Anterior Thalamic Nuclei Deep Brain Stimulation Inhibits Mossy Fiber Sprouting via 30,50-Cyclic Adenosine Monophosphate/Protein Kinase A Signaling Pathway in a Chronic Epileptic Monkey Model
Epilepsy is a prevalent neurological disorder affecting 0.5% to 1% of the global population, with nearly 30% of patients being refractory to existing medications. Not all patients with drug-resistant epilepsy are suitable candidates for resective surgery, especially those with multiple seizure foci, seizure foci that are hard to locate, or seizure foci in brain regions where damage could lead to severe functional deficits. Deep brain stimulation (DBS) has emerged as a novel technique for neuromodulation, particularly for epilepsy patients unsuitable for resective surgery. The anterior thalamic nuclei (ATN) are considered one of the best targets for controlling seizures due to their crucial role in seizure spread and the Papez circuit. A double-blind, randomized, multicenter study found that ATN-DBS produced a median percentage seizure reduction from baseline of 41% in the first year and 69% in the fifth year, with better seizure control achieved in patients with temporal lobe epilepsy (TLE).
Mossy fiber sprouting (MFS) is a common pathological hallmark of TLE, mediating reverberating excitation, reducing the threshold for granule cell synchronization, and playing a crucial role in the epileptic brain. MFS is regulated by multiple signaling pathways, among which 30,50-cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) appears to play a particularly important role. Ectopic granule cells are also closely associated with MFS in epilepsy. Although epileptogenesis in animal models is a continuous process, it can be divided into three stages: acute, latent, and chronic. Clinically, almost all patients undergo ATN-DBS several years after the onset of epilepsy, which corresponds to the chronic stage in animal models. The effect of ATN-DBS on ectopic granule cells and MFS in the chronic stage of epilepsy has not yet been investigated.
In this study, a kainic acid (KA)-induced epileptic model was used to investigate the effect of ATN-DBS on MFS and potential signaling pathways. Twenty-four male rhesus macaques were randomly divided into a control group, an epilepsy (EP) group, an EP-sham-DBS group, and an EP-DBS group. KA was injected into the left hippocampus and amygdalae to establish the chronic epileptic model. The left ATN was implanted with a DBS lead and stimulated for 8 weeks. Enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining were used to evaluate MFS and levels of potential molecular mediators in the hippocampus.
ATN-DBS significantly reduced seizure frequency in the chronic stage of epilepsy. The number of ectopic granule cells was reduced in monkeys that received ATN stimulation. Levels of cAMP and PKA in the hippocampus, together with Akt phosphorylation, were noticeably reduced in monkeys that received ATN stimulation. ATN-DBS also significantly reduced MFS scores in the hippocampal dentate gyrus and CA3 sub-regions.
Hilar ectopic granule cells are rare in normal adult animals but greatly increased in the TLE model, closely associated with epileptogenesis. NeuN is widely used as a marker of granule cells. In animals with KA-induced epilepsy, there were significantly more NeuN-positive cells in the dentate hilus than those in the dentate hilus of control animals. ATN stimulation significantly reduced the number of NeuN-positive cells in the dentate hilus of the epileptic monkeys, indicating that ATN-DBS reduces the number of hilar ectopic granule cells in the chronic stage in epileptic monkeys.
cAMP, which activates PKA, plays an indispensable role in the progression of MFS. A significant elevation of cAMP expression in the hippocampus was observed in the EP and EP-sham-DBS groups compared with the control group. This increase in cAMP expression was reversed in animals that received chronic ATN-DBS. A similar trend toward changing PKA expression was observed in these groups. The changes in cAMP and PKA expression suggest that ATN-DBS inhibits the cAMP/PKA signaling pathway in the chronic epileptic model.
Akt phosphorylation, mediated by the cAMP/PKA signaling pathway, influences hippocampal synaptic plasticity. Normal and low phosphorylated Akt/Akt was observed in the control group monkeys, but the ratio was increased by KA injection in the epileptic model. A significant decrease in the ratio was observed in monkeys receiving ATN stimulation compared with the EP and EP-sham-DBS groups.
Calbindin-D28k is a Ca2+-binding protein that shows a characteristic spatial pattern of expression in the hippocampus and is a marker of the mossy fibers. In the control group, no obvious calbindin-D28k granules were seen in the supragranular region of the dentate gyrus or in the pyramidal cell layer and stratum oriens of the CA3 region. In the EP and EP-sham-DBS groups, prominent MFS was observed in the inner molecular layer of the dentate gyrus. In the CA3 region, calbindin-D28k staining was noted primarily in the pyramidal cell layer, with obvious MFS. Consistently, MFS scores in the dentate gyrus and CA3 regions in the EP and EP-sham-DBS groups were significantly higher than those in the controls. In the EP-DBS group, less MFS was observed in the dentate gyrus and CA3, with a significant decrease in MFS scores compared with the EP and EP-sham-DBS groups. These results indicate that ATN-DBS inhibits MFS in the dentate gyrus and CA3 regions in the chronic epileptic monkey model.
In conclusion, ATN-DBS was shown to down-regulate the cAMP/PKA signaling pathway and Akt phosphorylation and to reduce the number of ectopic granule cells, which may contribute to the inhibition of MFS of chronic TLE. This study provides further insights into the mechanism by which ATN-DBS reduces epileptic seizures.
doi.org/10.1097/CM9.0000000000001302
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