Evaluation of a multiplex-qPCR for paediatric pleural empyema-An observational study in hospitalised children.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Therapeutic potential of stilbenes in neuropsychiatric and neurological disorders: A comprehensive review of preclinical and clinical evidence.

Neuropsychiatric disorders are anticipated to be a leading health concern in the near future, emphasizing an outstanding need for the development of new effective therapeutics to treat them. Stilbenes, with resveratrol attracting the most attention, are an example of multi-target compounds with promising therapeutic potential for a broad array of neuropsychiatric and neurological conditions. This review is a comprehensive summary of the current state of research on stilbenes in several neuropsychiatric and neurological disorders such as depression, anxiety, schizophrenia, autism spectrum disorders, epilepsy, traumatic brain injury, and neurodegenerative disorders. We describe and discuss the results of both in vitro and in vivo studies. The majority of studies concentrate on resveratrol, with limited findings exploring other stilbenes such as pterostilbene, piceatannol, polydatin, tetrahydroxystilbene glucoside, or synthetic resveratrol derivatives. Overall, although extensive preclinical studies show the potential benefits of stilbenes in various central nervous system disorders, clinical evidence on their therapeutic efficacy is largely missing.

Seizure progression is slowed by enhancing neurosteroid availability in the brain of epileptic rats.

Trilostane is a 3ß-hydroxysteroid dehydrogenase/Δ5-4 isomerase inhibitor able to produce a manyfold increase in brain levels of various neurosteroids, including allopregnanolone. We previously found that treatment with trilostane can slow down epileptogenesis in the kainic acid (KA) model of temporal lobe epilepsy. It is unknown whether trilostane may have a similar effect on the progression of epilepsy severity, as observed in KA-treated rats. Consequently, we investigated the effects of trilostane (50 mg/kg/day, 1 week) in epileptic rats, given 64 days after KA administration. Seizures were monitored by video-electrocorticographic recordings before and during the treatment with trilostane or vehicle (sesame oil), and neurosteroid levels were measured in serum and cerebral tissue using liquid chromatography-electrospray tandem mass spectrometry after treatment. Pregnenolone sulfate, pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone peripheral levels were massively increased by trilostane. With the only exception of hippocampal pregnenolone sulfate, the other neurosteroids augmented in both the neocortex and hippocampus. Only pregnanolone levels were not upregulated by trilostane. As expected, a significant increase in the seizure occurrence was observed in rats receiving the vehicle, but not in the trilostane group. This suggests that the increased availability of neurosteroids produced a disease-modifying effect in the brain of epileptic rats.

Evaluation of TIB Molbiol LightMix® assays for detection of Mycoplasma genitalium and key resistance mutations for macrolides and fluoroquinolones.

The LightMix® Modular Mycoplasma Macrolide and LightMix® Modular parC Fluoroquinolone Resistance assays (TIB Molbiol) were evaluated using sequential Mycoplasma genitalium positive (n = 125) and negative (n = 93) clinical samples. Results were compared to the results of an established commercial assay (ResistancePlus MG assay, SpeeDx Pty Ltd) or Sanger sequencing (for parC). Detection of M. genitalium by the TIB Molbiol assay had a high agreement with the reference assay, with a positive percent agreement (PPA) of 97.6 [95% confidence interval (CI): 93.1-99.5] and negative percent agreement (NPA) of 95.7 (95% CI: 89.5-98.8). From 105 positive samples, macrolide resistance detection had a PPA of 100% (95% CI: 93.7-100) and NPA of 81.3% (95% CI: 67.4-91.1). For the detection of fluroquinolone resistance mutation G248T/S83I or "other mutation" in the quinolone resistance determinant region, from 95 samples there was 100% (95% CI: 86.3-100) sensitivity and 100% (95% CI: 94.5-100) specificity. The understanding of the basis for fluoroquinolone treatment failure is still developing; it is therefore important to use the output of parC-based resistance assays with caution to avoid the inappropriate use of antibiotic therapies, especially considering the limited number of alternative treatments.

Antiepileptogenic effects of trilostane in the kainic acid model of temporal lobe epilepsy.

OBJECTIVE: Epileptogenesis after status epilepticus (SE) has a faster onset in rats treated to reduce brain levels of the anticonvulsant neurosteroid allopregnanolone with the 5α-reductase inhibitor finasteride; however, it still has to be evaluated whether treatments aimed at increasing allopregnanolone levels could result in the opposite effect of delaying epileptogenesis. This possibility could be tested using the peripherally active inhibitor of 3ß-hydroxysteroid dehydrogenase/Δ5-4 isomerase trilostane, which has been shown repeatedly to increase allopregnanolone levels in the brain. METHODS: Trilostane (50 mg/kg) was administered subcutaneously once daily for up to six consecutive days, starting 10 min after intraperitoneal administration of kainic acid (15 mg/kg). Seizures were evaluated by video-electrocorticographic recordings for 70 days maximum, and endogenous neurosteroid levels were assessed by liquid chromatography-electrospray tandem mass spectrometry. Immunohistochemical staining was performed to evaluate the presence of brain lesions. RESULTS: Trilostane did not alter the latency of kainic acid-induced SE onset or its overall duration. When compared to the vehicle-treated group, rats receiving six daily trilostane injections presented a remarkable delay of the first spontaneous electrocorticographic seizure and subsequent tonic-clonic spontaneous recurrent seizures (SRSs). Conversely, rats treated with only the first trilostane injection during SE did not differ from vehicle-treated rats in developing the SRSs. Notably, trilostane did not modify neuronal cell densities or the overall damage in the hippocampus. In comparison to the vehicle group, repeated administration of trilostane significantly decreased the activated microglia morphology in the subiculum. As expected, allopregnanolone and other neurosteroid levels were remarkably increased in the hippocampus and neocortex of rats treated for 6 days with trilostane, but pregnanolone was barely detectable. Neurosteroids returned to basal levels after a week of trilostane washout. SIGNIFICANCE: Overall, these results suggest that trilostane led to a remarkable increase in allopregnanolone brain levels, which was associated with protracted effects on epileptogenesis.

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses.

BACKGROUND: Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. OBJECTIVES: Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. STUDY DESIGN: Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. RESULTS: In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested. CONCLUSIONS: This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

Is the peroxisome proliferator-activated receptor gamma a putative target for epilepsy treatment? Current evidence and future perspectives.

The peroxisome proliferator-activated receptor gamma (PPARγ), which belongs to the family of nuclear receptors, has been mainly studied as an important factor in metabolic disorders. However, in recent years the potential role of PPARγ in different neurological diseases has been increasingly investigated. Especially, in the search of therapeutic targets for patients with epilepsy the question of the involvement of PPARγ in seizure control has been raised. Epilepsy is a chronic neurological disorder causing a major impact on the psychological, social, and economic conditions of patients and their families, besides the problems of the disease itself. Considering that the world prevalence of epilepsy ranges between 0.5% - 1.0%, this condition is the fourth for importance among the other neurological disorders, following migraine, stroke, and dementia. Among others, temporal lobe epilepsy (TLE) is the most common form of epilepsy in adult patients. About 65% of individuals who receive antiseizure medications (ASMs) experience seizure independence. For those in whom seizures still recur, investigating PPARγ could lead to the development of novel ASMs. This review focuses on the most important findings from recent investigations about the potential intracellular PPARγ-dependent processes behind different compounds that exhibited anti-seizure effects. Additionally, recent clinical investigations are discussed along with the promising results found for PPARγ agonists and the ketogenic diet (KD) in various rodent models of epilepsy.

Therapeutic Potential of Mesenchymal Stem Cells in the Treatment of Epilepsy and Their Interaction with Antiseizure Medications.

Epilepsy is a life-threatening neurological disease that affects approximately 70 million people worldwide. Although the vast majority of patients may be successfully managed with currently used antiseizure medication (ASM), the search for alternative therapies is still necessary due to pharmacoresistance in about 30% of patients with epilepsy. Here, we review the effects of ASMs on stem cell treatment when they could be, as expected, co-administered. Indeed, it has been reported that ASMs produce significant effects on the differentiation and determination of stem cell fate. In addition, we discuss more recent findings on mesenchymal stem cells (MSCs) in pre-clinical and clinical investigations. In this regard, their ability to differentiate into various cell types, reach damaged tissues and produce and release biologically active molecules with immunomodulatory/anti-inflammatory and regenerative properties make them a high-potential therapeutic tool to address neuroinflammation in different neurological disorders, including epilepsy. Overall, the characteristics of MSCs to be genetically engineered, in order to replace dysfunctional elements with the aim of restoring normal tissue functioning, suggested that these cells could be good candidates for the treatment of epilepsy refractory to ASMs. Further research is required to understand the potential of stem cell treatment in epileptic patients and its interaction with ASMs.

Antiseizure Effects of Cannabidiol Leading to Increased Peroxisome Proliferator-Activated Receptor Gamma Levels in the Hippocampal CA3 Subfield of Epileptic Rats.

We evaluated the effects of cannabidiol (CBD) on seizures and peroxisome proliferator-activated receptor gamma (PPARγ) levels in an animal model of temporal lobe epilepsy (TLE). Adult male Sprague-Dawley rats were continuously monitored by video-electrocorticography up to 10 weeks after an intraperitoneal kainic acid (15 mg/kg) injection. Sixty-seven days after the induction of status epilepticus and the appearance of spontaneous recurrent seizures in all rats, CBD was dissolved in medium-chain triglyceride (MCT) oil and administered subcutaneously at 120 mg/kg (n = 10) or 12 mg/kg (n = 10), twice a day for three days. Similarly, the vehicle was administered to ten epileptic rats. Brain levels of PPARγ immunoreactivity were compared to those of six healthy controls. CBD at 120 mg/kg abolished the seizures in 50% of rats (p = 0.033 vs. pre-treatment, Fisher's exact test) and reduced total seizure duration (p < 0.05, Tukey Test) and occurrence (p < 0.05). PPARγ levels increased with CBD in the hippocampal CA1 subfield and subiculum (p < 0.05 vs. controls, Holm−Sidák test), but only the highest dose increased the immunoreactivity in the hippocampal CA3 subfield (p < 0.001), perirhinal cortex, and amygdala (p < 0.05). Overall, these results suggest that the antiseizure effects of CBD are associated with upregulation of PPARγ in the hippocampal CA3 region.

Prospective Evaluation of Ghrelin and Des-Acyl Ghrelin Plasma Levels in Children with Newly Diagnosed Epilepsy: Evidence for Reduced Ghrelin-to-Des-Acyl Ghrelin Ratio in Generalized Epilepsies.

Children with epilepsy and identified as responders to antiseizure medications (ASMs) were found to present markedly higher ghrelin plasma levels when compared to drug-resistant patients. However, it was undetermined if this phenotype could be influenced by the ASMs. Here, we prospectively investigated total ghrelin and des-acyl ghrelin (DAG) plasma levels by enzyme-linked immunosorbent assay before and after ASM administration. Inclusion criteria were: (i) subject with a suspicion of epilepsy; (ii) age ranging from 0 to 16 years; and (iii) informed consent signed by parents or caregivers. Exclusion criteria were acute or chronic metabolic disorders with occasional convulsions but without epilepsy. Fifty patients were followed over a period of one year in Italian neuropediatric centers. Apart from a few exceptions, the majority of children were responsive to ASMs. No differences were found in total ghrelin and DAG levels before and after the treatment, but total ghrelin levels were significantly lower in children with generalized epilepsy compared to those with combined focal and generalized epilepsy. Moreover, the ghrelin-to-DAG ratio was also markedly lower in generalized epilepsies compared to all the other types of epilepsy. Finally, ghrelin was unchanged by ASMs, including the first (e.g., carbamazepine), second (levetiracetam), and third (lacosamide) generation of anticonvulsants.

Factors associated with severe respiratory syncytial virus disease in hospitalised children: a retrospective analysis.

BACKGROUND: Early recognition of children at risk of severe respiratory syncytial virus (RSV) lower respiratory tract infection is important as it informs management decisions. We aimed to evaluate factors associated with severe disease among young children hospitalised with RSV infection. METHODS: We conducted a retrospective cohort study of all children <2 years of age hospitalised for RSV lower respiratory tract infection at a single tertiary paediatric hospital over three RSV seasons (January 2017-December 2019). We classified children as having 'moderate' or 'severe' disease based on the level of respiratory intervention and used univariable and multivariable regression models to determine factors associated with severe disease. RESULTS: Of 970 hospitalised children, 386 (40%) were classified as having 'severe' and 584 (60%) as having 'moderate' RSV disease. On multivariable analyses, age <2 months (OR: 2.3, 95% CI 1.6 to 3.3, p<0.0001), prematurity (OR: 1.6, 95% CI 1.1 to 2.4, p=0.02) and RSV-parainfluenza virus type 3 (PIV3) codetection (OR: 2.6, 95% CI 1.05 to 6.5, p=0.04) were independently associated with severe disease. CONCLUSION: Younger age, prematurity and PIV3 codetection were associated with severe RSV disease in children <2 years of age hospitalised with RSV infection. The association between PIV3 and severe RSV disease is a novel finding and warrants further investigation.

Chlamydia trachomatis and Mycoplasma genitalium prevalence and associated factors among women presenting to a pregnancy termination and contraception clinic, 2009-2019.

BACKGROUND: Risk of pelvic inflammatory disease associated with Chlamydia trachomatis and Mycoplasma genitalium is increased after termination of pregnancy (TOP) and may be increased after insertion of intrauterine devices (IUDs). Screening prior to these procedures is recommended only for C. trachomatis. We examined C. trachomatis and M. genitalium prevalence and associated factors among women presenting to a pregnancy termination and contraception service over 10 years. METHODS: Retrospective analysis of clinical data collected from 17 573 women aged 15-45 years in 2009-2019 and for 266 M. genitalium positive women tested for macrolide resistance-associated mutations in 2016-2019. RESULTS: C. trachomatis and M. genitalium prevalence was 3.7% and 3.4%, respectively. In multivariable analyses, shared risk factors were younger age (p<0.001, for both C. trachomatis and M. genitalium), socioeconomic disadvantage (p=0.045 and p=0.008, respectively) and coinfection (p<0.001, for both sexually transmitted infections), with 10.1% of C. trachomatis positive women also positive for M. genitalium. Additional risk factors were earlier year of visit (p=0.001) for C. trachomatis and for M. genitalium residing outside a major city (p=0.013). The proportion of M. genitalium infections tested between 2016 and 2019 with macrolide resistance-associated mutations was 32.7%. CONCLUSIONS: Given the high level of antimicrobial resistance and the prevalence of coinfection, testing C. trachomatis positive women for M. genitalium could be considered in this setting to prevent further spread of resistant infections. Further research is required into the causal link between M. genitalium and pelvic inflammatory disease in women undergoing TOP and IUD insertion.

Antiseizure Effects of Fully Characterized Non-Psychoactive Cannabis sativa L. Extracts in the Repeated 6-Hz Corneal Stimulation Test.

Compounds present in Cannabis sativa L. preparations have recently attracted much attention in the treatment of drug-resistant epilepsy. Here, we screened two olive oil extracts from a non-psychoactive C. sativa variety, fully characterized by high-performance liquid chromatography and gas chromatography. Particularly, hemp oils with different concentrations of terpenes were administered at the same dose of cannabidiol (25 mg/kg/day orally), 1 h before the 6-Hz corneal stimulation test (44 mA). Mice were stimulated once a day for 5 days and evaluated by video-electrocorticographic recordings and behavioral analysis. Neuronal activation was assessed by FosB/ΔFosB immunoreactivity. Both oils significantly reduced the percentage of mice experiencing convulsive seizures in comparison to olive oil-treated mice (p < 0.050; Fisher's exact test), but only the oil enriched with terpenes (K2) significantly accelerated full recovery from the seizure. These effects occurred in the presence of reduced power of delta rhythm, and, instead, increased power of theta rhythm, along with a lower FosB/ΔFosB expression in the subiculum (p < 0.050; Duncan's method). The overall findings suggest that both cannabinoids and terpenes in oil extracts should be considered as potential therapeutic agents against epileptic seizures and epilepsy.

Adding saliva testing to oropharyngeal and deep nasal swab testing increases PCR detection of SARS-CoV-2 in primary care and children.

OBJECTIVE: To compare the concordance and acceptability of saliva testing with standard-of-care oropharyngeal and bilateral deep nasal swab testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in children and in general practice. DESIGN: Prospective multicentre diagnostic validation study. SETTING: Royal Children's Hospital, and two general practices (cohealth, West Melbourne; Cirqit Health, Altona North) in Melbourne, July-October 2020. PARTICIPANTS: 1050 people who provided paired saliva and oropharyngeal-nasal swabs for SARS-CoV-2 testing. MAIN OUTCOME MEASURES: Numbers of cases in which SARS-CoV-2 was detected in either specimen type by real-time polymerase chain reaction; concordance of results for paired specimens; positive percent agreement (PPA) for virus detection, by specimen type. RESULTS: SARS-CoV-2 was detected in 54 of 1050 people with assessable specimens (5%), including 19 cases (35%) in which both specimens were positive. The overall PPA was 72% (95% CI, 58-84%) for saliva and 63% (95% CI, 49-76%) for oropharyngeal-nasal swabs. For the 35 positive specimens from people aged 10 years or more, PPA was 86% (95% CI, 70-95%) for saliva and 63% (95% CI, 45-79%) for oropharyngeal-nasal swabs. Adding saliva testing to standard-of-care oropharyngeal-nasal swab testing increased overall case detection by 59% (95% CI, 29-95%). Providing saliva was preferred to an oropharyngeal-nasal swab by most participants (75%), including 141 of 153 children under 10 years of age (92%). CONCLUSION: In children over 10 years of age and adults, saliva testing alone may be suitable for SARS-CoV-2 detection, while for children under 10, saliva testing may be suitable as an adjunct to oropharyngeal-nasal swab testing for increasing case detection.

COVID-19 public health measures and respiratory viruses in children in Melbourne.

AIM: To describe the epidemiology of respiratory viruses in children before and during the 2020 SARS-CoV-2 pandemic and the relationship to public health measures instituted by the Victorian government. METHODS: Retrospective audit of respiratory viruses at a tertiary paediatric hospital in Melbourne from January 2015 up to week 47, 2020 in children under 18 years of age. The proportion of positive cases in weeks 1-47 in 2015-2019 (period 1) were compared to weeks 1-47, 2020 (period 2), and reviewed in the context of public health restrictions in Victoria. RESULTS: An annual average of 4636 tests were performed in period 1 compared to 3659 tests in period 2. Proportions of positive influenza A virus, influenza B virus, respiratory syncytial virus (RSV) and human parainfluenza virus were significantly reduced in period 2 compared to period 1: 77.3, 89.4, 68.6 and 66.9% reductions, respectively (all P < 0.001). From week 12-47, 2020, 28 893 SARS-CoV-2 tests were performed with a 0.64% positivity rate. Influenza viruses were not detected after week 17, RSV was not detected after week 35. CONCLUSIONS: Strict public health measures and border closures were successful in eliminating community transmission of SARS-CoV-2 in Melbourne. This was associated with a significant reduction in other respiratory virus infections in children. Identifying sustainable and effective ongoing public health interventions to reduce transmission of RSV and influenza could result in reduced morbidity and mortality in children and requires further research.

Rapid detection of human respiratory syncytial virus A and B by duplex real-time RT-PCR.

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.

Evaluation of E-Health Applications for Paediatric Patients with Refractory Epilepsy and Maintained on Ketogenic Diet.

E-health technologies improve healthcare quality and disease management. The aim of this study was to develop a ketogenic diet management app as well as a website about this dietary treatment and to evaluate the benefits of giving caregivers access to various web materials designed for paediatric patients with refractory epilepsy. Forty families participated in the questionnaire survey, from January 2016 to March 2016. All caregivers were exposed to paper-based materials about the ketogenic diet, whereas only 22 received the app, called KetApp, and videos produced by dieticians. Caregivers with free access to web materials were more satisfied than the others with the informative material provided by the centre (p ≤ 0.001, Mann-Whitney test). Indeed, they showed a better attitude towards treatment, and they became more aware of dietary management in comparison to the control group (p ≤ 0.001). Moreover, caregivers provided with web materials were stimulated to pursue the treatment (p = 0.002) and to introduce it to their children and other people (p = 0.001). Additionally, caregivers supplied with web materials were more willing to help other families in choosing the ketogenic diet (p = 0.004). Overall, these findings indicate that web materials are beneficial for caregivers of paediatric patients with refractory epilepsy in our centres. Thus, the use of e-health applications could be a promising tool in the daily aspects of ketogenic diet management, and it is especially of value in the attempt to start or maintain the diet during the ongoing COVID-19 pandemic crisis.

Relationship between Delta Rhythm, Seizure Occurrence and Allopregnanolone Hippocampal Levels in Epileptic Rats Exposed to the Rebound Effect.

Abrupt withdrawal from antiepileptic drugs is followed by increased occurrence of epileptic seizures, a phenomenon known as the "rebound effect". By stopping treatment with levetiracetam (LEV 300 mg/kg/day, n = 15; vs. saline, n = 15), we investigated the rebound effect in adult male Sprague-Dawley rats. LEV was continuously administered using osmotic minipumps, 7 weeks after the intraperitoneal administration of kainic acid (15 mg/kg). The effects of LEV were determined by comparing time intervals, treatments, and interactions between these main factors. Seizures were evaluated by video-electrocorticographic recordings and power band spectrum analysis. Furthermore, we assessed endogenous neurosteroid levels by liquid chromatography-electrospray-tandem mass spectrometry. LEV significantly reduced the percentage of rats experiencing seizures, reduced the seizure duration, and altered cerebral levels of neurosteroids. In the first week of LEV discontinuation, seizures increased abruptly up to 700% (p = 0.002, Tukey's test). The power of delta band in the seizure postictal component was related to the seizure occurrence after LEV withdrawal (r2 = 0.73, p < 0.001). Notably, allopregnanolone hippocampal levels were positively related to the seizure occurrence (r2 = 0.51, p = 0.02) and to the power of delta band (r2 = 0.67, p = 0.004). These findings suggest a role for the seizure postictal component in the rebound effect, which involves an imbalance of hippocampal neurosteroid levels.

Allopregnanolone and Pregnanolone Are Reduced in the Hippocampus of Epileptic Rats, but Only Allopregnanolone Correlates with Seizure Frequency.

BACKGROUND: Neurosteroids modulate epileptic activity by interacting with the γ-aminobutyric acid type A receptor, but their brain levels are still undetermined. OBJECTIVES: We aimed to establish neurosteroid levels in the neocortex and hippocampus by liquid chromatography/mass spectrometry in epileptic rats. METHODS: Kainic acid-treated rats were continuously monitored up to 9 weeks to determine seizure frequency by video electrocorticography (n = 23) and compared to age-matched controls monitored in the same manner (n = 11). RESULTS: Decreased allopregnanolone (-50%; p < 0.05, Mann-Whitney test) and pregnanolone levels (-64%; p < 0.01) were found in the hippocampus, whereas pregnenolone sulfate, pregnenolone, progesterone, and 5α-dihydroprogesterone were nonsignificantly reduced. No changes were found in the neocortex. Moreover, allopregnanolone (but not pregnanolone) levels were positively correlated with seizure frequency (r2 = 0.4606, p < 0.01). CONCLUSION: These findings indicate a selective reduction in hippocampal levels of 3α-reduced neurosteroids. This reduction was partially mitigated by seizures in the case of allopregnanolone.

Augmentation of endogenous neurosteroid synthesis alters experimental status epilepticus dynamics.

Neurosteroids can modulate γ-aminobutyric acid type A receptor-mediated inhibitory currents. Recently, we discovered that the neurosteroids progesterone, 5α-dihydroprogesterone, allopregnanolone, and pregnanolone are reduced in the cerebrospinal fluid of patients with status epilepticus (SE). However, it is undetermined whether neurosteroids influence SE. For this reason, first we evaluated whether the inhibitor of adrenocortical steroid production trilostane (50 mg/kg) could modify the levels of neurosteroids in the hippocampus and neocortex, and we found a remarkable increase in pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone levels using liquid chromatography tandem mass spectrometry. Second, we characterized the dynamics of SE in the presence of the varied neurosteroidal milieu by a single intraperitoneal kainic acid (KA; 15 mg/kg) injection in trilostane-treated rats and their controls. Convulsions started in advance in the trilostane group, already appearing 90 minutes after the KA injection. In contrast to controls, convulsions prevalently developed as generalized seizures with loss of posture in the trilostane group. However, this effect was transient, and convulsions waned 2 hours before the control group. Moreover, electrocorticographic traces of convulsions were shorter in trilostane-treated rats, especially at the 180-minute (P < .001) and 210-minute (P < .01) time points. These findings indicate that endogenous neurosteroids remarkably modulate SE dynamics.