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. 2017 Mar 17;8:131.
doi: 10.3389/fphar.2017.00131. eCollection 2017.

Protective Effects of Cannabidiol Against Seizures and Neuronal Death in a Rat Model of Mesial Temporal Lobe Epilepsy

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Free PMC article

Protective Effects of Cannabidiol Against Seizures and Neuronal Death in a Rat Model of Mesial Temporal Lobe Epilepsy

Raquel A Do Val-da Silva et al. Front Pharmacol. .
Free PMC article

Abstract

The present study reports the behavioral, electrophysiological, and neuropathological effects of cannabidiol (CBD), a major non-psychotropic constituent of Cannabis sativa, in the intrahippocampal pilocarpine-induced status epilepticus (SE) rat model. CBD was administered before pilocarpine-induced SE (group SE+CBDp) or before and after SE (group SE+CBDt), and compared to rats submitted only to SE (SE group), CBD, or vehicle (VH group). Groups were evaluated during SE (behavioral and electrophysiological analysis), as well as at days one and three post-SE (exploratory activity, electrophysiological analysis, neuron density, and neuron degeneration). Compared to SE group, SE+CBD groups (SE+CBDp and SE+CBDt) had increased SE latency, diminished SE severity, increased contralateral afterdischarge latency and decreased relative powers in delta (0.5-4 Hz) and theta (4-10 Hz) bands. Only SE+CBDp had increased vertical exploratory activity 1-day post SE and decreased contralateral relative power in delta 3 days after SE, when compared to SE group. SE+CBD groups also showed decreased neurodegeneration in the hilus and CA3, and higher neuron density in granule cell layer, hilus, CA3, and CA1, when compared to SE group. Our findings demonstrate anticonvulsant and neuroprotective effects of CBD preventive treatment in the intrahippocampal pilocarpine epilepsy model, either as single or multiple administrations, reinforcing the potential role of CBD in the treatment of epileptic disorders.

Keywords: animal model; cannabidiol; epilepsy; intrahippocampal pilocarpine; neuroprotection.

Figures

FIGURE 1
FIGURE 1
Cannabidiol (CBD) modulation of SE latency and severity. Rats submitted to CBD before SE induction (SE+CBD, green bar) had increased latency to SE onset (A) and decreased SE severity (B), compared to rats not injected with CBD (SE group, black bar). The indicates difference from SE group, and data are expressed as mean ± standard error.
FIGURE 2
FIGURE 2
CBD action on vertical activity of rats submitted to pilocarpine-induced SE. One day after pilocarpine-induced SE, SE rats had a reduced vertical exploratory activity (measured as number of rises in the rat’s hind limbs), when compared to the controls (VH and CBD), and with SE rats pretreated with CBD (SE+CBDp). Representative images from the activity monitor showing the location of each rise from a VH rat (B), a CBD rat (C), a SE rat (D), a SE+CBDp rat (E), and a SE+CBDt rat (F). The red dots in B–F indicate the places within the activity monitor where the rats rose on its hind limbs. Three days after SE, SE rats had low vertical exploratory activity only when compared to VH group (A). The indicates difference from SE group and data are expressed as mean ± standard error.
FIGURE 3
FIGURE 3
Cannabidiol effects on spontaneous local field potential (LFP) of the contralateral hippocampus during SE. CBD injection prior to SE (SE+CBD group, green bars) increased afterdischarge latency (A), compared to untreated rats (SE group, black bars). Relative power of frequencies 0.5–25 Hz from a representative SE rat (black line) and a SE+CBD rat (green line) and a raw LFP hippocampal recording from the same examples (B). Evaluating the LFP recording in time blocks, SE+CBD had a lower relative power in theta between 40 and 80 min and in the whole SE (C). As for delta oscillation, SE+CBD had a trend toward lower relative power in delta in the first 20 min of SE but no difference in the whole SE recording (D). The indicates difference from SE group, t indicates trend toward difference, and data are expressed as mean ± standard error.
FIGURE 4
FIGURE 4
Relative power in delta and theta frequencies 1 and 3 days after SE induction. CBD pretreatment (SE+CBDp, medium green bars) reduced the relative power in delta, compared to SE group 3 days after SE (black bar) (A; RM ANOVA followed by Sidak post hoc test). There was no difference between SE, SE+CBDp, and SE+CBDt in delta frequency 1 day post-SE and in theta frequency 1 or 3 days post-SE (B). The indicates difference from SE group and data are expressed as mean ± standard error.
FIGURE 5
FIGURE 5
Protective action of CBD over neuronal degeneration 1 day after pilocarpine-induced SE, evaluated by Fluoro-Jade B (FJB) staining. (A–F), representative micrographs from the contralateral (A–C) and ipsilateral (D–F) hilus of a SE rat (A,D), SE+CBDp rat (B,E), and a SE+CBDt rat (C,F). Whereas SE rats present higher number of FJB positive neurons in both hilus (arrows in A,D), CBD treated rats had only a few FJB positive neurons (B,C,E,F). Both the contralateral (G) and ipsilateral (H) hippocampus of SE+CBDp (medium green bars) and SE+CBDt (dark green bars) had a lower number of FJB positive neurons in the hilus and CA3, compared to SE group (black bars; ANOVA followed by Sidak post hoc test). The white bar in F indicates 100 μm and the indicates differences from SE group. Data were presented as mean ± standard error.
FIGURE 6
FIGURE 6
Protective action of CBD over neuronal degeneration 3 days after pilocarpine-induced SE, evaluated by Fluoro-Jade B (FJB) staining. (A–F), representative micrographs from the contralateral (A–C) and ipsilateral (D–F) hilus of a SE rat (A,D), SE+CBDp rat (B,E), and a SE+CBDt rat (C,F). Similar to 1 day evaluation, SE rats presented higher number of FJB positive neurons in both hilus (arrows in A,D), compared to CBD-treated rats (B,C,E,F). SE+CBDp (medium green bars) and SE+CBDt (dark green bars) had a lower number of FJB positive neurons in the contralateral hilus (G), compared to SE group (black bars), with no difference in CA3 and CA1 contralateral (G), or in the ipsilateral hippocampus (H; ANOVA followed by Sidak post hoc test). The white bar in F indicates 100 μm and the indicates differences from SE group. Data were presented as mean ± standard error.
FIGURE 7
FIGURE 7
Cannabidiol effects on hippocampal neuronal survival, 1 day after SE. Neuronal density, estimated in contralateral (A) and ipsilateral (B) hippocampal sections from VH (white bars), CBD (light green bars), SE (black bars), SE+CBDp (medium green bars), and SE+CBDt (dark green bars) groups. All groups had higher neuron density than SE group in the hilus, CA3, and CA1 subfields of contralateral (A) and ipsilateral (B) hippocampus (ANOVA followed by Sidak post hoc test). In the contralateral hippocampus, SE+CBDp group had higher neuron density than CBD and SE+CBDt in CA3 and CA1. SE+CBDp had also higher neuron density than VH group in the contralateral CA1 subfield. The groups VH, CBD, SE+CBDp, and SE+CBDt had also higher neuron density than SE group in the ipsilateral granule cell layer. GCL, granule cell layer; HIL, hilus. The indicates differences from SE group, the indicates difference from SE+CBDt group, the § indicates difference from CBD group, and the # indicates difference from VH group. Data were presented as mean ± standard error.
FIGURE 8
FIGURE 8
Cannabidiol effects on hippocampal neuronal survival, 3 days after SE. Neuronal density, estimated in contralateral (A) and ipsilateral (B) hippocampal sections from VH (white bars), CBD (light green bars), SE (black bars), SE+CBDp (medium green bars), and SE+CBDt (dark green bars) groups. The groups VH, CBD, SE+CBDp, and SE+CBDt had higher neuron density than SE group in contralateral (A) and ipsilateral (B) hilus, CA3, and CA1 subfields (ANOVA followed by Sidak post hoc test). In the contralateral granule cell layer, the groups VH, CBD, and SE+CBDp had higher neuron density than SE, and the groups CBD and SE+CBDp had higher neuron density than SE+CBDt. CBD group had also higher neuron density than SE+CBDt in contralateral CA1. In the ipsilateral granule cell layer (B), the group CBD had higher neuron density than SE, and the group SE+CBDp had higher neuron density than SE and SE+CBDt groups. In CA3, SE+CBDp group had higher neuron density than CBD and SE+CBDt groups, and in CA1 the groups VH, CBD, and SE+CBDp had higher neuron density than SE+CBDt group. GCL, granule cell layer; HIL, hilus. The indicates differences from SE group, the § indicates difference from CBD group, the indicates difference from SE+CBDt group. Data were presented as mean ± standard error.
FIGURE 9
FIGURE 9
Representative micrographs from contralateral (A–O) and ipsilateral (A’–O’) hilus (A–E,A’–E’), CA3 (F–J,F’–J’), and CA1 (K–O,K’–O’) of VH (A,A’,F,F’,K,K’), CBD (B,B’,G,G’,L,L’), SE (C,C’,H,H’,M,M’), SE+CBDp (D,D’,I,I’,N,N’), and SE+CBDt (E,E’,J,J’,O,O’) rats 3 days after SE, submitted to NeuN immunohistochemistry. See the intense neuron loss throughout the hippocampal subfields of a SE rat, both ipsilateral and contralateral to pilocarpine injection, compared to VH and CBD controls. The neuroprotective effect of CBD on SE-induced neuron loss is visible in the SE+CBD groups, as statistically confirmed in Figure 8. HIL, hilus; IPSI, ipsilateral side to pilocarpine injection; CONTRA, contralateral side to pilocarpine injection. The bar in O’ indicates 100 μm.

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