magnified images of a typical BrdU/Prox1+ EGC. Scale bar, 100 μm; inset, 25 μm. ns = not significant. **p ≤ 0.01.

Figure 5.

A model summarizing the observations in the study. A, A pictorial representation of seizure-induced neurogenesis in an epileptic dentate gyrus. B, Four weeks of ablation of post-seizure neurogenesis decreases neurogenesis but there is no change in seizure frequency. C, Greater than 4 weeks of ablation further reduces seizure-induced neurogenesis and consequently reduces SRS frequency. D, The reduction in SRS with 8 weeks of ablation of post-seizure neurogenesis does not persist until 20-WPSE possibly because of resumed neurogenesis, extrahippocampal factors and/or the role of other cell types in the epileptic brain.