Hippocampal Neuro-Networks and Dendritic Spine Perturbations in Epileptogenesis Are Attenuated by Neuroprotectin D1

<div><p>Purpose</p><p>Limbic epileptogenesis triggers molecular and cellular events that foster the establishment of aberrant neuronal networks that, in turn, contribute to temporal lobe epilepsy (TLE). Here we have examined hippocampal neuronal network activities in the pilocarpine post-status epilepticus model of limbic epileptogenesis and asked whether or not the docosahexaenoic acid (DHA)-derived lipid mediator, neuroprotectin D1 (NPD1), modulates epileptogenesis.</p><p>Methods</p><p>Status epilepticus (SE) was induced by intraperitoneal administration of pilocarpine in adult male C57BL/6 mice. To evaluate simultaneous hippocampal neuronal networks, local field potentials were recorded from multi-microelectrode arrays (silicon probe) chronically implanted in the dorsal hippocampus. NPD1 (570 μg/kg) or vehicle was administered intraperitoneally daily for five consecutive days 24 hours after termination of SE. Seizures and epileptiform activity were analyzed in freely-moving control and treated mice during epileptogenesis and epileptic periods. Then hippocampal dendritic spines were evaluated using Golgi-staining.</p><p>Results</p><p>We found brief spontaneous microepileptiform activity with high amplitudes in the CA1 pyramidal and stratum radiatum in epileptogenesis. These aberrant activities were attenuated following systemic NPD1 administration, with concomitant hippocampal dendritic spine protection. Moreover, NPD1 treatment led to a reduction in spontaneous recurrent seizures.</p><p>Conclusions</p><p>Our results indicate that NPD1 displays neuroprotective bioactivity on the hippocampal neuronal network ensemble that mediates aberrant circuit activity during epileptogenesis. Insight into the molecular signaling mediated by neuroprotective bioactivity of NPD1 on neuronal network dysfunction may contribute to the development of anti-epileptogenic therapeutic strategies.</p></div

Frequency analysis of microepileptiform activity in epileptogenesis.

<p>Frequency bands (delta, theta, beta and gamma) show reduced microepileptiform activity (MEA), except for theta and gamma, in all hippocampal layers, and reduced beta activity in the stratum oriens (OR) and dentate gyrus (DG) compared with controls. Bars indicate means and error bars represent S.E.M. P = p value (two sample t-test). B: Average of values of the frequency power of the signal of MEA (n = 7) showing peaks above 120 Hz synchronized in all hippocampal layers. Stratum radiatum, RAD; pyramidal layer, PYR; power spectral density, PSD.</p

Administration of neuroprotectin D1 during epileptogenesis attenuates micro-epileptiform activity.

<p>A: Hippocampal local field potentials from neuroprotectin D1 (NPD1)- and vehicle-treated mice (vehicle) one-week post-status epilepticus (epileptogenesis). Note NPD1-treated mice display attenuation of micro-epileptiform activity (MEA, below brackets) and reduction of the occurrence high frequency oscillation burst (circle) present in vehicle-treated mice. B: Averaged amplitude of MEA is reduced in all hippocampal layers in NPD1-treated mice (n = 7) compared with vehicle-treated mice (n = 7). NPD1-treated mice show attenuation of the number (C) and duration (D) of MEA compared with vehicle-treated mice. E: High frequency activity (HFA) is modulated by NPD1. Note NPD1-treated mice have a reduction of bursts of hippocampal high frequency oscillations (>200Hz, E) and high frequency activity at 240 Hz (F) compared with vehicle. Bars indicate means, and error bars represent S.E.M. P = p value, two sample t-test. * = p<0.05, *** = p<0.001, OR: stratum oriens, PYR: pyramidal layer; RAD: stratum radiatum; DG: dentate gyrus.</p

Experimental design to evaluate hippocampal aberrant neuronal networks in an epileptogenesis-induced post-status epilepticus model in mice.

<p>A: Algorithm depicting experimental design. Dashed line: control mice; thin line: mice in epileptogenesis non-treatment group; bold line: mice in epileptogenesis or epilepsy groups treated with NPD1 or vehicle. Right: Area of interest for dendritic spine analysis in hippocampal layers (Stratum oriens, SO; Stratum Lacunosum-Moleculare, L-M; and the DG). B: Silicon probe (red line) implanted in dorsal hippocampus. r: rostral; v: ventral; c: caudal; d: dorsal. Below: Representative simultaneous behavior and local field potential (LFP) recordings in freely-moving mice. LFPs were recorded from a silicon probe implanted in the dorsal CA1-dentate gyrus (DG) axis using Multiple Acquisition Processor (MAP) data acquisition system (Plexon). C: Silicon probe with 16 channels in the dorsal hippocampus, showing projections from CA1 pyramidal and granular cells from the DG using Golgi staining and hippocampal layers: stratum oriens (OR), pyramidal layer (PYR), stratum radiatum (RAD), and DG and representative LFPs (right traces). D: Representative ripple-sharp wave complex (circle) from CA1. E: Spontaneous LFP patterns from dorsal hippocampal regions (graphic in the background) of adult mice recorded from silicon probe before (control) and 14 days after SE (PSE) induced by pilocarpine administration. Control: Positive wave in OR associated with negative sharp wave in RAD, dentate spikes in DG, and ripples associated with sharp wave (oval). In PSE: fast ripple (square box); high frequency oscillation (HFO) in DG (circle) associated with positive spike in OR and interictal spikes with opposite phase in CA1 (rectangle). F: Seizures show a high frequency component (black arrow) preceded by an increase in gamma band (box), a reduced theta band. Interictal epileptiform activity with high frequency component (red arrows). G: Screenshot of automatic detection and quantification of spikes from seizures and a burst of HFO (H) using Offline Sorter V3 (Plexon). Rec: recording. I: Representative pictures (1–9) of one dendrite captured using the 100x/oil high magnification objective using Z-stack method of imaging (233–260 frames for each dendrites) at 0.3 μm between each consecutive image.</p

Neuroprotectin D1 induces sustained protection of dendritic spines during epileptogenesis.

<p>A: Representative dendrites from control (naïve) and a mouse at 7 days after status epilepticus (SE) from CA1, stratum radiatum (RAD), dentate gyrus (DG) and outer molecular layer (OM). B: Note loss of number of dendritic spines as a consequence of status epilepticus (PSE, n = 3) compared with control (n = 3). C: High-power light magnification of dendrite profiles of CA1 and dentate gyrus (DG) regions showing dendritic beadings in vehicle-treated mice (n = 4) (arrows) and lower dendritic spine profiles compared with NPD1-treated mice (n = 4) in epileptogenesis D: NPD1-treated mice show a higher number of dendritic spines in the hippocampal layers than vehicle. E: Number of dendritic beadings per dendrites is reduced in NPD1-treated mice compared with vehicle. Bars indicate means, and error bars represent S.E.M. p = p value. *: p<0.05, two sample t-test.</p

Hippocampal micro-epileptiform activities with high amplitude in the CA1 pyramidal-stratum radiatum region during epileptogenesis.

<p>A: Comparative local field potential (LFP) recordings of stratum oriens (OR), pyramidal layer (PYR), stratum radiatum (RAD), and dentate gyrus (DG) in the dorsal hippocampus during spontaneous immobility. Left is from a naïve mouse (control), which displays physiological electrical morphology of dentate spikes (DS) and sharp waves (SW), in contrast with a mouse in epileptogenesis (right) showing global low voltage activity (LVA) and ripples-sharp wave (red oval) associated with a marked reduction of amplitude in RAD. Below left: High frequencies activity (HFA) from DG at 150, 170 and above 215 are increased during LVA from mice in epileptogenesis (n = 7) compared with control mice (n = 7). Below right: representative trace from DG during LVA showing high frequency activity (box). Compare traces signed by brackets from control, epileptogenesis-LVA and epileptogenesis-HFA. PSD: Power spectral density from 100 to 300 Hz band, sec = seconds; mv = millivolts. * = p<0.05, One-way ANOVA); B: Representative micro-epileptiform activity (MEA, red traces) emerging from a LVA (black traces) in OR, PYR, RAD, and DG in epileptogenesis. Note MEA in hippocampal CA1 is characterized by successive spikes after a high amplitude fast spike (arrows) in RAD or initiated by a generalized burst of pathological HFO (pHFO, blue rectangle) followed by a high amplitude of synchronized spikes. Number (C) and duration (D) of micro-epileptiform activity (MEA) increases in mice during epileptogenesis (N = 7) compared with control mice (N = 7) #: p = 0.0001 ##: p = 0.001 two sample t-test. E: Average of square amplitude of LFP samples (n = 17 each group) of each hippocampal layer from periods of immobility in control mice (N = 7) and mice undergoing epileptogenesis (N = 7) during LVA and MEA periods. Note that MEA has a high amplitude in the pyramidal layer (PYR) and stratum radiatum (RAD) compared with LVA and control. Meanwhile LVA amplitude is reduced in RAD compared with control and MEA. Bars indicate means and error bars represent S.E.M.; #p = 0.0001; ##; p = 0.001, ###: p = 0.002 ####; p = 0.007, NS: no significant; one-way ANOVA. sec = seconds; mv = millivolts. OR: stratum oriens, DG: dentate gyrus.</p

Administration of neuroprotectin D1 during epileptogenesis reduces spontaneous recurrent epileptic seizures.

<p>A: Representative local field potential from the CA1-pyramidal layer three weeks after status epilepticus (epilepsy) from mice treated with NPD1 or vehicle during 5 days after status epilepticus. Note that NPD1-treated mice (NPD1) display an attenuation of spontaneous hippocampal epileptic electrical activity compared with vehicle-treated mouse (vehicle). Insets (red boxes from a-d) are traces from the vehicle: (a) initial discharges, followed by (b) synchronized poly-spike activity interrupted by bursts of high frequency discharge; (c) spike waves; and (d) post-ictal phase. Other insets (red boxes from e-g) are traces from NPD1-treated mice: (e) initial discharge; (f) synchronized spikes; and (g) post-ictal phase. B: Cumulative generalized seizures (Racine’s score > stage 3) per day and cumulative probability (C) at the third week after status epileptics are reduced in NPD1-treated mice (n = 5) vs. vehicle-treated mice (n = 6). D: Total cumulative seizures per week and duration of each locomotor seizure (E) are limited in NPD1-treated mice compared with vehicle. F: NPD1-treated mice (n = 4) show a reduced number of epileptic spikes compared with vehicle-treated mice (n = 4). Bars indicate means, and error bars represent S.E.M. p = p value (two sample t-test).</p

Frequency analysis of hippocampal layers in epileptogenesis.

<p>A: Analysis of frequency bands (delta, theta, beta and gamma) from different hippocampal layers during period of immobility in control (n = 7) and in epileptogenesis mice (n = 7). All frequency bands are reduced in epileptogenesis, except for theta in OR and PYR layers. Bars indicate means, and error bars represent S.E.M.; P: p values, two sample t-test. Power spectral density, PSD.</p

Protein phosphorylation in aged animals.

<p>Panel A: Schematic coronal brain diagram showing locations of regions of interest for Western blot analysis in the penumbra (regions 6, 7, and 8). Individual regions of the penumbra (6, 7, 8) were analyzed separately and also pooled together for Panel B. Treatment with NPD1 or DHA did not resulted in significantly different expression of phosphorylated p308 AKT or pGSK (ser9) over saline but resulted in higher levels of S6 (ser235) phosphorylation at 4 h when compared to saline-treated aged animals. Panel C: Representative Western blots of AKT p308, pGSK and pS6 trials in aged animals. Data are mean ± SEM. * indicates significant difference between groups (p<0.05; repeated measures ANOVA followed by Bonferroni tests).</p

Behavioral, histopathology and immunohistochemistry in young rats.

<p>Panel A: Total neurological score at 24, 48, 72 h, and 7 days after MCAo. Panel B: Cortical, subcortical and total infarct volumes on day 7 after stroke. Panel C: Computer-generated MosaiX-processed images of GFAP (green), ED-1 (red), and GFAP/ED-1 double staining at 4 h, 24 h and 7 days after stroke at magnification ×10. Panel D is a schematic coronal brain diagram (bregma level +1.2 mm) showing locations of regions for cell counts in ischemic core (1) and penumbra (2, 3 and 4). Panel E: Number of GFAP-positive astrocytes, ED-1-positive microglia cells, and NeuN-positive neurons at 4, 24 h, and day 7 after onset of stroke. Data are mean ± SEM. *indicates significant differences from saline (p<0.05; repeated measures ANOVA followed by Bonferroni tests).</p