Seizure Susceptibility and Sleep Disturbance as Biomarkers of Epileptogenesis after Experimental TBI.

Objectives: We investigated whether seizure susceptibility increases over weeks−months after experimental traumatic brain injury (TBI), and whether seizure susceptibility in rats predicts the development of post-traumatic epilepsy (PTE) or epileptiform activity. We further investigated whether rats develop chronic sleep disturbance after TBI, and whether sleep disturbance parameters­alone or in combination with pentylenetetrazol (PTZ) test parameters­could serve as novel biomarkers for the development of post-traumatic epileptogenesis. Methods: TBI was induced in adult male Sprague-Dawley rats with lateral fluid-percussion injury. Sham-operated experimental controls underwent craniectomy without exposure to an impact force. Seizure susceptibility was tested with a PTZ test (30 mg/kg, intraperitoneally) on day (D) 30, D60, D90, and D180 after TBI (n = 28) or sham operation (n = 16) under video electroencephalogram (vEEG). In the 7th post-injury month, rats underwent continuous vEEG monitoring to detect spontaneous seizures and assess sleep disturbances. At the end of the experiments, rats were perfused for brain histology. Results: In the TBI group, the percentage of rats with PTZ-induced seizures increased over time (adjusted p < 0.05 compared with D30). Combinations of three PTZ test parameters (latency to the first epileptiform discharge (ED), number of EDs, and number of PTZ-induced seizures) survived the leave-one-out validation for differentiating rats with or without epileptiform activity, indicating an area under the receiver operating curve (AUC) of 0.743 (95% CI 0.472−0.992, p = 0.05) with a misclassification rate of 36% on D90, and an AUC of 0.752 (95% CI 0.483−0.929, p < 0.05) with a misclassification rate of 32% on D180. Sleep analysis revealed that the number of transitions to N3 or rapid eye movement (REM) sleep, along with the total number of transitions, was increased in the TBI group during the lights-on period (all p < 0.05). The sleep fragmentation index during the lights-on period was greater in the TBI rats than in sham-operated rats (p < 0.05). A combination of sleep parameters showed promise as diagnostic biomarkers of prior TBI, with an AUC of 0.792 (95% CI 0.549−0.934, p < 0.01) and a misclassification rate of 28%. Rats with epilepsy or any epileptiform activity had more transitions from N3 to the awake stage (p < 0.05), and the number of N3−awake transitions differentiated rats with or without epileptiform activity, with an AUC of 0.857 (95% CI 0.651−1.063, p < 0.01). Combining sleep parameters with PTZ parameters did not improve the biomarker performance. Significance: This is the first attempt to monitor the evolution of seizure susceptibility over months in a well-described rat model of PTE. Our data suggest that assessment of seizure susceptibility and sleep disturbance can provide diagnostic biomarkers of prior TBI and prognostic biomarkers of post-traumatic epileptogenesis.

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.

Activation of D1/D5 receptors ameliorates decreased intrinsic excitability of hippocampal neurons induced by neonatal blockade of N-methyl-d-aspartate receptors.

BACKGROUND AND PURPOSE: Dysregulation of dopaminergic transmission combined with transient hypofunction of N-methyl-d-aspartate receptors (NMDARs) is a key mechanism that may underlie cognitive symptoms of schizophrenia. EXPERIMENTAL APPROACH: Therefore, we aimed to identify electrophysiologic alterations in animals neonatally treated with the NMDA receptor antagonist, MK-801, or with saline solution. KEY RESULTS: Patch-clamp whole-cell recordings from MK-801-treated animals revealed altered passive and active electrophysiologic properties compared with CA1 pyramidal cells from saline-treated animals, including up-regulation of the K+ inward-rectifier conductance and fast-inactivating and slow/non-inactivating K+ currents. Up-regulation of these membrane ionic currents reduced the overall excitability and altered the firing properties of CA1 pyramidal cells. We also explored the capability of cells treated with MK-801 to express intrinsic excitability potentiation, a non-synaptic form of hippocampal plasticity associated with cognition and memory formation. CA1 pyramidal cells from animals treated with MK-801 were unable to convey intrinsic excitability potentiation and had blunted synaptic potentiation. Furthermore, MK-801-treated animals also exhibited reduced cognitive performance in the Barnes maze task. Notably, activation of D1/D5 receptors with SKF-38,393 partially restored electrophysiologic alterations caused by neonatal treatment with MK-801. CONCLUSION AND IMPLICATIONS: Our results offer a molecular and mechanistic explanation based on dysregulation of glutamatergic transmission, in addition to dopaminergic transmission, that may contribute to the understanding of the cognitive deterioration associated with schizophrenia.

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 Downregulation of Novel Hypothalamic Protein Sushi Repeat-Containing Protein X-Linked 2 after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) causes damage to the hypothalamo-hypophyseal axis, leading to endocrine dysregulation in up to 40% of TBI patients. Hence, there is an urgent need to identify non-invasive biomarkers for TBI-associated hypothalamo-hypophyseal pathology. Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel hypothalamic protein expressed in both rat and human brain. Our objective was to investigate the effect of acquired brain injury on plasma SRPX2 protein levels and SRPX2 expression in the brain. We induced severe lateral fluid-percussion injury in adult male rats and investigated changes in SRPX2 expression at 2 h, 6 h, 24 h, 48 h, 72 h, 5 days, 7 days, 14 days, 1 month, and 3 months post-injury. The plasma SRPX2 level was assessed by Western blot analysis. Hypothalamic SRPX2-immunoreactive neuronal numbers were estimated from immunostained preparations. At 2 h post-TBI, plasma SRPX2 levels were markedly decreased compared with the naïve group (area under the curve = 1.00, p < 0.05). Severe TBI caused a reduction in the number of hypothalamic SRPX2-immunoreactive neurons bilaterally at 2 h post-TBI compared with naïve group (5032 ± 527 vs. 9440 ± 351, p < 0.05). At 1 month after severe TBI, however, the brain and plasma SRPX2 levels were comparable between the TBI and naïve groups (p > 0.05). Unsupervised hierarchical clustering using SRPX2 expression differentiated animals into injured and uninjured clusters. Our findings indicate that TBI leads to an acute reduction in SRPX2 protein expression and reduced plasma SRPX2 level may serve as a candidate biomarker of hypothalamic injury.

Propylparaben suppresses epileptiform activity in hippocampal CA1 pyramidal cells in vitro.

Epilepsy is a highly prevalent neurological disorder. Additionally, a percentage of patients do not respond to conventional antiepileptic drugs. Therefore, drugs for epilepsy control are still being developed. In the present study, the effect of propylparaben (PPB) in the epileptiform activity induced by 4-aminopyridine in hippocampal CA1 pyramidal neurons was evaluated using individual recordings in current-clamp mode. Results indicated that PPB suppressed the epileptiform activity in registered neurons. This effect disappeared when PPB was removed from the solution of incubation. In contrast, phenytoin only reduced the firing frequency without abolishing epileptiform activity. Our results indicate that PPB exerts an antiepileptic effect on CA1 pyramidal neurons in vitro. Therefore, PPB may represent an effective antiepileptic compound.

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels.

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (INa) amplitude, causing a hyperpolarizing shift in the inactivation curve of the INa, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (INaP). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner.