Introduction

Epilepsy is a severe, chronic neurological disorder that affects approximately 50 million individuals worldwide.1,2 There are several manifestations of epilepsy,3 of which focal epilepsy represents 60%. The pathogenesis of epilepsy is not yet completely elucidated. The imbalance of neural excitation and inhibition, which is the imbalance of glutamate (Glu) and γ-aminobutyric acid (GABA) neurotransmission, is one of the hypotheses for the pathogenesis of epilepsy.4 The role of genetic factors as endogenous factors in the pathogenesis of epilepsy is gaining increasing attention.5 Exploration of genes associated with epilepsy may be instrumental in elucidating the pathogenesis of epilepsy.

Seizures seriously damaged the physical and mental health of patients, diminishing their quality of life and placing tremendous stress on epilepsy patients’ families.3,6 Although a variety of anti-seizure medication (ASM) is available, many have similar efficacy and it is not possible to predict which drug is best for a given patient. Furthermore, only a few biomarkers can reliably forecast treatment response or the risk of epilepsy drug resistance.7 Effective prevention and treatment of epilepsy is therefore a current clinical priority, and it is essential to seek potential therapeutic markers, identify therapeutic targets for novel ASM, and develop novel drug candidates.

Neuregulin (NRG) is a family of growth factors that transmits signals via ErbB family receptors. Neuregulin-1 (NRG-1) is located on chromosome 8p and is the most extensively researched member of its family.8 Notably, chromosome 8 has been one of the more problematic chromosomes in terms of rearrangements and translocations,9 which may be one cause for its association with certain diseases. NRG-1 is divided into six isoforms (I–VI), with types III and II being the most prevalent in humans, followed by type I. In the brain, types I, II, and III are mainly expressed by neurons, and astrocytes also express type I, but not type IV, V, or VI.10 Type IV is more abundantly expressed in the fetal brain and may play an important role during early development.11 This gene produced numerous isoforms closely associated with synaptic plasticity, neuronal migration, regeneration, and repair.12–14 The receptor for NRG-1 is the ErbB tyrosine kinase receptor. There are four types of ErbB receptors: ErbB1, ErbB2, ErbB3, and ErbB4. NRG-1-mediated signaling is mainly via ErbB2, ErbB3, and ErbB4. NRG-1 has been found to activate signaling pathways by binding to ErbB2/ErbB3, ErbB2/ErbB4 heterodimers, and ErbB4 /ErbB4 homodimers.15,16

NRG-1 and ErbB4 have been identified as risk alleles for psychiatric disorders such as depression and schizophrenia, with type I and type IV NRG-1 possibly being especially associated with schizophrenia.17,18 Recent research demonstrates that NRG-1 rs35753505 is closely associated with temporal lobe epilepsy.19 Additionally, studies suggested that NRG-1/ErbB4 signaling was likely an endogenous inhibitor of epileptogenesis.20 Current ASM exerts its effects primarily through Glutamatergic or GABAergic neurotransmitters.21 In addition to inhibiting the glutamate receptor N-methyl-D-aspartic acid, NRG-1/ErbB4 signaling also increases GABA release, which inhibits neuronal excitability.22–24 To determine whether the NRG-1/ErbB4 signaling could be a viable therapeutic target for ASM, however, additional research is required.

Considering the potential role of NRG-1 in the pathogenesis of epilepsy and its therapeutic implications, we conducted a study to analyze the levels of NRG-1 mRNA and NRG-1 protein in the peripheral blood of epilepsy patients and healthy controls.

Materials and Methods Participants

This research included 39 people with focal first-episode epilepsy. Between December 2021 and May 2022, all patients were seen at the First Affiliated Hospital of Kunming Medical University. A neurologist or pediatrician diagnosed a patient with focal epilepsy based on the patient’s medical history, MRI scan results, and EEG, in accordance with the 2017 International League Against Epilepsy classification criteria. Two eligibility requirements were a confirmed diagnosis of focal epilepsy and a lack of prior ASM use. Exclusion criteria included patients with any form of epilepsy other than focal epilepsy, other neurological disorders, psychiatric diseases, a history of positive hepatitis B, hepatitis C, HIV antibody/antigen testing, and MRI lesions. In addition, we recruited 39 healthy controls from the health administration center of the First Affiliated Hospital of Kunming Medical University who did not have epilepsy or other neurological disorders, psychiatric diseases, or an infectious disease history. Our study complied with the Declaration of Helsinki. The Ethics Committee of the First Affiliated Hospital of Kunming Medical University granted approval for this study. All participants or their legal guardians signed a form indicating their informed consent. Patients with epilepsy were sampled twice: once before the ASM therapy and again on day 28, which marked the conclusion of the fourth week of treatment. Samples from healthy controls were collected only once. All samples were stored in the biobank of the First Affiliated Hospital of Kunming Medical University.

Measurement of NRG-1 mRNA Levels

Blood samples were collected in heparin-coated collection tubes and centrifuged at 1000g for 15 minutes. Blood cell samples were taken in a separate centrifuge tube and peripheral blood mononuclear cells (PBMCs) were selected by Lymphocyte Separation Medium (Solarbio, Beijing, China). PBMCs samples were stored at −80°C for backup. Total RNA was extracted from PBMCs using the Total RNA Extraction Kit (Solarbio, Beijing, China), and the RNA concentration and purity were measured by ultra-micro UV-Vis spectrophotometer (BioTeke, Beijing, China), followed by cDNA synthesis using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using TB Green® Premix Ex Taq™ II Kit (TaKaRa, Tokyo, Japan) based on the manufacturer’s recommended protocol. Gene expression was carried out on a LiKang CG-05. The sequences of the primers are shown in Table 1. GAPDH was used as an internal control to standardize gene expression. The relative expression of each gene was calculated using the 2^−ΔΔCt method.

Table 1 Sequences of the Primers Used for RT-qPCR

Measurement of NRG-1 Protein Levels

Blood samples are collected in additive-free collection tubes and centrifuged at 1000g for 15 minutes. Serum samples were stored at −80°C for backup. Using the Human NRG-1 ELISA Kit (MeiMian, Jiangsu, China) according to the manufacturer’s instructions, serum NRG-1 protein levels were measured. In brief, three microplate wells were filled with standards or samples. The detection range of the ELISA assay is 0.3 pg/mL to 12 pg/mL. At 450 nm, absorbance was measured using a microplate reader (PerkinElmer VICTOR Nivo). Based on the concentration and absorbance of the standards, the linear regression equation of the standard curve was calculated, and the absorbance of the samples was used to determine the concentration of NRG-1 in the serum samples.

Drug Efficacy Assessment

A total of 39 patients with first-episode focal epilepsy were treated with ASM and observed on day 28, the conclusion of the fourth week of medication administration. The duration of drug administration, the amount administered, the number of seizures, and the EEG results were recorded. The treatment’s efficacy was classified into four groups: control, significantly effective, effective, and ineffective. On the basis of the patient’s frequency of seizures every 28 days, the overall efficacy of the treatment was determined, with control denoting a 100% reduction in seizures, significantly effective denoting a 75% or greater reduction in seizures, effective denoting a 50% or greater reduction in seizures, and ineffective denoting a reduction of less than 50%. The relationship between efficacy, NRG-1 mRNA expression, and NRG-1 protein expression was analyzed.

Statistical Analysis

Statistical analysis was performed using IBM SPSS statistical software (IBM, version 26.0, Armonk, NY, USA). Each measurement was expressed as the mean ± standard deviation. Chi-square tests were used to analyze gender differences, while Student’s t-tests were used to compare age differences. One-way ANOVA was used to compare NRG-1 levels in patients before and after medication with healthy controls, and LSD post hoc tests were used to analyze experiments with two variables. We also examined the potential correlation between NRG-1 levels and efficacy in patients using Spearman correlation coefficient. A p-value of less than 0.05 was deemed statistically significant.

Results Levels of NRG-1 mRNA and Protein

Epilepsy patients and healthy controls were matched for age (P = 0.896) and sex (P = 0.820) (Table 2).

Table 2 Demographic Characteristics of All Participants

Among the four NRG-1 mRNA isoforms, type IV NRG-1 was not detected, therefore, this isoform will be excluded from further analysis. One-way ANOVA analysis revealed that type I NRG-1 (F = 26.045, P < 0.001), type II NRG-1 (F = 15.307, P < 0.001), and type III NRG-1 (F = 13.570, P < 0.001) mRNA levels were higher in epilepsy patients administered ASM than before medication, but still lower than in healthy controls. (Figure 1).

Figure 1 Levels of NRG-1 (type I, II, III) mRNA in first-episode focal epilepsy patients and healthy controls. Before treatment with ASM, type I, II, III NRG-1 mRNA levels (A–C) were lower in patients with first-episode focal epilepsy than in healthy controls. Type I, II, III NRG-1 mRNA levels (A–C) increased to some extent after drug administration, but remained lower than in healthy controls. (*: P < 0.05;**: P < 0.01;***: P < 0.001).

In contrast to the reduced levels of NRG-1 mRNA in first-episode epilepsy patients, we found that NRG-1 protein levels were higher in patients with epilepsy before and after treatment with ASM than in healthy controls (F = 17.728, P < 0.001), and there was no statistical difference in NRG-1 protein levels after four weeks of medication compared to before (P = 0.681) (Figure 2).

Figure 2 Levels of NRG-1 protein in first-episode focal epilepsy patients and healthy controls. Patients with first-episode focal epilepsy had higher NRG-1 protein levels before and after treatment with ASM than healthy controls, and there was no statistical difference between NRG-1 protein levels before and after dosing. (***: P < 0.001).

Relationship Between NRG-1 and Efficacy of ASM

After four weeks of continuous treatment with ASM in 39 patients with first-episode focal epilepsy (The medications used are listed in Table 3), with NRG-1 mRNA levels increased, seizures were effectively controlled and relieved, with an overall efficacy rate of 97.4% (Table 4). In addition, all patients showed varying degrees of EEG improvement (Figure 3). Spearman correlation analysis revealed that type I (rs = −0.46, P = 0.003), type II (rs = −0.563, P < 0.001), and type III (rs = −0.36, P = 0.034) NRG-1 mRNA were negatively correlated with the efficacy of ASM: as NRG-1 mRNA levels increased, the frequency of seizures decreased (Table 5). We found no correlation between NRG-1 protein and the effectiveness of ASM (rs = −0.234, P = 0.157).

Table 3 Medication Use in 39 Patients with Focal Epilepsy

Table 4 Efficacy Evaluation After Four Weeks of Medication in 39 Patients with Focal Epilepsy

Table 5 Relationship Between NRG-1 mRNA and Efficacy

Figure 3 EEG results of epilepsy patients before and following ASM treatment. (A and B) Before treatment with ASM, the patient’s VEEG revealed frequently synchronized paroxysm of short-range spike and wave complex, polyspike wave complex in both frontal regions, apparent in the right frontal area, which could spread to the adjacent leads. (C and D) After treatment with ASM, the patient’s AEEG revealed spike and wave complex in the right middle and posterior temporal areas during sleep.

Discussion

In this study, we evaluated the levels of NRG-1 in the peripheral blood of patients with first-episode focal epilepsy before and after ASM treatment. We discovered that NRG-1 levels differed between epilepsy patients and healthy controls and that ASM increased NRG-1 mRNA levels. These findings indicate that NRG-1 may play a role in the onset and progression of focal epilepsy.

Of the four NRG-1 mRNA isoforms we measured (I, II, III, and IV), type IV NRG-1 mRNA was not detected between epilepsy patients and healthy controls, consistent with a study conducted in schizophrenic patients that failed to detect this isoform in PBMCs.25 In the human brain, all six types of NRG-1 are detectable, but the abundance of each form varies greatly,26 with NRG-1-III being the most abundantly expressed,10 and unlike the other isoforms, NRG-1-IV is only expressed in the brain,11 which may be one of the reasons why we did not detect this isoform in peripheral blood. In addition, we attempted to detect ErbB4 mRNA expression, but it was not detected in almost all samples. This may be because ErbB4 is specifically expressed in GABAergic neurons in cortical and subcortical regions, but not in PBMCs.27,28 Furthermore, these probes and primers have been used successfully in other studies involving human brain tissue, but to our knowledge, they are rarely used in blood research. Future studies using blood samples should consider alternative probes and primers for type IV NRG-1 and ErbB4. Since NRG-1 activates multiple ErbB receptors, whether NRG-1 can cause changes in the expression of other ErbB receptors needs further investigation.

We first found that the levels of NRG-1 mRNA were lower in PBMCs in first-episode focal epilepsy patients than in healthy controls. After 4 weeks of drug administration, the levels of NRG-1 (type I, II, III) mRNA increased progressively and were distinctly different compared to before drug administration, but they remained lower than in healthy controls. Several studies have demonstrated a gradual increase in NRG-1 mRNA expression in epilepsy model mice treated with a ketogenic diet.29 Moreover, our findings are comparable to those of a study that found a progressive increase in NRG-1 mRNA expression in patients with first-episode schizophrenia treated with antipsychotic medication.30 NRG-1 mRNA expression was higher in schizophrenic patients than in healthy controls, according to a separate study.25 The schizophrenic patients included in this study were already on medication, and we hypothesize that the long-term effects of the medication may cause an increase in NRG-1 mRNA expression. Some evidence suggests that NRG-1 signaling needs to be within a certain range to function, and that exceeding this range may lead to disease-related consequences.31 NRG-1 signaling may function as a homeostatic regulator by maintaining NRG-1 mRNA levels within an optimal range, thereby protecting the brain from seizure discharges.32

In contrast to the decreased levels of NRG-1 mRNA in patients with first-episode focal epilepsy, we observed higher NRG-1 protein levels in epilepsy patients both before and after drug administration than in healthy controls. Over the longer follow-up time, there were no discernible differences in NRG-1 protein levels. One study revealed that unmedicated patients with autism spectrum disorders had higher NRG-1 protein levels than healthy controls.33 Additionally, another study on schizophrenia showed that NRG-1 mRNA and protein exhibited opposite alterations.25 This study suggested that smoking may be one of the factors influencing NRG-1 protein levels. We hypothesize that other factors may influence NRG-1 protein levels in epilepsy patients. Symptoms such as anxiety are common in epilepsy patients, and it remains to be determined whether these factors influence NRG-1 expression. In addition, mRNA and protein levels may differ due to the complexity of NRG-1 splicing and the influence of multiple post-transcriptional regulatory processes, such as protein stability and degradation, on protein levels. Moreover, weak correlations between transcripts and proteins are common in brain tissue. Since current NRG-1 antibodies cannot discriminate between isoforms, it is very difficult to compare transcripts and proteins from distinct isoforms.34

The majority of studies did not address the function of ASM. Our findings imply that ASM positively regulates NRG-1 mRNA levels. With the use of ASM, NRG-1 mRNA levels gradually increased, and seizures were reduced and controlled. Although long-term follow-up was not performed, transcriptional abnormalities of NRG-1 may be involved in the onset and progression of epilepsy.

Because the forms of seizures are complicated, only focal seizure patients were included in our study, which is not a large enough sample size. To determine whether other types of epilepsy also cause changes in NRG-1 mRNA and protein levels, additional sample expansion and strict subtype typing are required for more in-depth studies. Current ASM primarily acts on Glutamatergic or GABAergic neurotransmitters that directly target neurons.30 Glutamatergic and GABAergic neurotransmission imbalances may also be relevant to the pathophysiology and treatment of epilepsy.35 By interacting with the Glutamatergic or GABAergic transmitter system, NRG-1 may be involved in the pathophysiology of diseases such as epilepsy.36 A discussion of the combined action of NRG-1 with Glu or GABA may be more relevant to investigate new ASM mechanisms and drug targets. Since our study did not limit the categories of ASM, further research is required to determine which medications have the most pronounced effect on NRG-1. In addition, this study is a single-center study, so further validation in other regions is required.

Conclusions

The current study is the first to comprehensively investigate NRG-1 mRNA and protein expression in the peripheral blood of epilepsy patients treated with ASM. Our findings imply that NRG-1 transcription is dysregulated in focal epilepsy and that ASM can modulate it to some extent. NRG-1 may serve as a therapeutic marker for focal epilepsy and provide ideas for identifying therapeutic targets for novel ASM.

Funding

This research is funded by the Yunnan Applied Basic Research Projects (2017FE468(−037)), and the Yunnan Clinical Medical Center for Neurological and Cardiovascular Diseases (ZX2019-03-05), and the Major Science and Technology Special Project of Yunnan Province (202102AA100061).

Disclosure

The authors declare that they have no known competing interests that could affect the work reported in this paper.

References

1. Hesdorffer DC, Logroscino G, Benn EKT, Katri N, Cascino G, Hauser WA. Estimating risk for developing epilepsy. Neurology. 2011;76:23–27. doi:10.1212/WNL.0b013e318204a36a

2. Fiest KM, Sauro KM, Wiebe S, et al. Prevalence and incidence of epilepsy: a systematic review and meta-analysis of international studies. Neurology. 2017;88:296–303. doi:10.1212/WNL.0000000000003509

3. Devinsky O, Vezzani A, O’Brien TJ, et al. Epilepsy. Nat Rev Dis Primers. 2018:4. doi:10.1038/nrdp.2018.24.

4. Chen Z, Rollo B, Antonic-Baker A, et al. New era of personalised epilepsy management. BMJ. 2020;371:1–5. doi:10.1136/bmj.m3658

5. Niestroj LM, Perez-Palma E, Howrigan DP, et al. Epilepsy subtype-specific copy number burden observed in a genome-wide study of 17458 subjects. Brain. 2020;143:2106–2118. doi:10.1093/brain/awaa171

6. Jennum P, Debes NMM, Ibsen R, Kjellberg J. Long-term employment, education, and healthcare costs of childhood and adolescent onset of epilepsy. Epilepsy Behav. 2021;114:107256. doi:10.1038/s41598-018-26492-4

7. Marchiò M, Roli L, Giordano C, et al. High plasma levels of ghrelin and des-acyl ghrelin in responders to antiepileptic drugs. Neurology. 2018;91:e62–e66. doi:10.1212/WNL.0000000000005741

8. Mata I, Perez-Iglesias R, Roiz-Santiañez R, et al. Additive effect of NRG1 and DISC1 genes on lateral ventricle enlargement in first episode schizophrenia. Neuroimage. 2010;53:1016–1022. doi:10.1007/s12035-014-8876-5

9. Giglio S, Broman KW, Matsumoto N, et al. Olfactory receptor-Gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements. Am J Hum Genet. 2001;68:874–883. doi:10.1093/brain/awaa171

10. Hu X, Fan Q, Hou H, Yan R. Neurological dysfunctions associated with altered BACE1-dependent Neuregulin-1 signaling. J Neurochem. 2016;136:234–249. doi:10.1111/jnc.13395

11. Tan W, Wang Y, Gold B, et al. Molecular cloning of a brain-specific, developmentally regulated neuregulin 1 (NRG1) isoform and identification of a functional promoter variant associated with schizophrenia. J Biol Chem. 2007;282:24343–24351. doi:10.1074/jbc.M702953200

12. Spahic H, Parmar P, Miller S, et al. Dysregulation of erbb4 signaling pathway in the dorsal hippocampus after neonatal hypoxia-ischemia and late deficits in pv+ interneurons, synaptic plasticity and working memory. Int J Mol Sci. 2023;24. doi:10.3390/ijms24010508

13. Lysko DE, Talbot WS. Unmyelinated sensory neurons use Neuregulin signals to promote myelination of interneurons in the CNS. Cell Rep. 2022;41. doi:10.1016/j.celrep.2022.111669

14. Fornasari BE, El Soury M, De Marchis S, Perroteau I, Geuna S, Gambarotta G. Neuregulin1 alpha activates migration of neuronal progenitors expressing ErbB4. Mol Cell Neurosci. 2016;77:87–94. doi:10.1016/j.mcn.2016.10.008

15. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127–137. doi:10.1038/35052073

16. Bublil EM, Yarden Y. The EGF receptor family: spearheading a merger of signaling and therapeutics. Curr Opin Cell Biol. 2007;19:124–134. doi:10.1016/j.ceb.2007.02.008

17. Le-Niculescu H, Roseberry K, Gill SS, et al. Precision medicine for mood disorders: objective assessment, risk prediction, pharmacogenomics, and repurposed drugs. Mol Psychiatry. 2021;26:2776–2804. doi:10.1038/s41380-021-01061-w

18. Harrison PJ, Law AJ. Neuregulin 1 and schizophrenia: genetics, gene expression, and neurobiology. Biol Psychiatry. 2006;60:132–140. doi:10.1016/j.biopsych.2005.11.002

19. Zhu WY, Jiang P, He X, et al. Contribution of NRG1 gene polymorphisms in temporal lobe epilepsy. J Child Neurol. 2016;31:271–276. doi:10.1177/0883073815589757

20. Tan GH, Liu YY, Hu XL, Yin DM, Mei L, Xiong ZQ. Neuregulin 1 represses limbic epileptogenesis through ErbB4 in parvalbumin-expressing interneurons. Nat Neurosci. 2012;15:258–266. doi:10.1038/nn.3005

21. Miziak B, Borowicz-Reutt K, Rola R, Blaszczyk B, Czuczwar M, Czuczwar SJ. The prophylactic use of antiepileptic drugs in patients scheduled for neurosurgery. Curr Pharm Des. 2018;23:6411–6427. doi:10.2174/1381612823666171027125408

22. Nakazawa K, Sapkota K. The origin of NMDA receptor hypofunction in schizophrenia. Pharmacol Ther. 2020;205:107426. doi:10.1016/j.pharmthera.2019.107426

23. Rahman-Enyart A, Lai C, Prieto AL. Neuregulins 1, 2, and 3 promote early neurite outgrowth in ErbB4-expressing cortical GABAergic interneurons. Mol Neurobiol. 2020;57:3568–3588. doi:10.1007/s12035-020-01966-7

24. Wen L, Lu YS, Zhu XH, et al. Neuregulin 1 regulates pyramidal neuron activity via ErbB4 in parvalbumin-positive interneurons. Proc Natl Acad Sci U S A. 2010;107:1211–1216. doi:10.1073/pnas.0910302107

25. Mostaid MS, Lee TT, Chana G, et al. Elevated peripheral expression of neuregulin-1 (NRG1) mRNA isoforms in clozapine-treated schizophrenia patients. Transl Psychiatry. 2017;7. doi:10.1038/s41398-017-0041-2

26. Liu X, Bates R, Yin DM, et al. Specific regulation of NRG1 isoform expression by neuronal activity. J Neurosci. 2011;31:8491–8501. doi:10.1523/JNEUROSCI.5317-10.2011

27. Fazzari P, Paternain AV, Valiente M, et al. Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature. 2010;464:1376–1380. doi:10.1038/nature08928

28. Bean JC, Lin TW, Sathyamurthy A, et al. Genetic labeling reveals novel cellular targets of schizophrenia susceptibility gene: distribution of gaba and non-gaba erbb4-positive cells in adult mouse brain. J Neurosci. 2014;34:13549–13566. doi:10.1523/JNEUROSCI.2021-14.2014

29. Wang J, Huang J, Yao S, et al. The ketogenic diet increases Neuregulin 1 expression via elevating histone acetylation and its anti-seizure effect requires ErbB4 kinase activity. Cell Biosci. 2021;11:1–14. doi:10.1186/s13578-021-00611-7

30. Zhang HX, Zhao JP, Lv LX, et al. Explorative study on the expression of neuregulin-1 gene in peripheral blood of schizophrenia. Neurosci Lett. 2008;438:1–5. doi:10.1016/j.neulet.2007.09.051

31. Dominguez SL, Hegde GV, Hanson JE, et al. Antibody-mediated stabilization of NRG1 induces behavioral and electrophysiological alterations in adult mice. Sci Rep. 2018;8:1–13. doi:10.1038/s41598-018-26492-4

32. Zhu JM, Li KX, Cao SX, et al. Increased NRG1-ErbB4 signaling in human symptomatic epilepsy. Sci Rep. 2017;7:1–7. doi:10.1038/s41598-016-0028-x

33. Esnafoglu E. Levels of peripheral Neuregulin 1 are increased in non-medicated autism spectrum disorder patients. J Clin Neurosci. 2018;57:43–45. doi:10.1016/j.jocn.2018.08.043

34. Paterson C, Cumming B, Law AJ. Temporal dynamics of the neuregulin-ErbB network in the murine prefrontal cortex across the lifespan. Cereb Cortex. 2020;30:3325–3339. doi:10.1093/cercor/bhz312

35. Amini E, Rezaei M, Mohamed Ibrahim N, et al. A molecular approach to epilepsy management: from current therapeutic methods to preconditioning efforts. Mol Neurobiol. 2015;52:492–513. doi:10.1007/s12035-014-8876-5

36. Li KX, Lu YM, Xu ZH, et al. Neuregulin 1 regulates excitability of fast-spiking neurons through Kv1.1 and acts in epilepsy. Nat Neurosci. 2012;15:267–273. doi:10.1111/jnc.13395