Ultrasonic vocalizations - Novel seizure-related manifestation in rats.

OBJECTIVE: Seizures of frontal or temporal lobe origin can associate with vocalizations in humans. Our objective was to assess whether rats emit specific seizure-related patterns of ultrasonic vocalizations (USVs) during seizures and epileptiform activity. METHODS: Adult male Sprague-Dawley rats were treated with a single administration of pentylenetetrazol (PTZ, 50 mg/kg, i.p.) and monitored with simultaneous USV and video-electroencephalogram recordings for up to 15 min. USVs were detected using a deep learning algorithm (DeepSqueak-Screener) and manually annotated into the 15 previously described subcategories. The number, frequency, duration, sonographic structure, and temporal relationship of the USVs to seizures and epileptiform activity were assessed. RESULTS: A total of 2147 USVs were recorded in 12 rats that expressed a total of 22 PTZ-induced seizures. Of the USVs, 77% were in the 50-kHz range (i.e., appetitive state) and 23% in the 22-kHz ( i.e., aversive state) range. More than a third (37%) of the USVs could be classified into 1 of the 15 call subcategories; the remaining 63% belonged to a novel "multiform" USV category with a complex sonographic structure. Of the 2147 USVs, 23% occurred during the PTZ-induced seizures and 77% during other types of PTZ-induced epileptiform activity. Almost all (19/22) of seizures were associated with USVs. In each rat, the first seizure was always associated with a USV. The shorter the latency to the first USV, the shorter the latency to the onset of the first electrographic seizure (r = 0.995, p < 0.001). The greater the number of USVs, the greater the number of seizures (r = 0.916, p < 0.001) and the longer the total seizure duration in a given rat (r = 0.750, p < 0.05). SIGNIFICANCE: Like in humans, vocalizations are a seizure-related behavioral feature in rats and recording USVs provides a novel noninvasive tool for detecting experimental seizures. Further studies are needed to explore USV occurrence during spontaneous seizures and their potential for screening novel anti-seizure drugs.

Biomarkers for posttraumatic epilepsy.

A biomarker is a characteristic that can be objectively measured as an indicator of normal biologic processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. Biomarker modalities include molecular, histologic, radiographic, or physiologic characteristics. To improve the understanding and use of biomarker terminology in biomedical research, clinical practice, and medical product development, the Food and Drug Administration (FDA)-National Institutes of Health (NIH) Joint Leadership Council developed the BEST Resource (Biomarkers, EndpointS, and other Tools). The seven BEST biomarker categories include the following: (a) susceptibility/risk biomarkers, (b) diagnostic biomarkers, (c) monitoring biomarkers, (d) prognostic biomarkers, (e) predictive biomarkers, (f) pharmacodynamic/response biomarkers, and (g) safety biomarkers. We hypothesize some potential overlap between the reported biomarkers of traumatic brain injury (TBI), epilepsy, and posttraumatic epilepsy (PTE). Here, we tested this hypothesis by reviewing studies focusing on biomarker discovery for posttraumatic epileptogenesis and epilepsy. The biomarker modalities reviewed here include plasma/serum and cerebrospinal fluid molecular biomarkers, imaging biomarkers, and electrophysiologic biomarkers. Most of the reported biomarkers have an area under the receiver operating characteristic curve greater than 0.800, suggesting both high sensitivity and high specificity. Our results revealed little overlap in the biomarker candidates between TBI, epilepsy, and PTE. In addition to using single parameters as biomarkers, machine learning approaches have highlighted the potential for utilizing patterns of markers as biomarkers. Although published data suggest the possibility of identifying biomarkers for PTE, we are still in the early phase of the development curve. Many of the seven biomarker categories lack PTE-related biomarkers. Thus, further exploration using proper, statistically powered, and standardized study designs with validation cohorts, and by developing and applying novel analytical methods, is needed for PTE biomarker discovery.

Acute Non-Convulsive Status Epilepticus after Experimental Traumatic Brain Injury in Rats.

Severe traumatic brain injury (TBI) induces seizures or status epilepticus (SE) in 20-30% of patients during the acute phase. We hypothesized that severe TBI induced with lateral fluid-percussion injury (FPI) triggers post-impact SE. Adult Sprague-Dawley male rats were anesthetized with isoflurane and randomized into the sham-operated experimental control or lateral FPI-induced severe TBI groups. Electrodes were implanted right after impact or sham-operation, then video-electroencephalogram (EEG) monitoring was started. In addition, video-EEG was recorded from naïve rats. During the first 72 h post-TBI, injured rats had seizures that were intermingled with other epileptiform EEG patterns typical to non-convulsive SE, including occipital intermittent rhythmic delta activity, lateralized or generalized periodic discharges, spike-and-wave complexes, poly-spikes, poly-spike-and-wave complexes, generalized continuous spiking, burst suppression, or suppression. Almost all (98%) of the electrographic seizures were recorded during 0-72 h post-TBI (23.2 ± 17.4 seizures/rat). Mean latency from the impact to the first electrographic seizure was 18.4 ± 15.1 h. Mean seizure duration was 86 ± 57 sec. Analysis of high-resolution videos indicated that only 41% of electrographic seizures associated with behavioral abnormalities, which were typically subtle (Racine scale 1-2). Fifty-nine percent of electrographic seizures did not show any behavioral manifestations. In most of the rats, epileptiform EEG patterns began to decay spontaneously on Days 5-6 after TBI. Interestingly, also a few sham-operated and naïve rats had post-operation seizures, which were not associated with EEG background patterns typical to non-convulsive SE seen in TBI rats. To summarize, our data show that lateral FPI-induced TBI results in non-convulsive SE with subtle behavioral manifestations; this explains why it has remained undiagnosed until now. The lateral FPI model provides a novel platform for assessing the mechanisms of acute symptomatic non-convulsive SE and for testing treatments to prevent post-injury SE in a clinically relevant context.

T-cell infiltration into the perilesional cortex is long-lasting and associates with poor somatomotor recovery after experimental traumatic brain injury.

BACKGROUND: T-lymphocyte (T-cell) invasion into the brain parenchyma is a major consequence of traumatic brain injury (TBI). However, the role of T-cells in the post-TBI functional outcome and secondary inflammatory processes is unknown. We explored the dynamics of T-cell infiltration into the cortex after TBI to establish whether the infiltration relates to post-injury functional impairment/recovery and progression of the secondary injury. METHOD: TBI was induced in rats by lateral fluid-percussion injury, and the acute functional impairment was assessed using the neuroscore. Animals were killed between 1-90 d post-TBI for immunohistochemical analysis of T-cell infiltration (CD3), chronic macrophage/microglial reaction (CD68), blood-brain barrier (BBB) dysfunction (IgG), and endophenotype of the cortical injury. Furthermore, the occurrence of spontaneous seizures and spike-and-wave discharges were assessed using video-electroencephalography. RESULTS: The number of T-cells peaked at 2-d post-TBI, and then dramatically decreased by 7-d post-TBI (5% of 2-d value). Unexpectedly, chronic T-cell infiltration at 1 or 3 months post-TBI did not correlate with the severity of chronic inflammation (p > 0.05) or BBB dysfunction (p > 0.05). Multiple regression analysis indicated that inflammation and BBB dysfunction is associated with 48% of the perilesional T-cell infiltration even at the chronic time-point (r = 0.695, F = 6.54, p < 0.05). The magnitude of T-cell infiltration did not predict the pathologic endophenotype of cortical injury, but the higher the number of T-cells in the cortex, the poorer the recovery index based on the neuroscore (r = - 0.538, p < 0.05). T-cell infiltration was not associated with the number or duration of age-related spike-and-wave discharges (SWD). Nevertheless, the higher the number of SWD, the poorer the recovery index (r = - 0.767, p < 0.5). CONCLUSIONS: These findings suggest that acute infiltration of T-cells into the brain parenchyma after TBI is a contributing factor to poor post-injury recovery.

Pharmacoresistant temporal lobe epilepsy modifies histamine turnover and H3 receptor function in the human hippocampus and temporal neocortex.

Experiments were designed to evaluate the tissue content of tele-methylhistamine (t-MeHA) and histamine as well as H3 receptor (H3 Rs) binding and activation of the heterotrimeric guanine nucleotide binding αi/o proteins (Gαi/o) coupled to these receptors in the hippocampus and temporal neocortex of patients (n = 10) with pharmacoresistant mesial temporal lobe epilepsy (MTLE). Patients with MTLE showed elevated tissue content of t-MeHA in the hippocampus. Analyses revealed that a younger age at seizure onset was correlated with a higher tissue content of t-MeHA, lower H3 R binding, and lower efficacy of Gαi/o protein activation in the hippocampus. We conclude that the hippocampus shows a reduction in the H3 R function associated with enhanced histamine. In contrast, the temporal neocortex displayed a high efficacy of H3 Rs Gαi/o protein activation that was associated with low tissue contents of histamine and t-MeHA. These results indicate an overactivation of H3 Rs leading to decreased histamine in the temporal neocortex. However, this situation was lessened in circumstances such as a longer duration of epilepsy or higher seizure frequency. It is concluded that decrease in H3 Rs function and enhanced levels of histamine may contribute to the epileptic activity in the hippocampus and temporal neocortex of patients with pharmacoresistant MTLE.

Transcranial focal electrical stimulation reduces the convulsive expression and amino acid release in the hippocampus during pilocarpine-induced status epilepticus in rats.

The aim of the present study was to evaluate the effects of transcranial focal electrical stimulation (TFS) on γ-aminobutyric acid (GABA) and glutamate release in the hippocampus under basal conditions and during pilocarpine-induced status epilepticus (SE). Animals were previously implanted with a guide cannula attached to a bipolar electrode into the right ventral hippocampus and a concentric ring electrode placed on the skull surface. The first microdialysis experiment was designed to determine, under basal conditions, the effects of TFS (300 Hz, 200 µs biphasic square pulses, for 30 min) on afterdischarge threshold (ADT) and the release of GABA and glutamate in the hippocampus. The results obtained indicate that at low current intensities (<2800 µA), TFS enhances and decreases the basal extracellular levels of GABA and glutamate, respectively. However, TFS did not modify the ADT. During the second microdialysis experiment, a group of animals was subjected to SE induced by pilocarpine administration (300 mg/kg, i.p.; SE group). The SE was associated with a significant rise of GABA and glutamate release (up to 120 and 182% respectively, 5h after pilocarpine injection) and the prevalence of high-voltage rhythmic spikes and increased spectral potency of delta, gamma, and theta bands. A group of animals (SE-TFS group) received TFS continuously during 2h at 100 µA, 5 min after the establishment of SE. This group showed a significant decrease in the expression of the convulsive activity and spectral potency in gamma and theta bands. The extracellular levels of GABA and glutamate in the hippocampus remained at basal conditions. These results suggest that TFS induces anticonvulsant effects when applied during the SE, an effect associated with lower amino acid release. This article is part of a Special Issue entitled "Status Epilepticus".

Transcranial focal electrical stimulation reduces seizure activity and hippocampal glutamate release during status epilepticus.

Previously we demonstrated that noninvasive transcranial focal electrical stimulation (TFS) with sub-effective doses of diazepam reduces status epilepticus (SE)-induced neuronal damage. However, it was unclear if this neuroprotective effect is a consequence of the decrease in the glutamate release. The aim of the present study was to evaluate the effects of TFS on γ-Aminobutyric acid (GABA) and glutamate release in the hippocampus during pilocarpine-induced SE. After pilocarpine administration, the rats showed progressive behavioral changes that culminated in SE with a significant increase of GABA and glutamate (95 and 128% respectively), even more evident at the end of the experiment (120 and 182% respectively), 5 hours after pilocarpine injection and was associated with the prevalence of high-voltage rhythmic spikes and increased spectral power in the 4-90 Hz bands. The TFS application during the SE decreased the convulsive expression, the prevalence of high-voltage rhythmic spikes and spectral power in 4-8 Hz and 30-90 Hz bands. These effects were associated with lower release of GABA and glutamate in the hippocampus. These results support the anticonvulsive and neuroprotective effects induced by TFS.

Role of histaminergic system in blood-brain barrier dysfunction associated with neurological disorders.

Blood-brain barrier (BBB) disruption has been associated with several acute and chronic brain disorders such as Alzheimer's disease, Parkinson's disease and epilepsy. This represents a critical situation because damaged integrity of the BBB is related to the influx of immune mediators, plasma proteins and other outside elements from blood to the central nervous system (CNS) that may trigger a cascade of events that leads to neuroinflammation. In this review, evidence that mast cells and the release of factors such as histamine play an important role in the neuroinflammatory process associated with brain disorders such as Alzheimer's disease, Parkinson's disease and epilepsy is presented.

P-glycoprotein contributes to cell membrane depolarization of hippocampus and neocortex in a model of repetitive seizures induced by pentylenetetrazole in rats.

P-glycoprotein (P-gp) has been associated with pharmacoresistance and mechanisms regulating the membrane potential. However, at present it is unknown if P-gp overexpression in brain is associated with changes in membrane depolarization in refractory epilepsy. Experiments were designed to evaluate the membrane depolarization and P-gp overexpression induced by repetitive pentilenetetrazole (PTZ)-induced-seizures. Wistar rats were daily treated with PTZ during 4 to 7 days (PTZ4 and PTZ7 groups), and the brain was used to evaluate membrane potential by in vitro electrophysiological procedures and using bis-oxonol dye, [bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)], a fluorescence dye voltage-sensitive to membrane potentials. Rats with repetitive PTZ-induced seizures demonstrated lower phenytoin-induced anticonvulsant effects, increased number of DiBAC4(3) fluorescence cells and P-gp overexpression in hippocampus and neocortex, as well as augmentation of the induced fEPSP in CA1 field. These changes were more evident in PTZ7 group. Phenytoin or phenytoin plus nimodipine (a P-gp antagonist) avoided the enhanced fEPSP and decreased DiBAC4(3) fluorescence in animals from PTZ4 group. However, in PTZ7 group these effects were evident only when phenytoin was combined with nimodipine. An additional flow cytometry study demonstrated increased intracellular accumulation of DiBAC4(3) in K562 leukemic cells that overexpress MDR-1 and COX-2 genes, and are refractory to specific cytotoxic agents. These results represent the first evidence supporting the notion that brain P-gp overexpression contributes to a progressive seizure-related membranes depolarization in hippocampus and neocortex. Further experiments should be carried out to confirm the role of P-gp on membrane depolarization and epileptogenesis process.