TY - JOUR
AU - P, Wongvravit, Joann
AB - Abstract Anticonvulsant effects of cannabidiol (CBD), a nonpsychoactive cannabinoid, have not been investigated in the juvenile brain. We hypothesized that CBD would attenuate epileptiform activity at an age when the brain first becomes vulnerable to neurotoxicity and social/cognitive impairments. To induce seizures, kainic acid (KA) was injected either into the hippocampus (KAih) or systemically (KAip) on postnatal (P) day 20. CBD was coadministered (KA + CBDih, KA + CBDip) or injected 30 minutes postseizure onset (KA/CBDih, KA/CBDip). Hyperactivity, clonic convulsions, and electroencephalogram rhythmic oscillations were attenuated or absent after KA + CBDih and reduced after KA + CBDip. NeuN immunohistochemistry revealed neuroprotection. Augmented reactive glia number and expression were reversed in CA1 but persisted deep within the dentate hilus. Parvalbumin-positive (PV+) interneurons were reduced in both models, whereas immunolabeling was dramatically increased within ipsilateral and contralateral dendritic/neuropilar fields following KA + CBDih. Cannabinoid receptor 1 (CB1) expression was minimally affected after KAih contrasting elevations observed after KAip. Intracranial coadministration data suggest that CBD has higher efficacy in epilepsy with hippocampal focus rather than when extrahippocampal amygdala/cortical structures are triggered by systemic treatments. Inhibition of surviving PV+ and CB1+ interneurons may be facilitated by CBD implying a protective role in regulating hippocampal seizures and neurotoxicity at juvenile ages. Anticonvulsant, Cannabidiol, Development, Hippocampus, Kainic acid, Seizures INTRODUCTION Cannabinoids have gained medicinal interest worldwide for drug resistant epilepsies and a number of other neurological disorders. They have high-affinity interactions with G-protein-coupled receptors to exert their therapeutic effect via receptor types: CB1 and CB2, which bind to natural (Δ9-tetrahydrocannabinol [THC]) and synthetic cannabinoid receptor agonists (1–3). Endogenous CB1 receptors are responsible for reducing presynaptic neurotransmitter release at excitatory and inhibitory synapses, a negative feedback mechanism (4–7). This negative feedback mechanism appears to underlie neuroprotective effects of the endocannabinoid system following seizures partly due to coupling of N-methyl-D-aspartate (NMDA) receptors to cannabinoid receptors causing abrogation of downstream toxic events such as nitric oxide (NO) overproduction and Zn2+ mobilization (8). Indeed, elevated endocannabinoid responses attenuated kainic acid (KA)-induced seizures and NO synthesis as well as the associated brain injury (8, 9). In contrast, the CB2 receptor is associated with the immune system and anti-inflammatory responses (10). The isomer, cannabidiol (CBD), the major nonpsychoactive constituent of Cannabis sativa, has low affinity to either cannabinoid receptor but nevertheless has anticonvulsant, anti-inflammatory, and neuroprotective properties (2, 3). It can produce a low-affinity inverse agonist action upon CB1 receptors as well as interact with 5HT1A and GABAA receptors to influence neurotransmission (11, 12). Additionally, CBD can reduce voltage-gated Na+ (NaV) channel activity (13). There is a paucity of research on how CBD affects provoked or unprovoked seizure activity in development. A cohort study showed CBD “enriched” plants reduced seizure frequency in drug treatment resistant pediatric epilepsy cases (14). Oral or artinasal treatment of CBD (Epidiolex, GW Pharmaceuticals, Cambridge, UK) had success on its own to reduce seizure frequency or in combination with other anticonvulsants in children with difficult seizures associated with Dravet syndrome (SCN1+ mutation) or Lennox-Gastaut Syndrome (15–17). A recent meta-analysis on the effects of CBD on alterations in hemodynamics showed acute and sustained clinical treatments was safe without adverse side-effects (18). In rat pups or piglets, following hypoxia-induced neonatal ischemia, electroencephalographic (EEG) epileptiform abnormalities and subsequent injury were attenuated in the presence of CBD resulting in short- and long-term neuroprotection (19, 20). In rats, the period of maximal seizure susceptibility coincides with the second and third weeks of postnatal life, a time when NMDA receptors are transiently overexpressed (21, 22). This is a critical period when neuroanatomical connections are maturing and neurotoxicity, gene expression (RNA and protein), and function (EEG) in response to anticonvulsant treatment protocols may be differentially affected (22, 23). In addition, differential regulation of GABAA receptor subtypes on nonprincipal interneurons was shown to protect principal excitatory neurons from seizure-induced injury due to their inhibitory role in balancing excitation (24). In adult rodents following systemic KA-induced seizures, delayed reductions in mRNA and immunostaining of the GABAA receptor α1 subunit and calcium-binding protein parvalbumin (PV)-basket type interneurons were observed in hippocampal CA1-3/hilar subfields suggesting acute degeneration of these types of neurons occurs after continuous seizures and subsequently reduces inhibitory control (25–28). A retrospective developmental study illustrated that PV+ immunoreactive neurons do not appear before P7, whereas GAD+ interneuronal expression appears earlier, by P2, indicating there is a preprogrammed latent inhibitory effect of PV-type interneurons (29). This is likely due to lack of PV mRNA abundance that is directly correlated to protein expression. Although incomplete migration to the hippocampus, which is not well studied, cannot be ruled out, no clear-cut radial gradients were observed (30). Moreover, research has suggested that certain interneurons have dissimilar sensitivity among seizure models that can differentially affect retrograde cannabinoid signaling at immature ages. However, no study has yet examined the fate of PV-type interneurons after sustained seizures provoked during the early juvenile period and whether CBD can protect them from hippocampal epileptiform discharges. Therefore, this study employed an intrahippocampal KA model, predominantly conducted in adult rodents by utilizing a unilateral intrahippocampal microinfusion of KA during the third postnatal week. A major advantage of intrahippocampal drug screening herein was to accurately regulate the local doses of KA ± CBD and resulting seizure activities. Both immediate and delayed EEG epileptiform activity of the hippocampus could be simultaneously monitored. In contrast, with peripheral treatment, dosing is less accurate and only the delayed components can be measured which are often mixed with confounding movement artifacts. Coadministration was more effective than posttreatment in preventing clonic convulsions and providing neuroprotection in both KA models tested, which appears important for interpretation of its clinical potential. Upregulation of PV-type interneuron neuropillar/dendritic field expression in the immature rodent hippocampus occurred in the contralateral hippocampus after intrahippocampal seizures, and also within the ipsilateral hippocampus but only in the presence of CBD. There was also steady CB1 receptor expression. Maintaining interneuronal expression may contribute to the resistance of the immature rodent hippocampus to the development of seizures and neuronal cell death. MATERIALS AND METHODS Animals Immature male Sprague-Dawley P20 rats (40–50 g) (Charles River, St. Louis, MO) were used for intrahippocampal and intraperitoneal drug delivery. Juvenile rat pups were housed in single cages with their lactating mother and were given food and water ad libitum until sacrifice. All animals were kept on a 12-hour light/dark cycle at room temperature (55% humidity) in our New York Medical College (NYMC) accredited animal facility. The work described has been carried out in accordance with The Code of Ethics of the World Medical Association and all animal procedures were in agreement with NIH guidelines approved by NYMC internal Animal Ethical Committees. All possible steps were taken to avoid animals’ suffering at each stage of the experiment. Electrode/Cannula Surgical Implantation Surgical procedures were carried out on P18 to allow a 48-hour recovery period prior to treatment(s). Rat pups were unilaterally stereotaxically implanted with either dual bipolar electrode/cannula assemblies (Plastics One, San Diego, CA) into the right hippocampus for local injection experiments or tungsten bipolar electrodes to record from systemically treated animals. To prevent pain, rat pups were anesthetized with a mixture of 70 mg/kg ketamine and 6 mg/kg of xylazine. The level of anesthesia was checked by hind limb pinch reflex. The scalp was exposed, and small holes were gently prepared manually with a 21 G1/2 needle for placement of holding screws and electrode insertion. Placement was perpendicularly oriented (coordinates in mm with respect to bregma: AP: −3.2; L: 2.4; D: −2.4; incisor bar at −3.5) (31). After implantation, dental acrylic was used to close the wound and stabilize the electrode assembly. Animals had 100% recovery from anesthesia maintained at 33°C in a clean cage box under an incandescent lamp. They became active 30–40 minutes following surgery and then returned to their lactating mother until experimental testing. Drug Administration CBD was purchased predissolved in methanol at 1 mg/ml (Millipore Sigma-Aldrich, St. Louis, MO). KA stock solution was also suspended in 50% methanol then diluted to same final concentration as the CBD final suspension, ≤10%. Seizure activity was induced with KA by a single saturating intrahippocampal dose of 1.2 µg/0.5 µL/minute (5 µM) that was microinfused unilaterally via the bipolar/cannula assembly into the CA1 region with a microsyringe (5 µL Hamilton syringe, Millipore, Sigma-Aldrich). Animals were divided into vehicle and 4 experimental groups: CBD was coadministered with KA in the same cocktail, also at a saturating concentration, 1 µg/0.5 µL/minutes (3.18 µM) before and after onset to rhythmic spike activity. CBD was administered either together or 30 minutes after systemic injection of KA (Control groups received 1 microinjection of vehicle (ih: n = 5, ip: n = 11) and experimental groups received intrahippocampal KA (KAih, n = 7), intrahippocampal KA + CBD (KA + CBDih) (n = 7), intrahippocampal KA then CBD (KA/CBDih, n = 4), intraperitoneal KA (KAip, 7 mg/kg), n = 12), KA + CBDip (n = 8, 10 mg/kg of CBD) (n = 5, 4, respectively) or after onset to behavioral symptoms (KA/CBDip, n = 3:8 mg/kg, n = 2:4 mg/kg), and CBD alone (n = 4). Behavioral Seizure Scoring To assess seizure severity in the age group of our study (P20), our modified Racine method was used for both models to calculate seizure severity scores as described in (32). Scoring was on a scale of 0–6, with 0 representing normal behavior and 6 representing death so that highest score would be 5. Stage 1 = mild scratching and grooming; stage 2 = continuous scratching, wet dog shakes (WDSs), hyperexcitability; stage 3 = periodic body stiffness, tail stiffness and wagging; stage 4 = prolonged immobility, standing body tonus, salivation; stage 5 = 4 or more bouts of rearing and/or standing tonus with and without bilateral fore limb clonus (FLC), foaming salivation, circling, bouncing, and/or loss of postural control during the 2-hour observation period. Seizure behaviors commenced after early signs emerged (e.g. scratching), and seizure behavior scores were calculated for each animal. Stages 1–2 were classified as nonconvulsive; stages 3–5 were classified as convulsive as these stages were associated with epileptiform events in the EEG. After the onset to behavioral seizures which varied according to model used, frequency and duration of different seizure behavior manifestations were recorded every 2 minutes for 2 hours. The total raw score was equal to the sum of these scored numbers, which was then divided by the number of intervals. All KAih and KAip injected animals in the absence of CBD reached stage 4–5 seizures within 30 or 60 minutes, respectively. Controls were injected with vehicle for both intracranial and systemic delivery methods. EEG Recordings KA intrahippocampal administration was combined with in vivo electrographic recording of neuronal activity to produce a reliable and less variable seizure model when compared with systemic administration. Acute EEG recordings were obtained from KA- and KA + CBD-treated groups from both delivery models. KAih and KA + CBDih-treated animals were paired, placed in an insulated chamber, and connected from a head stage (FHC) to the recording set up through flexible low noise leads (Plastics One), which permitted free movement as described in (31, 32). For online EEG acquisition, the head stage was connected to Xcell 3 × 4 amplifier that interfaces with Data Wave ADC (Analog to USB converter Digital Converter) hardware and a Dell computer. EEG continuous data recordings were obtained with bipolar electrodes to measure potential differences within the hippocampal CA1 from experimental animals for 2 hours following drug administrations via 2 or 3 separate channels at the same sampling rate. For offline analysis, a SciWorks plugin for real-time data acquisition and analysis was employed to quantify wave frequency, amplitude, and number of spike and burst events (32, 33). The software program uses digital filtering with a Butterworth 3 pole filter and a 6 dB per octave roll-off to screen a range of frequencies for post experiment data analysis. All recordings were time stamped to 1 microsecond resolution with a high-resolution clock. The spectral power was monitored from 6 frequency bands: delta (0.1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta (12–30 Hz), low-gamma (30–70 Hz), and high-gamma (70–180 Hz). Epileptic spike discharges were recorded in the low and high gamma range. EEG traces were filtered prior to being subjected to burst, Fourier, and spectral analyses to quantify changes in oscillation frequency and duration. Vehicle-injected control rats were also used for baseline comparison. Electrode placement into the CA1 was verified histologically with Nissl staining. Open Field Test Postictal locomotion of the juvenile rats was examined in the open field test at the 72-hour time point. Testing the animals was performed by placing the pups, one at a time, into the center of an open field without visual cues for 5 minutes. Area dimensions were 42.5 × 42.50 cm. The inside area of the arena was 26.56 × 26.56 cm (39% of total area). Movements were automatically recorded with Activity Monitor program software, splitting movements between the inside area and periphery (Med-Associates, St. Albans, VT) as described previously in (32). The software program was set to monitor distance traveled, number of zone entries, time ambulant, counts and time of ambulation, stereotypic behaviors (e.g. grooming), time resting, vertical counts, time vertical, and average velocity. Histology Nissl staining with cresyl violet was carried out on fixed serial air-dried sections (30 µm) from serial vibratome fixed sections in experimental and control groups. Histology included different levels of the hippocampus along the septotemporal axis and associated areas for qualitative analysis of injury in the CA1, CA3/hilar, and dentate gyrus (DG) subregions. NeuN immunohistochemistry was simultaneously assessed in adjacent sections to monitor injury and/or acute cell loss at 24 and 72 hours in both models. P20 rats were first anesthetized with a lethal dose of sodium pentobarbital (50 mg/kg) to minimize pain and discomfort, and then perfused intraaortically with ice-cold 0.9% saline followed by 200 mL 4% paraformaldehyde in phosphate buffered saline (PBS). Brains were removed and postfixed for 16 hours at 4°C. Vibratome hippocampal sections (40 µm) were cut and preincubated with 5% normal goat serum in 0.5% bovne serum albumin (BSA)/PBS for 45 minutes. The sections were incubated with NeuN and PV monoclonal (Millipore, Sigma-Aldrich) and glial fibrillary acidic protein (GFAP) polyclonal (Millipore, Sigma-Aldrich) antibodies overnight (1:1000) shaking at 4°C for 24 hours. Secondary biotinylated goat antimouse and rabbit antibodies were added (Vector Laboratories, Burlingame, CA) and then sections were developed with avidin biotin complex and reacted with peroxidase-reduced diaminobenzidine (DAB) (Millipore, Sigma-Aldrich). Nissl- and NeuN-labeled sections were mounted, processed through graded ethanol, and cleared with 3 changes of xylene for 15 minutes each. Sections were viewed and photographed under bright-field optics and phase contrast according to the rat atlas (31). First, objectives were calibrated with a 1-mm ruler (100 × 0.01). Multiple sections per animal were photographed with a CCD camera interfaced to a Dell computer equipped with Pro Image software. An estimate of injured surface neurons within the ipsilateral hippocampus was made at the acute time point by counting neurons that were irregular shaped and highly hyperbasophilic under bright-field microscopy with a manual counter and grid reticule in the CA1 and hilar subfields. Cannula/electrode placement was verified and the extent of neuronal injury with respect to immunohistochemical labeling was estimated. Electrode placement was accepted if the tip of the electrode lesion was located anywhere in the CA1 region of the hippocampus. Immunohistochemistry Rat pups were anesthetized with a lethal dose of sodium pentobarbital (25 mg/kg) and quickly perfused intraaortically with 50 mL of ice-cold 0.9% saline followed by 200 mL of 4% paraformaldehyde in 0.1 m PBS. Fresh free-floating fixed coronal vibratome sections (40 μm) derived from control and experimental animals at the level of the hippocampus were processed for immunohistochemistry with several antibodies: NeuN, a neuronal marker (Millipore, Sigma-Aldrich, Cat No. MAB377); GFAP, an astrocytic marker (Millipore Sigma-Aldrich, Cat No. 69269); CB1, detects a protein band of ∼60 kDa and can be blocked with cannabinoid receptor I peptide (ab50542) (Abcam, Cambridge, MA, Cat No. 23703); and PV a selective noncannabinoid interneuronal marker that recognizes a protein of 12 kDa and specifically stains the Ca2+-bound form), (Millipore Sigma-Aldrich, Cat No. MAB1572). Cut sections were first submerged in quenching solution (0.1% H2O2 in PBS) for 30 minutes, rinsed 4 times with PBS, and then incubated in 1% BSA and 0.2% Triton X-100 for 45 minutes. Sections were then incubated with NeuN or GFAP at an antibody dilution of 1:1000 in 0.1% BSA, and PV was diluted at 1:500 in 0.1% BSA. Immunodensity Measurements After mounting sections on slides, dehydration in graded ethanol, clearing and cover slipping, hippocampal sections were scanned with a digital spot camera attached to an Olympus BX51 microscope interfaced with a Pentium IV DELL computer. All camera settings were held constant during image capturing. NIH-Scion Image program was used to quantify protein expression in selected areas of interest with densitometry measurements. All camera settings were held constant during image capturing. Densitometry measurements averaged from 4 to 11 animals per group were used to quantify NeuN, GFAP, PV, and CB1 protein expression in gray scale from selected areas of interest (CA1, CA3, DG, and hilar subregions of the hippocampus). Using stereological methods, this study estimated the total numbers of GFAP stained astrocytes and PV-stained interneurons within a defined area of 4 consecutive 0.7-mm2 sectors in the middle of the CA1 as described in (32, 33). A grid reticule (100 μm × 0.10 mm), inserted into the eyepiece, with a 40 × objective was aligned along the CA1 and dentate hilus from both hemispheres, and a manual cell counter was used by 2 investigators blind to the experimental conditions. To avoid overlap, sections were first randomized from 4 hippocampal levels (every 4th and 12th section between −2.4 and −4.6 mm from bregma [31]. Sections were scanned into a Nikon E600 microscope interfaced with a Pentium IV DELL computer. Counts were averaged into a grand mean per group. For densitometry, values were averaged after background subtraction and sections were then viewed at a higher power to capture images of individual cells. Estimating the level of protein distribution was accomplished by either evaluating the number of pixels within all of the sublayers in one mean area sweep per subarea for CB1 and PV immunodensity labeling around cell bodies of the hippocampal subfields. For single cell analysis of interneurons and glia optical densities were measured with an NIH Image point to point area tool. Since GFAP staining grouped in optical density (OD) measurements such that they were either “light” or “dark”, after subtracting background, (range OD ∼50 vs ∼120), a cut-off OD reading was used for separate analysis. Astrocytes were considered as high-density dark when they exceeded an OD threshold of ≥80, whereas light density was considered <80. Percentages of labeled cell numbers and optical intensities were then calculated for each area and antibody relative to respective controls. Statistics Results are presented as means ± SEM for each animal group. Significant differences were determined from numbers assessed from hyperbasophilic and NeuN+ cell counting. Optical immunodensity measurements of neurons, interneurons and glia for all groups were compared with parametric statistics using Sigma Stat Software 12.0. Immunodensity values were normalized against the control group. Variations in the immunodensity of experimental groups were also represented in percentages. For multiple comparisons 1-way analysis of variance (ANOVAs) followed by Holm-Sidak post-hoc test was used to analyze parametric histological and EEG data as well as immunodensity values for the 5 groups. One-way ANOVA on ranks was used to analyze the frequency of spikes counted in the EEG and rhythmic burst duration with seizure severity. The probability level interpreted as significant was p < 0.05 for all tests. RESULTS Seizure Behavior At P20, no changes in behavior were noted by acute administration of CBD at 4 or 8–10 mg/kg alone. To evaluate the anticonvulsant effect of CBD on acute seizures, KAih- and KA + CBDih-treated animals were attached to long leads so they were freely moving and simultaneously monitored in a 2-chamber cage. Being that P20 rats are more resistant to KA than adults (33, 34), a single saturating microinjection of KA was administered to the CA1, which resulted in rapid scratching and hyperactivity (circling and twitching) lasting 1–2 minutes followed by a quiet, sleepy period. After 10–30 minutes, seizure scoring stages appeared at a faster rate than systemic administration but the behavioral phenotype resembled KAip. Advanced motor symptoms included continuous circling, rearing with forelimb clonus with drooling salivation, FLC and body tonus with or without postural control as previously described in (33). Coadministration of intrahippocampal CBD prevented the development of stage 4-5 seizures for the duration of the observation period, consistent with a recent study conducted in adult male rodents (35). In contrast, presentation of WDS was prominent in 90% of the animals of this treatment group at a rate of 1.99 ± 0.68 minutes compared with control, 0.617 ± 0.197 events per minute (F = 19.834, p < 0.001) (Fig. 1A). Accordingly, when CBD 8 mg/kg was administered after animals reached stage 4–5 seizures (KA/CBDih), behavioral manifestations quickly calmed (Fig. 1A;Table). In addition, coadministration of KA + CBDip (8 or 10 mg/kg) attenuated KA induced seizure behavior, clonic convulsions were not observed. However, seizure behavioral symptoms appeared after 2 hours, which corresponded with epileptiform discharges in the EEG (Fig. 1B–H). When CBD was injected systemically after onset to FLC, seizure behavioral seizures were not significantly attenuated (Fig. 1A;Table). Lower doses of 4 mg/kg had no inhibitory effect on KA-induced seizures. TABLE Behavior Onset and EEG Quantification Treatment Groups Seizure Score WDS Onset (min) FLC Onset (min) Spike Onset (min) Spike Frequency (Hz) Spike Amplitude (mV) Burst Onset (min) Burst Frequency (Hz) Burst Duration (min) KAih (n = 7) 4.2 ± 0.2 24.14 ± 6.0 12.6 ± 0.5 0.10 ± 0.03 1.66 ± 0.52 4.25 ± 0.67 14.21 ± 3.22 1.66 ± 0.52 1.16 ± 0.23 KA+CBDih (n = 7) 2.1 ± 0.4** 27 ± 8.2 0 13.57 ± 6.3** 0.03 ± 0.21** 0.391 ± 0.30** 106.0 ± 2.89** 0.02 ± 0.21** 0.022 ± 85** KA/CBDih (n = 4) 3.88 ± 1.3 22.5 ± 3.0 63 ± 4.2 15 ±3.3 0.30 ± 0.16** 0.263 ± 1.66* 120.7 ± 4.08** 0.3 ± 0.21** 0.349 ± 0.11* KAip (n = 12) 4.0 ± 0.1 30.2 ± 2.4 60 ± 2.7** 22.6 ± 3.8 1.27 ± 3.4 11.25 ± 0.67 26.68 ± 6.37 1.27 ± 0.36 1.84 ± 0.34 KA+CBDip (n = 5) 3.14 ± 0.4 29.1 ±4.2 70.67±10 36 ±5.2* 0.18 ± 0.30** 2.1 ± 2.0* 90.2 ± 14.2** 0.1 ± 0.31** 0.30 ± 0.21* KA/CBDip (n = 3) 3.7 ± 0.14 34.6 ±3.8 63 ±4.1 19.8 ±4.4 0.89 ± 0.90* 2.2 ± 2.4* 98.2 ± 16.2** 0.23 ± 0.07** 0.194 ± 0.07** Treatment Groups Seizure Score WDS Onset (min) FLC Onset (min) Spike Onset (min) Spike Frequency (Hz) Spike Amplitude (mV) Burst Onset (min) Burst Frequency (Hz) Burst Duration (min) KAih (n = 7) 4.2 ± 0.2 24.14 ± 6.0 12.6 ± 0.5 0.10 ± 0.03 1.66 ± 0.52 4.25 ± 0.67 14.21 ± 3.22 1.66 ± 0.52 1.16 ± 0.23 KA+CBDih (n = 7) 2.1 ± 0.4** 27 ± 8.2 0 13.57 ± 6.3** 0.03 ± 0.21** 0.391 ± 0.30** 106.0 ± 2.89** 0.02 ± 0.21** 0.022 ± 85** KA/CBDih (n = 4) 3.88 ± 1.3 22.5 ± 3.0 63 ± 4.2 15 ±3.3 0.30 ± 0.16** 0.263 ± 1.66* 120.7 ± 4.08** 0.3 ± 0.21** 0.349 ± 0.11* KAip (n = 12) 4.0 ± 0.1 30.2 ± 2.4 60 ± 2.7** 22.6 ± 3.8 1.27 ± 3.4 11.25 ± 0.67 26.68 ± 6.37 1.27 ± 0.36 1.84 ± 0.34 KA+CBDip (n = 5) 3.14 ± 0.4 29.1 ±4.2 70.67±10 36 ±5.2* 0.18 ± 0.30** 2.1 ± 2.0* 90.2 ± 14.2** 0.1 ± 0.31** 0.30 ± 0.21* KA/CBDip (n = 3) 3.7 ± 0.14 34.6 ±3.8 63 ±4.1 19.8 ±4.4 0.89 ± 0.90* 2.2 ± 2.4* 98.2 ± 16.2** 0.23 ± 0.07** 0.194 ± 0.07** There was either no clonic seizure behavior or a delayed onset to as well a significant reduction in seizure severity with intrahippocampal cotreatment, KA + CBDih. Epileptiform activity was attenuated in both intrahippocampal and systemic seizure models. Peripheral cotreatment was more effective than posttreatment. WDS, wet dog shake; FLC, forelimb clonus. Values represent means ± SEM for each group. *p < 0.05; **p ≤ 0.01. TABLE Behavior Onset and EEG Quantification Treatment Groups Seizure Score WDS Onset (min) FLC Onset (min) Spike Onset (min) Spike Frequency (Hz) Spike Amplitude (mV) Burst Onset (min) Burst Frequency (Hz) Burst Duration (min) KAih (n = 7) 4.2 ± 0.2 24.14 ± 6.0 12.6 ± 0.5 0.10 ± 0.03 1.66 ± 0.52 4.25 ± 0.67 14.21 ± 3.22 1.66 ± 0.52 1.16 ± 0.23 KA+CBDih (n = 7) 2.1 ± 0.4** 27 ± 8.2 0 13.57 ± 6.3** 0.03 ± 0.21** 0.391 ± 0.30** 106.0 ± 2.89** 0.02 ± 0.21** 0.022 ± 85** KA/CBDih (n = 4) 3.88 ± 1.3 22.5 ± 3.0 63 ± 4.2 15 ±3.3 0.30 ± 0.16** 0.263 ± 1.66* 120.7 ± 4.08** 0.3 ± 0.21** 0.349 ± 0.11* KAip (n = 12) 4.0 ± 0.1 30.2 ± 2.4 60 ± 2.7** 22.6 ± 3.8 1.27 ± 3.4 11.25 ± 0.67 26.68 ± 6.37 1.27 ± 0.36 1.84 ± 0.34 KA+CBDip (n = 5) 3.14 ± 0.4 29.1 ±4.2 70.67±10 36 ±5.2* 0.18 ± 0.30** 2.1 ± 2.0* 90.2 ± 14.2** 0.1 ± 0.31** 0.30 ± 0.21* KA/CBDip (n = 3) 3.7 ± 0.14 34.6 ±3.8 63 ±4.1 19.8 ±4.4 0.89 ± 0.90* 2.2 ± 2.4* 98.2 ± 16.2** 0.23 ± 0.07** 0.194 ± 0.07** Treatment Groups Seizure Score WDS Onset (min) FLC Onset (min) Spike Onset (min) Spike Frequency (Hz) Spike Amplitude (mV) Burst Onset (min) Burst Frequency (Hz) Burst Duration (min) KAih (n = 7) 4.2 ± 0.2 24.14 ± 6.0 12.6 ± 0.5 0.10 ± 0.03 1.66 ± 0.52 4.25 ± 0.67 14.21 ± 3.22 1.66 ± 0.52 1.16 ± 0.23 KA+CBDih (n = 7) 2.1 ± 0.4** 27 ± 8.2 0 13.57 ± 6.3** 0.03 ± 0.21** 0.391 ± 0.30** 106.0 ± 2.89** 0.02 ± 0.21** 0.022 ± 85** KA/CBDih (n = 4) 3.88 ± 1.3 22.5 ± 3.0 63 ± 4.2 15 ±3.3 0.30 ± 0.16** 0.263 ± 1.66* 120.7 ± 4.08** 0.3 ± 0.21** 0.349 ± 0.11* KAip (n = 12) 4.0 ± 0.1 30.2 ± 2.4 60 ± 2.7** 22.6 ± 3.8 1.27 ± 3.4 11.25 ± 0.67 26.68 ± 6.37 1.27 ± 0.36 1.84 ± 0.34 KA+CBDip (n = 5) 3.14 ± 0.4 29.1 ±4.2 70.67±10 36 ±5.2* 0.18 ± 0.30** 2.1 ± 2.0* 90.2 ± 14.2** 0.1 ± 0.31** 0.30 ± 0.21* KA/CBDip (n = 3) 3.7 ± 0.14 34.6 ±3.8 63 ±4.1 19.8 ±4.4 0.89 ± 0.90* 2.2 ± 2.4* 98.2 ± 16.2** 0.23 ± 0.07** 0.194 ± 0.07** There was either no clonic seizure behavior or a delayed onset to as well a significant reduction in seizure severity with intrahippocampal cotreatment, KA + CBDih. Epileptiform activity was attenuated in both intrahippocampal and systemic seizure models. Peripheral cotreatment was more effective than posttreatment. WDS, wet dog shake; FLC, forelimb clonus. Values represent means ± SEM for each group. *p < 0.05; **p ≤ 0.01. FIGURE 1. View largeDownload slide (A) Graphical analysis of seizure severity scoring and rate of WDS. (B) Baseline activity of electrographic recording epochs from pups prior to treatment. (C) Intrahippocampal KA-induced acute and delayed spikes and burst oscillations. (D) Intrahippocampal coadministration reduced or prevented KA-induced epileptiform activity for the duration of the recording period at acute and delayed time points; however, WDS were prominently expressed. (E, F) When CBD microinjection followed KA-induced sharp wave oscillations, spike amplitude was quickly reduced and clustered burst activity was diminished but to a lesser degree than cotreatment. (G) Peripheral KA induced tight burst oscillations followed by low amplitude activity, not observed in intrahippocampal injected animals, appeared after 70 minutes. The higher dose of CBD (8 mg/kg) reduced spike and burst discharges within minutes and (H) EEG traces were flattened at 90 minutes; sharp wave oscillations returned at 120 minutes. Bars represent mean averages ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. FIGURE 1. View largeDownload slide (A) Graphical analysis of seizure severity scoring and rate of WDS. (B) Baseline activity of electrographic recording epochs from pups prior to treatment. (C) Intrahippocampal KA-induced acute and delayed spikes and burst oscillations. (D) Intrahippocampal coadministration reduced or prevented KA-induced epileptiform activity for the duration of the recording period at acute and delayed time points; however, WDS were prominently expressed. (E, F) When CBD microinjection followed KA-induced sharp wave oscillations, spike amplitude was quickly reduced and clustered burst activity was diminished but to a lesser degree than cotreatment. (G) Peripheral KA induced tight burst oscillations followed by low amplitude activity, not observed in intrahippocampal injected animals, appeared after 70 minutes. The higher dose of CBD (8 mg/kg) reduced spike and burst discharges within minutes and (H) EEG traces were flattened at 90 minutes; sharp wave oscillations returned at 120 minutes. Bars represent mean averages ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. EEG Activity: Intrahippocampal (ih) Versus Systemic (ip) Models Representative EEG recordings of 2 epochs following KAih in the presence and absence of CBD are illustrated and compared with KAip administration (Fig. 1B–H). Baseline EEG activity was first obtained prior to intrahippocampal KA injections and was of low amplitude and arrhythmic (Fig. 1B). After KAih, sharp wave spikes appeared within seconds, 6.13 ± 1.9 seconds, and initial rhythmic oscillations initially occurred within 52 ± 0.14 seconds, while typical delayed spike and burst discharges of high amplitude were observed at 14.21 ± 3.22 (Fig. 1C;Table). Although high amplitude spiking appeared at times, these were not associated with severe seizure behavior such as FLC and frothing salivation. Intracranial coadministration, KA + CBDih, resulted in delayed spike and burst onset (F = 5.64, p = 0.015 and F = 47.3, p = 0.001, respectively), and attenuated spike frequency and amplitude of initial and delayed epileptiform discharges (Fig. 1D;Table). After KAip (7 mg/kg), spike onset and bursting oscillations were delayed (Fig. 1G;Table). High-frequency burst oscillations followed by low amplitude activity, not observed in KAih-treated animals at this age, appeared after ∼60–70 minutes. Systemic doses of CBD (8 mg/kg) reduced spike and burst discharges within 10 minutes of the injection (Fig. 2G, H); EEG wave form traces were flattened at 90 minutes but sharp wave oscillations returned at ∼120 minutes (Fig. 1H). Peripheral CBD injection could not block intrahippocampal KA evoked seizures. FIGURE 2. View largeDownload slide Open field activity 72 hours after KA ± CBD treatments. (A) Control juvenile animals show a low level of exploration and mainly stay near the walls with few entries to the center of the field. (B) After KAih, open field activity increased compared with controls. (C) After KAih + CBD, low levels of open field activity were seen. (D) Following KAip, juvenile rat pups were active, roaming around the field entering center zones. (E) Changes in locomotor activity were attenuated by CBDip treatment. (F–I) Increases in total distance traveled, entries into the center, vertical movements, and reduced resting time were attenuated in the presence of CBD. Bars represent the mean averages ± SEM of n = 5 per group. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. FIGURE 2. View largeDownload slide Open field activity 72 hours after KA ± CBD treatments. (A) Control juvenile animals show a low level of exploration and mainly stay near the walls with few entries to the center of the field. (B) After KAih, open field activity increased compared with controls. (C) After KAih + CBD, low levels of open field activity were seen. (D) Following KAip, juvenile rat pups were active, roaming around the field entering center zones. (E) Changes in locomotor activity were attenuated by CBDip treatment. (F–I) Increases in total distance traveled, entries into the center, vertical movements, and reduced resting time were attenuated in the presence of CBD. Bars represent the mean averages ± SEM of n = 5 per group. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. Open Field Test Control and experimental groups were tested in an open field apparatus to quantify general differences in locomotor activity (Fig. 2). Entry was initiated from the center of the apparatus. Control vehicle-treated rats quickly left the center and clung to the walls or spent most of the time in one corner of the apparatus (Fig. 2A). After KAih, or KAip, the total distance traveled and altered pattern of total movement were significantly higher, particularly after systemic administration (Fig. 2B, D, F). Accordingly, resting time was decreased, entries into the center zone increased, and vertical movements also increased (Fig. 2B, D, G–I). Changes in locomotion, resting, and other measures were attenuated or reversed by CBD treatment in both intracranial and systemic KA administration models (Fig. 2C, E–I). Neuronal Assessment NeuN immunohistochemistry and Nissl staining were used to estimate changes in cellular morphology or cell loss in KAih, KAip, and KA ± CBD-treated groups at 24 hours (not illustrated) and 72 hours after seizure-induced insult (Fig. 3A–L). In intrahippocampal vehicle control pups, NeuN immunolabelling was robust. The cytoplasm and nuclei of CA1-labeled neurons were densely stained, round and healthy in appearance; proximal dendrites were prominent in label (Fig. 3A, B). Nissl staining with cresyl violet was uniform; pyramidal and DG cell bodies were round bearing healthy morphology (Fig. 3C, D). At 24 hours after KAih, there was a lack of NeuN label just near, but not away, from the infusion site; no obvious changes in histology were noted (not illustrated). At 72 hours after KAih, reduced NeuN label was also near, but not away, from the infusion site (Fig. 3E, F). Upon close inspection, lightly stained CA1 neurons were relatively spared, but Nissl staining detected hyperbasophilic cells that were of irregular elongated or shrunken shape in and around the CA1 layer, possibly injured interneurons (Fig. 3G, H, M, N). After KA + CBDih, NeuN immunolabeling resembled controls; reduced label was restricted to the infusion site; control labeling was observed away from the infusion site (Fig. 3I, J). Nissl staining revealed healthy pyramidal and granule cells, relatively few hyperbasophilic cells appeared outside of the cell body layer (Fig. 3K, L). Within the DG, at 24 hours after KAih, granular cells did not exhibit any injury under bright-field microscopy. However, at 72 hours there was an increase in injured appearing cells as observed in the CA1 that had hyperbasophilic, elongated, flattened, or shrunken shaped characteristics. Irregular stained cells were counted under bright-field microscopy within the polymorphic region of the dentate hilus (Fig. 3H). FIGURE 3. View largeDownload slide NeuN immunohistochemistry and Nissl staining 72 hours after KAih, KAip, and KA ± CBD treatments at the level of the hippocampus in P20 rat pups. (A) Control NeuN labeling was intense and uniform throughout the hippocampal subfields (40×). (B) High magnification (400×) of control CA1-labeled neurons showed the cytoplasm and nuclei were densely stained with round, healthy appearance (400×). (C) Nissl staining with cresyl violet was also uniform; cell bodies were round bearing healthy morphology. (D) The DG granule cells and hilar cells were also uniformly stained with healthy appearance. (E, F) KAih resulted either in a lack of NeuN label near (box) from the infusion site, or control labeling away from the infusion site. (G) Hyperbasophilic neurons presented on the edge or outside of the pyramidal layer and appeared to be interneurons (arrows). (H) Hyperbasophilic neurons were also detected along the GCL facing the hilus and within the polymorphic region of the hilus (arrows). (I, J) Pups with KA + CBDih co-treatment showed NeuN immunolabeling resembled KA-treated animals, weak label was near the infusion site and control labeling was away from the infusion site (box). (K, L) Nissl staining revealed healthy pyramidal cells; few hyperbasophilic cells with elongated irregular shape appeared outside of the cell body layer (arrows). Quantification of the number of darkened cells with compromised morphology was measured. Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. FIGURE 3. View largeDownload slide NeuN immunohistochemistry and Nissl staining 72 hours after KAih, KAip, and KA ± CBD treatments at the level of the hippocampus in P20 rat pups. (A) Control NeuN labeling was intense and uniform throughout the hippocampal subfields (40×). (B) High magnification (400×) of control CA1-labeled neurons showed the cytoplasm and nuclei were densely stained with round, healthy appearance (400×). (C) Nissl staining with cresyl violet was also uniform; cell bodies were round bearing healthy morphology. (D) The DG granule cells and hilar cells were also uniformly stained with healthy appearance. (E, F) KAih resulted either in a lack of NeuN label near (box) from the infusion site, or control labeling away from the infusion site. (G) Hyperbasophilic neurons presented on the edge or outside of the pyramidal layer and appeared to be interneurons (arrows). (H) Hyperbasophilic neurons were also detected along the GCL facing the hilus and within the polymorphic region of the hilus (arrows). (I, J) Pups with KA + CBDih co-treatment showed NeuN immunolabeling resembled KA-treated animals, weak label was near the infusion site and control labeling was away from the infusion site (box). (K, L) Nissl staining revealed healthy pyramidal cells; few hyperbasophilic cells with elongated irregular shape appeared outside of the cell body layer (arrows). Quantification of the number of darkened cells with compromised morphology was measured. Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. After KAip, NeuN and Nissl staining were indistinguishable from controls at 24 hours after status epilepticus (Fig. 4A–C). In contrast, at 72 hours, the CA1 layer thinned out with fewer NeuN+ cells; many survivors appeared dystrophic with irregular shape with enhanced nuclear staining (Fig. 4D, E). Typical CA1 hippocampal pyknotic cell damage with widespread shrunken cell morphology to the inner layer including cell loss was observed consistent with our prior report at this postnatal age (33) (Fig. 4F). Attenuating KA-induced seizures with CBDip posttreatment also recovered NeuN+ cell counts and pyramidal cell morphology in CA1 and CA3 subfields, similar to controls (Fig. 4G–I). Graphical analysis of NeuN+ cell counts from several CA1 sectors after KA or CBD confirmed insignificant differences at 24 hours and neuroprotection at 72 hours (Fig. 4J). The number of injured cells of the inner CA1 layer at 72 hours following KAip in the absence of CBD was significant, reproducible (497 ± 78.8 vs 155 ± 63.8, F = 6.89, p = 0.01) and as previously observed (23, 33). FIGURE 4. View largeDownload slide (A, B) Following KAip, NeuN staining was indistinguishable from controls at 24 hours after status epilepticus; Nissl staining was uniform, few hyperbasophilic cells appeared in and out of the CA1 layer (arrows) (C). (D, E) In contrast, after 72 hours, the CA1 and hilus lost cells and many survivors appeared dystrophic with irregular shape with enhanced nuclear staining. (F) Typical CA1 hippocampal pyknotic cell damage including cell loss was observed in the inner layer (at 72 hours) (arrows). (G, H, Animals with peripheral CBD post-treatment also had improved cell morphology resembling controls. (J) Averaged NeuN+ Cell counts from several CA1 sectors further supported insignificant differences at 24 hours but neuroprotection at 72 hours predominated with co-treatment (KA + CBDip). Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. FIGURE 4. View largeDownload slide (A, B) Following KAip, NeuN staining was indistinguishable from controls at 24 hours after status epilepticus; Nissl staining was uniform, few hyperbasophilic cells appeared in and out of the CA1 layer (arrows) (C). (D, E) In contrast, after 72 hours, the CA1 and hilus lost cells and many survivors appeared dystrophic with irregular shape with enhanced nuclear staining. (F) Typical CA1 hippocampal pyknotic cell damage including cell loss was observed in the inner layer (at 72 hours) (arrows). (G, H, Animals with peripheral CBD post-treatment also had improved cell morphology resembling controls. (J) Averaged NeuN+ Cell counts from several CA1 sectors further supported insignificant differences at 24 hours but neuroprotection at 72 hours predominated with co-treatment (KA + CBDip). Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA and Holm-Sidak multiple comparisons. Astrocyte Assessment To estimate glial cell proliferation and/or reactivity in response to CBD treatment, immunohistochemistry with a GFAP antibody was examined at the level of the hippocampus at 24 and 72 hours in both KA models. Under control conditions, glial cells were star-like in appearance with thin fibers; staining was moderate and uniform across the hippocampal subfields with the highest OD measurements being outside of the principle cell layers, in structures such as the hilus, stratum radiatum and stratum lacunosum moleculare of the pyramidal fields (Fig. 5A–D). Three magnifications are illustrated. At 24 hours after KAih or KAip, little or no change in glia cell immunoreactivity was observed in the CA1, even near the infusion site, unless local cell loss was caused by the injection itself (not illustrated). In contrast, within the dentate hilus, mild changes in astrocyte morphology and increases of astrocytic immunolabeling were detectable under bright-field microscopy which was reversed in the presence of CBD (not illustrated). At 72 hours, after KAih, there was only a moderate increase in GFAP staining throughout the subfields (Fig. 5E). However, within the CA1, morphological changes in astrocytic structure occurred. Many glial cells were very dark, less star-like in appearance, and instead had thick stubby projections (Fig. 5E, F). Dark GFAP+ cells were also observed deep within the dentate hilus proliferative zones exhibiting large cell bodies and long processes (Fig. 5G, H). After KA + CBDih, overall GFAP-staining intensity and morphology of astrocytes within the CA1 layers was “light” and resembled controls (Fig. 5I, J). In contrast, “dark” reactive glia continued to appear very deep near the vertex of the dentate hilar blades; some processes were long extending into the granule cell layer (GCL) (Fig. 5K, L). FIGURE 5. View largeDownload slide GFAP immunohistochemistry was examined at the level of the hippocampus 24 or 72 hours after KA ± CBD intrahippocampal or systemic treatments, respectively. (A–D) In controls, glial cells were star-like in appearance with thin fibers; staining was moderate and uniform across the hippocampal subfields with the highest OD being outside the principle cell layers, in structures such as the hilus, stratum radiatum and stratum lacunosum moleculare; 2 or 3 magnifications are shown for CA1 and dentate hilar subfields, respectively (boxes). (E–H) After KAih, there was a moderate increase in GFAP staining throughout the subfields. In the CA1, morphological changes in astrocytic structure occurred; many glial cells were dark, less star-like but with thick stubby projections; these features were more prominent and pronounced near the infusion site of the CA1 and deep within the dentate hilus across proliferative zones. (I–L) After KA + CBDih, staining intensity and morphology of the CA1 resembled the control groups, but reactive glia still appeared deep within the hilar blades; some processes were long extending into the GCL. (M, N), After KAip there was a marked increase in GFAP staining throughout the subfields. Morphological changes in astrocytic structure were most pronounced in the CA1; many glial cells were dark with thick long or short stubby projections; deep within the dentate hilus many reactive glia were observed with long processes that extended through the GCL into the molecular layers. (Q–T) Following KA/CBDip, reactive gliosis of the CA1 was not significantly different from the controls but also not from the other KA-treated groups. Reactive gliosis was still apparent within deep hilar regions along proliferative zones but to a lesser degree than the KAip-treated animals. (U–W) Graphical analyses monitored 2 populations of cells, dark and light. Significant differences were by rises in number of darkly stained glia; light numbers were constant. Results are expressed as means ± SEM; *p < 0.05, **p < 0.01. FIGURE 5. View largeDownload slide GFAP immunohistochemistry was examined at the level of the hippocampus 24 or 72 hours after KA ± CBD intrahippocampal or systemic treatments, respectively. (A–D) In controls, glial cells were star-like in appearance with thin fibers; staining was moderate and uniform across the hippocampal subfields with the highest OD being outside the principle cell layers, in structures such as the hilus, stratum radiatum and stratum lacunosum moleculare; 2 or 3 magnifications are shown for CA1 and dentate hilar subfields, respectively (boxes). (E–H) After KAih, there was a moderate increase in GFAP staining throughout the subfields. In the CA1, morphological changes in astrocytic structure occurred; many glial cells were dark, less star-like but with thick stubby projections; these features were more prominent and pronounced near the infusion site of the CA1 and deep within the dentate hilus across proliferative zones. (I–L) After KA + CBDih, staining intensity and morphology of the CA1 resembled the control groups, but reactive glia still appeared deep within the hilar blades; some processes were long extending into the GCL. (M, N), After KAip there was a marked increase in GFAP staining throughout the subfields. Morphological changes in astrocytic structure were most pronounced in the CA1; many glial cells were dark with thick long or short stubby projections; deep within the dentate hilus many reactive glia were observed with long processes that extended through the GCL into the molecular layers. (Q–T) Following KA/CBDip, reactive gliosis of the CA1 was not significantly different from the controls but also not from the other KA-treated groups. Reactive gliosis was still apparent within deep hilar regions along proliferative zones but to a lesser degree than the KAip-treated animals. (U–W) Graphical analyses monitored 2 populations of cells, dark and light. Significant differences were by rises in number of darkly stained glia; light numbers were constant. Results are expressed as means ± SEM; *p < 0.05, **p < 0.01. After KAip, there was a marked increase in GFAP immunostaining throughout the subfields as previously observed (36). Morphological changes in astrocyte structure were most pronounced in the vulnerable CA1 subregion. Similar to KAih, many glial cells were very dark with thick, long and/or short stubby projections. In addition, deep within the dentate hilus, dark reactive glial cells were observed, also containing long thin processes that extended through the GCL and into the molecular layers (Fig. 5M, N). After KA/CBDip, astrocytic staining of the CA1 was not significantly different from the controls, but also not from the other KA-treated groups, only showing a trend of increased expression since status epilepticus was induced for at least 30 minutes prior to the CBD posttreatment (F = 2.62, p = 0.08) (Fig. 5Q, R). Reactive gliosis also was apparent deep and laterally within proliferative hilar zones but to a lesser degree that KAih-treated pups (Fig. 5S, T). High-density measurements were compared with control and experimental groups as well as with surrounding glia that had lower OD intensities (Fig. 5U, W). There was an increase in the immuno-intensity of darkly stained glial cells which also bared altered morphology in both KA models, but was exclusively attenuated in the KA + CBDih treatment group (Fig. 5U). The OD of lightly stained glia was relatively uniform across the groups and was significantly less intense than dark cell intensity cells calculated from controls (33.8% ± 4.8%/0.1 mm2) as well as from experimental groups suggesting a large population of glia were unaffected or unreactive at the acute time points examined. Within the CA1, the majority of astrocytes (≥70%), expressed GFAP above the optical intensity threshold cut-off for KAih, KAip, and KA/CBDip treatment groups, whereas, in control sections, less than one third (23.7 ± 10.3%/0.1 mm2) expressed values over the OD threshold cut-off (Fig. 5V, W). PV Interneuron Immunohistochemistry To estimate the acute effect of CBD treatment on inhibitory neurotransmission, immunohistochemical analysis of a selective calcium-binding protein and interneuronal marker, PV, was used in both KA models. In control animals, the cell bodies of PV+ interneurons were intensely labeled throughout the subfields of the immature hippocampus. In control animals, the highest numbers were found in the CA1 > CA3 > GCL > hilus (Figs. 6A–C and 7A). Cell body labeling was had the highest intensity and their immunodensity measurements were very steady and similar throughout the hippocampus (Figs. 6A–C and 7A). Within the DG, dense bands of neuropillar/dendritic field immunoreactivity were observed juxtaposed to pyramidal cell layers and in proximal dendritic areas of the inner molecular layer of dorsal DG (DGdorsal) and ventral DG (DGventral) blades, rich in medial perforant path synapses (Figs. 6A–C and 7A, C). At 24 hours after KAih, the number of PV+ interneurons declined in CA1, CA3, and GCL subfields (Fig. 7A). The number of PV+ interneurons after KA + CBD was recovered and KAip did not affect PV cell counts at this early time point (Fig. 7A). In contrast, at 72 hours after KAih, the number of PV immunoreactive cells on the ipsilateral side significantly decreased within all hippocampal subfields (Figs. 6D–F and 7B–F, C). The percentage of PV+ interneurons was halved after KAih or KA/CBDih in CA1 (55% ± 8.4% and 50% ± 5% of control, respectively, F = 9.7, p = 0.001); a partial recovery in cell counts was observed after KA + CBDih (Fig. 7B, C). FIGURE 6. View largeDownload slide PV immunohistochemistry and Nissl staining of the DG after KA ± CBD treatments (KA: 7 mg/kg and CBD: 8–10 mg/kg). (A–C) Control PV+ interneurons labeled intensely; dense bands of dendritic immunoreactivity were observed juxtaposed to pyramidal cell layers (box) and in proximal dendritic areas of the molecular layer of dorsal and ventral DG blades and along the outer cell body inner molecular layer of DGdorsal. (D–F) After KAih, reduced staining was observed in the CA1 and DG. (G–I) After KA + CBD labeling was most similar to controls; however, dendritic elevations of PV expression were observed throughout the inner molecular layer of the DG exhibiting defined processes. (J–L), Dendritic elevation of PV expression was further intensified in the contralateral hippocampal subfields; cell body immunodensities were similar to controls but dendritic expression was highly elevated. (M–O) Rat pups treated only with CBD exhibited control labeling. (P–R) At 72 hours after systemic KA treatment significant decreases in expression of interneuronal labeling in the CA1 and dentate hilus resulted; dendritic labeling was restricted to the proximal DG inner molecular layer. (T–V) KA + CBDip-treated animals had recovered expression in the CA1, DG and hilus. FIGURE 6. View largeDownload slide PV immunohistochemistry and Nissl staining of the DG after KA ± CBD treatments (KA: 7 mg/kg and CBD: 8–10 mg/kg). (A–C) Control PV+ interneurons labeled intensely; dense bands of dendritic immunoreactivity were observed juxtaposed to pyramidal cell layers (box) and in proximal dendritic areas of the molecular layer of dorsal and ventral DG blades and along the outer cell body inner molecular layer of DGdorsal. (D–F) After KAih, reduced staining was observed in the CA1 and DG. (G–I) After KA + CBD labeling was most similar to controls; however, dendritic elevations of PV expression were observed throughout the inner molecular layer of the DG exhibiting defined processes. (J–L), Dendritic elevation of PV expression was further intensified in the contralateral hippocampal subfields; cell body immunodensities were similar to controls but dendritic expression was highly elevated. (M–O) Rat pups treated only with CBD exhibited control labeling. (P–R) At 72 hours after systemic KA treatment significant decreases in expression of interneuronal labeling in the CA1 and dentate hilus resulted; dendritic labeling was restricted to the proximal DG inner molecular layer. (T–V) KA + CBDip-treated animals had recovered expression in the CA1, DG and hilus. FIGURE 7. View largeDownload slide Quantification of the number of PV+ cells in hippocampal subregions. (A) Control sections had differential amounts of PV+ cell numbers; the highest concentration was in the CA1/CA3 pyramidal fields followed by the GCL then hilus. At 24 hours, reduced counts were seen only after KAih so that the control pattern was lost and was reversed in the presence of CBD. (B, C) At 72 hours following CBD treatments, significant decreases in cell numbers occurred throughout all subfields after KAih, and in the CA1 and GCL after KAip. (D) Immunodensity values of the somata were unchanged after 72 hours under all conditions. (E) Immunodensity values from the neuropillar/dendritic proximal field regions revealed significant elevations in the CA1 and CA3 subfields on contralateral side after KAih and ipsilateral and contralateral sides after KA + CBDih. (F) Within the dorsal and ventral DG blades, ipsilateral and contralateral hilar dendritic/neuropillar expression elevations were significant after KAih or KA/CBDih; systemic treatment group OD readings were unchanged. Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. ***p < 0.001. One-way ANOVA and Holm-Sidak multiple comparisons. FIGURE 7. View largeDownload slide Quantification of the number of PV+ cells in hippocampal subregions. (A) Control sections had differential amounts of PV+ cell numbers; the highest concentration was in the CA1/CA3 pyramidal fields followed by the GCL then hilus. At 24 hours, reduced counts were seen only after KAih so that the control pattern was lost and was reversed in the presence of CBD. (B, C) At 72 hours following CBD treatments, significant decreases in cell numbers occurred throughout all subfields after KAih, and in the CA1 and GCL after KAip. (D) Immunodensity values of the somata were unchanged after 72 hours under all conditions. (E) Immunodensity values from the neuropillar/dendritic proximal field regions revealed significant elevations in the CA1 and CA3 subfields on contralateral side after KAih and ipsilateral and contralateral sides after KA + CBDih. (F) Within the dorsal and ventral DG blades, ipsilateral and contralateral hilar dendritic/neuropillar expression elevations were significant after KAih or KA/CBDih; systemic treatment group OD readings were unchanged. Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. ***p < 0.001. One-way ANOVA and Holm-Sidak multiple comparisons. Although fewer cell bodies in number were immunoreactive for the PV antibody, survivors were of a similar intensity as controls (Fig. 6D–F). Under all conditions, the cell body immunodensity measurements were steady and greater than measurements from neuropillar/dendritic fields (Fig. 6D). Following KA + CBDih, ipsilateral neuropillar/dendritic field labeling was reduced by 45.5% ± 4.8% in the CA1 pyramidal fields relative to controls (Figs. 6G–I and 7E, F). Although the highest OD measurements were in PV+ cell bodies throughout the hippocampus, followed by the CA1 neuropillar/dendritic fields, the largest percent increases calculated were in the DGventral in the KAih model but only in the presence of CBD (79.9% ± 9% increase) when comparing against the ih groups or when all 5 groups were analyzed with One-way ANOVA (KAih: residual df = 20, F = 7.3, p = 0.004 vs KAih + ip: df = 29, F = 4.28, p = 0.008). Interestingly, the contralateral side of KAih and KA + CBDih treatment animals also had high PV OD measurements in the inner DG molecular layers but it was restricted to the DGdorsal, (df = 29, F = 13.8, p = 0.001) (Figs. 6J–L and 7E, F). In the DGventral, significant increases were seen only after KAih and KA + CBDih within the contralateral side, likely due to seizure activity in the absence of infusion artifacts (F = 23.6; t = 5.7 p = 0.001). When CBD was administered in the absence of KA, PV expression was unaltered (Fig. 6M–O). After KAip at 72 hours, reduced PV+ cell number was readily detected in CA1, CA3, and hilar subfields, but these reductions in OD were significantly less than expression quantified after KAih (Figs. 6M–R and 7B, C). However, akin KAih, the DGventral was significantly elevated in PV dendritic field expression of the molecular layer with systemic KA (Figs. 6P–R and 7F). KA + CBDip-treated rat pups recovered expression in the CA1, DG and hilus (Fig. 6T–V). In contrast, in aged adult rats, faster and more robust deficits in PV expression were reported after seizures (37). CB1 Receptor Immunohistochemistry To examine the expression of CB1 receptor protein distribution following KA-induced status epilepticus in the presence and absence of CBD treatment during the juvenile period, serial sections from control and experimental animals at the level of the hippocampus were analyzed with a CB1 specific antibody and then developed with a DAB chromogen at 72 hours. In PBS-injected controls, CB1 immunoreactivity had a laminated pattern exhibiting dense and punctuate labeling throughout all layers of the hippocampus, particularly around the CA1 cell bodies as described in (38, 39). Following systemic administration of KA, increased expression of the CB1 receptor within the stratum oriens of the CA1 subregion was observed similar to prior research (38, 39). In contrast, after KAih, CB1 expression was relatively steady throughout the hippocampal subfields or a significant reduction in the ipsilateral CA1/hilus was observed compared with controls despite similar stages of Racine scaled seizures (Fig. 8). After KA + CBDih, CB1 immunostaining was also steady and uniform or reduced to a similar extent as KA alone (Fig. 8). CB1 expression was further reduced very near the infusion site (not illustrated). After CBD/KAih, CB1 expression was steady or increased in areas of injury (Fig. 8). After systemic KA injection, CB1 immunostaining was steady or increased in the CA1 stratum oriens and only reduced in associated areas of CA1 injury as described in (38, 40). FIGURE 8. View largeDownload slide Cannabinoid CB1 receptor immunohistochemistry is illustrated at the level of the hippocampus 72 hours after intrahippocampal or systemic KA ± CBD treatments. (A, B) Control hippocampal CB1 labeling was dense and uniform throughout the cell body layers, illustrated at 40× and 400× magnifications. (C, D) Following KAih, KAih + CBD (E, F), or KAih/CBD (G, H), the overall density of CB1 immunostaining was relatively stable and dense throughout the ipsilateral hippocampus relative to control labeling; however, less labeling was observed in the CA1 outer layer of stratum oriens (SO) and subiculum (arrows) relative to elevations observed with systemic KA treatment (I, J). (K) Quantitative analyses of the CA1 SO and stratum radiatum (SR) showed significant elevations in expression were only observed after KAip and that lower levels observed after KAih + CBD were only significantly different from the elevated KAip group. (L) Data normalization also showed that the SO: SR ratio was only significantly elevated after KAip and decreases in the ratio did not differ from control groups. (M) Graphic analyses also showed significant elevations of CB1 expression in the CA3, DG, and SR neuropil were only observed after KAip. Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. ***p < 0.001. One-way ANOVA and Holm-Sidak multiple comparisons. FIGURE 8. View largeDownload slide Cannabinoid CB1 receptor immunohistochemistry is illustrated at the level of the hippocampus 72 hours after intrahippocampal or systemic KA ± CBD treatments. (A, B) Control hippocampal CB1 labeling was dense and uniform throughout the cell body layers, illustrated at 40× and 400× magnifications. (C, D) Following KAih, KAih + CBD (E, F), or KAih/CBD (G, H), the overall density of CB1 immunostaining was relatively stable and dense throughout the ipsilateral hippocampus relative to control labeling; however, less labeling was observed in the CA1 outer layer of stratum oriens (SO) and subiculum (arrows) relative to elevations observed with systemic KA treatment (I, J). (K) Quantitative analyses of the CA1 SO and stratum radiatum (SR) showed significant elevations in expression were only observed after KAip and that lower levels observed after KAih + CBD were only significantly different from the elevated KAip group. (L) Data normalization also showed that the SO: SR ratio was only significantly elevated after KAip and decreases in the ratio did not differ from control groups. (M) Graphic analyses also showed significant elevations of CB1 expression in the CA3, DG, and SR neuropil were only observed after KAip. Bars represent the mean averages ± SEM. *p < 0.05; **p < 0.01. ***p < 0.001. One-way ANOVA and Holm-Sidak multiple comparisons. DISCUSSION This is the first study to examine whether a nonpsychoactive constituent of cannabis sativa, CBD, could attenuate KA-induced epileptiform discharges of the immature brain. On P20, anticonvulsant effects of CBD were associated with reduced hyperlocomotor activity and neuroprotection. Intrahippocampal coadministration was most effective in inhibiting KA-induced seizures, which appears important for interpretation of its effective action and clinical potential. Upregulation of PV-type interneuron dendritic expression in the immature rodent hippocampus, particularly after intrahippocampal seizures, appears to be a result of seizure activity. However, the saturating intracranial doses used, and long half-life of CBD noted with systemic treatment (41, 42), may further stabilize PV expression and contribute to anticonvulsant effects and delayed neuroprotection observed. Except for the subiculum, the level and distribution of CB1 receptor immunodensity was minimally affected after intrahippocampal treatments, contrasting the complex alterations observed after KAip. Differences between intrahippocampal and systemic models are likely due to simultaneous triggering of high-affinity KA binding sites of extrahippocampal brain regions such as the amygdala complex, which matures around P17–19, in addition to activation of high-affinity sites expressed on CA3 hippocampal neurons (43). Consistent with neural assessments with NeuN and Nissl stains, the KA + CBDih group showed a significant reduction in reactive astroglia within the vulnerable CA1 indicating less inflammation occurred with cannabinoid treatment at this juvenile age. Cannabinoids and Development The cannabinoid receptors and endocannabinoids express a mature phenotype in prenatal and early postnatal life and they are thought to play critical roles in normal brain development and maturation (44–45). Several studies have emerged concerning the anticonvulsant effect of CB1 receptor agonists in development. Physiology studies originally conducted in adult hippocampal slice preparations showed that depolarization of CA1 neurons provokes an endocannabinoid-mediated inhibition of GABA release, also known as depolarized-induced suppression of inhibition (DSI) involves Group I metabotropic receptors (46). Acute DSI was more pronounced in slices prepared from juvenile rats, similar to the ages of our study, whereas the suppression of excitatory transmission by the exogenous cannabinoid receptor agonist (R+)WIN 55 212 was indistinguishable indicating that inhibitory synaptic function of the immature brain is more sensitive to cannabinoids compared with adults (47). In keeping with physiology studies, both acute and chronic treatment of (R+)WIN 55 212 induced more severe behavioral object/social recognition deficits in pubescent rats than in mature animals (48). In P20 rat pups, lower doses of (R+)WIN 55 212 (0.5 mg/kg) had minimal or no behavioral effect but had potent anticonvulsant effects, whereas higher doses induced a drunken state and was proconvulsant in the KA model of juvenile seizures (38). Accordingly, low doses of CB1 receptor agonists, (R+)WIN55 212 or anandamide (AEA) provided the best protection from cerebral ischemia or oxygen deprivation to cultured neurons, consistent with our observations (49). In addition, acute THC or (R+)WIN55 212 treatment markedly enhanced proapoptotic properties of ethanol plus NMDA antagonist, MK801, during the first postnatal week which then declined thereafter as a function of age further supporting that inhibitory synaptic function of the immature brain is more sensitive to cannabinoids (50). In adults, chronic treatment of THC or (R+)WIN55 212 protected neurons and promoted tolerance to seizure-induced cognitive deficits. These were associated with reduced phosphorylated-ERK (extracellular signal-regulated kinase) activation (51). Finally, chronic exposure of a different CB1 receptor agonist, CP 55 940, led to increased anxiety and persistent memory dysfunction in adolescent but not adult rats confirming the heightened sensitivity of cannabinoids in development (52). Much less is known about how a low-affinity receptor interacting cannabinoid, CBD, exerts its anticonvulsant effects. Aside from proposed antiinflammatory mechanisms, we question whether CBD can also affect CB1 receptors indirectly due to its long half-life and its effect on the metabolism of fatty acid amino-hydrolase (FAAH). Since CBD in rodents binds to FAAH in rodents and to fatty acid binding proteins in humans, endogenous cannabinoid levels will be will elevated for prolonged periods by either preventing hydrolysis (degradation), or by inhibiting endocannabinoid transport to the ribosome to prevent degradation (53). In keeping with this, additional nonΔ9-THC cannabinoids have micromolar potency on enzyme inhibitors of endocannabinoid uptake that will result in elevated extracellular AEA levels (and 2-AG). For example, cannabidavarin (CBDV) inhibits DAGLα while CBD inhibits FAAH activity to increase endocannabinoid concentration (53, 54). Another protective mechanism of CBD and CBDV can be via their ability to activate or desensitize transient receptor potential cation channel subfamily V member 1 (TRPV1), as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1) channels that cause intracellular calcium elevations to regulate neurotransmitter release (55, 56). Together, CBD treatment may indirectly allow a slower activation of retrograde feedback signaling compared with depolarization currents, due to elevated levels of endogenous postsynaptically synthesized endocannabinoids (AEA, 2-AG) which subsequently bind to CB1 receptors to further exert anticonvulsant effects. In reference to development, the rodent FAAH enzyme is also abundant pre- and postnatally to regulate neurotransmission at times when GABA has depolarizing properties (57). It is also important to note that cellular and subcellular distributions of FAAH varies as a function of age and within specific brain regions such as the hippocampus; mature patterns are not observed until after P20, which could explain maturational differences in sensitivity to cannabinoids (58, 59). The developmental stability of cannabinoid receptor expression in both seizure models implies that they play important protective roles in regulating hippocampal seizure activity at young ages before neurotoxicity becomes progressive and irreversible. Neurotoxicity and Neuroprotection Behavioral recovery produced by CBD treatment was correlated with KA + CBDih cotreatment and lowest level of hyperactivity. The KAih model used herein at P20 did not cause remarkable morphological changes or cell loss of CA1 or CA3 hippocampal neurons as compared with juvenile rat pups with KAip or adult rodents receiving KAih, suggesting that pharmokinetics is not the only factor responsible for differences in neuronal vulnerability. Instead, intrahippocampal KA injection causes increased neuropathogenicity with increasing age due to maturation of limbic networks and specific receptor subunit composition (ie glutamatergic and GABAergic) (27, 60, 61). Neuroprotection at the young age studied was characterized by morphological appearance and reversal of dark hyperbasophilic neuronal staining when KA and CBD were coadministered irrespective of central versus peripheral treatment. Moreover, CBD improved neurological function scores, motor coordination, reduced hyper-locomotor behavior, and reduced gliosis seen at 72 hours consistent with an ischemic brain insult in rats or gerbils indicating that CBD is most effective when administered quickly following a neurological insult (62, 63). A recent longitudinal cohort study showed CBD-enriched cannabis extract given to children at lower doses (11 mg/kg/d), similar to doses used in our systemic part of the study, and adolescents at higher doses (>11 mg/kg/d), as an adjunctive therapy, can significantly reduce seizure frequency in patients with refractory epilepsy; however, a large percentage of patients (46%) could not tolerate the regimen, suggesting cross reactivity and drug–drug interactions with CBD need further testing (64). Astroglia Since signaling from astrocytes is required for postnatal maturation of interneurons expressing PV (65), acute inflammation and an estimate of GABAergic activity was evaluated with PV and GFAP immunolabeling of the hippocampus in both KA models. In our study, in P20 rats, a significant percentage of reactive astroglia were less intense and fewer in number within the vulnerable CA1 at 72 hours, but alterations persisted deep within proliferative dentate hilus, suggesting CBD-reduced seizure-associated inflammation, but without affecting reactive glia that may be involved with cell proliferation. This supports that less inflammation occurs in vulnerable areas while proliferation zones of the dentate hilus remain active despite attenuation of seizure activity by the CBD treatment. PV Interneurons PV-expressing (PV+) basket cells are fast-spiking and nonaccommodating in their spiking properties, have fast membrane time constants and release GABA synchronously, allowing precise timed oscillations within a network of pyramidal cells (66). In certain adult seizure models, loss of PV neurons follows is thought to result in an imbalance of excitatory and inhibitory neurotransmission to contribute to development of spontaneous seizures (67, 68). In keeping, adult rats receiving unilateral intrahippocampal injection of KA, which resulted in both acute rapid discharges as well as a delayed onset to seizures, caused selective PV+ and calbindin-D28k+ cell depletion within the CA1 and DG, whereas calretinin positive interneurons were spared (69). Neuropilin 2-deficient mice, vulnerable to KA-induced seizures, also exhibited reduced number of PV+ interneurons (70). In contrast, in adult awake rats, PV+ interneurons were resistant to cell death following prolonged stimulation of the perforant pathway, while somatostatin containing interneurons were vulnerable (71). Interestingly, many PV+ interneurons are often spared and intensely stained in human resected hippocampi from mesial temporal lobe epilepsy patients (67, 66, 72, 73). In the pilocarpine model of juvenile status epilepticus, PV+ interneurons were reduced in number when evaluated after 4 weeks, but survivors had increased expression of mRNA levels particularly in the pyramidal cell layer of the subiculum and throughout the entorhinal cortex (74). Accordingly, in both KA models of our study, in P20 rats, many PV+ expressing neurons were spared and PV immunodensity was selectively increased. Together, data suggest that GABAergic activity alterations may be facilitated by epileptiform events and also by the long half-life of CBD. Since PV expression was not altered by CBD alone suggests that the seizure activity is responsible for the increased expression. On the other hand, the lack of regression in the number of WDS due to reduced clonic seizures in the presence of CBD may also contribute to elevations in PV expression, which could improve neuronal cell survival and prevent development of spontaneous seizures in the immature brain. Finally, learning and social deficits were subtle and recovered quickly in young rats compared with adults, which may in part be due to loss of SCN1a channels in PV-expressing cells that would impair their inhibitory control and hippocampal neuroplasticity (75). Conclusion Intrahippocampal coadministration of CBD provided the best anticonvulsant effects that lead to reduced hyper-locomotor activity and neuroprotection. 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TI - Anticonvulsant and Neuroprotective Effects of Cannabidiol During the Juvenile Period
JF - Journal of Neuropathology & Experimental Neurology
DO - 10.1093/jnen/nly069
DA - 2018-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/anticonvulsant-and-neuroprotective-effects-of-cannabidiol-during-the-bY5IaPHdpl
SP - 904
VL - 77
IS - 10
DP - DeepDyve
ER -