Subchronic Cannabidiol (CBD) Treatment During the Silent Period Fails to Prevent the Increased Seizure Susceptibility Following Lithium Pilocarpine-Induced Status Epilepticus

1. Introduction

Cannabidiol (CBD), a phytocannabinoid without hallucinogenic effects, has been widely reported to have anticonvulsant and anti-inflammatory properties, highlighting its potential use in the treatment of drug-resistant epilepsy. Several clinical trials have shown that CBD as an adjuvant treatment can reduce the frequency and intensity of seizures in a wide range of patients with epileptic syndromes. These studies also demonstrated that CBD was well tolerated by most patients, with minimal adverse effects [1,2,3]. Based on this, the Food and Drug Administration approved its use as an adjunct to anti-seizure medication (ASM) treatment in Dravet and Lennox-Gastaut syndromes [4], although its mechanism of action has not been elucidated. Information from pharmacokinetic monitoring and ASM interactions shows that CBD treatment promotes an increase in ASM plasma concentrations [5], presumably by blocking cytochrome activity [6]and/or reducing the activity of ABC family transporters (ABC-t) [7,8,9]. CBD has been shown to interact with a variety of molecular targets, including cannabinoid, dopamine, serotonin, and opioid receptors, PPARγ, and ion channels such as TRPV1 and TRPV2 [10]. It has recently been suggested that CBD may enhance the effect of ASMs acting on the GABAergic system, while it may have a paradoxical effect when acting in combination with sodium channel blockers [11]. The complexity of interactions between CBD and its molecular targets suggests a multiplicity of effects that could regulate various cellular processes. In addition, several publications have demonstrated the anti-inflammatory potential of CBD and other cannabinoids through receptor activation, inhibition of the synthesis and release of inflammatory cytokines, inhibition of cell proliferation, and even induction of apoptosis [12,13,14]. Furthermore, it has been established that treatment with CBD decreases the cellular function of human neutrophils, monocytes, and eosinophils [15,16,17,18]. It has been suggested that neuroinflammation is a trigger for the development of neurodegenerative diseases [19]. Epileptogenesis is the process by which epilepsy develops into a chronic disease, increasing the susceptibility to seizure development [20]. It can be triggered by both genetic causes and acquired factors [20]. These factors include stroke, traumatic brain injury (TBI), and status epilepticus (SE), with different rates of epilepsy development in humans. Cases involving SE have a risk rate greater than 40% within the first few years after the insult. Insults such as epileptic seizures without SE, stroke, and severe TBI are associated with a 10–20% risk of developing epilepsy, while mild TBI has a risk of less than 5% [21,22,23]. Despite its effectiveness in controlling seizures and its anti-inflammatory properties, it is unclear whether treatment with CBD can prevent epilepsy from becoming a chronic disease. Few experimental studies have addressed the role of CBD in epileptogenesis. When CBD was administered prior to the induction of SE by pilocarpine or during the development of kindling with pentylenetetrazol (PTZ), a decrease in the incidence of seizures was reported [24,25]. Similarly, chronic treatment with CBD during the development of audiogenic kindling in audiogenic rats prevented the recruitment of limbic structures [26] However, all these results may be due to a decrease in the intensity of the insult rather than a direct action on the development of epileptogenesis. Using rats with fully kindled amygdala, partial seizure suppression was observed at high doses of CBD (>250mg/kg) administered prior to testing [27]. On the other hand, using Scn1a +/− mice in which seizures are triggered by hyperthermia, subchronic treatment with CBD did not alter the frequency of spontaneous seizures [28]. Similar results were obtained in a model of cobalt-induced seizures [29]. In contrast, a recent article showed that chronic treatment with high doses of CBD after pilocarpine-induced SE produces a decrease in seizure burden; however, the experimental design included treatment with CBD prior to SE. [30]

The aim of this study was to determine whether subchronic treatment with CBD can affect the susceptibility to seizure development. Given the variability in experimental approaches, routes of administration, doses, and duration of CBD treatment found in the literature, we used the lithium-pilocarpine SE induction model. CBD was administered orally at the maximum daily dose recommended for clinical treatment (20 mg/kg) for 14 days, and susceptibility to seizure development was evaluated by the administration of subconvulsive PTZ.

2. Materials and Methods

2.1. Animal and Experimental Model

Male Sprague Dawley rats weighing approximately 250 grams at the start of the experiment were used. The animals were housed under standard conditions one week prior to the start of the experiment (12 h light/12 h dark, temperature 22 ± 2 °C, food and water ad libitum). Status epilepticus (SE) was induced by the lithium-pilocarpine paradigm as described previously [31]. Briefly, each animal was administered lithium chloride (127 mg/kg i.p.), since lithium potentiates and facilitates the induction of pilocarpine-induced epileptic seizures, reducing 10 times the pilocarpine doses. After 18 hours, pilocarpine (30 mg/kg i.p.) was administered to induce SE, and convulsive behavior was assessed using the Racine scale. The control group was treated with a similar volume of saline solution i.p. instead of pilocarpine. SE was considered achieved when the animals presented generalized tonic-clonic seizures (GTCS) for at least 5 minutes. Thirty minutes after the onset of SE, diazepam (DZP, 50 mg/kg i.p.) was administered. SE was considered terminated when seizures ceased. The animals were monitored for a period of 48 hours, during which vital signs were assessed to ensure recovery from SE Animals exhibiting prolonged respiratory distress, weight loss exceeding 50% of their initial body weight, severe dehydration, or visible signs of necrosis (cyanotic discoloration) on the tail or hind limbs were euthanized in accordance with resolution REDEC-2025-2661-E-UBA-DCT_FFYB of the Institutional Committee for the Care and Use of Laboratory Animals of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires. The control group was treated with lithium and an equivalent volume of saline solution, followed one hour later by a single dose of DZP.

2.2. Experimental Design

In this study, we used 31 rats, 10 of which formed the control group, while the remaining rats were subjected to the lithium-pilocarpine paradigm. Only 14 rats developed SE and were divided into three experimental groups. Four SE rats and 5 control rats were reserved for the epileptic threshold proof-of-concept test at 7 days post-SE (7DPSE). The other groups, meanwhile, were treated orally for 14 days post-SE (14DPSE). The control group (n=5) was administered an equivalent volume of water. The CBD group (n=5) was treated with a dose of 20 mg/kg CBD every 24 hours (Kanbis^®, CBD 100 mg/ml, Elea). Finally, the vehicle group (n=5) was treated with a proportional volume of sesame oil every 24 hours. All treatments were administered via gavage after recording the weights and the handling index.

2.3. Evaluation of the Discretely Measure the Seizure Threshold (DMST)

After 24 hs at the end of the experimental period for each group (7 or 14 DPSE), the seizure threshold was evaluated as the DMST protocol adapted from the rapid kindling protocol [31]. PTZ is rapidly absorbed (1–3 min i.p.) with a half-life of 116 ± 25 minutes, a clearance of 5.36 ± 0.34 ml/min/kg, and very low protein binding [32]. In addition, PTZ exhibits a typically sigmoidal dose-response relationship, with subthreshold doses in the range of <20–30 mg/kg (Löscher, 2017). Finally, the DMST protocol consists of the administration of successive subconvulsive doses (15 mg/kg) of pentylenetetrazol (PTZ) intraperitoneally every 10 minutes. During the interval between doses, the animals were observed, and the seizure threshold was established when they presented with GTCS (Figure 3A). For each animal, the cumulative dose and latency to GTCS were recorded.

2.4. Monitoring Animal Development

A. Weight evolution. The body weight of each animal was recorded daily. The weight measured on the day of SE induction was established as the reference value (100%), and the values for consecutive days were expressed as a percentage of that baseline weight. Based on these data, a sixth-degree polynomial was fitted to describe the temporal fluctuations in weight. The parameters defining the growth model of the animals after SE were defined as $T_{min}$: (time at which maximum weight loss occurred); $T_{rec}$ (time at which the animals reached 100% of their initial weight); $T_{max}$ (time at which the animals reached their maximum weight); ${Weight}_{loss}$ (difference between initial and minimum weight); ${Weight}_{gain}$ (difference between maximum and initial weight). The parameters for each animal were obtained from the models and then averaged, and the adjusted average growth curve was plotted with its confidence interval.

B. Manipulation Score: The reaction to daily handling was assessed using a behavioral scale designed based on previous handling score [33,34], adapted to our observations of the animals following exposure to the Li-Pilo paradigm. Each animal was assigned a daily score from 1 to 10, depending on its behavior during the weighing procedure. Reactivity to handling was always recorded at the same time and under the same conditions. The levels of the scale were defined as follows:

1: Normal, active, and docile in the cage before and during handling.

2: Active but alert, elusive, responds well to handling.

3: Decreased activity but alert, responds to handling.

4: Lethargic, unresponsive to handling.

5: Lethargic, responsive to handling.

6: Slight difficulty handling inside the cage (exaggerated response to minor noises, difficult to remove from cage).

7: Moderate difficulty handling inside the cage (exaggerated response to minor noises, jumps uncontrollably, difficult to remove from cage).

8: Moderate difficulty handling outside the cage (flight response, jumps uncontrollably).

9: Severe difficulty handling inside and outside the cage (jumps uncontrollably, spins around on its tail).

10: Maximum difficulty handling (jumps uncontrollably, spins around on its tail, bites).

2.5. Statistics

All statistical tests were performed using Graphpad Prism 8. Parameters obtained from weight evolution fitting and seizure threshold (control vs 7DPSE and 7DPSE vs 14DPSE) were compared using two-tailed Student t-test with a significance level of 0.05. Correlation analyses were made by two-tailed linear regression with the same significance level. The seizure threshold to explore the effects of CBD was conducted by One-way ANOVA following Tukey’s multiple comparisons post-test with a significant level of 0.05. The behavioral analysis was carried out by Two-way ANOVA and difference for each treatment as well as interaction were analyzed with a p-value of 0.05.

3. Results

To begin investigating whether CBD treatment contributes to mitigating susceptibility to seizures after SE, out of a total of 31 male rats, 21 rats were subjected to lithium-pilocarpine-induced SE while the remaining ten rats remained as a control group (Figure 1A). Of the total number of rats subjected to SE, 16 developed SE and five showed tonic-clonic seizures without developing SE (Figure 1A, NoSE group). The SE mortality rate was 12.5% (2/16 rats that develop SE - Figure 1B). Thirty minutes after the onset of SE, rats received a single dose of DZP, and the time until the animals stopped showing convulsive activity was recorded. Figure 1C shows the individual seizure duration for each animal that survival to SE induction (14/16), which ranged from 103 to 234 minutes with a mean of 171.2 ± 36.02 minutes and a mean coefficient of variation of 21.04%.

Take into account that aggressive behavior was reported after SE, we recorded the rats responses to daily manipulation during the postictal period and the beginning of the silent period (7 days after SE induction). To describe this behavior, it was necessary to design an ad-hoc scale (Manipulation Score) that described behaviors ranging from normal to lethargic and exacerbated as a result of SE. Figure 2A shows the individual Manipulation Score for each rat in the control, 7DPSE, and NoSE groups. Rats that developed SE were more reactive to manipulation than the control group (p=0.005). We observed a heterogeneous response in the SE group, as some rats showed a high Manipulation Score in the first few days after SE, while another subgroup of animals showed exacerbated responses from the third day after SE (pscore=0.008; ptime > 0.05; interaction=ns). On the other hand, the animals in the NoSE group showed a Manipulation Score as low as that of the control group, suggesting that reactive behavior could be related to the intensity of the seizures rather than the time elapsed since SE induction.

The effect of SE was also reflected in weight evolution, showing on average a ${Weight}_{loss}$ = 16.5 ± 2.7% from their initial weight, within a time elapsed to reach minimum weight $T_{min}$ =1.19±0.65 days (Figure 2B). Time to recover that initial weight varied between 2.08 and 4.94 days ($T_{rec}$=3.75±1.12 days), and subsequent weight gain ranged between 7.6 and 10.4% ( ${Weight}_{gain}$=9.3±1.2%). This behavior differed from the control group, which showed continuous growth during the same period. Animals that did not develop SE showed behavior similar to the control group (data not shown).

Considering the variability in both the duration of SE and the parameters derived from the evolution of the animals’ weight, we examined whether there is a correlation between seizure duration and the animals’ response after SE. We did not observe a significant correlation between the SE duration and the assessed parameters (Figure 2C-E), suggesting that each response to SE was individual, even though they show similar profiles.

3.1. Seizure Threshold Measure

Our next step was to confirm whether susceptibility to seizures increased as a result of SE. To do this, we designed a DMST protocol in which we administered subconvulsive doses of PTZ every ten minutes until the rats developed GTCS (Figure 3A). The animals that suffered SE required 30% less PTZ dosage to generate GTCS (Control: 75±12.25mg/kg vs.7DPSE: 50±8.6mg/kg, Figure 3B). Figure 3C shows a similar change in the latency for the development of GTCS from 42.5±8.2 minutes vs. 29±3.6 minutes. Taken together, these results suggest that SE increases susceptibility to developing seizures 7 days after SE.

3.2. Evaluation of the Effect of CBD on Susceptibility to Develop Seizures After SE

Since CBD has well-documented anticonvulsant and anti-inflammatory effects, we evaluated whether treatment with CBD can modify susceptibility to seizures development after SE. To do this, animals that developed SE were divided into two groups and treated with CBD or vehicle for 14 days, and susceptibility to seizures development was evaluated using the DMST protocol (Figure 1A-3A). We studied individual behavior during treatment using the Manipulation Score. Figure 4A shows the behavioral evolution of the control groups and those treated with vehicle or CBD. The control group maintained normal behavior throughout the experimental period, with scores between 1 and 2 on the scale, as they did not develop SE. As expected, rats treated with vehicle showed a period of decreasing reactivity during the 2-3 days post SE, although we did not observe heterogeneous behavior as was the case with animals at 7 DPSE. Strikingly, the group treated with CBD showed homogeneous and atypical behavior. During the first two days, the animals remained as unresponsive as the control group; however, from day 3 onwards, the animals exhibited markedly increased reactivity that persisted for a longer period compared to those treated with vehicle (pscore<0.0001; ptime=0.0002; interaction<0.0001). Despite these noticeable changes in the rats’ behavior, weight changes did not show significant differences in the parameters obtained from the adjustments for the vehicle and CBD treatments, as shown in Figure 4B. As observed in the analysis of parameters after SE, the growth parameters obtained for each of the treatments did not show a correlation with the duration of SE (Figure 4C-D). Conversely, ${Weight}_{loss}$ was positively correlated with $T_{min}$ for both vehicle and CBD treatments (p=0.026 and p=0.0129 respectively).

Given that the animals showed a distinctive behavioral profile for each treatment, we evaluated whether treatment with CBD could produce a shift in the growth parameters established after SE. Given the time difference between treatments (7 vs. 14DPSE), we only evaluated $T_{min}$, $T_{rec}$, and ${Weight}_{loss}$. We observed that CBD treatment produced an increase in $T_{min}$ (1.61±0.57 days vs. 2.59±1.43 days; p=0.036) and $T_{rec}$ (3.76±1.20 days vs. 8.02±3.96 days; p=0.039) and a tendency to increase ${Weight}_{loss}$ (16.54±2.66% vs. 21.34±5.53%; p=0.079). Taken together, these results suggest that CBD modifies the parameters established for SE and may have no effect on seizure susceptibility. For this reason, we evaluated the seizure threshold after each treatment using DMST. Figure 5A shows a decrease in the PTZ doses required for the development of GTCS for both vehicle and CBD treatment. This was also reflected in the latency to GTCS (Figure 5B), suggesting that CBD treatment does not affect susceptibility to seizures development after pilocarpine-induced SE.

Figure legends

Figure 5. CBD treatment fails to improve the seizure susceptibility after SE. A) accumulative doses of PTZ needed to trigger GTCS. B) latency to the onset of GCTS. Results are presented as mean±SD. Differences were analyzed by One-way ANOVA following by Tukey’s multiple comparisons post test (*p<0.05; **p<0.01).

Figure 5. CBD treatment fails to improve the seizure susceptibility after SE. A) accumulative doses of PTZ needed to trigger GTCS. B) latency to the onset of GCTS. Results are presented as mean±SD. Differences were analyzed by One-way ANOVA following by Tukey’s multiple comparisons post test (*p<0.05; **p<0.01).

4. Discussion

In this study, we evaluated whether CBD administered orally at a human maximum therapeutic daily dose (20 mg/kg) can prevent epilepsy from becoming a chronic disease. In this context, few studies have experimentally addressed this issue, and certain experimental approaches may lead to misinterpretation of results, confusing anticonvulsant activity with antiepileptogenic potential.

Several mechanisms, such as loss of GABAergic neurons, abnormal axon growth, changes in the number of synaptic terminals, changes in the composition and/or gene variants in ion channels, have been proposed to explain the process by which epilepsy becomes a chronic disease after brain injury [25,35,36,37,38]. However, none of these mechanisms has been entirely conclusive. In this regard, processes such as hypoxia, oxidative stress, and neuroinflammation have been described as key interconnected factors in the development of neurodegenerative diseases[19] . This is where CBD could play an important role, as it has been described as having anticonvulsant and anti-inflammatory activity [12,13,14]

Considering that SE is the most prevalent cause of epilepsy becoming chronic [20]we induced SE using the lithium-pilocarpine model, and we measured the animals’ response post-SE. We observed a state of hypersensitivity represented by an increase in aggressive behavior, as described in the literature on epilepsy animal models and patients [39,40]. These behavioral changes were accompanied with a marked decrease in weight (Figure 2), as previously we reported [31]. Strikingly, rats that did not develop SE showed a similar behavior to the control group, even though they experienced GTCS, suggesting that the intensity and/or duration of the seizure affects this hypersensitivity state. This is directly in line with previously reported results, which showed a positive correlation between weight loss and the duration of SE [31]. Given that prolonged SE is associated with a high mortality rate, we administer DZP 30 minutes after the onset of SE to improve the animals’ survival. However, the animals exhibit spontaneous recurrent seizures (SRS) over a highly variable time frame [41,42]; therefore, we developed the DMST as a way to assess susceptibility to seizures without having to wait for the onset of SRS. Ideally, seizures could be monitored using EEG recordings. The lack of such recordings could represent a significant limitation in our study; however, measuring the seizure threshold in conjunction with behavioral observations may provide a good approximation. Compared to the traditional method of measuring the epileptic threshold, which involves administering a continuous flow of PTZ through the rat’s tail vein, the DMST does not use PTZ administration via low-flow peristaltic pumps. Infusion rates as low as 0.1–0.5 ml/min are difficult to maintain. Furthermore, direct infusion can interfere with the animal’s free movement, causing mechanical problems with the infusion flow, damage to the catheter by the animal and even disconnections of the line.

On the other hand, DMST may not show such a direct relationship between the PTZ dose and seizure activity; however, the published pharmacokinetic data for PTZ suggest that this issue will be minimal, since its PTZ absorption by the intraperitoneal route is rapid (1–3 minutes) and its half-life is approximately 2 hours [32]. Furthermore, to minimize this effect, we consider the onset of GTCS as the cutoff point for determining the epileptic threshold. Given the experimental design of the DMST, both the time required to determine the seizure threshold (approximately one hour versus a few minutes) and repeated handling of the animals can affect the determination of the threshold. In a seminal study conducted by our laboratory, we tested these variables and found that they did not affect the determination of the threshold in either normal mice or mice susceptible to seizures [43]. Using DMST, we showed a greater susceptibility to developing seizures in 7 DPSE rats, reflected by a lower PTZ dose and a decrease in the latency to develop GTCS. These results are consistent with those reported by other researchers using continuous measurement of the seizure threshold, who observed a decrease in the seizure threshold within 1 day to 6 months after the SE [44,45,46]

Our next step was to investigate whether oral treatment with CBD oil after SE induction could improve the seizure threshold. This experimental scheme was used to prevent the anticonvulsant action of CBD from interfering with the injury caused by SE. It should be noted that experiments in which CBD has been administered before or during the brain injury have pointed to a potential antiepileptogenic effect of CBD [24,26,30,47,48]. However, these experimental designs, in addition to increasing the probability of false positives, are also far removed from clinical situations where CBD could be applied, i.e., once brain injury has already occurred. Surprisingly, we did not observe an increase in the seizure threshold after administering CBD for 14 days at a daily dose of 20 mg/kg. Also, aggressive behavior and weight loss were sustained. Strikingly, treatment with CBD deepened irritability and prolonged post-SE recovery times ($T_{min}$ and $T_{rec}$) (Figure 4), suggesting also that CBD treatment could contribute to a decrease in the seizure threshold. Furthermore, one might assume that the weight loss is due to a disruption in food intake caused by the oily composition of the CBD carrier (sesame oil). However, weight loss is a characteristic of SE that was also observed in 7DPSE animals that did not receive additional treatment following the SE. However, our findings may be limited by the CBD administration regimen used, as its pharmacokinetics differ between humans and rodents. While the bioavailability of orally administered CBD is low in humans, it is typically higher in rodents, as absorption is generally faster (0.5–2 hours in rodents vs. 1–4 hours in humans), although these values are comparable to those of a high-fat diet [49,50,51]. On the other hand, the half-life of a single oral doses of CBD oils is shorter (2–6 hours in rodents vs. 1-10 hours in humans) [50,52] These differences may be even more pronounced in subchronic treatment, where the half-life in rats can be as long as 12 hours, while in humans it ranges from 2 to 5 days[49,52] , because rodents have a much faster metabolism, and the accumulation of CBD in fat has less of an impact than it does in humans. Therefore, it is expected that regimens with more daily applications and/or higher doses will yield different results. In this regard, Colasanti and colleagues administered two daily doses of 60 mg/kg of CBD following brain injury and found no changes in the threshold [29]. Similarly, Anderson and colleagues demonstrated that doses of 100 mg/kg of CBD, as well as doses of 0.1 mg/kg of THC—either alone or in combination—had an anticonvulsant effect over hyperthermic seizure-induced in mice, whereas chronic treatment with doses of 500–1000 mg/kg/day of CBD did not alter the frequency of SRS. Even the co-administration of 130 mg/kg/day of CBD + 5 mg/kg/day of THC administered orally increased seizure severity and the mortality rate[28].

Since CBD inhibits the cytochromes CYP 3A4 and CYP 2C19 (Herdegen and Cascorbi, 2023), subchronic treatment could lead to drug-drug interactions with PTZ, altering the DMST result and producing a false-negative result. For this reason, the DMST measurement was performed 24 hours after the last administration of CBD, which corresponds to 2–4 half-lives of CBD. In this context, the result obtained with the DMST protocol was statistically indistinguishable between the vehicle and CBD treatments (Figure 5) and showed a level similar to that obtained with 7DPSE rats.

It is known that CBD has an inverted U-shaped dose-response curve for effects on anxiety, sleep disturbance, substance use disorder, arthritis, between others [53,54,55,56]. However, in response to seizures, CBD presents a traditional sigmoid curve [57,58], and it could be expected that over susceptibility to seizure development effect would have a similar response. A recent meta-analysis found that prolonged use of CBD for seizure control was associated with a higher risk of adverse effects compared to placebo and emphasized the importance of adjusting doses to avoid them [59]. For these reasons, acute treatment with high doses of CBD could be effective in reducing susceptibility to seizures. However, high concentrations of CBD (at µM range) have been shown to have a cytotoxic effect [16,60,61,62], therefore determining the therapeutic window for the prophylactic treatment of epileptogenesis after brain injury is key to working with high concentrations of CBD. Finally, another factor that could influence our results is the route of administration. A recent article has shown that inhaled CBD is much more potent than the oral route for controlling kainic acid-induced seizures, reducing the expression of IL-6, IL-33, and BDNF both in the brain and peripherally (Bhandari et al., 2025). In this regard, absorption via inhalation is much faster than via oral administration (5 minutes vs. 0.5–2 hours), while the peak concentration is 4–6 times higher with the inhalation route[50] In summary, our results suggest that CBD 20 mg/kg orally administered has no protective effects susceptibility to seizure development after SE.