Insulin-like growth factor-1 promotes synaptogenesis signaling, a major dysregulated pathway in malformation of cortical development, in a rat model

Insulin-like growth factor-1 promotes synaptogenesis signaling, a major dysregulated pathway in malformation of cortical development, in a rat model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insulin-like growth factor-1 promotes synaptogenesis signaling, a major dysregulated pathway in malformation of cortical development, in a rat model Minyoung Lee, Eun-Jin Kim, Min-Jee Kim, Mi-Sun Yum, Jeonghun Yeom, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1598872/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2023 Read the published version in Molecular Neurobiology → Version 1 posted 5 You are reading this latest preprint version Abstract Malformation of cortical development (MCD) is one of the main causes of intractable epilepsy in childhood. We explored a treatment based on molecular changes using an infant rat model of methylazoxymethanol (MAM)-induced MCD established by injecting MAM at gestational day 15. The offspring were sacrificed on postnatal day (P) 15 for proteomic analysis, which revealed significant downregulation in the synaptogenesis signaling pathway in the cortex of MCD rats. Recombinant human insulin-growth factor-1 (rhIGF-1) was injected from P12 to P14 twice daily and the effect of IGF1 on N-methyl-D-aspartate (NMDA)-induced spasms (15 mg/kg of NMDA, i.p.) was tested; the onset of P15 single spasm was significantly delayed (p = 0.002) and the number of spasms decreased (p < 0.001) in rhIGF1-pretreated rats (n = 17) as compared to those in VEH-treated rats (n = 18). Electroencephalographic monitoring during spasms showed significantly reduced spectral entropy and event-related spectral dynamics of fast oscillation in rhIGF-1 treated rats. Magnetic resonance spectroscopy of the retrosplenial cortex showed decreased glutathione (GSH) (p = 0.039) and significant developmental changes in GSH, phosphocreatine (PCr), and total creatine (tCr) (p = 0.023, 0.042, 0.015, respectively) after rhIGF1 pretreatment. rhIGF1 pretreatment significantly upregulated expression of cortical synaptic proteins such as PSD95, AMPAR1, AMPAR4, NMDAR1, and NMDAR2A (p < 0.05). Thus, early rhIGF-1 treatment could promote synaptic protein expression, which was significantly downregulated by prenatal MAM exposure, and effectively suppress NMDA-induced spasms. Early IGF1 treatment should be further investigated as a therapeutic strategy in infants with MCD-related epilepsy. methylazoxymethanol acetate (MAM) animal model synaptic protein recombinant human insulin-like growth factor-1 (rhIGF-1) malformation of cortical development (MCD) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The development of the cerebral cortex is accomplished through well-orchestrated neurogenesis, neuronal migration, cell proliferation, and organization involving various proteins and transcription factors[1,2]. Any dysregulation results in a broad-spectrum disorder, termed as malformation of cortical development (MCD). Many genetic or environmental fcators[3,4] are associated with the pathogenesis of MCD. Patients with MCD suffer from developmental problems, neurological deficits, and epilepsy[1,5,6]. In particular, MCD is the most common cause of intractable epilepsy in pediatric populations[5,7,8,2]. The earlier the epilepsy begins, the more frequent and severe cognitive impairment occurs[2]. Despite many clinical and translational researches[9,10], epileptogenesis of the malformed brain is still unclear. The afterbirth diagnosis of fetal brain development makes these structural alterations in brain development permanent, ultimately leaving physicians with the choice of only symptomatic treatment. The offspring from methylazoxymethanol (MAM)-treated rats have developmental brain anomalies, including migration failure, ventricular enlargement, and disorganization of neocortical and hippocampal structures[9,11], which resemble the pathologic findings observed in patients with MCD[11,5]. Our previous studies have reported cognitive impairment and increased seizure susceptibility with increased fast oscillation (FO) as well as decreased dendritic arborization or cortical neurons during infancy in this MAM-induced MCD rat model[5] [12,13]. Using this MAM-induced MCD model[4,14], we aimed to identify the most severely deteriorated canonical pathway in the early postnatal period. Insulin-like growth factor-1 (IGF-1), produced by all central nervous system (CNS) cell types, plays important roles in brain development and neuroplasticity. IGF-1 is involved in cell organization, neural circuitry formation, and maturation of synaptic efficacy in the early brain, CNS development, and neuronal cell growth and proliferation[15–17]. In the developing brain, IGF-1 modulates the axonal development and synapse formation through the phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) pathway[15,16]. Several studies[18–21] have reported that the application of IGF-1 could improve outcomes after brain injury; however, the role of IGF-1 in epilepsy or early brain development is still controversial[17]. In this study, we investigated whether recombinant human IGF-1 (rhIGF-1) pretreatment at early postnatal period inhibits N-methyl-D-aspartate (NMDA)-induced spasms in a rat MCD model[4,14] and if IGF-1 pretreatment reverses the key pathway involved in pathologic MCD brain. 2. Materials And Methods 2.1 Animal experiments Animal experiments were approved by the Institutional Animal Care and Use Committee and conformed to the Revised Guide for the Care and Use of Laboratory Animals (8th Edition, 2011). Timed-pregnant Sprague-Dawley rats were purchased (Orient Bio Inc., Seoul, Korea) at gestational day 14 (G14) and housed under a 12 h light/dark cycle with free access to food and water. On G15, two doses of MAM (15 mg/kg intraperitoneally, MRIGlobal, Missouri) or normal saline were injected into pregnant rats at 8:00 AM and 6:00 PM. Delivery occurred consistently on gestational day 21 for all the rats, which was considered postnatal day (P) 0 for the offspring. 2.2 Proteomics analysis Prenatally MAM-exposed rats (n = 4) and control rats (n = 4) were sacrificed on P15 and their cortex were separated for proteomic analysis. Sample preparation and Nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis Brain tissues were carefully washed in phosphate-buffered saline (PBS) on ice to remove blood. The cortices of prenatally MAM-exposed rats and controls were individually cryopulverized using a Cryoprep device (CP02, Covaris) as previously described (PMID: 24678027). Peptide separation was performed using Dionex UltiMate 3000 RSLCnano system (Thermo-Fisher Scientific). Mass spectra were acquired in a data-dependent mode with an automatic switch between a full scan with 20 data-dependent MS/MS scans. Database searching and label-free quantitation (LFQ) and functional enrichment and gene ontology analysis The acquired MS/MS spectra were searched using the SequestHT on Proteome discoverer (version 2.2, Thermo Fisher Scientific) against the SwissProt database (July 2019). False discovery rates (FDRs) were set for 1% for each analysis. For the differential analysis of the relative abundance of proteins between samples, Perseus (version 1.6.13.0) was used. Proteins with a q -value of < 0.05 and log2 fold change ± 1 were considered as differentially regulated proteins. Gene ontology analysis was performed through ShinyGO v0.60 (PMID: 31882993, http://bioinformatics.sdstate.edu/go60/ ) and data were further analyzed using IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis ). 2.3 rhIGF-1 experiments Prenatally MAM-exposed rats were pretreated with rhIGF-1 (0.5 mg/kg for each dose, P12 to P14, twice a day at 8:00 AM and 6:00 PM) or vehicle (VEH, 0.1% bovine serum albumin [BSA]). The overall experimental schedule is described in Figure S1. 2.4 Western blot analysis For western blot analysis, bilateral cortical tissues from bregma to posterior hippocampal areas without hippocampus (anterior posterior 0 to -5 mm) were obtained from control rats (n = 12), MAM-exposed rats (n = 12), rhIGF-1–treated MAM-exposed rats (n = 11) and VEH-treated MAM-exposed rats (n = 9) at P15. The following primary antibodies were used: anti-AMPA receptor 1 (AMPAR1), AMPAR2, AMPAR3, and AMPAR4 (Cell signaling, Technology, Inc.); anti-calcium/calmodulin-dependent protein kinase II (CaMKII) (Cell signaling, Technology, Inc.); anti-glutamic acid decarboxylase 67 (GAD67) and GAD65 (Millipore, Technology, Inc.); anti-neuronal nuclei (NeuN; Millipore, Technology, Inc.); anti-N-methyl-D-aspartate receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B (Cell signaling, Technology, Inc.); anti-PSD95 (Cell signaling, Technology, Inc.); Anti-β-actin (Santa Cruz Biotechnology, Inc.). 2.5 In vivo magnetic resonance imaging (MRI) studies MAM-induced MCD rats were maintained under anesthesia with 1% isoflurane in a 1:2 mixture of O 2 :N 2 O and their respiratory rate, electrocardiogram, and rectal temperature were monitored. MRI was performed using a 7.0 T/160-mm bore animal MRI system (Bruker Pharmascan, Ettlingen, Germany). 1 H-MRS was performed at P8 and P15 in rhIGF-1–treated rats (n = 13) and VEH-treated rats (n = 12). The MR spectra were acquired through a signal voxel (from bregma to -4.0 mm in a coronal section, 1.5 × 1 × 4 mm 3 ; Fig. 3 A) in the retrosplenial cortex (RSC). All MR spectra were processed using the linear combination analysis method (LC Model ver. 6.0, Los Angeles, CA) to calculate the metabolite concentrations.[22] The following brain metabolites were included in the metabolite basis set: alanine (Ala), aspartate (Asp), creatine (Cr), ɣ-aminobutyric acid (GABA), glucose, glutamate, glutamine, glycerophosphorylcholine, phosphorylcholine, myo-inositol (mIns), lactate (Lac), phosphocreatine (PCr), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), taurine, macromolecules (MMs), and lipids. The in vivo proton spectra were considered to have an acceptable value if the standard deviation of the fit for the metabolite was less than 20%. 2.6 Cortical electroencephalography (EEG) recording and analysis For intracranial EEG recording, two cortical electrodes were surgically implanted in each of five rats treated with rhIGF-1 or VEH under ketamine/xylazine sedation (50/7 mg/kg in 10 mL/kg saline i.p.) at P12. At P15, spasms were triggered by a single dose of NMDA (15 mg/kg i.p.; Sigma), and EEGs of the two groups of rats were recorded with simultaneous videos using the Twin EEG system (Grass Technologies) for 90 min before NMDA injection (pre-ictal period) and 120 min after injection or until the end of spasms. The sampling rate was 400 Hz with a 0.1 Hz high‐pass filter, and 5 min of artifact‐free data were collected for each. Before analysis, the data were preprocessed using the EEGLAB toolbox of MATLAB 2015b. From each rats, 100 epochs (1 s duration) of pre‐ictal and spasms periods per rat were extracted from the EEG data. For quantitative estimation, averaged spectral entropy (SE) and event-related spectral dynamics (ERSP) of FO (25–200 Hz), power spectral density (PSD) from each epoch were calculated using the EEGLAB toolbox of MATLAB 2017b. 2.7 Behavioral assessment Behavioral assessments were performed in rhIGF-1– or VEH-treated MAM-exposed rats at P40–P43. Each experiment was conducted in a standard behavioral testing room during the light phase (8–20 h) of the 12 h light-dark cycle. Every test was monitored by a blinded observer. We used 70% ethanol as cleaning agent before each session. Y-maze test at P40 On P40, the Y-maze test was performed as previously described[23] with a computerized motion-tracking software (SMART 3.0; Panlab S.L.U., Barcelona, Spain). An arm entry was counted when all four limbs were inside the arm, and the number of novel arm entries divided by the total number of entries (i.e., the number of novel and familiar arm entries) was multiplied by 100 to calculate the novelty preference (NP) index. Open-field test at P41 The locomotive activity of each rat was assessed for 5 min. The chamber comprised black plastic boxes with bottom (60 × 60 cm) bordered by 30 cm high sidewalls. The duration of the resting, slow and fast activities, and the moving distances in peripheral (20 cm from each of the four walls) or central zones (20 × 20 cm) were measured using a computerized motion tracking apparatus and software equipped with a CCD camera (SMART 3.0, Panlab. S.L.U., Spain). Fear conditioning at P42 and P43 Fear conditioning tests were performed in an observation chamber (25 × 25 × 25 cm; Panlab S.L.U.) built with aluminum (two side walls and ceiling) and Plexiglas® (rear wall and hinged front door) inside a soundproof box. The chamber delivers a shock and tone through the floor and light (conditioned stimulus) under computerized system control (Panlab S.L.U.). On P42, tone conditioning was performed using a modified protocol adopted from a previous study[24]. Immediately after the conditioning trial on P42, the chamber was cleaned; the rats were returned to the chamber for 5 min to measure the response to the context. On P43, a fear response to the conditioned stimulus was induced with a replaced wall and floor to reduce tactile and visual cues. Following a 2 min period without a conditioned stimulus, five tone parings were presented to the rats without a foot shock. 2.8 NMDA-induced spasms after rhIGF-1 pretreatment To evaluate the response of NMDA-induced single spasm to rhIGF-1 pretreatment, prenatally MAM-exposed rats were pretreated with rhIGF-1 (0.5 mg/kg, P12 to P14, twice a day at 8:00 AM and 6:00 PM) or VEH (0.1% BSA) and the number and latency to onset of spasms were monitored for 90 min after NMDA (15 mg/kg i.p.) injection at P15. To generate multiple spasms, prenatally MAM-exposed rats were injected with NMDA at P12 and then randomly assigned into rhIGF-1 or VEH group. Rats were treated with rhIGF-1 or VEH from P12 at 6:00 PM to P15 at 8:00 AM. All rats received additional spasm triggers on P13 and P15. The rats were monitored on P15 to determine the effects of rhIGF-1 pretreatment on spasms. 2.9 Statistical analysis Statistical analysis was performed using IBM SPSS (ver. 22.0; IBM Corp., Armonk, NY, USA). Level of significance was preset to p < 0.05. Two-group comparisons of the concentrations of neuro-metabolites, cortical protein expression, behavioral assessments, and spasms data were performed using the Mann–Whitney U test. Repeated measure-analysis of variance (RM-ANOVA) with Bonferroni correction was used to test the difference between two groups on the time course data of freezing behaviors and developmental changes of neuro-metabolites. Intracranial EEG recording data was analyzed using a linear mixed model. 3. Results 3.1 Synaptogenesis in infant rats with MCD Proteome changes in the cortices of prenatally MAM-exposed rats at P15 We found a total of 3,943 proteins, of which 3,736 proteins had quantitative information. IPA analysis identified the top 30 enriched canonical pathways in MAM-exposed rat cortex (Table S1). Synaptogenesis signaling was the most significantly downregulated pathway in rats with MAM-induced MCD as compared to that in normal controls (-Log 10 [ P value] = 21, z-score = -1.455). The significantly altered proteins of synaptogenesis signaling pathway are presented in Supplementary Data 1. MAM-exposed rats showed significant activation of IGF-1 signaling pathway affecting neurogenesis (PMID: 26879907) (-Log 10 [ P value] = 8.1, z-score = 1.789). The IPA of total dataset showed general decrement of neurotransmission in prenatally MAM-exposed rat cortices at their infancy (Fig. 1 ). HOMER1, GRIN2A, and CAMK2A levels were significantly downregulated with the predicted inhibition of the upstream regulator, NMDAR, in MAM-exposed rat cortices as compared with those in controls (Fig. 1 ). Alteration in synaptic proteins in infant rats with MCD Infant rats with MCD (n = 12) showed significantly lower protein expression levels of CaMKII and AMPAR2 than that in the controls (n = 12; CaMKII, 0.78 ± 0.27 vs. 1.0 ± 0.23, p = 0.038; AMPAR2, 0.77 ± 0.08 vs. 0.86 ± 0.13, p = 0.021). There were no changes in the expression of glutamate receptors, including other subunits of AMPAR and NMDAR (Fig. 2 ). 3.2 Cortical changes after rhIGF-1 pretreatment in infant rats with MCD Neuro-metabolic changes after rhIGF-1 pretreatment in infant rats with MCD MRS analysis focusing on the RSC (Fig. 3 A) showed significantly lower levels of the neuro-metabolite GSH in infant rats subjected to rhIGF-1 pretreatment (n = 13) than in VEH rats (n = 12, 1.12 ± 0.35 vs. 1.46 ± 0.38, p = 0.039) (Fig. 3 B). After rhIGF-1 pretreatment, developmental patterns of neuro-metabolites, including GSH (RM-ANOVA, F(1, 23) = 5.938, p = 0.023), PCr (RM-ANOVA, F(1, 23) = 4.614, p = 0.042), and tCr (RM-ANOVA, F(1, 23) = 6.965, p = 0.015) were significantly different between the two groups (Fig. 3 C). rhIGF-1 pretreatment involved in synaptic protein maturation in the brain cortex The cortical protein expression level of NeuN, but not GAD67 and GAD65, significantly increased after rhIGF-1 pretreatment in MCD infant rats (n = 11) as compared with that in the VEH control group (n = 9) (1.02 ± 0.12 vs. 0.90 ± 0.1, p = 0.044) (Fig. 4 A). The expression of AMPAR1, AMPAR4, NMDAR1, NMDAR2A, and PSD95 increased and that of CaMKII decreased in the P15 cortex of rhIGF-1 pretreatment group as compared with that in the control group (AMPAR1, 0.87 ± 0.11 vs. 0.64 ± 0.19, p = 0.003; AMPAR4, 0.82 ± 0.11 vs. 0.67 ± 0.14, p = 0.020; NMDAR1, 0.95 ± 0.1 vs. 0.8 ± 0.16, p = 0.025; NMDAR2A, 0.89 ± 0.23 vs. 0.68 ± 0.14, p = 0.037; PSD95, 0.98 ± 0.14 vs. 0.75 ± 0.15, p = 0.004; CaMKII, 1.43 ± 0.08 vs. 1.53 ± 0.09, p = 0.030, Fig. 4 B, C). 3.3 Behavioral assessment on rhIGF-1 pretreatment in malformed brain development There was no difference in the distance traveled and times in central and peripheral zone between rhIGF-1–pretreated rats and controls at P41 (Fig. 5 A). In the short-term memory assessment through the Y-maze test, no significant difference was observed between rhIGF-1–treated rats and controls (Fig. 5 B). No significant difference was reported in the freezing duration during the conditioning, context tests, and tests for the conditioned stimuli between the rhIGF-1 pretreatment group (n = 14) and VEH group (n = 15, Fig. 5 C). 3.4 Successful reduction in spasms in infant MCD rats after rhIGF-1 pretreatment rhIGF-1 pretreatment significantly reduced the number of spasms induced by a single dose of NMDA at P15 in MAM-induced MCD rats (n = 17) as compared to that in VEH control rats (n = 18, 17.8 ± 11.8 vs. 45.61 ± 16.8, p < 0.001). In addition, rhIGF-1 pretreatment significantly delayed the onset of tailing, first spasms, and full spasms (tailing, 1107.9 ± 207.4 vs. 970.3 ± 143.0, p = 0.021; first spasms, 1386.8 ± 237.3 vs. 1157.1 ± 174.4, p = 0.006; full spasms, 1853.9 ± 575.4 vs. 1329.7 ± 230.1, p = 0.002). In contrast, rhIGF-1 pretreatment had little effect on the body weight (Fig. 6 A). In experiments using multiple NMDA administrations (Fig. 6 B), the onset of tailing and spasms was not significantly different after rhIGF-1 or VEH pretreatment. However, the number of spasms, which was not different before rhIGF-1 treatment, significantly decreased after rhIGF-1 pretreatment (n = 6) as compared to that after VEH treatment (n = 6, 17.3 ± 9.5 vs. 38.2 ± 13.3, p = 0.016, RM-ANOVA, F(1, 10) = 5.374, p = 0.043). We performed quantitative measurement of FO from EEGs after single-dose NMDA-induced spasms (Fig. 6 C). At baseline before spasm, the fast oscillation-spectral entropy (FO-SE) was significantly lower in rhIGF-1 pretreatment group than in the VEH group (n = 5, 0.39 ± 0.04 vs. n = 5, 0.42 ± 0.02, p < 0.001, linear mixed model analysis). During single-dose NMDA-induced spasms, the FO-SE and FO-event–related spectral dynamics (FO-ERSP) of rhIGF-1 group were significantly lower than those of the VEH group (FO-SE, n = 5, 0.38 ± 0.02 vs. n = 5, 0.44 ± 0.02, p < 0.001; FO-ERSP, n = 5, 4.43 ± 0.35 vs. n = 5, 5.04 ± 3.29, p < 0.001, linear mixed model analysis). Both FO-SE and FO-ERSP significantly increased during ictal period only in VEH group when compared to those of during inter-ictal period (FO-SE, n = 5, 0.42 ± 0.02 vs. n = 5, 0.44 ± 0.02, p < 0.001; FO-ERSP, n = 5, 4.42 ± 0.28 vs. n = 5, 5.04 ± 3.29, p < 0.001, linear mixed model analysis). There were no significant changes in FO-SE/FO-ERSP of rhIGF-1 group between inter-ictal and ictal period (FO-SE, n = 5, 0.39 ± 0.04 vs. n = 5, 0.38 ± 0.02, p = 0.347; FO-ERSP, n = 5, 4.45 ± 0.45 vs. n = 5, 4.43 ± 0.35, p = 0.253, linear mixed model analysis). 4. Discussion Development of the cerebral cortex is very intricate and mediated by various factors and processes[1]. Any dysregulation in these processes or factors causes malformation in the brain development[3,4,25]. Abnormal brain development, especially MCD, is the main cause of intractable epilepsy in pediatric[25,26] patients, leading to sequelae such as cognitive impairment affecting whole life[5,2,27]. However, the precise pathogenetic mechanism underlying epileptogenesis in MCD brain is yet unknown[10,9]. Hence, treatment is usually focused only on symptom relief or surgical resection of the dysplastic cortex. To identify the epileptogenetic mechanism of MCD, we conducted studies on a rat model using MAM and found clinical phenotypes similar to those in patients with MCD[5,12]. Using this model, we aimed to determine the major disrupted pathway in the MCD cortex. Quantitative proteome analysis revealed that synaptogenesis signaling was the most significantly downregulated canonical pathway in the malformed cortex of prenatal rats exposed to MAM at P15 (Table S1), consistent with the decrease in CaMKIIA along with an increase in the susceptibility to NMDA-induced spasms[5]. Our previous work demonstrated the poor dendritic spine development and reduced neuronal population in the RSC of prenatally MAM-exposed rats[12]. Altered early synaptogenesis is reported in neurodevelopmental disorders, including epilepsy, intellectual disability, and autism spectrum disorders[28]. The neuronal connections of the CNS comprise both inhibitory and excitatory synapses where GABA and glutamate are involved in the major inhibitory and excitatory actions. In this experimental model, the second trimester of gestation when the fetus is exposed to MAM is the time of the formation of the excitatory synapses[29] and overexpression of the genes associated with neurodevelopmental disorder[30]. To confirm the changes in the excitatory/inhibitory synaptic development in rats with malformed cortex, the profiles of glutamate receptors, CaMKII, and PSD95 were examined. We observed a significant decrease in the expression of AMPAR2 and CaMKII in the MAM-exposed rat cortex at P15 (Fig. 2 A, 2 C). CaMKII is abundantly found in the brain and plays a crucial role in synaptic plasticity and function, including synaptic spine formation[31]. Alterations in CaMKII activity and expression have been confirmed in various neuropsychiatric diseases[31]. In particular, the reduced activity of CaMKII is known to be related to epilepsy[31,32]. This decreased expression of cortical CaMKII in malformed brain may be related to the previously reported dendritic arborization[12] and increased spasm susceptibility[5]. IGF-1, a member of the insulin-like peptides (ILPs) family, is a polypeptide that plays an essential role in early brain development. We observed a marked increase in IGF-1 signaling pathway in prenatal MAM-exposed rat cortex at P15 (Table S1). IGF-1 is produced in all cell types in the CNS and is involved in neuronal growth, polarity, maturation, and neuroplasticity[15,33]. However, the function of IGF-1 is ambivalent in relation to neurological diseases[17] and understudied. Some studies suggest that IGF-1 and IGF-1 signaling pose a risk of epilepsy with increasing seizure activity[17,34]. Further, various effects of IGF-1 on synapses were reported[35,33] that IGF-1 application increases the AMPAR-mediated synaptic transmission and increases excitatory postsynaptic potentials (EPSP)[18] or increases the expression of NMDAR2A and NMDAR2B in addition to increasing the complexity of synapses[35–37]. However, most results were limited to aged rats with disorders other than epilepsy. In these rats with malformed cortices, early postnatal systemic rhIGF-1 treatment increased the expression of some subunits of AMPARs (AMPAR1 and AMPAR4) and NMDARs (NMDAR1 and NMDAR2A) as well as PSD-95 (Fig. 4 B, C). In addition to AMPAR and NMDAR, which play important roles in neurotransmission[38], the increase in the expression of PSD-95, a post-synaptic density protein that promotes synaptic maturation[33], suggests that IGF-1 treatment during early developmental period can contribute to the modulation in synaptogenesis. Moreover, we demonstrated the increase in NeuN expression in early rhIGF-1–treated rats (Fig. 4 A), which is consistent with previous study in transgenic mice with IGF-1 overexpression[37] or in neuronal cell culture study observing neuronal growth and migration[39] or study of IGF-1 on hippocampal neurogenesis in old rats[40]. Despite these neuronal changes, there is no behavioral improvement, including short-term and long-term memories, in rhIGF-treated adolescent rats with MCD (Fig. 5 ). To demonstrate the in vivo anti-seizure efficacy of rhIGF-1 pretreatment, we tested NMDA-induced spasm susceptibility after rhIGF-1 pretreatment or randomized treatment protocols using this infant rat model[13,12,5]. Both pretreatment or randomized treatment with rhIGF-1 could effectively reduce the number or delay the onset of spasms (Fig. 6 A, B). EEG also supported the neuronal changes after rhIGF-1 pretreatment, as evident from the reduced FO in MCD infant rats (Fig. 6 C). The FO plays a crucial role in the integration of neuronal networks and is related to the synchronized activation of interconnected excitatory pyramidal neurons and inhibitory interneurons[41,13]. In our previous study, we reported increased FO-ERSP, a time-related shift of the FO band frequency[42,13], consistent with an increase in seizure susceptibility in these MCD rats at P15 [5,13]. In the present study, rhIGF-1 pretreatment significantly reduced the ictal FO-ERSP that suggested attenuation of neuronal network dysregulation. Further, the SE-FO significantly reduced in rats with rhIGF-1 pretreatment as compared to that in rats subjected to VEH treatment during inter-ictal and ictal periods. SE is a measure of the irregularity of neuronal network signals, and higher SE was reported in patients with drug-resistant epilepsy than in healthy controls[43]. Previous reports have shown that IGF-1 reduces excitatory post-synaptic currents and partially rescues immature synaptic functions in MePC2 mutant mice[44]. Similarly, rhIGF-1 pretreatment could suppress the overwhelming pathologic FO in rats with MCD in this study. After rhIGF-1 pretreatment in MAM-induced MCD rats, GSH level significantly decreased after rhIGF-1 pretreatment, and there were significant developmental changes in Cr and GSH concentration after rhIGF-1 pretreatment (Fig. 3 B, C). GSH, a tripeptide composed of glutamate, glycine, and cysteine, is an antioxidant that protects cells from the damage caused by reactive oxygen species (ROS)[45–47]. Changes in GSH levels are known to be related to neurological disorders; in particular, reductions in GSH levels are closely associated with an increase in oxidative stress and are related to epilepsy[47,45]. Although there are the studies of GSH in epilepsy patients or patients with focal cortical dysplasia[48,47], the role of GSH in epileptic brain is unclear. Creatine (Cr) is a marker for energy metabolism[49,50], and recent studies have reported the elevation of Cr levels in malformed cortices of patients with epilepsy and suggests Cr as hypometabolic marker during inter-ictal period[49,50]. Thus, stabilization of cortical Cr and GSH after rhIGF-1 treatment may add evidence of GSH/Cr as a marker of neuronal stabilization in the MCD cortex. Epileptogenesis in MCD is intricately intertwined with the timing of insult, etiology, extent of disease, and patient’s age[2]. In this model of MCD, which experiences a mid-gestation insult, synaptogenesis signaling was markedly disrupted. Early rhIGF-1 pretreatment could attenuate the spasms susceptibility induced by NMDA at P15, along with alterations of in synaptic protein expression. These results suggest that rhIGF-1 can potentially serve as a therapeutic agent in patients with MCD-associated epilepsy and may modulate early synapse formation, one of the main target pathways of epilepsy. Declarations Funding This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A6A3A01090600). Mi-Sum Yum and Eun-Jin Kim were supported by Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2021R1A2C100447111). We gratefully acknowledge technical support from Biomedical Imaging Infrastructure, Department of Radiology, Asan Medical Center. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Author Contributions M. Lee contributed to the conception, design of the study, and drafting the manuscript and figures. M. Lee, E.J. Kim and M.J. Kim were involved in data acquisition, interpretation, and analysis. M. Lee contributed to the editing the manuscript and the figures. M.J. Kim conducted the EEG experiments. M.S. Yum, J. Yeom, and K. Kim performed proteomic analysis. M. Lee and M.S. Yum reviewed the submitted version of manuscript and supervised the study. Data Availability The datasets generated and analyzed in the current study are available from the corresponding author on reasonable request. Ethics approval All experiments were approved by the Institutional Animal Care and Use Committee of the Ulsan University College of Medicine and conducted in accordance with the Revised Guide for the Case and Use of Laboratory Animals [NIH GUIDE, 8 th Edition, 2011]. Consent to participate Not applicable. Consent to publish Not applicable. References Pang T, Atefy R, Sheen V (2008) Malformations of cortical development. 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Seizure 32:23-29. doi:10.1016/j.seizure.2015.08.008 Supplementary Files FigureSupplement1.tif Sup.AnimalN.tif Sup.LitterN.tif Sup.WBAb.tif TableS1.docx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2023 Read the published version in Molecular Neurobiology → Version 1 posted Reviewers agreed at journal 19 Aug, 2022 Reviewers invited by journal 23 May, 2022 Editor invited by journal 21 May, 2022 Editor assigned by journal 28 Apr, 2022 First submitted to journal 26 Apr, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1598872","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":102112247,"identity":"ca2841e7-8088-48df-a0ab-bbe4529e31c2","order_by":0,"name":"Minyoung Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYNACAwYGfgkwS0KGWC0GDJIzIFp4gARjA1HWGNyAsAhrMTjeY/jgR8EfeePbzc8e3aix4GGQbj7+AK+WM2eMDXsMDAy33TlmbpxzDOgwmWOJeG0xu5FjJsFjYMC47UaCmXQOG1CLRI4hIS3mP/8YGNhvnpH+TTrnH0hL/keCtjADbUncIJFjJp3bBrYFv/ftzxwrlpYxME6ecSOnTDq3T4KHTSLNcAY+LZLtzRs/vvkjZ9s/I32bdM63Ojl+ieQHH/BpYWDgMEDls+FXDgLsDwirGQWjYBSMgpENAL3tRWT0CYc3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6636-6054","institution":"University of Ulsan College of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Minyoung","middleName":"","lastName":"Lee","suffix":""},{"id":102112248,"identity":"7c5be336-50ed-43d5-be59-8c0382294e19","order_by":1,"name":"Eun-Jin Kim","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eun-Jin","middleName":"","lastName":"Kim","suffix":""},{"id":102112249,"identity":"9960abd1-807c-4df8-ad4b-f8d8b064e1db","order_by":2,"name":"Min-Jee Kim","email":"","orcid":"","institution":"Asan Medical Center Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min-Jee","middleName":"","lastName":"Kim","suffix":""},{"id":102112250,"identity":"eaecaa40-3be8-40cd-b1b6-78dbdf00f701","order_by":3,"name":"Mi-Sun Yum","email":"","orcid":"https://orcid.org/0000-0002-5986-5258","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mi-Sun","middleName":"","lastName":"Yum","suffix":""},{"id":102112251,"identity":"afab1620-6864-4266-ad20-218f0d5ff8bb","order_by":4,"name":"Jeonghun Yeom","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeonghun","middleName":"","lastName":"Yeom","suffix":""},{"id":102112252,"identity":"d5f193ac-e34a-4b3a-a993-e4950cfbaba2","order_by":5,"name":"Kyunggon Kim","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyunggon","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2022-04-27 00:33:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1598872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1598872/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-023-03256-4","type":"published","date":"2023-02-27T19:29:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":21116495,"identity":"35fe6059-7b58-41ce-9bcc-d2601dc4f28e","added_by":"auto","created_at":"2022-05-05 15:50:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109419,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical summary of ingenuity pathway analysis (IPA). A graphical summary of IPA of the total dataset (left) and graphical representation of significantly inhibited upstream regulators of synaptic transmission found from the dataset (right).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/fc52fb7a66af7fedee2e9c2f.jpg"},{"id":21115769,"identity":"a3d6692b-fea4-46dc-9a17-2eeb7431d20e","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156411,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis of synaptic protein expression in rats treated with MCD at P15. (A) In infant rats with MCD (n = 12), the expression of AMPAR2 subunit is significantly decreased in the cortex (0.77 ± 0.08 vs. 0.86 ± 0.13, p = 0.021). (B) There is no significant change in the expression of NMDAR subunits between infant rats with MCD and controls (n = 12). (C) The infant rats with MCD show significant reduction in CaMKII expression as compared to controls (0.78 ± 0.27 vs. 1.0 ± 0.23, p = 0.038).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/65069761eeafbb59bfb6b8a4.jpg"},{"id":21115771,"identity":"954ba21e-86b3-449d-9e45-cc9ff730b3ce","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e neurometabolic changes in the cortex after rhIGF-1 pretreatment. (A) At P15, retrosplenial regions of interest (ROI) for magnetic resonance spectroscopy (MRS) data acquisition is depicted in the coronal (upper right), and an example of MR spectra is shown (left). (B) In rhIGF-1 pretreatment group (n = 13), GSH level is significantly decreased (1.12 ± 0.35 vs. 1.46 ± 0.38, p = 0.039) as compared with that in the VEH pretreatment group (n = 12) during infancy. (C) The developmental changes in GSH (RM-ANOVA, F(1, 23) = 5.938, p = 0.023), PCr (RM-ANOVA, F(1, 23) = 4.614, p = 0.042), and tCr (RM-ANOVA, F(1, 23) = 6.965, p = 0.015) are significantly different between rhIGF-1 pretreatment and VEH control rats.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/c10d1f66c6ed97e792603e2f.jpg"},{"id":21115768,"identity":"6b2fd770-9cfe-4959-be95-725aa941768e","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121067,"visible":true,"origin":"","legend":"\u003cp\u003eSynaptic protein expression in infant rats with MCD after rhIGF-1 pretreatment. (A) In the western blot analysis (rhIGF-1 group vs. VEH group, n = 11 vs. n = 9), there is significant increase in neuronal nuclei (NeuN) (1.02 ± 0.12 vs. 0.90 ± 0.1, p = 0.044) but not in GABAergic interneuron. (B) AMPAR1 (0.87 ± 0.11 vs. 0.64 ± 0.19, p = 0.003) and AMPAR4 (0.82 ± 0.11 vs. 0.67 ± 0.14, p = 0.020) subunits are markedly elevated in the cortex after rhIGF-1 pretreatment during infancy. (C) The expression of NMDAR1 (0.95 ± 0.1 vs. 0.8 ± 0.16, p = 0.025), NMDAR2A (0.89 ± 0.23 vs. 0.68 ± 0.14, p = 0.037), and PSD95 (0.98 ± 0.14 vs. 0.75 ± 0.15, p = 0.004) is significantly increased in infant rats with MCD after rhIGF-1 pretreatment. CaMKII expression is significantly reduced after rhIGF-1 pretreatment (1.43 ± 0.08 vs. 1.53 ± 0.09, p = 0.030). (D) Predictive schematic diagram of synaptic protein expression in infant rats with MCD after rhIGF-1 pretreatment. Based on western blot results, alterations in the cortical protein expression, including increase in PSD-95, NMDAR, and AMPAR subunits and decrease in CaMKII, are observed after rhIGF-1 pretreatment in the malformed brain.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/ca7cd45c18adce329c3a5dd2.jpg"},{"id":21115778,"identity":"629e51f2-4da2-4b8f-85bb-558aff87fb3c","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":199468,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral analysis of adolescent rats with MCD subjected to postnatal rhIGF-1 treatment (P12–P14). (A) In the open-field test, there is no significant difference between the rhIGF-1 pretreatment group (n = 14) and VEH group (n = 15). (B) No difference is reported in the movement pattern through the Y-maze test. (C) The rhIGF-1 pretreatment group did not show any difference in freezing duration as compared to the VEH group during fear conditioning.\u0026nbsp;\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/f593b01b47a39f986806e5f0.png"},{"id":21115773,"identity":"3645f12d-8e4d-47d0-a53f-ddf130be7943","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":391912,"visible":true,"origin":"","legend":"\u003cp\u003erhIGF-1 pretreatment effect on NMDA-induced spasms in infant rats with MCD. rhIGF-1 (n = 17) or VEH (n = 18) pretreatment was administered to rats from postnatal day 12 to 14, and spasms were induced by NMDA at postnatal day 15. (A) There is no change in the body weight between the two groups, but the number of spasms decreased (7.8 ± 11.8 vs. 45.61 ± 16.8, p \u0026lt; 0.001) and onset of tailing and spasms was delayed after rhIGF-1 pretreatment (tailing, 1107.9 ± 207.4 vs. 970.3 ± 143.0, p = 0.021; first spasms, 1386.8 ± 237.3 vs. 1157.1 ± 174.4, p = 0.006; full spasms, 1853.9 ± 575.4 vs. 1329.7 ± 230.1, p = 0.002). (B) Although the onset of tailing and spasms was not significantly different between rhIGF-1 pretreatment group and VEH controls (n = 6), the number of spasms is fewer in rhIGF-1 pretreatment group (n = 6, 17.3 ± 9.5 vs. 38.2 ± 13.3, p = 0.016, RM-ANOVA, F(1, 10) = 5.374, p = 0.043). (C) The fast oscillation-event-related spectral dynamics (FO-ERSP) is significantly decreased (4.43 ± 0.35 vs. 5.04 ± 3.29, p \u0026lt; 0.001, linear mixed model analysis) in rhIGF-1 pretreatment group (n = 5) as compared to that in VEH group during spasms (n = 5). In addition, fast oscillation-spectral entropy (FO-SE) is reduced in rhIGF-1 pretreatment group at inter-ictal and ictal periods as compared to that in VEH (inter-ictal, 0.39 ± 0.04 vs. n = 5, 0.42 ± 0.02, p \u0026lt; 0.001; ictal, 0.38 ± 0.02 vs. 0.44 ± 0.02, p \u0026lt; 0.001, linear mixed model analysis). Although there is no difference in rhIGF-1 pretreatment group, the FO-SE and FO-ERSP increased in ictal periods than in inter-ictal periods in VEH group (FO-SE, 0.42 ± 0.02 vs. 0.44 ± 0.02, p \u0026lt; 0.001; FO-ERSP, 4.42 ± 0.28 vs. 5.04 ± 3.29, p \u0026lt; 0.001, linear mixed model analysis).\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/49ae7b3f0800149ed78e4ca8.png"},{"id":44721342,"identity":"02e8f24d-1ed8-4a3f-9bf4-0eb2dd446659","added_by":"auto","created_at":"2023-10-16 19:33:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1156484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/866836bf-5747-4f0d-8b82-3e5f2d11bb70.pdf"},{"id":21115775,"identity":"07bdd60f-fdac-4c6c-9da1-32ac2b901fab","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1102828,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupplement1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/73fb9e0ffbf83ceb94cd7c58.tif"},{"id":21117108,"identity":"a5d21e6b-0b7d-4819-bffd-c822a4d78589","added_by":"auto","created_at":"2022-05-05 15:55:12","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":179384,"visible":true,"origin":"","legend":"","description":"","filename":"Sup.AnimalN.tif","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/1402d6fbc2b1bffbebc2b207.tif"},{"id":21115772,"identity":"59b04865-2e0f-425e-8811-3c2b433980a3","added_by":"auto","created_at":"2022-05-05 15:45:12","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":158668,"visible":true,"origin":"","legend":"","description":"","filename":"Sup.LitterN.tif","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/10835cd46c48e9e919bd99ed.tif"},{"id":21116496,"identity":"050aaae7-dc48-4e5a-8d20-cd4e2ce6bc53","added_by":"auto","created_at":"2022-05-05 15:50:12","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":163416,"visible":true,"origin":"","legend":"","description":"","filename":"Sup.WBAb.tif","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/90ada2d5cb2910ea83de840d.tif"},{"id":21116497,"identity":"9dd6e6a3-660e-4a5e-933a-74819ed0b2ba","added_by":"auto","created_at":"2022-05-05 15:50:12","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":21220,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1598872/v1/cfd4dd60f2f2e92b865570af.docx"}],"financialInterests":"","formattedTitle":"Insulin-like growth factor-1 promotes synaptogenesis signaling, a major dysregulated pathway in malformation of cortical development, in a rat model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe development of the cerebral cortex is accomplished through well-orchestrated neurogenesis, neuronal migration, cell proliferation, and organization involving various proteins and transcription factors[1,2]. Any dysregulation results in a broad-spectrum disorder, termed as malformation of cortical development (MCD). Many genetic or environmental fcators[3,4] are associated with the pathogenesis of MCD. Patients with MCD suffer from developmental problems, neurological deficits, and epilepsy[1,5,6]. In particular, MCD is the most common cause of intractable epilepsy in pediatric populations[5,7,8,2]. The earlier the epilepsy begins, the more frequent and severe cognitive impairment occurs[2]. Despite many clinical and translational researches[9,10], epileptogenesis of the malformed brain is still unclear. The afterbirth diagnosis of fetal brain development makes these structural alterations in brain development permanent, ultimately leaving physicians with the choice of only symptomatic treatment.\u003c/p\u003e \u003cp\u003eThe offspring from methylazoxymethanol (MAM)-treated rats have developmental brain anomalies, including migration failure, ventricular enlargement, and disorganization of neocortical and hippocampal structures[9,11], which resemble the pathologic findings observed in patients with MCD[11,5]. Our previous studies have reported cognitive impairment and increased seizure susceptibility with increased fast oscillation (FO) as well as decreased dendritic arborization or cortical neurons during infancy in this MAM-induced MCD rat model[5] [12,13]. Using this MAM-induced MCD model[4,14], we aimed to identify the most severely deteriorated canonical pathway in the early postnatal period.\u003c/p\u003e \u003cp\u003eInsulin-like growth factor-1 (IGF-1), produced by all central nervous system (CNS) cell types, plays important roles in brain development and neuroplasticity. IGF-1 is involved in cell organization, neural circuitry formation, and maturation of synaptic efficacy in the early brain, CNS development, and neuronal cell growth and proliferation[15\u0026ndash;17]. In the developing brain, IGF-1 modulates the axonal development and synapse formation through the phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) pathway[15,16]. Several studies[18\u0026ndash;21] have reported that the application of IGF-1 could improve outcomes after brain injury; however, the role of IGF-1 in epilepsy or early brain development is still controversial[17].\u003c/p\u003e \u003cp\u003eIn this study, we investigated whether recombinant human IGF-1 (rhIGF-1) pretreatment at early postnatal period inhibits N-methyl-D-aspartate (NMDA)-induced spasms in a rat MCD model[4,14] and if IGF-1 pretreatment reverses the key pathway involved in pathologic MCD brain.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal experiments\u003c/h2\u003e \u003cp\u003eAnimal experiments were approved by the Institutional Animal Care and Use Committee and conformed to the Revised Guide for the Care and Use of Laboratory Animals (8th Edition, 2011). Timed-pregnant Sprague-Dawley rats were purchased (Orient Bio Inc., Seoul, Korea) at gestational day 14 (G14) and housed under a 12 h light/dark cycle with free access to food and water. On G15, two doses of MAM (15 mg/kg intraperitoneally, MRIGlobal, Missouri) or normal saline were injected into pregnant rats at 8:00 AM and 6:00 PM. Delivery occurred consistently on gestational day 21 for all the rats, which was considered postnatal day (P) 0 for the offspring.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Proteomics analysis\u003c/h2\u003e \u003cp\u003ePrenatally MAM-exposed rats (n\u0026thinsp;=\u0026thinsp;4) and control rats (n\u0026thinsp;=\u0026thinsp;4) were sacrificed on P15 and their cortex were separated for proteomic analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSample preparation and Nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBrain tissues were carefully washed in phosphate-buffered saline (PBS) on ice to remove blood. The cortices of prenatally MAM-exposed rats and controls were individually cryopulverized using a Cryoprep device (CP02, Covaris) as previously described (PMID: 24678027). Peptide separation was performed using Dionex UltiMate 3000 RSLCnano system (Thermo-Fisher Scientific). Mass spectra were acquired in a data-dependent mode with an automatic switch between a full scan with 20 data-dependent MS/MS scans.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDatabase searching and label-free quantitation (LFQ) and functional enrichment and gene ontology analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe acquired MS/MS spectra were searched using the SequestHT on Proteome discoverer (version 2.2, Thermo Fisher Scientific) against the SwissProt database (July 2019). False discovery rates (FDRs) were set for 1% for each analysis. For the differential analysis of the relative abundance of proteins between samples, Perseus (version 1.6.13.0) was used. Proteins with a \u003cem\u003eq\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 and log2 fold change\u0026thinsp;\u0026plusmn;\u0026thinsp;1 were considered as differentially regulated proteins.\u003c/p\u003e \u003cp\u003eGene ontology analysis was performed through ShinyGO v0.60 (PMID: 31882993, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go60/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go60/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and data were further analyzed using IPA (QIAGEN Inc., \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis\u003c/span\u003e\u003cspan address=\"https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 rhIGF-1 experiments\u003c/h2\u003e \u003cp\u003ePrenatally MAM-exposed rats were pretreated with rhIGF-1 (0.5 mg/kg for each dose, P12 to P14, twice a day at 8:00 AM and 6:00 PM) or vehicle (VEH, 0.1% bovine serum albumin [BSA]). The overall experimental schedule is described in Figure S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot analysis\u003c/h2\u003e \u003cp\u003eFor western blot analysis, bilateral cortical tissues from bregma to posterior hippocampal areas without hippocampus (anterior posterior 0 to -5 mm) were obtained from control rats (n\u0026thinsp;=\u0026thinsp;12), MAM-exposed rats (n\u0026thinsp;=\u0026thinsp;12), rhIGF-1\u0026ndash;treated MAM-exposed rats (n\u0026thinsp;=\u0026thinsp;11) and VEH-treated MAM-exposed rats (n\u0026thinsp;=\u0026thinsp;9) at P15. The following primary antibodies were used: anti-AMPA receptor 1 (AMPAR1), AMPAR2, AMPAR3, and AMPAR4 (Cell signaling, Technology, Inc.); anti-calcium/calmodulin-dependent protein kinase II (CaMKII) (Cell signaling, Technology, Inc.); anti-glutamic acid decarboxylase 67 (GAD67) and GAD65 (Millipore, Technology, Inc.); anti-neuronal nuclei (NeuN; Millipore, Technology, Inc.); anti-N-methyl-D-aspartate receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B (Cell signaling, Technology, Inc.); anti-PSD95 (Cell signaling, Technology, Inc.); Anti-β-actin (Santa Cruz Biotechnology, Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cem\u003eIn vivo\u003c/em\u003e magnetic resonance imaging (MRI) studies\u003c/h2\u003e \u003cp\u003eMAM-induced MCD rats were maintained under anesthesia with 1% isoflurane in a 1:2 mixture of O\u003csub\u003e2\u003c/sub\u003e:N\u003csub\u003e2\u003c/sub\u003eO and their respiratory rate, electrocardiogram, and rectal temperature were monitored. MRI was performed using a 7.0 T/160-mm bore animal MRI system (Bruker Pharmascan, Ettlingen, Germany). \u003csup\u003e1\u003c/sup\u003eH-MRS was performed at P8 and P15 in rhIGF-1\u0026ndash;treated rats (n\u0026thinsp;=\u0026thinsp;13) and VEH-treated rats (n\u0026thinsp;=\u0026thinsp;12). The MR spectra were acquired through a signal voxel (from bregma to -4.0 mm in a coronal section, 1.5 \u0026times; 1 \u0026times; 4 mm\u003csup\u003e3\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) in the retrosplenial cortex (RSC). All MR spectra were processed using the linear combination analysis method (LC Model ver. 6.0, Los Angeles, CA) to calculate the metabolite concentrations.[22] The following brain metabolites were included in the metabolite basis set: alanine (Ala), aspartate (Asp), creatine (Cr), ɣ-aminobutyric acid (GABA), glucose, glutamate, glutamine, glycerophosphorylcholine, phosphorylcholine, myo-inositol (mIns), lactate (Lac), phosphocreatine (PCr), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), taurine, macromolecules (MMs), and lipids. The \u003cem\u003ein vivo\u003c/em\u003e proton spectra were considered to have an acceptable value if the standard deviation of the fit for the metabolite was less than 20%.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cortical electroencephalography (EEG) recording and analysis\u003c/h2\u003e \u003cp\u003eFor intracranial EEG recording, two cortical electrodes were surgically implanted in each of five rats treated with rhIGF-1 or VEH under ketamine/xylazine sedation (50/7 mg/kg in 10 mL/kg saline i.p.) at P12. At P15, spasms were triggered by a single dose of NMDA (15 mg/kg i.p.; Sigma), and EEGs of the two groups of rats were recorded with simultaneous videos using the Twin EEG system (Grass Technologies) for 90 min before NMDA injection (pre-ictal period) and 120 min after injection or until the end of spasms. The sampling rate was 400 Hz with a 0.1 Hz high‐pass filter, and 5 min of artifact‐free data were collected for each. Before analysis, the data were preprocessed using the EEGLAB toolbox of MATLAB 2015b. From each rats, 100 epochs (1 s duration) of pre‐ictal and spasms periods per rat were extracted from the EEG data.\u003c/p\u003e \u003cp\u003eFor quantitative estimation, averaged spectral entropy (SE) and event-related spectral dynamics (ERSP) of FO (25\u0026ndash;200 Hz), power spectral density (PSD) from each epoch were calculated using the EEGLAB toolbox of MATLAB 2017b.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Behavioral assessment\u003c/h2\u003e \u003cp\u003eBehavioral assessments were performed in rhIGF-1\u0026ndash; or VEH-treated MAM-exposed rats at P40\u0026ndash;P43. Each experiment was conducted in a standard behavioral testing room during the light phase (8\u0026ndash;20 h) of the 12 h light-dark cycle. Every test was monitored by a blinded observer. We used 70% ethanol as cleaning agent before each session.\u003c/p\u003e \u003cp\u003e \u003cem\u003eY-maze test at P40\u003c/em\u003e \u003c/p\u003e \u003cp\u003eOn P40, the Y-maze test was performed as previously described[23] with a computerized motion-tracking software (SMART 3.0; Panlab S.L.U., Barcelona, Spain). An arm entry was counted when all four limbs were inside the arm, and the number of novel arm entries divided by the total number of entries (i.e., the number of novel and familiar arm entries) was multiplied by 100 to calculate the novelty preference (NP) index.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOpen-field test at P41\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe locomotive activity of each rat was assessed for 5 min. The chamber comprised black plastic boxes with bottom (60 \u0026times; 60 cm) bordered by 30 cm high sidewalls. The duration of the resting, slow and fast activities, and the moving distances in peripheral (20 cm from each of the four walls) or central zones (20 \u0026times; 20 cm) were measured using a computerized motion tracking apparatus and software equipped with a CCD camera (SMART 3.0, Panlab. S.L.U., Spain).\u003c/p\u003e \u003cp\u003e \u003cem\u003eFear conditioning at P42 and P43\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFear conditioning tests were performed in an observation chamber (25 \u0026times; 25 \u0026times; 25 cm; Panlab S.L.U.) built with aluminum (two side walls and ceiling) and Plexiglas\u0026reg; (rear wall and hinged front door) inside a soundproof box. The chamber delivers a shock and tone through the floor and light (conditioned stimulus) under computerized system control (Panlab S.L.U.).\u003c/p\u003e \u003cp\u003eOn P42, tone conditioning was performed using a modified protocol adopted from a previous study[24]. Immediately after the conditioning trial on P42, the chamber was cleaned; the rats were returned to the chamber for 5 min to measure the response to the context. On P43, a fear response to the conditioned stimulus was induced with a replaced wall and floor to reduce tactile and visual cues. Following a 2 min period without a conditioned stimulus, five tone parings were presented to the rats without a foot shock.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 NMDA-induced spasms after rhIGF-1 pretreatment\u003c/h2\u003e \u003cp\u003eTo evaluate the response of NMDA-induced single spasm to rhIGF-1 pretreatment, prenatally MAM-exposed rats were pretreated with rhIGF-1 (0.5 mg/kg, P12 to P14, twice a day at 8:00 AM and 6:00 PM) or VEH (0.1% BSA) and the number and latency to onset of spasms were monitored for 90 min after NMDA (15 mg/kg i.p.) injection at P15.\u003c/p\u003e \u003cp\u003eTo generate multiple spasms, prenatally MAM-exposed rats were injected with NMDA at P12 and then randomly assigned into rhIGF-1 or VEH group. Rats were treated with rhIGF-1 or VEH from P12 at 6:00 PM to P15 at 8:00 AM. All rats received additional spasm triggers on P13 and P15. The rats were monitored on P15 to determine the effects of rhIGF-1 pretreatment on spasms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using IBM SPSS (ver. 22.0; IBM Corp., Armonk, NY, USA). Level of significance was preset to p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Two-group comparisons of the concentrations of neuro-metabolites, cortical protein expression, behavioral assessments, and spasms data were performed using the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test. Repeated measure-analysis of variance (RM-ANOVA) with Bonferroni correction was used to test the difference between two groups on the time course data of freezing behaviors and developmental changes of neuro-metabolites. Intracranial EEG recording data was analyzed using a linear mixed model.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Synaptogenesis in infant rats with MCD\u003c/h2\u003e \u003cp\u003e \u003cem\u003eProteome changes in the cortices of prenatally MAM-exposed rats at P15\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe found a total of 3,943 proteins, of which 3,736 proteins had quantitative information. IPA analysis identified the top 30 enriched canonical pathways in MAM-exposed rat cortex (Table S1). Synaptogenesis signaling was the most significantly downregulated pathway in rats with MAM-induced MCD as compared to that in normal controls (-Log\u003csub\u003e10\u003c/sub\u003e [\u003cem\u003eP\u003c/em\u003e value]\u0026thinsp;=\u0026thinsp;21, z-score = -1.455). The significantly altered proteins of synaptogenesis signaling pathway are presented in Supplementary Data 1. MAM-exposed rats showed significant activation of IGF-1 signaling pathway affecting neurogenesis (PMID: 26879907) (-Log\u003csub\u003e10\u003c/sub\u003e [\u003cem\u003eP\u003c/em\u003e value]\u0026thinsp;=\u0026thinsp;8.1, z-score\u0026thinsp;=\u0026thinsp;1.789).\u003c/p\u003e \u003cp\u003eThe IPA of total dataset showed general decrement of neurotransmission in prenatally MAM-exposed rat cortices at their infancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). HOMER1, GRIN2A, and CAMK2A levels were significantly downregulated with the predicted inhibition of the upstream regulator, NMDAR, in MAM-exposed rat cortices as compared with those in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAlteration in synaptic proteins in infant rats with MCD\u003c/em\u003e \u003c/p\u003e \u003cp\u003eInfant rats with MCD (n\u0026thinsp;=\u0026thinsp;12) showed significantly lower protein expression levels of CaMKII and AMPAR2 than that in the controls (n\u0026thinsp;=\u0026thinsp;12; CaMKII, 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 vs. 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23, p\u0026thinsp;=\u0026thinsp;0.038; AMPAR2, 0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 vs. 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, p\u0026thinsp;=\u0026thinsp;0.021). There were no changes in the expression of glutamate receptors, including other subunits of AMPAR and NMDAR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cortical changes after rhIGF-1 pretreatment in infant rats with MCD\u003c/h2\u003e \u003cp\u003e \u003cem\u003eNeuro-metabolic changes after rhIGF-1 pretreatment in infant rats with MCD\u003c/em\u003e \u003c/p\u003e \u003cp\u003eMRS analysis focusing on the RSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) showed significantly lower levels of the neuro-metabolite GSH in infant rats subjected to rhIGF-1 pretreatment (n\u0026thinsp;=\u0026thinsp;13) than in VEH rats (n\u0026thinsp;=\u0026thinsp;12, 1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 vs. 1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38, p\u0026thinsp;=\u0026thinsp;0.039) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). After rhIGF-1 pretreatment, developmental patterns of neuro-metabolites, including GSH (RM-ANOVA, F(1, 23)\u0026thinsp;=\u0026thinsp;5.938, p\u0026thinsp;=\u0026thinsp;0.023), PCr (RM-ANOVA, F(1, 23)\u0026thinsp;=\u0026thinsp;4.614, p\u0026thinsp;=\u0026thinsp;0.042), and tCr (RM-ANOVA, F(1, 23)\u0026thinsp;=\u0026thinsp;6.965, p\u0026thinsp;=\u0026thinsp;0.015) were significantly different between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cem\u003erhIGF-1 pretreatment involved in synaptic protein maturation in the brain cortex\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe cortical protein expression level of NeuN, but not GAD67 and GAD65, significantly increased after rhIGF-1 pretreatment in MCD infant rats (n\u0026thinsp;=\u0026thinsp;11) as compared with that in the VEH control group (n\u0026thinsp;=\u0026thinsp;9) (1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 vs. 0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, p\u0026thinsp;=\u0026thinsp;0.044) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The expression of AMPAR1, AMPAR4, NMDAR1, NMDAR2A, and PSD95 increased and that of CaMKII decreased in the P15 cortex of rhIGF-1 pretreatment group as compared with that in the control group (AMPAR1, 0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 vs. 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, p\u0026thinsp;=\u0026thinsp;0.003; AMPAR4, 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 vs. 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, p\u0026thinsp;=\u0026thinsp;0.020; NMDAR1, 0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, p\u0026thinsp;=\u0026thinsp;0.025; NMDAR2A, 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 vs. 0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, p\u0026thinsp;=\u0026thinsp;0.037; PSD95, 0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 vs. 0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, p\u0026thinsp;=\u0026thinsp;0.004; CaMKII, 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 vs. 1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, p\u0026thinsp;=\u0026thinsp;0.030, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Behavioral assessment on rhIGF-1 pretreatment in malformed brain development\u003c/h2\u003e \u003cp\u003eThere was no difference in the distance traveled and times in central and peripheral zone between rhIGF-1\u0026ndash;pretreated rats and controls at P41 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn the short-term memory assessment through the Y-maze test, no significant difference was observed between rhIGF-1\u0026ndash;treated rats and controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). No significant difference was reported in the freezing duration during the conditioning, context tests, and tests for the conditioned stimuli between the rhIGF-1 pretreatment group (n\u0026thinsp;=\u0026thinsp;14) and VEH group (n\u0026thinsp;=\u0026thinsp;15, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Successful reduction in spasms in infant MCD rats after rhIGF-1 pretreatment\u003c/h2\u003e \u003cp\u003erhIGF-1 pretreatment significantly reduced the number of spasms induced by a single dose of NMDA at P15 in MAM-induced MCD rats (n\u0026thinsp;=\u0026thinsp;17) as compared to that in VEH control rats (n\u0026thinsp;=\u0026thinsp;18, 17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8 vs. 45.61\u0026thinsp;\u0026plusmn;\u0026thinsp;16.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, rhIGF-1 pretreatment significantly delayed the onset of tailing, first spasms, and full spasms (tailing, 1107.9\u0026thinsp;\u0026plusmn;\u0026thinsp;207.4 vs. 970.3\u0026thinsp;\u0026plusmn;\u0026thinsp;143.0, p\u0026thinsp;=\u0026thinsp;0.021; first spasms, 1386.8\u0026thinsp;\u0026plusmn;\u0026thinsp;237.3 vs. 1157.1\u0026thinsp;\u0026plusmn;\u0026thinsp;174.4, p\u0026thinsp;=\u0026thinsp;0.006; full spasms, 1853.9\u0026thinsp;\u0026plusmn;\u0026thinsp;575.4 vs. 1329.7\u0026thinsp;\u0026plusmn;\u0026thinsp;230.1, p\u0026thinsp;=\u0026thinsp;0.002). In contrast, rhIGF-1 pretreatment had little effect on the body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn experiments using multiple NMDA administrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), the onset of tailing and spasms was not significantly different after rhIGF-1 or VEH pretreatment. However, the number of spasms, which was not different before rhIGF-1 treatment, significantly decreased after rhIGF-1 pretreatment (n\u0026thinsp;=\u0026thinsp;6) as compared to that after VEH treatment (n\u0026thinsp;=\u0026thinsp;6, 17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5 vs. 38.2\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3, p\u0026thinsp;=\u0026thinsp;0.016, RM-ANOVA, F(1, 10)\u0026thinsp;=\u0026thinsp;5.374, p\u0026thinsp;=\u0026thinsp;0.043).\u003c/p\u003e \u003cp\u003eWe performed quantitative measurement of FO from EEGs after single-dose NMDA-induced spasms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). At baseline before spasm, the fast oscillation-spectral entropy (FO-SE) was significantly lower in rhIGF-1 pretreatment group than in the VEH group (n\u0026thinsp;=\u0026thinsp;5, 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs. n\u0026thinsp;=\u0026thinsp;5, 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, linear mixed model analysis). During single-dose NMDA-induced spasms, the FO-SE and FO-event\u0026ndash;related spectral dynamics (FO-ERSP) of rhIGF-1 group were significantly lower than those of the VEH group (FO-SE, n\u0026thinsp;=\u0026thinsp;5, 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs. n\u0026thinsp;=\u0026thinsp;5, 0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; FO-ERSP, n\u0026thinsp;=\u0026thinsp;5, 4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 vs. n\u0026thinsp;=\u0026thinsp;5, 5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, linear mixed model analysis). Both FO-SE and FO-ERSP significantly increased during ictal period only in VEH group when compared to those of during inter-ictal period (FO-SE, n\u0026thinsp;=\u0026thinsp;5, 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs. n\u0026thinsp;=\u0026thinsp;5, 0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; FO-ERSP, n\u0026thinsp;=\u0026thinsp;5, 4.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 vs. n\u0026thinsp;=\u0026thinsp;5, 5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, linear mixed model analysis). There were no significant changes in FO-SE/FO-ERSP of rhIGF-1 group between inter-ictal and ictal period (FO-SE, n\u0026thinsp;=\u0026thinsp;5, 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs. n\u0026thinsp;=\u0026thinsp;5, 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.347; FO-ERSP, n\u0026thinsp;=\u0026thinsp;5, 4.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 vs. n\u0026thinsp;=\u0026thinsp;5, 4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35, p\u0026thinsp;=\u0026thinsp;0.253, linear mixed model analysis).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDevelopment of the cerebral cortex is very intricate and mediated by various factors and processes[1]. Any dysregulation in these processes or factors causes malformation in the brain development[3,4,25]. Abnormal brain development, especially MCD, is the main cause of intractable epilepsy in pediatric[25,26] patients, leading to sequelae such as cognitive impairment affecting whole life[5,2,27]. However, the precise pathogenetic mechanism underlying epileptogenesis in MCD brain is yet unknown[10,9]. Hence, treatment is usually focused only on symptom relief or surgical resection of the dysplastic cortex. To identify the epileptogenetic mechanism of MCD, we conducted studies on a rat model using MAM and found clinical phenotypes similar to those in patients with MCD[5,12]. Using this model, we aimed to determine the major disrupted pathway in the MCD cortex.\u003c/p\u003e \u003cp\u003eQuantitative proteome analysis revealed that synaptogenesis signaling was the most significantly downregulated canonical pathway in the malformed cortex of prenatal rats exposed to MAM at P15 (Table S1), consistent with the decrease in CaMKIIA along with an increase in the susceptibility to NMDA-induced spasms[5]. Our previous work demonstrated the poor dendritic spine development and reduced neuronal population in the RSC of prenatally MAM-exposed rats[12]. Altered early synaptogenesis is reported in neurodevelopmental disorders, including epilepsy, intellectual disability, and autism spectrum disorders[28]. The neuronal connections of the CNS comprise both inhibitory and excitatory synapses where GABA and glutamate are involved in the major inhibitory and excitatory actions. In this experimental model, the second trimester of gestation when the fetus is exposed to MAM is the time of the formation of the excitatory synapses[29] and overexpression of the genes associated with neurodevelopmental disorder[30].\u003c/p\u003e \u003cp\u003eTo confirm the changes in the excitatory/inhibitory synaptic development in rats with malformed cortex, the profiles of glutamate receptors, CaMKII, and PSD95 were examined. We observed a significant decrease in the expression of AMPAR2 and CaMKII in the MAM-exposed rat cortex at P15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). CaMKII is abundantly found in the brain and plays a crucial role in synaptic plasticity and function, including synaptic spine formation[31]. Alterations in CaMKII activity and expression have been confirmed in various neuropsychiatric diseases[31]. In particular, the reduced activity of CaMKII is known to be related to epilepsy[31,32]. This decreased expression of cortical CaMKII in malformed brain may be related to the previously reported dendritic arborization[12] and increased spasm susceptibility[5].\u003c/p\u003e \u003cp\u003eIGF-1, a member of the insulin-like peptides (ILPs) family, is a polypeptide that plays an essential role in early brain development. We observed a marked increase in IGF-1 signaling pathway in prenatal MAM-exposed rat cortex at P15 (Table S1). IGF-1 is produced in all cell types in the CNS and is involved in neuronal growth, polarity, maturation, and neuroplasticity[15,33]. However, the function of IGF-1 is ambivalent in relation to neurological diseases[17] and understudied. Some studies suggest that IGF-1 and IGF-1 signaling pose a risk of epilepsy with increasing seizure activity[17,34]. Further, various effects of IGF-1 on synapses were reported[35,33] that IGF-1 application increases the AMPAR-mediated synaptic transmission and increases excitatory postsynaptic potentials (EPSP)[18] or increases the expression of NMDAR2A and NMDAR2B in addition to increasing the complexity of synapses[35\u0026ndash;37]. However, most results were limited to aged rats with disorders other than epilepsy. In these rats with malformed cortices, early postnatal systemic rhIGF-1 treatment increased the expression of some subunits of AMPARs (AMPAR1 and AMPAR4) and NMDARs (NMDAR1 and NMDAR2A) as well as PSD-95 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). In addition to AMPAR and NMDAR, which play important roles in neurotransmission[38], the increase in the expression of PSD-95, a post-synaptic density protein that promotes synaptic maturation[33], suggests that IGF-1 treatment during early developmental period can contribute to the modulation in synaptogenesis. Moreover, we demonstrated the increase in NeuN expression in early rhIGF-1\u0026ndash;treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which is consistent with previous study in transgenic mice with IGF-1 overexpression[37] or in neuronal cell culture study observing neuronal growth and migration[39] or study of IGF-1 on hippocampal neurogenesis in old rats[40]. Despite these neuronal changes, there is no behavioral improvement, including short-term and long-term memories, in rhIGF-treated adolescent rats with MCD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo demonstrate the \u003cem\u003ein vivo\u003c/em\u003e anti-seizure efficacy of rhIGF-1 pretreatment, we tested NMDA-induced spasm susceptibility after rhIGF-1 pretreatment or randomized treatment protocols using this infant rat model[13,12,5]. Both pretreatment or randomized treatment with rhIGF-1 could effectively reduce the number or delay the onset of spasms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). EEG also supported the neuronal changes after rhIGF-1 pretreatment, as evident from the reduced FO in MCD infant rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The FO plays a crucial role in the integration of neuronal networks and is related to the synchronized activation of interconnected excitatory pyramidal neurons and inhibitory interneurons[41,13]. In our previous study, we reported increased FO-ERSP, a time-related shift of the FO band frequency[42,13], consistent with an increase in seizure susceptibility in these MCD rats at P15 [5,13]. In the present study, rhIGF-1 pretreatment significantly reduced the ictal FO-ERSP that suggested attenuation of neuronal network dysregulation. Further, the SE-FO significantly reduced in rats with rhIGF-1 pretreatment as compared to that in rats subjected to VEH treatment during inter-ictal and ictal periods. SE is a measure of the irregularity of neuronal network signals, and higher SE was reported in patients with drug-resistant epilepsy than in healthy controls[43]. Previous reports have shown that IGF-1 reduces excitatory post-synaptic currents and partially rescues immature synaptic functions in MePC2 mutant mice[44]. Similarly, rhIGF-1 pretreatment could suppress the overwhelming pathologic FO in rats with MCD in this study.\u003c/p\u003e \u003cp\u003eAfter rhIGF-1 pretreatment in MAM-induced MCD rats, GSH level significantly decreased after rhIGF-1 pretreatment, and there were significant developmental changes in Cr and GSH concentration after rhIGF-1 pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). GSH, a tripeptide composed of glutamate, glycine, and cysteine, is an antioxidant that protects cells from the damage caused by reactive oxygen species (ROS)[45\u0026ndash;47]. Changes in GSH levels are known to be related to neurological disorders; in particular, reductions in GSH levels are closely associated with an increase in oxidative stress and are related to epilepsy[47,45]. Although there are the studies of GSH in epilepsy patients or patients with focal cortical dysplasia[48,47], the role of GSH in epileptic brain is unclear. Creatine (Cr) is a marker for energy metabolism[49,50], and recent studies have reported the elevation of Cr levels in malformed cortices of patients with epilepsy and suggests Cr as hypometabolic marker during inter-ictal period[49,50]. Thus, stabilization of cortical Cr and GSH after rhIGF-1 treatment may add evidence of GSH/Cr as a marker of neuronal stabilization in the MCD cortex.\u003c/p\u003e \u003cp\u003eEpileptogenesis in MCD is intricately intertwined with the timing of insult, etiology, extent of disease, and patient\u0026rsquo;s age[2]. In this model of MCD, which experiences a mid-gestation insult, synaptogenesis signaling was markedly disrupted. Early rhIGF-1 pretreatment could attenuate the spasms susceptibility induced by NMDA at P15, along with alterations of in synaptic protein expression. These results suggest that rhIGF-1 can potentially serve as a therapeutic agent in patients with MCD-associated epilepsy and may modulate early synapse formation, one of the main target pathways of epilepsy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A6A3A01090600). Mi-Sum Yum and Eun-Jin Kim were supported by Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2021R1A2C100447111). We gratefully acknowledge technical support from Biomedical Imaging Infrastructure, Department of Radiology, Asan Medical Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. Lee contributed to the conception, design of the study, and drafting the manuscript and figures. M. Lee, E.J. Kim and M.J. Kim were involved in data acquisition, interpretation, and analysis. M. Lee contributed to the editing the manuscript and the figures. M.J. Kim conducted the EEG experiments. M.S. Yum, J. Yeom, and K. Kim performed proteomic analysis. M. Lee and M.S. Yum reviewed the submitted version of manuscript and supervised the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Institutional Animal Care and Use Committee of the Ulsan University College of Medicine and conducted in accordance with the Revised Guide for the Case and Use of Laboratory Animals [NIH GUIDE, 8\u003csup\u003eth\u003c/sup\u003e Edition, 2011].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePang T, Atefy R, Sheen V (2008) Malformations of cortical development. 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Seizure 32:23-29. doi:10.1016/j.seizure.2015.08.008\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"methylazoxymethanol acetate (MAM), animal model, synaptic protein, recombinant human insulin-like growth factor-1 (rhIGF-1), malformation of cortical development (MCD)","lastPublishedDoi":"10.21203/rs.3.rs-1598872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1598872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalformation of cortical development (MCD) is one of the main causes of intractable epilepsy in childhood. We explored a treatment based on molecular changes using an infant rat model of methylazoxymethanol (MAM)-induced MCD established by injecting MAM at gestational day 15. The offspring were sacrificed on postnatal day (P) 15 for proteomic analysis, which revealed significant downregulation in the synaptogenesis signaling pathway in the cortex of MCD rats. Recombinant human insulin-growth factor-1 (rhIGF-1) was injected from P12 to P14 twice daily and the effect of IGF1 on N-methyl-D-aspartate (NMDA)-induced spasms (15 mg/kg of NMDA, i.p.) was tested; the onset of P15 single spasm was significantly delayed (p\u0026thinsp;=\u0026thinsp;0.002) and the number of spasms decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in rhIGF1-pretreated rats (n\u0026thinsp;=\u0026thinsp;17) as compared to those in VEH-treated rats (n\u0026thinsp;=\u0026thinsp;18). Electroencephalographic monitoring during spasms showed significantly reduced spectral entropy and event-related spectral dynamics of fast oscillation in rhIGF-1 treated rats. Magnetic resonance spectroscopy of the retrosplenial cortex showed decreased glutathione (GSH) (p\u0026thinsp;=\u0026thinsp;0.039) and significant developmental changes in GSH, phosphocreatine (PCr), and total creatine (tCr) (p\u0026thinsp;=\u0026thinsp;0.023, 0.042, 0.015, respectively) after rhIGF1 pretreatment. rhIGF1 pretreatment significantly upregulated expression of cortical synaptic proteins such as PSD95, AMPAR1, AMPAR4, NMDAR1, and NMDAR2A (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, early rhIGF-1 treatment could promote synaptic protein expression, which was significantly downregulated by prenatal MAM exposure, and effectively suppress NMDA-induced spasms. Early IGF1 treatment should be further investigated as a therapeutic strategy in infants with MCD-related epilepsy.\u003c/p\u003e","manuscriptTitle":"Insulin-like growth factor-1 promotes synaptogenesis signaling, a major dysregulated pathway in malformation of cortical development, in a rat model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-05 15:45:10","doi":"10.21203/rs.3.rs-1598872/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-08-19T16:26:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-23T17:18:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Neurobiology","date":"2022-05-21T23:51:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-28T10:59:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2022-04-26T20:31:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b1a2e886-d0d8-4349-9f38-89d67f8f46a6","owner":[],"postedDate":"May 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T19:31:53+00:00","versionOfRecord":{"articleIdentity":"rs-1598872","link":"https://doi.org/10.1007/s12035-023-03256-4","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2023-02-27 19:29:35","publishedOnDateReadable":"February 27th, 2023"},"versionCreatedAt":"2022-05-05 15:45:10","video":"","vorDoi":"10.1007/s12035-023-03256-4","vorDoiUrl":"https://doi.org/10.1007/s12035-023-03256-4","workflowStages":[]},"version":"v1","identity":"rs-1598872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1598872","identity":"rs-1598872","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}