Our principal findings were that IGF-1 is neuroprotective immediately after injury, but prolonged treatment with IGF-1 leads to an increase in electrographic seizures. Short-term IGF-1 treatment had no effect on ictal events. Prolonged IGF-1 treatment led to increased phosphorylation of Akt and S6, and increased protein synthesis, while inhibition of Akt or mTOR prevented an increase in ictal activity caused by IGF-1.

In organotypic hippocampal cultures, the initial period after injury, DIV 0–3, is characterized by a high amount of cell death that decreases by DIV 4–736,37, mirroring the time course in animal models of traumatic brain injury38,39,40. Our finding that IGF-1 is neuroprotective during this early period is in agreement with results obtained from short-term IGF-1 treatments in animal models of brain injury10,11,41. Interestingly, increased phosphorylation of IGF-1R/InR, Akt, and S6 in −IGF-1 organotypic cultures after first 24 hours in vitro (Fig. 5a,b) closely mirrors increased phosphorylation of these proteins in animal models of traumatic brain injury10,19. This finding suggests the presence of injury-activated autocrine or paracrine growth factor signaling in the hippocampal slice cultures up to 72 hours after slicing.

The initial period is followed by a latent period when there is little cell death, and neural activity includes single and multiple units and population spikes. The population spikes become more frequent by DIV 6, when spontaneous ictal events begin to occur36 (Fig. 1). To examine the role of IGF-1 in epileptogenesis, we used three experimental groups (Fig. 3a). We included +/−IGF-1 group to control for differences in the number of surviving neurons in +IGF-1 and −IGF-1 groups by DIV 3. We found that cultures in all 3 groups developed epilepsy, demonstrating that externally applied IGF-1 is not necessary for development of spontaneous population spikes or ictal events. The fraction of –IGF-1 cultures with ictal events was smaller than corresponding fraction of +IGF-1 cultures, but −IGF-1 cultures also had fewer surviving neurons (Figs 2 and 3f,g), and this may have affected epileptogenesis. However, we discovered that prolonged treatment with IGF-1 increased ictal activity not only when compared to −IGF-1 cultures, but also when compared to +/−IGF-1 cultures that entered this period with same number of neurons as +IGF-1 cultures (Fig. 3f,g). It was recently reported that acute application of IGF-1 enhances chemically induced seizures42, in contrast to our finding that acute application of IGF-1 has no effect (Fig. 4). This difference is likely due to different mechanisms of IGF-1 effect on seizures induced by chemoconvulsants versus spontaneously occurring seizures in our model.

Ictal activity in organotypic cultures leads to a second wave of cell death that peaks around DIV 14–17, and that is significantly attenuated by addition of glutamate antagonist kynurenic acid or anticonvulsant phenytoin to culture medium36,37. Interestingly, phosphorylation of MAPK in −IGF-1 cultures increased during this period, suggesting that MAPK signaling may be activated by ictal cell death (Fig. 5a,b). We found that IGF-1 increased cell death during this time in vitro, in direct contrast to neuroprotective role of early IGF-1 treatment. This cell death was likely caused by increased ictal activity rather than directly by IGF-1, since addition of anticonvulsant phenytoin to IGF-1 treated cultures eliminated the increase in toxicity (Fig. 6d).

We examined the potential downstream effectors of IGF-1 signaling in this model by looking at phosphorylation of Akt, MAPK, and S6 at the conclusion of the latent period (DIV 6). At this time, epileptogenic processes are actively ongoing43, but ictal activity and concomitant cell death have not yet occurred. Thus, we can separate IGF-1 effects from potential changes in intracellular signaling that may be introduced by epileptiform activity or cell death at later time points. We found that IGF-1 treatment during the latent period increases phosphorylation of Akt and S6, but not MAPK. It was previously found that promotion of neuronal survival by IGF-1 is mediated by Akt44. In contrast, our results suggest that IGF-1 - Akt signaling can lead to neuronal death through increased ictal activity. and that effects of IGF-1 signaling are context-dependent as has been previously found in other models13,45.

Akt is an upstream activator of mTOR, while S6, a ribosomal protein that is involved in translation, is activated by mTOR through S6 kinase20,46,47. We previously found that mTOR inhibition in organotypic hippocampal cultures is antiepileptic31 and we also found that mTOR inhibition is sufficient to prevent an increase in ictal activity and subsequent neuron death caused by IGF-1 in this work. It should be noted that some S6 phosphorylation was present without any externally applied IGF-1 (Fig. 5c), suggesting that other pathways may also play a role in activation of mTOR signaling in this model. However, increased activation of Akt and S6 due to IGF-1 treatment, and increased protein synthesis (hallmark of mTOR activation48), suggest that the mTOR pathway may mediate pro-epileptic effects of IGF-1.

Prolonged IGF-1 application resulted in increased amount of protein by DIV 6, when both +IGF-1 and −IGF-1 (with no IGF-1 since DIV 3) cultures have had similar levels of cell death (Fig. 3b), suggesting that the increased protein in tissue lysates on DIV 6 is due to increased protein synthesis, and not to improved neuronal survival. It has been previously suggested that IGF-1 receptor is involved in regulation of protein synthesis and neural metabolism3. IGF-1 plays a major role in central nervous system development, from proliferation, differentiation, brain size49 and establishment of neuronal polarity50 to postnatal neurogenesis and synaptogenesis51,52, and axon outgrowth53,54. It has been proposed that the high IGF-1 mRNA expression in neurons that undergo dendritic and synaptic remodeling plays a role in trophic support of neurite outgrowth and synaptogenesis8. Axonal sprouting, dendritic plasticity, and neurogenesis have been identified as structural changes that play a role in epileptogenesis after brain insult55. Inhibition of mTOR in models of acquired epilepsy led to reduction in mossy fiber sprouting, as well as a decrease in seizures in some but not all preparations31,46,47,56,57,58,59. Addition of external IGF-1 to organotypic hippocampal culture, which is injured brain tissue that is undergoing axon sprouting, dendritogenesis and synaptogenesis31,60,61,62 may alter these processes through activation of anabolic mTOR signaling63, potentially exacerbating epileptogenesis. In addition, we must consider that neuroprotection may sometimes save neurons that would be “better off dead”. That is to say, it may be better not to rescue some populations of neurons, such as those that have been severely injured or that are receiving abnormal inputs as a consequence of erroneous synaptogenesis. This is only a speculation at this point, but this possibility emphasizes the need to understand in more detail what populations of neurons are salvaged by different neuroprotective strategies. For example, it may be that IGF-1 should only be administered during a specific time window after injury.

On the other hand, both recovery and epileptogenesis may be enhanced by nonspecific anabolic enhancement after injury. Axon remodeling, neurogenesis, and synaptogenesis have been identified as potential targets for pharmacological interventions to promote brain repair and regeneration after traumatic brain injury64. Long-term (4 to 21 days) addition of IGF-1 was found to be beneficial in alleviating neurobehavioral deficits after traumatic brain injury12,65 and ischemic injury66. To reconcile the beneficial and pro-epileptic effects of IGF-1, varying incidence rates and time-courses of epileptogenesis after different types of brain injury need to be considered. Stroke increases epilepsy risk 20-fold, while increase in risk due to traumatic brain injury ranges from less than 2-fold increase in cases of mild civilian head injury to over 500-fold for military penetrating head injury32. Animal models of brain injury also have variable incidence of epilepsy and latency to first occurrence of spontaneous seizures that span the range from 6 to 50% of animals that develop epilepsy after 4 weeks to 6 months67. On the other hand, epileptogenesis occurs in most of organotypic cultures after 1 to 2 weeks36. Therefore, in this model, pro-epileptic effects of long-term IGF-1 treatment are apparent after 2 weeks, while same effects in animal models may be detectable only after several months. Also, we found that while IGF-1 is not necessary for epileptogenesis, it increases severity of epileptic events in tissue that is already epileptic. It may be possible that prolonged IGF-1 treatment does not increase incidence of epilepsy, and animals that will not develop spontaneous seizures may still derive neurobehavioral benefit from increased IGF-1. If this is confirmed by future studies, clinical implication will be that long-term treatment with IGF-1 or its agonists may be beneficial in cases where brain injury carries relatively low risk of epilepsy, but harmful in cases where epilepsy risk is high.

In conclusion, this study provides evidence that IGF-1 may play a role in epileptogenesis. Effects of IGF-1 application were strongly context-dependent, with IGF-1 increasing neuronal survival immediately after injury, but contributing to epileptogenesis after prolonged application. Chronic exposure to IGF-1 led to an increase in ictal event-mediated cell death, in contrast to early IGF-1 neuroprotection. Pro-epileptogenic effects of IGF-1 were mediated by Akt-mTOR, but not MAPK signaling.