The vigabatrin-associated brain abnormalities on MRI and their differential diagnosis.

Vigabatrin is an anti-epileptic drug that inhibits the enzyme γ-aminobutyric acid (GABA)-transaminase. The anticonvulsant effect of vigabatrin involves increasing GABA levels and attenuating glutamate-glutamine cycling. Vigabatrin indications include infantile spasms and refractory focal seizures. Despite having a significant role in paediatric epileptology, vigabatrin has adverse effects, such as retinal toxicity, in up to 30% of patients after 1 year of use and brain abnormalities on magnetic resonance imaging (MRI). The percentage of patients with brain abnormalities on MRI varies between 22-32% of children using vigabatrin to treat infantile spasms. Risk factors for presenting these imaging abnormalities are cryptogenic infantile spasms, age <12 months old, high dosage, and possible concomitant hormonal therapy. Clinically, these abnormalities are usually asymptomatic. Histopathological analysis reveals white matter vacuolation and intramyelinic oedema. The typical findings of vigabatrin-associated brain abnormalities on MRI are bilateral and have a symmetrical hyperintense signal on T2-weighted imaging, with diffusion restriction, that often compromise the globi pallidi, thalami, subthalamic nuclei, cerebral peduncles, midbrain, dorsal brainstem, including the medial longitudinal fasciculi, and dentate nuclei of the cerebellum. In this article, the authors intend to review the clinical manifestations, histopathological features, imaging aspects, and differential diagnosis of vigabatrin-associated brain abnormalities on MRI.

Novel neurosteroid pregnanolone pyroglutamate suppresses neurotoxicity syndrome induced by tetramethylenedisulfotetramine but is ineffective in a rodent model of infantile spasms.

BACKGROUND: Neurosteroids are investigated as effective antidotes for the poisoning induced by tetramethylenedisulfotetramine (TMDT) as well as treatments for epileptic spasms during infancy. Both these conditions are quite resistant to pharmacotherapy; thus, a search for new treatments is warranted. METHODS: In this study, we determined the efficacy of two novel neurosteroids, pregnanolone glutamate (PAG) and pregnanolone pyroglutamate (PPG), and tested these drugs in doses of 1-10 mg/kg (ip) against the TMDT syndrome and in our rodent model of infantile spasms. RESULTS: Only PPG in doses 5 and 10 mg/kg suppressed the severity of the TMDT syndrome and TMDT-induced lethality, while the 1 mg/kg dose was without an effect. Interestingly, the 1 mg/kg dose of PPG in combination with 1 mg/kg of diazepam was also effective against TMDT poisoning. Neither PAG nor PPG were effective against experimental spasms in the N-methyl-D-aspartate (NMDA)-triggered model of infantile spasms. CONCLUSIONS: While evidence suggests that PAG can act through multiple actions which include allosteric inhibition of NMDA-induced and glycine receptor-evoked currents as well as augmentation of É£-aminobutyric acid subtype A (GABAA) receptor-induced currents, the agent appears to neither have the appropriate mechanistic signature for activity in the infantile spasm model, nor the adequate potency, relative to PPG, for ameliorating the TMDT syndrome. The full mechanisms of action of PPG, which may become a potent TMDT antidote either alone or in combination with diazepam are yet unknown and thus require further investigation.

Response to sequential treatment with prednisolone and vigabatrin in infantile spasms.

AIM: To report response to first treatment in infants with infantile spasms (IS), including incremental benefit of prednisolone 60 mg/day and vigabatrin following prednisolone 40 mg/day failure in infants commenced on the United Kingdom Infantile Spasms Study (UKISS) treatment sequence. METHODS: In this retrospective analysis, we compared effectiveness of prednisolone, vigabatrin and nonstandard treatments as first treatment for IS. In infants who commenced the UKISS treatment sequence, we evaluated response to each step. Primary outcome was spasm cessation after 42 days. Secondary outcomes were severe side effects and spasm relapse after 42 days. RESULTS: Treatment response data were available for 151 infants. First treatment was prednisolone in 99 infants, vigabatrin in 18 and nonstandard treatment in 34. The rate of spasm cessation with first treatment was significantly higher with prednisolone (62/99, 63%) than vigabatrin (5/18, 28%, P = 0.01) or nonstandard treatment (2/34, 5.9%, P < 0.01). Of 112 infants who commenced the UKISS treatment sequence, 71/112 (63%) responded to prednisolone 40 mg/day. Among non-responders, 12/29 (41%) subsequently responded to prednisolone 60 mg/day, and 10/22 (45%) to vigabatrin. Severe side effects and spasm relapse were not significantly different between each treatment. CONCLUSION: We confirm higher rates of spasm cessation with initial treatment with prednisolone than vigabatrin and nonstandard therapy. Non-use of prednisolone as first treatment in over one third of infants highlights a concerning treatment gap. The UKISS treatment sequence has high overall treatment response (total 93/112; 83%), with similar benefit of subsequent prednisolone 60 mg/day and vigabatrin in prednisolone 40 mg/day non-responders.

A phase 2, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of soticlestat as adjunctive therapy in pediatric patients with Dravet syndrome or Lennox-Gastaut syndrome (ELEKTRA).

OBJECTIVE: Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS) are rare treatment-resistant childhood epilepsies classed as developmental and epileptic encephalopathies. ELEKTRA investigated the efficacy and safety of soticlestat (TAK-935) as adjunctive therapy in children with DS or LGS (NCT03650452). METHODS: ELEKTRA was a phase 2, randomized, double-blind, placebo-controlled study of soticlestat (≤300 mg twice daily, weight-adjusted) in children (aged 2-17 years) with DS, demonstrating three or more convulsive seizures/month, or with LGS, demonstrating four or more drop seizures/month at baseline. The 20-week treatment period comprised an 8-week dose-optimization period and a 12-week maintenance period. Efficacy endpoints included change from baseline in seizure frequency versus placebo. Safety assessments included incidence of treatment-emergent adverse events (TEAEs). RESULTS: ELEKTRA enrolled 141 participants; 126 (89%) completed the study. The modified intent-to-treat population included 139 participants who received one or more doses of study drug and had one or more efficacy assessments (DS, n = 51; LGS, n = 88). ELEKTRA achieved its primary endpoint: the combined soticlestat-treated population demonstrated a placebo-adjusted median reduction in seizure frequency of 30.21% during the maintenance period (p = .0008, n = 139). During this period, placebo-adjusted median reductions in convulsive and drop seizure frequencies of 50.00% (p = .0002; patients with DS) and 17.08% (p = .1160; patients with LGS), respectively, were observed. TEAE incidences were similar between the soticlestat (80.3%) and placebo (74.3%) groups and were mostly mild or moderate in severity. Serious TEAEs were reported by 15.5% and 18.6% of participants receiving soticlestat and placebo, respectively. TEAEs reported in soticlestat-treated patients with ≥5% difference from placebo were lethargy and constipation. No deaths were reported. SIGNIFICANCE: Soticlestat treatment resulted in statistically significant, clinically meaningful reductions from baseline in median seizure frequency (combined patient population) and in convulsive seizure frequency (DS cohort). Drop seizure frequency showed a nonstatistically significant numerical reduction in children with LGS. Soticlestat had a safety profile consistent with previous studies.

Adrenal insufficiency among children treated with hormonal therapy for infantile spasms.

OBJECTIVE: Hormonal therapy is a standard treatment for children with infantile spasms. However, the high doses given and long treatment duration expose patients to the risk of adrenal insufficiency (AI). This study aims to quantify the cumulative incidence of AI among children with infantile spasms treated with high-dose corticosteroids and/or adrenocorticotropic hormone. METHODS: A retrospective chart review of patients treated for infantile spasms was performed between January 2009 and March 2020 in one pediatric specialized hospital. Variables collected include patient and treatment characteristics, risk factors of AI, and adrenal function testing. Analysis included descriptive statistics such as incidence and bivariate analysis. RESULTS: Thirty-one patients were included and received a total of 33 courses of treatment (17 corticosteroids [prednisone/prednisolone], 12 adrenocorticotropic hormone, and four combined). Physiologic hydrocortisone replacement therapy with stress supplementation was received after 32 of 33 (97%) courses of treatment. Adrenal function was assessed in 32 of 33 (97%) and AI occurred in 25 of 33 (76%, 95% confidence interval = 58-89). No predictive factor of AI was identified after hormonal treatment. No drug regimen was found to be safe. The two patients who developed an acute adrenal crisis presented to the emergency room within the days (between 2 and 7) following weaning off of hormonal treatment. They were the youngest children of the cohort, and both received prednisolone. SIGNIFICANCE: Adrenal insufficiency is frequent and can potentially lead to an adrenal crisis in this population. This study highlights the necessity of hydrocortisone replacement therapy until AI has been excluded in a patient who has received hormonal therapy to treat infantile spasms. As such, routine laboratory assessment of adrenal function should be done in all patients.

A Role for Insulin-like Growth Factor 1 in the Generation of Epileptic Spasms in a murine model.

OBJECTIVE: Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries. We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms. METHODS: We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1's role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile spasms patients. We used viral injections in neonatal conditional IGF-1R knock-out mice to show that an IGF-1-derived tripeptide (1-3)IGF-1, acts through the IGF-1 receptor to abolish spasms. RESULTS: Immunohistochemical methods revealed widespread loss of IGF-1 from cortical neurons, but an increase in IGF-1 in the reactive astrocytes in the TTX-induced lesion. Very similar changes were observed in the neocortex from patients with spasms. In animals, we observed reduced signaling through the IGF-1 growth pathways in areas remote from the lesion. To show the reduction in IGF-1 expression plays a role in spasm generation, epileptic rats were treated with (1-3)IGF-1. We provide 3 lines of evidence that (1-3)IGF-1 activates the IGF-1 signaling pathway by acting through the receptor for IGF-1. Treatment with (1-3)IGF-1 abolished spasms and hypsarrhythmia-like activity in the majority of animals. INTERPRETATION: Results implicate IGF-1 in the pathogenesis of infantile spasms and IGF-1 analogues as potential novel therapies for this neurodevelopmental disorder. ANN NEUROL 2022;92:45-60.

Risk of vigabatrin-associated brain abnormalities on MRI: A retrospective and controlled study.

OBJECTIVE: Vigabatrin (VGB) is the first-line treatment for infantile spasms (IS). Previous studies have shown that VGB exposure may cause vigabatrin-associated brain abnormalities on magnetic resonance imaging (MRI) (VABAM). Based on previous studies, this study aimed to go further to explore the possible risk factors and the incidence of VABAM. In addition, diffusion-weighted imaging (DWI) and T2-weighted imaging (T2WI) were compared to explore whether DWI should be used as a routine examination sequence when MRI is performed in children receiving VGB. METHODS: Children with IS receiving VGB were selected as the study subjects. Whether VABAM occurred or not was categorized as the VABAM group and the non-VABAM group, respectively. Their general clinical data and medication exposure were collected. The possible risk factors of VABAM and different MRI sequences were compared and statistically analyzed. RESULTS: A total of 77 children with IS were enrolled in the study, of which 25 (32.5%) developed VABAM. Twenty-three of the 25 VABAM cases have a peak dosage of VGB between 50 and 150 mg/kg/day. The earliest observation time of VABAM was 30 days. Regression analysis of relevant risk factors showed that the peak dosage of VGB was the risk factor for VABAM. Comparison between different MRI sequences showed that DWI is more sensitive than T2WI to the evaluation of VABAM. SIGNIFICANCE: In our study, the occurrence of VABAM was 32.5%, indicating a higher incidence than in most previous reports. In addition, we once again verified that the peak dosage of VGB was the risk factor of VABAM. Caution should be exercised that our data also suggest that VABAM may occur even using the conventional dosage of VGB (ie, 50-150 mg/kg/day). Therefore, even when using the conventional dosage of VGB, regular MRI examination should be required. Furthermore, DWI sequence should be used as a routine examination sequence when MRI is performed in children with IS who are receiving VGB.

Acthar® Gel (repository corticotropin injection) dose-response relationships in an animal model of epileptic spasms.

Studies were undertaken to evaluate the effectiveness of Acthar® Gel (repository corticotropin injection [RCI]) in the tetrodotoxin (TTX) model of early-life-induced epileptic spasms. Repository corticotropin injection (RCI) is widely used in the United States to treat infantile spasms. A major component of RCI is N25 deamidated ACTH. Additionally, we hoped to provide some insight into the possible role circulating corticosteroids play in spasm cessation by comparing the RCI dose-response relationships for spasm suppression to RCI-induced corticosterone release from the adrenal gland. Spasms were induced by chronic TTX infusion into the neocortex beginning on postnatal day 11. Repository corticotropin injection (RCI) dosages were between 8 and 32 IU/kg/day. Drug titration protocols were used, and comparisons were made to injections of a vehicle gel. Video/EEG recordings (24/7) monitored the drug's effects continuously for up to 2 months. Tetrodotoxin (TTX)-infused control rats were monitored for the same period of time. In separate experiments, the same dosages of RCI were given to rats and 1 h later plasma was collected and assayed for corticosterone. A parallel study compared the effects of 1-day and 10-day RCI treatments on circulating corticosterone. Results showed that RCI was ineffective at dosages of 8, 12, and 16 IU/kg/day but eliminated spasms in 66% of animals treated with 24 or 32 IU/kg/day. Treating animals with 32 IU/kg/day alone produced the same degree of spasms suppression as observed during the titration protocols. In rats that had hypsarrhythmia-like activity, RCI eliminated this abnormal interictal EEG pattern in all rats that became seizure-free. In terms of plasma corticosterone, 1- and 10-day treatments with RCI produced similar increases in this hormone and the levels increased linearly with increasing dosages of RCI. This stood in sharp contrast to the sigmoid-like dose-response curve for decreases in spasm counts. Our results further validate the TTX model as relevant for the study of infantile spasms. The model should be useful for investigating how RCI acts to eliminate seizures and hypsarrhythmia. Dose-response results suggest that either very high concentrations of circulating corticosteroids are required to abolish spasms or RCI acts through a different mechanism.

Potential induction of epileptic spasms by nonselective voltage-gated sodium channel blockade: Interaction with etiology.

PURPOSE: Epileptic spasms are often preceded by focal (or multifocal) seizures. Based on a series of case reports suggesting that carbamazepine and oxcarbazepine may induce epileptic spasms, we set out to rigorously evaluate the potential association between exposure to voltage-gated sodium channel blockade and latency to epileptic spasms. METHODS: We identified 50 cases (children with focal seizures and evolution to epileptic spasms) and 50 controls (children with focal seizures without evolution to epileptic spasms). For each patient, we reviewed all sequential neurology encounters between onset of epilepsy and emergence of epileptic spasms. For each encounter we recorded seizure-frequency and all anti-seizure therapy exposures. Using multivariable Cox proportional hazards regression, we evaluated the association between voltage-gated sodium channel exposure (carbamazepine, oxcarbazepine, lacosamide, or phenytoin) and latency to epileptic spasms onset, with adjustment for etiology and seizure-frequency. RESULTS: Latency to epileptic spasms onset was independently associated with exposure to sodium channel blockade (hazard ratio = 2.4; 95% CI 1.1-5.2; P = 0.03) and high-risk etiology (hazard ratio = 2.8; 95% CI 1.5-5.1; P = 0.001). With assessment for interaction between sodium channel blockade and etiology, we identified an estimated 7-fold increased risk of epileptic spasms with the combination of sodium channel blockade and high-risk etiology (hazard ratio = 7.0, 95% CI 2.5-19.8; P < 0.001). CONCLUSION: This study suggests that voltage-gated sodium channel blockade may induce epileptic spasms among children at risk on the basis of etiology. Further study is warranted to replicate these findings, ascertain possible drug- and dose-specific risks, and identify potential mechanisms of harm.

Neocortical Slow Oscillations Implicated in the Generation of Epileptic Spasms.

OBJECTIVE: Epileptic spasms are a hallmark of severe seizure disorders. The neurophysiological mechanisms and the neuronal circuit(s) that generate these seizures are unresolved and are the focus of studies reported here. METHODS: In the tetrodotoxin model, we used 16-channel microarrays and microwires to record electrophysiological activity in neocortex and thalamus during spasms. Chemogenetic activation was used to examine the role of neocortical pyramidal cells in generating spasms. Comparisons were made to recordings from infantile spasm patients. RESULTS: Current source density and simultaneous multiunit activity analyses indicate that the ictal events of spasms are initiated in infragranular cortical layers. A dramatic pause of neuronal activity was recorded immediately prior to the onset of spasms. This preictal pause is shown to share many features with the down states of slow wave sleep. In addition, the ensuing interictal up states of slow wave rhythms are more intense in epileptic than control animals and occasionally appear sufficient to initiate spasms. Chemogenetic activation of neocortical pyramidal cells supported these observations, as it increased slow oscillations and spasm numbers and clustering. Recordings also revealed a ramp-up in the number of neocortical slow oscillations preceding spasms, which was also observed in infantile spasm patients. INTERPRETATION: Our findings provide evidence that epileptic spasms can arise from the neocortex and reveal a previously unappreciated interplay between brain state physiology and spasm generation. The identification of neocortical up states as a mechanism capable of initiating epileptic spasms will likely provide new targets for interventional therapies. ANN NEUROL 2021;89:226-241.

Disparate effects of hormones and vigabatrin on sleep slow waves in patients with West syndrome - An indication of their mode of action?

Synaptic downscaling during sleep, a physiological process to restore synaptic homeostasis and maintain learning efficiency and healthy brain development, has been related to a reduction of the slope of sleep slow waves (SSW). However, such synaptic downscaling seems not to be reflected in high-amplitude SSW. Recently we have shown reduced SSW slopes during hormonal treatment (adrenocorticotrophic hormone, prednisolone) in patients with West syndrome (WS). Yet, whether this reduction was related to successful treatment or reflects a specific effect of hormone therapy is unknown. Thus, we retrospectively analysed nap electroencephalograms of 61 patients with WS successfully treated with hormones, vigabatrin (VGB), or both. The slope of SSW during treatment (T1) and 2-7 months later (T2) when hormonal treatment was tapered off were compared between the treatment groups and healthy, age-matched controls. At T1 hormone treatment reduced the slope of low-amplitude SSW, whereas VGB increased the slope of high-amplitude SSW (linear mixed effect model: FGroup  = 7.04, p < 0.001; FAmplitude  = 1,646.68, p < 0.001; FGroup*Amplitude  = 3.38, p < 0.001). At T2, untreated patients did not differ anymore from healthy controls, whereas those still under VGB showed the same alterations as those with VGB at T1. This result indicates a disparate effect of VGB and hormone on the SSW slope. In particular, hormones seem to reduce the slope of cortical generated low-amplitude SSW, similar to the physiological synaptic downscaling during sleep. Thus, a loss of functional neuronal connectivity might be an alternative explanation of the antiepileptic effect of hormonal treatment.

AQB-565 shows promise in preclinical testing in the model of epileptic spasms during infancy: Head-to-head comparison with ACTH.

Epileptic spasms during infancy (infantile spasms) represent a serious treatment and social problem despite their rare occurrence. Current treatments include hormonal therapy (adrenocorticotropin-ACTH or corticosteroids) or vigabatrin (per se or in the combination). These treatments are partially effective and with potentially significant adverse effects. Thus, the search for new effective drugs is warranted. We tested efficacy of a novel fusion peptide AQB-565 developed by Aequus Biopharma in a model of infantile spasms consisting of prenatal exposure to betamethasone and repeated postnatal trigger of spasms with N-methyl-d-aspartic acid (NMDA). AQB-565 molecule includes the first 24 amino acids of ACTH, a ten amino acid linker and a modified melanocyte-stimulating hormone molecule. In contrast to ACTH with almost uniform activity over all peripheral and central melanocortin receptor isoforms, AQB is preferentially active on central melanocortin receptors MC3 and MC4. Here, we used equivalent doses of rat ACTH (full molecule) and AQB-565 and compared their efficacy in a prospective randomized test against of repeated bouts of spasms on postnatal days (P)12, P13 and P15 in the rat model. All doses of ACTH (range 0.02-1.0 mg/kg s.c.) and all doses but one of AQB-565 in the same range suppressed spasms in P15 rats (treatment stopped on P14). There was no dose-dependent effect and both compounds had all-or-none effect that is similar to clinical outcome of hormonal treatment of infantile spasms in children. Thus, AQB-565 may represent a novel treatment of infantile spasms similarly effective as ACTH but with potentially limited side effects.

Preclinical Screening for Treatments for Infantile Spasms in the Multiple Hit Rat Model of Infantile Spasms: An Update.

Infantile spasms are the typical seizures of West syndrome, an infantile epileptic encephalopathy with poor outcomes. There is an increasing need to identify more effective and better tolerated treatments for infantile spasms. We have optimized the rat model of infantile spasms due to structural etiology, the multiple-hit rat model, for therapy discovery. Here, we test three compounds administered after spasms induction in the multiple hit model for efficacy and tolerability. Specifically, postnatal day 3 (PN3) male Sprague-Dawley rats were induced by right intracerebral injections of doxorubicin and lipopolysaccharide. On PN5 p-chlorophenylalanine was given intraperitoneally (i.p.). Daily monitoring of weights and developmental milestones was done and rats were intermittently video monitored. A blinded, randomized, vehicle-controlled study design was followed. The caspase 1 inhibitor VX-765 (50-200 mg/kg i.p.) and the GABAB receptor inhibitor CGP35348 (12.5-100 mg/kg i.p.) each was administered in different cohorts as single intraperitoneal injections on PN4, using a dose- and time-response design with intermittent monitoring till PN5. 17ß-estradiol (40 ng/g/day subcutaneously) was given daily between PN3-10 and intermittent monitoring was done till PN12. None of the treatments demonstrated acute or delayed effects on spasms, yet all were well tolerated. We discuss the implications for therapy discovery and challenges of replication trials.

Infantile spasms in down syndrome: Rescue by knockdown of the GIRK2 channel.

OBJECTIVE: The Ts65Dn (Ts) mouse model of Down syndrome (DS) is exquisitely sensitive to an infantile spasms phenotype induced by γ-aminobutyric acidB receptor (GABAB R) agonists. The Ts mouse contains the core genomic triplication of the DS critical region, which includes 3 copies of the Kcnj6 gene that encodes the GABAB R-coupled G protein-coupled inward rectifying potassium channel subunit 2 (GIRK2) channel. We test the hypothesis that GIRK2 is necessary for the GABAB R agonist-induced infantile spasms phenotype in Ts. METHODS: We assessed the result of either genetic or pharmacological knockdown of the GIRK2 channel in Ts brain upon the GABAB R agonist-induced infantile spasms phenotype in the Ts mouse model of DS. As well, we examined GABAB R currents in hippocampal neurons prepared from GIRK2-trisomic Ts control mice and GIRK2-disomic Ts mice in which Kcnj6 had been genetically knocked down from 3 to 2 copies. RESULTS: The reduction of the copy number of Kcnj6 in Ts mice rescued the GABAB R agonist-induced infantile spasms phenotype. There was an increase in GABAB R-mediated GIRK2 currents in GIRK2-trisomic Ts mouse hippocampal neurons, which were normalized in the GIRK2-disomic Ts mice. Similarly, pharmacological knockdown of the GIRK2 channel in Ts brain using the GIRK antagonist tertiapin-Q also rescued the GABAB R agonist-induced infantile spasms phenotype in Ts mutants. INTERPRETATION: The GABAB R-coupled GIRK2 channel is necessary for the GABAB R agonist-induced infantile spasms phenotype in the Ts mouse and may represent a novel therapeutic target for the treatment of infantile spasms in DS. Ann Neurol 2016;80:511-521.

Estradiol does not affect spasms in the betamethasone-NMDA rat model of infantile spasms.

OBJECTIVE: This study attempted to validate the effects of neonatal estradiol in ameliorating the spasms in the prenatally betamethasone-primed N-methyl-d-aspartate (NMDA) model of infantile spasms in rats as shown previously in a mouse Arx gene knock-in expansion model of infantile spasms. METHODS: Neonatal rats prenatally exposed to betamethasone (on day 15 of pregnancy) were treated with subcutaneous 40 ng/g estradiol benzoate (EB) between postnatal days (P)3-P10 or P0-P5. A synthetic estrogen analogue, diethylstilbestrol, was used between P0 and P5 (2 µg per rat, s.c.). On P12, P13, and P15, the rats were subjected to NMDA-triggered spasms, and latency to onset and number of spasms were evaluated. Rats with EB on P3-P10 were tested after spasms in the open field, novel object recognition, and elevated plus maze to determine effects of treatment on behavior. Additional rats with P3-P10 or P0-P5 EB were investigated for γ-aminobutyric acid (GABA)ergic neurons (glutamate decarboxylase [GAD]67 expression) in the neocortex. As a positive control, a group of rats received either subcutaneous adrenocorticotropic hormone (ACTH) (2 × 0.3 mg/kg on P12 and 3 × 0.3 mg/kg on P13 and P14) or vehicle after the first episode of spasms on P12. RESULTS: Neither EB treatment nor diethylstilbestrol consistently affected expression of spasms in this model, although we found a significant increase in GAD67-immunopositive cells in the neocortex after P3-P10 and P0-P5 EB treatment, consistent with a study in mice. Behavioral tests showed increase in lateralization in male rats treated with P3-P10 EB, a behavioral trait usually associated with female sex. Diethylstilbestrol treatment in male rats resulted in arrested pubertal descent of testes. ACTH had robust effects in suppressing spasms. SIGNIFICANCE: Treatment of infantile spasms (IS) using neonatal EB may be justified in those cases of IS that present with detectable deficits in GABAergic neurons. In other types of IS, the efficacy of neonatal EB and its analogues is not supported.

Development of a mouse model of infantile spasms induced by N-methyl-D-aspartate.

Using N-methyl-D-aspartate (NMDA) injection, we attempt to develop a mouse model for infantile spasms (IS). Experiments were performed in postnatal 11- to 13-day-old C57 and Balbc mice. In the pilot experiment, mice were injected with different doses of NMDA (7, 15, and 30 mg/kg) to determine the optimal age and convulsant doses of NMDA. In further experiment optimal age mice were divided into five groups: group A, control group that received intraperitoneal injection of physiological saline; group B, convulsion group that was given intraperitoneal NMDA; group C, pretreatment group that received adrenocorticotropin (ACTH) injection (100 IU/kg) 30 min before NMDA administration; group D, electroencephalogram (EEG) group that received EEG recording, group E, performance group that received motor and learning test at different time point after NMDA administration. The behaviors of each group were observed continuously for 3h, the latency and the total numbers of spasms were recorded. Pilot experiments showed that a 15 mg/kg dose of NMDA could induce typical spasm-like seizures in P13 C57 mice, NMDA administration caused anxiety and deficits in motor and cognitive functions at early time and that large doses of ACTH reduced the number of seizures and rating scale (P<0.05). The NMDA mouse model has the following characteristics: age dependency, spasm-like seizures, cognitive impairment and response to ACTH, which fulfills the criteria of an IS model.

ß-Hydroxybutyrate attenuates NMDA-induced spasms in rats with evidence of neuronal stabilization on MR spectroscopy.

BACKGROUND: Infantile spasms (IS) is a devastating epileptic encephalopathy. The ketogenic diet (KD) has been successfully used as a treatment for IS. This study was designed to test whether beta-hydroxybutyrate (BHB), a major metabolite of the KD, is effective in an animal model of IS. METHODS: Pregnant rats received betamethasone on gestational day 15. The offspring received either single [30min prior to NMDA-triggered spasms on postnatal day (P) 15] or prolonged (three per day from P12 to P15) i.p. BHB. An additional experiment used repeated bouts of spasms on P12, P13, and P15 with randomized prolonged BHB treatment initiated after the first spasms. We determined the latency to onset of spasms and the number of spasms after the NMDA injection on P15. The rats that received randomized BHB treatment were also monitored with open field, sociability, and fear-conditioning tests and underwent in vivo (1)H MR imaging on a 9.4T MR system after NMDA-induced spasms. The acquired (1)H MR spectra were quantified using LC model. RESULTS: Single-dose BHB pretreatment had no effect on spasms. In contrast, prolonged pretreatment with BHB significantly delayed the onset and decreased the frequency of spasms. In addition, randomized prolonged BHB treatment resulted in a significant reduction in number of spasms at P15. BHB treatment had no significant effect on motor activities, but significantly decreased the interactions with strangers and increased the contextual memory. On MR spectroscopic analysis of randomized prolonged BHB-treated rats at 24h after the cluster of spasms, the elevation of GABA, glutamine, glutamate, total creatine, macromolecule-plus lipids, and N-acetylaspartate levels after spasms were significantly attenuated by randomized BHB treatment (p<0.05). SIGNIFICANCE: Prolonged administration of BHB directly suppresses development of spasms in a rat model of IS with acute stabilization of brain metabolites. Additionally, BHB appears to decrease the interests to other rats and improve memory responses.

The GIRK2 subunit is involved in IS-like seizures induced by GABA(B) receptor agonists.

OBJECTIVE: Infantile spasms (or IS) is a catastrophic childhood epilepsy that is particularly prevalent in children with Down syndrome. Previously, we have shown that the Ts65Dn (Ts) mouse model of Down syndrome is a useful substrate upon which to develop an animal model of infantile spasms. Specifically, the Ts mouse is exquisitely sensitive to the electroencephalography (EEG) and behavioral effects of γ-aminobutyric acid (GABA) B receptor (GABA(B)R) agonists with a resultant phenotype that bears behavioral, EEG, and pharmacologic semblance to infantile spasms in humans. The G protein-coupled inward rectifying potassium channel subunit 2 (GIRK2) gene, KCNJ6, is overexpressed in Ts mice, and the GABA(B)R-mediated GIRK2 current is significantly increased in these mutant animals as well. Therefore, we formulated the hypothesis that the GIRK2 channel plays a significant role in the behavioral (measured by acute extensor spasms quantification) and EEG (measured by the electrodecremental response duration) phenotype induced in the Ts mice by GABA(B)R agonists. METHODS: GIRK2(-/-), (+/-), and (+/+) mice were treated with γ-butyrolactone (GBL), a pro-drug of the GABA(B)R agonist γ-hydroxybutyric acid, and the specific GABA(B)R agonist baclofen (BAC) under continuous EEG monitoring. These drugs induce epileptiform bursts, extensor spasms, and an electrodecremental response (EDR) in Ts mice at low doses, and in wild-type mice at high doses. A dose-response curve was ascertained with two treatment groups: GBL (100, 200, and 400 mg/kg) and BAC (4, 8, 12, and 16 mg/kg). We determined the baseline, the presence and duration of electrodecremental epochs (EDEs), and quantified acute epileptic extensor spasms. RESULTS: Analysis of EEG and behavior of GIRK2(-/-), (+/-), and (+/+) mice after treatment with GABA(B)R agonists and antagonists, indicate that GIRK2(-/-) mice are highly resistant to GABA(B)R agonist-induced EEG and behavioral changes. SIGNIFICANCE: These data increase the possibility that GIRK2 channel function plays a major role in the genesis of infantile spasms.

A potential effect of ganaxolone in an animal model of infantile spasms.

RATIONALE: Infantile spasms (IS), a devastating epileptic encephalopathy of infancy, involve various etiologies associated with an unknown underlying common pathophysiology. The efficacy of adrenocorticotropic hormone (ACTH) as an IS therapy suggests a role for steroid hormones in treating IS. This study used an animal model of IS to test the efficacy of ganaxolone, a synthetic neurosteroid, promoting tonic GABAA inhibition. METHODS: The model of cryptogenic IS used in this study involved prenatal priming of rats with betamethasone (0.4 mg/kg i.p. at 08:30 and 18:30) on gestational day 15. To test the acute effects of ganaxolone, rats were pretreated with ganaxolone (10, 25, or 50mg/kg i.p.) or vehicle (ß-cyclodextrin, i.p.) 30 min prior to N-methyl-d-aspartate (NMDA)-induced spasms at postnatal day 15 (P15). To mimic human conditions, another group of rats was randomly divided and repeatedly treated with ganaxolone (20mg/kg at 9:00 and 18:00 from P13-15) or vehicle after experiencing NMDA-triggered spasms at P12. Additional spasms were triggered on P13 and P15. We determined latency to the onset of spasms and the total number of spasms per 90-min observation period after the trigger at P15. On P19 and P21, behavioral tests were performed in rats with randomized repeated treatments. RESULTS: The 25mg/kg and 50mg/kg doses of ganaxolone significantly delayed the onset of spasms compared with the controls, and significantly decreased the number of spasms or suppressed their incidence. Ganaxolone had significant side effects in terms of sedation: all animals with the 50mg/kg dose were sleeping during the test. Randomized ganaxolone treatment for 3 days also significantly delayed the onset and decreased the number of spasms triggered by NMDA on P15, and decreased exploratory behavior after multiple NMDA triggered spasms. CONCLUSION: Ganaxolone significantly suppresses the development of spasms in the rat model of cryptogenic IS. This synthetic neurosteroid active in an animal model of IS might contribute to the current armamentarium to treat human IS.

Proteomic analysis on infantile spasm and prenatal stress.

Infantile spasms (IS) are an age-dependent epileptic encephalopathy with severe cognitive dysfunction. Prenatal stress (PS) has been reported to increase the risk for IS through clinical and animal studies. We aim to investigate the mechanism of brain damage caused by IS and the effect of PS. Animals were divided into 4 groups: PS-spasm model, PS-saline control, NS-spasm model, and saline control. N-methyl-d-aspartate (NMDA) was used to induce spasm and swimming in cold water was used to induce PS. A proteomics-based approach was used to compare the NS-spasm model vs. saline control, and PS-spasm model vs. NS-spasm model. Gel image analysis was followed by mass spectrometric protein identification and bioinformatics analysis. We observed an increased spasm frequency (t=8.65, P<0.001), and a shorter latency period (t=3.96, P<0.001) in the PS-spasm model vs. the NS-spasm model. In the NS-spasm model vs. saline control, the main differentially expressed proteins were CFL1, PKM2, PRPS2, DLAT, CKB, DPYSL3, and SNAP25. In the PS-spasm model vs. NS-spasm model, MDH1 and YWHAZ were differentially expressed. YWHAZ was directly connected with CFL1 in protein networks. YWHAZ and CFL1 were further validated by Western blot analysis. The biological function of differentially expressed proteins indicates the pathogenesis of IS maybe relevant to energy metabolism, brain development, and neural remodeling. PS aggravated seizures in the NMDA-induced spasm model, YWHAZ, and CFL1 may be involved.