Development of an antiseizure drug screening platform for Dravet syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program.

OBJECTIVE: Dravet syndrome (DS) is a rare but catastrophic genetic epilepsy, with 80% of patients carrying a mutation in the SCN1A gene. Currently, no antiseizure drug (ASD) exists that adequately controls seizures. In the clinic, individuals with DS often present first with a febrile seizure and, subsequently, generalized tonic-clonic seizures that can continue throughout life. To facilitate the development of ASDs for DS, the contract site of the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) has evaluated a mouse model of DS using the conditional knock-in Scn1aA1783V/WT mouse. METHODS: Survival rates and temperature thresholds for Scn1aA1783V/WT were determined. Prototype ASDs were administered via intraperitoneal injections at the time-to-peak effect, which was previously determined, prior to the induction of hyperthermia-induced seizures. ASDs were considered effective if they significantly increased the temperature at which Scn1aA1783V/WT mice had seizures. RESULTS: Approximately 50% of Scn1aA1783V/WT survive to adulthood and all have hyperthermia-induced seizures. The results suggest that hyperthermia-induced seizures in this model of DS are highly refractory to a battery of ASDs. Exceptions were clobazam, tiagabine, levetiracetam, and the combination of clobazam and valproic acid with add-on stiripentol, which elevated seizure thresholds. SIGNIFICANCE: Overall, the data demonstrate that the proposed model for DS is suitable for screening novel compounds for the ability to block hyperthermia-induced seizures and that heterozygous mice can be evaluated repeatedly over the course of several weeks, allowing for higher throughput screening.

Chronic scrotal hyperthermia induces azoospermia and severe damage to testicular tissue in mice.

Scrotal hyperthermia leads to altered spermatogenesis due to heat-related oxidative stress. One of the main causes of infertility in men is oxidative stress, which refers to an imbalance in the levels of reactive oxygen species (ROS) and antioxidants. Therefore, this study aimed to evaluate the effects of chronic scrotal hyperthermia on testicular tissue structure, sperm parameters, and oxidative stress in adult mice. Thirty adult NMRI male mice were divided into three groups: Control (n = 10), Sham (n = 10), and Hyperthermia (n = 10). At the end of the study animals were sacrificed for evaluations of biochemical, cellular and histological analysis. The Hyperthermia group revealed a significant reduction in sperm count and weight of testis when compared to the control and sham groups. Also, succinate dehydrogenase (SDH) activity, ROS, ATP production, glutathione disulfide (GSH), tiols metabolism and stereological parameters in the hyperthermia group showed a significant reduction compared to the control and sham groups. Our results also revealed that scrotal hyperthermia significantly increases ROS production, mitochondrial membrane permeability (MMP), malondialdehyde (MDA), oxidized glutathione (GSSG) and apoptotic cells in testicular tissue in the hyperthermia groups in comparison with the control and sham groups. Overall, our result indicated that chronic scrotal hyperthermia causes complete spermatogenic arrest, probably mainly throughout the induction of oxidative stress.

Caffeine Exacerbates Hyperventilation and Reductions in Cerebral Blood Flow in Physically Fit Men Exercising in the Heat.

INTRODUCTION: Caffeine is an exercise performance enhancer widely used by individuals engaged in training or competition under heat-stressed conditions. Caffeine ingestion during exercise in the heat is believed to be safe because it does not greatly affect body temperature responses, heart rate, or body fluid status. However, it remains unknown whether caffeine affects hyperthermia-induced hyperventilation or reductions in the cerebral blood flow index. We tested the hypothesis that under conditions inducing severe hyperthermia, caffeine exacerbates hyperthermia-induced hyperventilation and reduces the cerebral blood flow index during exercise. METHODS: Using a randomized, single-blind, crossover design, 12 physically active healthy young men (23 ± 2 yr) consumed a moderate dose of caffeine (5 mg·kg-1) or placebo in the heat (37°C). Approximately 60 min after the ingestion, they cycled for ~45 min at a workload equal to ~55% of their predetermined peak oxygen uptake (moderate intensity) until their core temperature increased to 2.0°C above its preexercise baseline level. RESULTS: In both trials, ventilation increased and the cerebral blood flow index assessed by middle cerebral artery mean blood velocity decreased as core temperature rose during exercise (P < 0.05), indicating that hyperthermia-induced hyperventilation and lowering of the cerebral blood flow occurred. When core temperature was elevated by 1.5°C or more (P < 0.05), ventilation was higher and the cerebral blood flow was lower throughout the caffeine trial than the placebo trial (P < 0.05). CONCLUSIONS: A moderate dose of caffeine exacerbates hyperthermia-induced hyperventilation and reductions in the cerebral blood flow index during exercise in the heat with severe hyperthermia.

Scn1a and Cacna1a mutations mutually alter their original phenotypes in rats.

This study aimed to examine the effects of Cacna1a mutation on the phenotype of Scn1a-associated epilepsy in rats. We used rats with an N1417H missense mutation in the Scn1a gene and others with an M251K mutation in the Cacna1a gene. Scn1a/Cacna1a double mutant rats were generated by mating both Scn1a and Cacna1a mutants. We investigated general health and the epileptic phenotype in all these genotypes. The onset threshold of hyperthermia-induced seizures was examined at 5 weeks and spontaneous seizures were monitored using video-EEG recordings from 6 to 12 weeks of age. Scn1a/Cacna1a double mutants showed significantly reduced threshold for hyperthermia-sensitive seizures onset compared with the Scn1a mutants and had absence seizures having 6-7 c/s spike-wave bursts with changes in the spike-wave pattern, whereas Cacna1a mutants had regular 6-7 c/s spike-wave bursts. In Scn1a/Cacna1a double mutants, 6-7 c/s spike-wave bursts were accompanied with eyelid myoclonia and continuously shifting generalized clonic seizures, which were not observed in either Scn1a or Cacna1a mutants. Although a curvature of the spine was observed in rats of all these genotypes, the degree of curvature was more pronounced in Scn1a/Cacna1a double mutants, followed by Cacna1a and Scn1a mutants. Our results indicate that Cacna1a and Scn1a mutations mutually alter their original phenotypes in rats. The phenotype of absence seizures with eyelid myoclonia, generalized clonic seizures, and of spine curvature in the Scn1a/Cacna1a double mutants were similar to that observed in patients with Dravet syndrome.

Cannabidiol inhibits febrile seizure by modulating AMPA receptor kinetics through its interaction with the N-terminal domain of GluA1/GluA2.

Cannabidiol (CBD) is a major phytocannabinoid in Cannabis sativa. CBD is being increasingly reported as a clinical treatment for neurological diseases. Febrile seizure is one of the most common diseases in children with limited therapeutic options. We investigated possible therapeutic effects of CBD on febrile seizures and the underlying mechanism. Use of a hyperthermia-induced seizures model revealed that CBD significantly prolonged seizure latency and reduced the severity of thermally-induced seizures. Hippocampal neuronal excitability was significantly decreased by CBD. Further, CBD significantly reduced the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated evoked excitatory postsynaptic currents (eEPSCs) and the amplitude and frequency of miniature EPSCs (mEPSCs). Furthermore, CBD significantly accelerated deactivation in GluA1 and GluA2 subunits. Interestingly, CBD slowed receptor recovery from desensitization of GluA1, but not GluA2. These effects on kinetics were even more prominent when AMPAR was co-expressed with γ-8, the high expression isoform 8 of transmembrane AMPAR regulated protein (TARPγ8) in the hippocampus. The inhibitory effects of CBD on AMPAR depended on its interaction with the distal N-terminal domain of GluA1/GluA2. CBD inhibited AMPAR activity and reduced hippocampal neuronal excitability, thereby improving the symptoms of febrile seizure in mice. The putative binding site of CBD in the N-terminal domain of GluA1/GluA2 may be a drug target for allosteric gating modulation of AMPAR.

Effect of transient scrotal hyperthermia on human sperm: an iTRAQ-based proteomic analysis.

BACKGROUND: Through this prospective study, we aimed to explore the change of molecular modification after the transient scrotal hyperthermia on human sperm. METHODS: Ten healthy subjects selected with strict screening criteria underwent testicular warming in a 43 °C water bath for 30 min a day for 10 consecutive days. Semen samples were collected 2 weeks before the first heat treatment and 6 weeks after the first heat treatment. Proteins from the samples were labeled with isobaric tags for relative and absolute quantitation and analyzed by two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS: In contrast to the control, of the 3446 proteins identified, 61 proteins were deregulated: 28 were up-regulated and 33 were down-regulated. Approximately 95% of the differentially expressed proteins were found to participate in spermatogenesis, fertilization, or other aspects of reproduction. In particular, the expression of sperm motility and energy metabolism-related proteins AKAP4, SPESP1, ODF1, ODF2, GAPDHS, and ACTRT2, validated by western blotting of the proteins obtained from human and mouse samples, tended to be reduced under scrotal hyperthermia. CONCLUSIONS: The results indicated that the proteins AKAP4, ODF1, ODF2, GAPDHS, SPESP1, and ACTRT2, play an important role in the heat-induced reversible reduction in sperm concentration and motility and have the potential to be the biomarkers and clinical targets for scrotal heat treatment induced male infertility.

Protective effects of maternal administration of curcumin and hesperidin in the rat offspring following repeated febrile seizure: Role of inflammation and TLR4.

Neuroinflammation has a key role in seizure generation and perpetuation in the neonatal period, and toll-like receptor 4 (TLR4) pathway has a prominent role in neuroinflammatory diseases. Administration of antioxidants and targeting TLR4 in the embryonic period may protect rat offspring against the next incidence of febrile seizure and its harmful effects. Curcumin and hesperidin are natural compounds with anti-inflammatory and antioxidant properties and have an inhibitory action on TLR4 receptors. We evaluated the effect of maternal administration of curcumin and hesperidin on infantile febrile seizure and subsequent memory dysfunction in adulthood. Hyperthermia febrile seizure was induced on postnatal days 9-11 on male rat pups with 24 h intervals, in a Plexiglas box that was heated to ~45 °C by a heat lamp. We used enzyme-linked immunosorbent assay, Western blotting, malondialdehyde (MDA), and glutathione (GSH) assessment for evaluation of inflammatory cytokine levels, TLR4 protein expression, and oxidative responses in the hippocampal tissues. For assessing working memory and long-term potentiation, the double Y-maze test and Schaffer collateral-CA1 in vivo electrophysiological recording were performed, respectively Our results showed that curcumin and hesperidin decreased TNF-α, IL-10, and TLR4 protein expression and reversed memory dysfunction. However, they did not provoke a significant effect on GSH content or amplitude and slope of recorded fEPSPs in the hippocampus. In addition, curcumin, but not hesperidin, decreased interleukin-1ß (IL-1ß) and MDA levels. These findings imply that curcumin and hesperidin induced significant protective effects on febrile seizures, possibly via their anti-inflammatory and antioxidant properties and downregulation of TLR4.

Hyperthermia Increases Neurotoxicity Associated with Novel Methcathinones.

Hyperthermia is one of the severe acute adverse effects that can be caused by the ingestion of recreational drugs, such as methcathinones. The effect of hyperthermia on neurotoxicity is currently not known. The primary aim of our study was therefore to investigate the effects of hyperthermia (40.5 °C) on the neurotoxicity of methcathinone (MC), 4-chloromethcathinone (4-CMC), and 4-methylmethcathinone (4-MMC) in SH-SY5Y cells. We found that 4-CMC and 4-MMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) under both hyper- (40.5 °C) and normothermic conditions (37 °C), whereby cells were more sensitive to the toxicants at 40.5 °C. 4-CMC and 4-MMC impaired the function of the mitochondrial electron transport chain and increased mitochondrial formation of reactive oxygen species (ROS) in SH-SY5Y cells, which were accentuated under hyperthermic conditions. Hyperthermia was associated with a rapid expression of the 70 kilodalton heat shock protein (Hsp70), which partially prevented cell death after 6 h of exposure to the toxicants. After 24 h of exposure, autophagy was stimulated by the toxicants and by hyperthermia but could only partially prevent cell death. In conclusion, hyperthermic conditions increased the neurotoxic properties of methcathinones despite the stimulation of protective mechanisms. These findings may be important for the understanding of the mechanisms and clinical consequences of the neurotoxicity associated with these compounds.

Pain-modulating effects of oxytocin in patients with chronic low back pain.

The neuropeptide oxytocin (OT) has been shown to play a modulatory role in nociception. However, analgesic effects of OT in chronic pain conditions remain elusive and the neural underpinnings have not yet been investigated in humans. Here, we conducted an exploratory, randomized, placebo-controlled, cross-over study to examine effects of intranasal OT in male patients suffering from chronic low back pain (CBP) versus healthy controls (HC). N = 22 participants with CBP and 22 HCs were scanned using functional magnetic resonance imaging (fMRI) while they continuously rated either spontaneously occurring back pain or acute thermal pain stimuli applied to the lower back. During heat pain processing we found that OT versus PL attenuated pain intensity ratings and increased BOLD responses in the caudate nucleus of the striatum in CBP versus HCs. Spontaneously experienced pain in contrast to heat pain was associated with activation changes in the medial frontal cortex (MFC) and the anterior cingulate cortex (ACC) as reported in previous studies. However, we did not observe OT effects on spontaneously experienced pain in CBP patients. Overall, our preliminary data may suggest that the striatum is a key structure underlying the pain-modulating effects of OT in patients with chronic pain and adds to the growing evidence linking the neuropeptide to pain modulation in humans. Further studies on neuronal OT effects in larger samples of chronic back pain patients are needed to understand probable mechanisms of OT effects in chronic pain. This article is part of the special issue on Neuropeptides.