Ketogenic diet-mediated seizure reduction preserves CA1 cell numbers in epileptic Kcna1-null mice: An unbiased stereological assessment.

There is growing evidence for the disease-modifying potential of metabolic therapies, including the ketogenic diet (KD), which is used to treat medically intractable epilepsy. However, it remains unclear whether the KD exerts direct effects on histopathological changes in epileptic brain, or whether the changes are a consequence of diet-induced reduction in seizure activity. Here, we used unbiased stereological techniques to quantify the seizure-induced reduction in cell number in the CA1 region of the hippocampus of epileptic Kcna1-null mice and compared the effects of the KD with that of phenobarbital (PB), a widely employed anti-seizure drug. Our data suggest that the anti-seizure activity of the KD or PB was similar. However, CA1 cell numbers of KD-treated hippocampi were not significantly different from those seen in wild-type (WT) mice, whereas CA1 cell counts in standard diet and PB-treated Kcna1-null mice were 23% and 31% lower than WT animals, respectively. These results support the notion that structural protection of cells may involve more than seizure attenuation, and that the KD engages mechanisms that also promote or restore hippocampal morphological integrity.

Clinical application of cell-free next-generation sequencing for infectious diseases at a tertiary children's hospital.

BACKGROUND: Children affected by infectious diseases may not always have a detectable infectious etiology. Diagnostic uncertainty can lead to prolonged hospitalizations, inappropriately broad or extended courses of antibiotics, invasive diagnostic procedures, and difficulty predicting the clinical course and outcome. Cell-free plasma next-generation sequencing (cfNGS) can identify viral, bacterial, and fungal infections by detecting pathogen DNA in peripheral blood. This testing modality offers the ability to test for many organisms at once in a shotgun metagenomic approach with a rapid turnaround time. We sought to compare the results of cfNGS to conventional diagnostic test results and describe the impact of cfNGS on clinical care in a diverse pediatric population at a large academic children's hospital. METHODS: We performed a retrospective chart review of hospitalized subjects at a tertiary pediatric hospital to determine the diagnostic yield of cfNGS and its impact on clinical care. RESULTS: We describe the clinical application of results from 142 cfNGS tests in the management of 110 subjects over an 8-month study period. In comparison to conventional testing as a reference standard, cfNGS was found to have a positive percent agreement of 89.6% and negative percent agreement of 52.3%. Furthermore, 32.4% of cfNGS results were directly applied to make a clinical change in management. CONCLUSIONS: We demonstrate the clinically utility of cfNGS in the management of acutely ill children. Future studies, both retrospective and prospective, are needed to clarify the optimal indications for testing.

Community-acquired pneumonia in children: cell-free plasma sequencing for diagnosis and management.

Community-acquired pneumonia (CAP) is a common cause of pediatric hospital admission. Empiric antibiotic therapy for hospitalized children with serious CAP now targets the most likely pathogen(s), including those that may demonstrate significant antibiotic resistance. Cell-free plasma next-generation sequencing (CFPNGS) was first made available for Pediatric Infectious Diseases physicians in June 1, 2017, to supplement standard-of-care diagnostic techniques. A retrospective chart review was performed for children hospitalized with CAP between June 1, 2017, and January 22, 2018, to evaluate the impact of CFPNGS. We identified 15 hospitalized children with CAP without other underlying medical conditions for whom CFPNGS was performed. CFPNGS identified a pathogen in 13 of 15 (86%) children compared with 47% for those using standard culture and PCR-based methods alone. Changes in antibiotic management were made in 7 of 15 (47%) of children as a result of CFPNGS.

Ketone bodies mediate antiseizure effects through mitochondrial permeability transition.

OBJECTIVE: Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism. METHODS: We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multielectrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria. RESULTS: KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice. INTERPRETATION: The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.

Anticonvulsant effects of the selective melatonin receptor agonist ramelteon.

OBJECTIVE: The endogenous hormone melatonin has previously been shown to exert anticonvulsant effects in a variety of experimental models. Accordingly, we asked whether ramelteon, a synthetic and selective melatonin receptor agonist, might also possess anticonvulsant and/or antiepileptogenic properties. METHODS: The effects of ramelteon (30 or 100 mg/kg intraperitoneally twice daily for 5 days) were evaluated in two animal models of epilepsy. In the rat rapid kindling model, baseline hippocampal afterdischarge properties, kindling progression, and hippocampal excitability in kindled animals were measured. Anti-ictogenic efficacy was assessed after acute administration in untreated kindled rats. In the spontaneously epileptic Kcna1-null mouse model, we determined seizure frequency and periodicity using continuous video/EEG monitoring over 72 hours. Further, circadian rest-activity rhythms in ramelteon-treated animals were studied with actigraphy. RESULTS: In kindled animals, ramelteon reversed kindling-induced hippocampal excitability; however, it did not modify baseline afterdischarge properties, the progression and establishment of the kindled state in the rapid kindling model. However, in Kcna1-null mice, ramelteon (200 mg/kg/day) significantly attenuated seizure periodicity and frequency and improved circadian rest-activity rhythms compared with control animals. CONCLUSIONS: The selective melatonin receptor agonist ramelteon possesses anticonvulsant properties in a chronic epilepsy model. Our findings provide further support for melatonin receptors being potential novel targets for anticonvulsant drug development.

Ketogenic diet treatment abolishes seizure periodicity and improves diurnal rhythmicity in epileptic Kcna1-null mice.

INTRODUCTION: Seizures are known to perturb circadian rhythms in humans as well as in animal models of epilepsy. However, it is unknown whether treatment of the underlying epilepsy restores normal biologic rhythms. We asked whether: (1) seizure activity is characterized by diurnal rhythmicity, (2) chronically epileptic mice exhibit impaired rest-activity rhythms, and (3) treatment with the anticonvulsant ketogenic diet (KD) improves such perturbations. METHODS: Chronically epileptic Kcna1-null mice were fed either a standard diet (SD) or KD for 4 weeks and subjected to continuous video-EEG (electroencephalography) and actigraphy monitoring for 3-5 days to assess seizure activity and rest-activity cycles. RESULTS: Seizure activity in Kcna1-null mice demonstrated diurnal rhythmicity, peaking at zeitgeber (ZT)2.30 +/- 1.52. Rest-activity rhythms of epileptic mice were significantly disrupted. Whereas locomotor activity for wild-type mice peaked at ZT15.45 +/- 0.28 (ZT14:26-ZT16:51), peak activity of epileptic mice was more unpredictable, occurring over a 12.4 h range (ZT06:33-ZT18:57). In six of nine epileptic mice, peak activity was delayed to ZT17.42 +/- 0.38, whereas peak activity was advanced to ZT10.00 +/- 1.26 in the remaining mice. Treatment with the KD abolished seizure periodicity and restored the rest-activity rhythm to values resembling those of wild-type mice (i.e., activity peaking at ZT16.73 +/- 0.67). CONCLUSIONS: Kcna1-null mice experience seizures with 24-h periodicity and impaired circadian behavior. KD reduces the number and periodicity of seizures and restores normal behavioral rhythms, suggesting that this nonpharmacologic therapy may benefit biologic rhythm disturbances in epileptic patients.