Experimental animals’ seizures are often defined arbitrarily based on duration, which may lead to misjudgement of the syndrome and failure to develop a cure. We employed a functional definition of seizures based on the clinical practice of observing epileptiform electrocorticography and simultaneous ictal behaviour, and examined post-traumatic epilepsy induced in rats by rostral parasagittal fluid percussion injury and epilepsy patients evaluated with invasive monitoring. We showed previously that rostral parasagittal fluid percussion injury induces different types of chronic recurrent spontaneous partial seizures that worsen in frequency and duration over the months post injury. However, a remarkable feature of rostral parasagittal fluid percussion injury is the occurrence, in the early months post injury, of brief (<2 s) focal, recurrent and spontaneous epileptiform electrocorticography events (EEEs) that are never observed in sham-injured animals and have electrographic appearance similar to the onset of obvious chronic recurrent spontaneous partial seizures. Simultaneous epidural-electrocorticography and scalp-electroencephalography recordings in the rat demonstrated that these short EEEs are undetectable by scalp electrocorticography. Behavioural analysis performed blinded to the electrocorticography revealed that (i) brief EEEs lasting 0.8–2 s occur simultaneously with behavioural arrest; and (ii) while behavioural arrest is part of the rat's behavioural repertoire, the probability of behavioural arrest is greatly elevated during EEEs. Moreover, spectral analysis showed that EEEs lasting 0.8–2 s occurring during periods of active behaviour with dominant theta activity are immediately followed by loss of such theta activity. We thus conclude that EEEs lasting 0.8–2 s are ictal in the rat. We demonstrate that the assessment of the time course of fluid percussion injury-induced epileptogenesis is dramatically biased by the definition of seizure employed, with common duration-based arbitrary definitions resulting in artificially prolonged latencies for epileptogenesis. Finally, we present four human examples of electrocorticography capturing short (<2 s), stereotyped, neocortically generated EEEs that occurred in the same ictal sites as obvious complex partial seizures, were electrographically similar to rat EEEs and were not noted during scalp electroencephalography. When occurring in the motor cortex, these short EEEs were accompanied by ictal behaviour detectable with simultaneous surface electromyography. These data demonstrate that short (<2 s) focal recurrent spontaneous EEEs are seizures in both rats and humans, that they are undetectable by scalp electroencephalography, and that they are typically associated with subtle and easily missed behavioural correlates. These findings define the earliest identifiable markers of progressive post-traumatic epilepsy in the rat, with implications for mechanistic and prophylactic studies, and should prompt a re-evaluation of the concept of post-traumatic silent perio...

While past neuroimaging methods have contributed greatly to our understanding of brain function after traumatic brain injury (TBI), resting state functional MRI (rsfMRI) connectivity methods have more recently provided a far more unbiased approach with which to monitor brain circuitry compared to task-based approaches. However, current knowledge on the physiologic underpinnings of the correlated blood oxygen level dependent signal, and how changes in functional connectivity relate to reorganizational processes that occur following injury is limited. The degree and extent of this relationship remain to be determined in order that rsfMRI methods can be fully adapted for determining the optimal timing and type of rehabilitative interventions that can be used post-TBI to achieve the best outcome. Very few rsfMRI studies exist after experimental TBI and therefore we chose to acquire rsfMRI data before and at 7, 14 and 28 days after experimental TBI using a well-known, clinically-relevant, unilateral controlled cortical impact injury (CCI) adult rat model of TBI. This model was chosen since it has widespread axonal injury, a well-defined time-course of reorganization including spine, dendrite, axonal and cortical map changes, as well as spontaneous recovery of sensorimotor function by 28 d post-injury from which to interpret alterations in functional connectivity. Data were co-registered to a parcellated rat template to generate adjacency matrices for network analysis by graph theory. Making no assumptions about direction of change, we used two-tailed statistical analysis over multiple brain regions in a data-driven approach to access global and regional changes in network topology in order to assess brain connectivity in an unbiased way. Our main hypothesis was that deficits in functional connectivity would become apparent in regions known to be structurally altered or deficient in axonal connectivity in this model. The data show the loss of functional connectivity predicted by the structural deficits, not only within the primary sensorimotor injury site and pericontused regions, but the normally connected homotopic cortex, as well as subcortical regions, all of which persisted chronically. Especially novel in this study is the unanticipated finding of widespread increases in connection strength that dwarf both the degree and extent of the functional disconnections, and which persist chronically in some sensorimotor and subcortically connected regions. Exploratory global network analysis showed changes in network parameters indicative of possible acutely increased random connectivity and temporary reductions in modularity that were matched by local increases in connectedness and increased efficiency among more weakly connected regions. The global network parameters: shortest path-length, clustering coefficient and modularity that were most affected by trauma also scaled with the severity of injury, so that the corresponding regional measures were correlated to the injury severity most notably at 7 and 14 days and esp...

The current dogma to explain the extent of injury related changes following rodent controlled cortical impact (CCI) injury is a focal injury with limited axonal pathology. However, there is in fact good, published histologic evidence to suggest that axonal injury is far more widespread in this model than generally thought. One possibility that might help to explain this is the often-used region-of-interest data analysis approach taken by experimental traumatic brain injury (TBI) diffusion tensor imaging (DTI) or histologic studies that might miss more widespread damage, when compared to the whole brain, statistically robust method of tract based analysis used more routinely in clinical research. To determine the extent of DTI changes in this model, we acquired in vivo DTI data before and at 1 and 4 weeks after CCI injury in 17 adult, male rats and analyzed parametric maps of fractional anisotropy (FA), axial, radial and mean diffusivity (AD, RD, MD), tensor mode (MO), and fiber tract density (FTD) using tract based spatial statistics. Contusion volume was used as a surrogate marker of injury severity and as a covariate for investigating severity dependence of the data. Mean fiber tract length was also computed from seeds in the cortical spinal tract regions. In parallel experiments (n=3–5/group) we investigated corpus callosum neurofilaments and demyelination using immunohistochemistry (IHC) at 3 days and 6 weeks, callosal tract patency using dual-label retrograde tract-tracing at 5 weeks, and the contribution of gliosis to DTI parameter maps using GFAP IHC at 4 weeks post-injury. The data show widespread ipsilateral regions of significantly reduced FA at 1 week post-injury, driven by temporally changing values of AD, RD and MD that persist to 4 weeks. Demyelination, retrograde label tract loss, and reductions in MO (tract degeneration) and FTD were shown to underpin these data. Significant FA increases occurred in subcortical and corticospinal tract regions that were spatially distinct from regions of FA decrease, grossly affected gliotic areas and from MO changes. However, there was good spatial correspondence between regions of increased FA and areas of increased FTD and mean fiber length. We discuss these widespread changes in DTI parameters in terms of axonal degeneration and potential reorganization, with reference to a resting state fMRI companion paper (Harris et al, 2016, Exp. Neurol.227:124–138) that demonstrated altered functional connectivity data acquired from the same rats used in this study.

SUMMARY A club-shaped, tachykinin-immunopositive structure first described nearly two decades ago in the commissural ganglion (CoG) of three species of decapod crustaceans has remained enigmatic, as its function is unknown. Here, we use a combination of anatomical, mass spectrometric and electrophysiological techniques to address this issue in the crab Cancer productus. Immunohistochemistry using an antibody to the vertebrate tachykinin substance P shows that a homologous site exists in each CoG of this crab. Confocal microscopy reveals that its structure and organization are similar to those of known neuroendocrine organs. Based on its location in the anterior medial quadrant of the CoG, we have named this structure the anterior commissural organ (ACO). Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry shows that the ACO contains the peptide APSGFLGMRamide,commonly known as Cancer borealis tachykinin-related peptide Ia(CabTRP Ia). Using the same technique, we show that CabTRP Ia is also released into the hemolymph. As no tachykinin-like labeling is seen in any of the other known neuroendocrine sites of this species (i.e. the sinus gland, the pericardial organ and the anterior cardiac plexus), the ACO is a prime candidate to be the source of CabTRP Ia present in the circulatory system. Our electrophysiological studies indicate that one target of hemolymph-borne CabTRP Ia is the foregut musculature. Here, no direct CabTRP Ia innervation is present, yet several gastric mill and pyloric muscles are nonetheless modulated by hormonally relevant concentrations of the peptide. Collectively,our findings show that the C. productus ACO is a neuroendocrine organ providing hormonal CabTRP Ia modulation to the foregut musculature. Homologous structures in other decapods are hypothesized to function similarly.

The use of electrocorticography (ECoG) with etiologically realistic epilepsy models promises to facilitate the discovery of better anti-epileptic drugs (AEDs). However, this novel approach is labor intensive, and must be optimized. To this end, we employed rostral parasaggital fluid percussion injury (rpFPI) in the adolescent rat, which closely replicates human contusive closed head injury and results in posttraumatic epilepsy (PTE). We systematically examined variables affecting the power to detect antiepileptic effects by ECoG and used a non-parametric bootstrap strategy to test several different statistics, study designs, statistical tests, and impact of nonresponders. We found that logarithmically transformed data acquired in repeated-measures experiments provided the greatest statistical power to detect decreases in seizure frequencies of pre-clinical interest with just 8 subjects and with up to ~40% non-responders. We then used this optimized design to study the antiepileptic effects of acute exposure to halothane, and chronic (1 week) exposures to carbamazepine (CBZ) and valproate (VPA) one month post-injury. While CBZ was ineffective in all animals, VPA induced, during treatment, a progressive decrease in seizure frequency in animals primarily suffering from non-spreading neocortical seizures, but was ineffective in animals with high frequency of spreading seizures. Halothane powerfully blocked all seizure activity. The data show that rpFPI and chronic ECoG can conveniently be employed for evaluation of AEDs, suggest that VPA may be more effective than CBZ to treat some forms of PTE and support the theory that pharmacoresistance may depend on the severity of epilepsy. The data also demonstrate the utility of chronic exposures to experimental drugs in preclinical studies and highlight the need for greater attention to etiology in clinical studies of AEDs.

Objective Posttraumatic epilepsy is prevalent, often difficult to manage, and currently cannot be prevented. While cooling is broadly neuroprotective, cooling-induced prevention of chronic spontaneous recurrent seizures has never been demonstrated. We examined the effect of mild passive focal cooling of the perilesional neocortex on the development of neocortical epileptic seizures after head injury in the rat. Methods Rostral parasagittal fluid percussion injury in rats reliably induces a perilesional, neocortical epileptic focus within weeks after injury. Epileptic seizures were assessed by 5-electrode video-electrocorticography (ECoG) 2–16 weeks post-injury. Focal cooling was induced with ECoG headsets engineered for calibrated passive heat dissipation. Pathophysiology was assessed by GFAP immunostaining, cortical sclerosis, gene expression of inflammatory cytokines IL-1α and IL-1β, and ECoG spectral analysis. All animals were formally randomized to treatment groups and data were analyzed blind. Results Cooling by 0.5–2°C inhibited the onset of epileptic seizures in a dose dependent fashion. The treatment induced no additional pathology or inflammation, and normalized the power spectrum of stage N2 sleep. Cooling by 2°C for 5.5 weeks beginning 3 days after injury virtually abolished ictal activity. This effect persisted through the end of the study, over ten weeks after cessation of cooling. Rare remaining seizures were shorter than in controls. Interpretation These findings demonstrate potent and persistent prevention and modification of epileptic seizures after head injury with a cooling protocol that is neuroprotective, compatible with the care of head-injury patients, and conveniently implemented. The required cooling can be delivered passively without Peltier cells or electrical power.

Astrocytic inwardly rectifying K(+) currents (I(KIR)) have an important role in extracellular K(+) homeostasis, which influences neuronal excitability, and serum extravasation has been linked to impaired K(IR)-mediated K(+) buffering and chronic hyperexcitability. Head injury induces acute impairment in astroglial membrane I(KIR) and impaired K(+) buffering in the rat hippocampus, but chronic spontaneous seizures appear in the perilesional neocortex--not the hippocampus--in the early weeks to months after injury. Thus we examined astrocytic K(IR) channel pathophysiology in both neocortex and hippocampus after rostral parasaggital fluid percussion injury (rpFPI). rpFPI induced greater acute serum extravasation and metabolic impairment in the perilesional neocortex than in the underlying hippocampus, and in situ whole cell recordings showed a greater acute loss of astrocytic I(KIR) in neocortex than hippocampus. I(KIR) loss persisted through 1 mo after injury only in the neocortical epileptic focus, but fully recovered in the hippocampus that did not generate chronic seizures. Neocortical cell-attached recordings showed no loss or an increase of I(KIR) in astrocytic somata. Confocal imaging showed depletion of KIR4.1 immunoreactivity especially in processes--not somata--of neocortical astrocytes, whereas hippocampal astrocytes appeared normal. In naïve animals, intracortical infusion of serum, devoid of coagulation-mediating thrombin activity, reproduces the effects of rpFPI both in vivo and at the cellular level. In vivo serum infusion induces partial seizures similar to those induced by rpFPI, whereas bath-applied serum, but not dialyzed albumin, rapidly silenced astrocytic K(IR) membrane currents in whole cell and cell-attached patch-clamp recordings in situ. Thus both acute impairment in astrocytic I(KIR) and chronic spontaneous seizures typical of rpFPI are reproduced by serum extravasation, whereas the chronic impairment in astroglial I(KIR) is specific to the neocortex that develops the epileptic focus.

Carisbamate (CRS) exhibits broad acute anticonvulsant activity in conventional anticonvulsant screens, genetic models of absence epilepsy and audiogenic seizures, and chronic spontaneous motor seizures arising after chemoconvulsant-induced status epilepticus. In add-on phase III trials with pharmacoresistant patients CRS induced Ͻ30% average decreases in partial-onset seizure frequency. We assessed the antiepileptogenic and antiepileptic performance of subchronic CRS administration on posttraumatic epilepsy (PTE) induced by rostral parasaggital fluid percussion injury (rpFPI), which closely replicates human contusive closed head injury. Studies were blind and randomized, and treatment effects were assessed on the basis of sensitive electrocorticography (ECoG) recordings. Antiepileptogenic effects were assessed in independent groups of control and CRS-treated rats, at 1 and 3 months postinjury, after completion of a 2-week prophylactic treatment initiated 15 min after injury. The antiepileptic effects of 1-week CRS treatments were assessed in repeated measures experiments at 1 and 4 months postinjury. The studies were powered to detect ϳ50 and ϳ40% decreases in epilepsy incidence and frequency of seizures, respectively. Drug/vehicle treatment, ECoG analysis, and [CRS] plasma determination all were performed blind. We detected no antiepileptogenic and an equivocal transient antiepileptic effects of CRS despite [CRS] plasma comparable with or higher than levels attained in previous preclinical and clinical studies. These findings contrast with previous preclinical data demonstrating large efficacy of CRS, but agree with the average effect of CRS seen in clinical trials. The data support the use of rpFPI-induced PTE in the adolescent rat as a model of pharmacoresistant epilepsy for preclinical development.