Description of the Characteristic Behavior Related to Camphor-Induced Convulsion

After the administration of camphorated oil, the animals showed a rapid evolution of the seizure, displaying six behaviors. The change in behavior begins with immobility (akinesia) with an average latency of 78.89 ± 18.73 s, followed by head and neck tremors.

(135.9 ± 17.03 s) and tail stiffening (178 ± 17.59 s). The next behaviour involves the somatic system more intensely, revealing itself as clonias of the forelimbs (219.6 ± 37.37 s), the crisis evolving into a generalized clonic convulsion without loss of the posture reflex (281 ± 38.70 s) and with a worsening of the crises causing generalized clonic convulsions with loss of the posture reflex (384.9 ± 74.41 s). Each component of the behaviors recorded above is part of the subsequent behaviors (Fig. 1).

Fig. 1

- Description of camphor-induced seizure-related behavior according to the latency for observing the behavior (n = 9)

There are reports in the literature of Cinnamomum camphora inducing or provoking tonic-clonic seizures in patients with and without a history of seizures. Camphor induced generalized tonic-clonic seizures in 95% of the cases in a study with human patients with no previous history of seizures (p = 22) and caused generalized tonic- clonic seizures in 27.3% and localized or bilateral seizures in 15% of the other group of patients in the study, with a history of epilepsy or drug seizures (p = 33) (Mathew et al. 2020).

Camphor-Induced Seizures have Similar Characteristics to the PTZ-Induced Chemoconvulsant Model

The readings of the ECoG recordings for the control group showed low amplitudes (0.05 mV), which keeps the recording regular, so the spectrogram shows higher levels of energy distributed at frequencies below 10 Hz (Fig. 2A and B). The amplification of the recording shows morphographic components related to the activity of the rat’s motor cortex, characterized by low amplitude with a predominance of low-frequency brain oscillations (Fig. 2C).

Fig. 2

- ECoG recordings of the motor cortex of the control group (A), spectrogram of energy distribution up to 40 Hz (B), amplification of the recordings showing a duration of one second (899 to 900s) (C), recordings lasting 20 min

The PTZ-treated group showed changes in the ECoG tracing with cyclic activity reaching amplitudes above 0.5 mV during ictal status, which are characteristic of seizures, and interictal status, which was characterized by a decrease in the amplitude of the recording to the range of 0.05 mV. After the application of PTZ, the latency for the onset of seizures was 48 ± 24s. The frequency spectrogram shows an increase in power in oscillations distributed up to 40 Hz (Fig. 3A).

The group treated with camphor oil showed intercalated ECoG tracings between ictal and interictal status, characterized by neuronal recruitment with an increase in field potential during ictal status, which showed an increase in the power of the recordings, and interictal status with a decrease in power; the latency period for occurrence was 78.89 ± 18.73 s. The spectrogram shows a greater distribution of energy during ictal status (Fig. 3B).

Fig. 3

Demonstrations of ECoG recordings lasting 30 min. ECoG tracing of the group treated with pentylenetetrazole (PTZ) (left), amplification showing graphic components of ictal status (top center), amplification showing graphical elements of interictal status (bottom center) and spectrogram of energy distribution up to 40 Hz (right) (A). ECoG recording lasting 30 min of the camphorated oil-treated group (left), amplification of ictal status (top center), amplification of interictal status (bottom center) and power distribution spectrogram at frequencies up to 40 Hz (B)

The spectral power distribution revealed greater amplitude in all brain waves in the camphor and PTZ groups compared to the control group (Fig. 4A). Power in the delta, theta and alpha frequencies was greater in the PTZ group, while gamma oscillations were more powerful in the camphor group. In this way, the average power in linear oscillations up to 40 Hz for the control group was 0.366 ± 0.086 mV² / Hz x 10-³ was lower than the groups treated with PTZ 9.11 ± 1.78 mV² / Hz x 10-³ and camphorated oil 6.46 ± 1.77 mV² / Hz x 10-³ (p = 0.001). There was a difference between the group treated with PTZ and camphor (p = 0.02) ( F(2, 24) = 85.77; p < 0.0001) (Fig. 4B).

Mean analyses of the delta, theta, alpha, beta and gamma brain frequency bands showed an increase compared to the control group. Thus, PTZ and camphorated oil were able to induce disturbances in all brain waves, but the animals treated with camphorated oil showed greater power in gamma oscillations compared to the PTZ group (Fig. 4A).

For delta oscillations, the control group had a lower mean power (0.109 ± 0.022 mV² / Hz x 10-³) than the other groups PTZ (0.814 ± 0.171 mV² / Hz x 10-³) ( p = 0.001) and camphorated oil (0.271 ± 0.0620 mV² / Hz x 10-³) (p = 0.009). The group treated with PTZ had a higher mean than the group treated with camphorated oil ( p = 0.001) ( F(2, 24) = 109.2; p < 0.0001) (Fig. 4C).

The mean power in theta oscillations for the control group (0.723 ± 0.017 mV² / Hz x 10-³) was lower than the PTZ (2.141 ± 0.263 mV² / Hz x 10-³) (p = 0.001) and camphorated oil (0.321 ± 0.048 mV² / Hz x 10-³) (p = 0.006) groups. The group treated with PTZ was superior to the group treated with camphorated oil (p = 0.001) ( F(2, 24) = 478.5; p < 0.0001) (Fig. 4D).

In alpha oscillations, the control group had a mean of 0.033 ± 0.007 mV²/Hz x 10-.

³, which was lower than the groups treated with PTZ (2.35 ± 0.51 mV²/Hz x 10− 3) (p = 0.001) and camphorated oil (0.60 ± 0.153 mV²/Hz x 10− 3). The group treated with camphorated oil was inferior to the group treated with PTZ (p = 0.001) ( F(2, 24) = 136; p < 0.0001) (Fig. 4E).

For beta oscillations, the control group had a mean of 0.013 ± 0.006 mV² / Hz x 10-³, which was lower than the treated groups (p = 0.001). The PTZ group had a mean power of 2.13 ± 0.354 mV²/Hz x 10-³ and was similar to the group treated with camphorated oil (p = 0.1928) ( F(2, 24) = 112.1; p < 0.0001) (Fig. 4F).

For gamma oscillations, the control group (0.0059 ± 0.0011 mV²/Hz x 10-³) was lower than the other treated groups (p = 0.001). The camphor oil-treated group (0.730 ± 0.141 mV²/Hz x 10-³) was superior to the PTZ-treated group (0.471 ± 0.141 mV²/Hz x 10-³) (p = 0.001) ( F(2, 24) = 118.7; p < 0.0001) (Fig. 4G).

Fig. 4

Spectral distribution of power at frequencies up to 40 Hz for the control, PTZ and camphor recordings (A); Graph representing the average linear power up to 40 Hz for the groups (B); Graphs of power distribution in brain oscillations up to 40 Hz: Delta ( 1–4 Hz) ( red line) (C), Theta (4–8 Hz) (black line) (D), Alpha (8–12 Hz) (yellow line) (E), Beta ( 12–28 Hz) (purple line) (F) and Gamma (28–40 Hz) (green line) (G). After ANOVA followed by Tukey, * P < 0.05 ** P < 0.01 ***P < 0.001, n = 9)

The spectral distribution graph of power at frequencies up to 40 Hz in the recordings of camphorated oil-induced seizures was divided into two distinct periods during the recording that occur in a cyclical manner, in the ictal status period a higher level of power was observed and in the interictal status period a lower level of power was observed in the oscillations (Fig. 5A). The average power for the control group (0.366 ± 0.086 mV²/Hz x 10-³) was similar to the interictal status group (p = 0.075). However, all groups had lower mean power during ictal status (10.25 ± 1.661 mV²/Hz x 10-³) ( F(3, 32) = 116.4; p < 0.0001) (Fig. 5B).

For delta oscillations (1–4 Hz), the control group (0.109 ± 0.022 mV²/Hz x 10-³) had a mean power similar to the interictal period (p = 0.092), but was lower than the camphor oil (0.271 ± 0.062 mV²/Hz x 10-³) and ictal status (0.351 ± 0.081 mV²/Hz x 10-³) groups. The ictal status group was superior to all groups( F(3, 32) = 32.94; p < 0.0001) (Fig. 5C).

The theta oscillations in the status ictal period (0.433 ± 0.114 mV²/Hz x 10-³) were higher than in the other groups. The interictal period had a higher mean (0.197 ± 0.100 mV²/Hz x 10-³) than the control group (0.072 ± 0.017 mV²/Hz x 10-³) ( F(3, 32) = 33.76; p < 0.0001) (Fig. 5D).

For alpha oscillations, the mean power during ictal status (0.872 ± 0.308 mV²/Hz x 10-³) was higher than in the other groups. The average power for interictal status (0.225 ± 0.063 mV²/Hz x 10-³) was similar to the control group (p = 0.1129) ( F(3, 32) = 41.72; p < 0.0001) (Fig. 5E).

The average power of beta oscillations during the ictal status period (3.18 ± 0.364 mV²/Hz x 10-³) was higher than the other groups. The control group (0.013 ± 0.0065 mV²/Hz x 10-³) was lower than the interictal status period (0.762 ± 0.212 mV²/Hz x 10-³)( F(3, 32) = 184.5; p < 0.0001) (Fig. 5F).

For gamma oscillations, the control group (0.005 ± 0.001 mV²/Hz x 10-³) was lower than the other groups. The average power for the ictal status period (1.20 ± 0.25 mV²/Hz x 10-³) was higher than the other groups. The camphorated oil group (0.73 ± 0.141 mV²/Hz x 10-³) was superior to the interictal status group ( F(3, 32) = 118.1; p < 0.0001) (Fig. 5G).

Fig. 5

Spectral power distribution graph at frequencies up to 40 Hz in ECoG recordings during the seizure in the camphor oil-treated group and in the ictal status and interictal status periods (A); Linear power averages during ictal status and interictal status (B); Linear power average between groups with frequency in delta oscillations (1–4 Hz) (red line) (C); Average power distribution in theta frequency (4–8 Hz) (black line) (D); Graph of linear power distribution in the recordings between the groups in alpha oscillations (8–12 Hz) (yellow line) (E); Average power distribution in beta frequency (12–28 Hz) (purple line) (F); Graph of linear power distribution between the groups in gamma frequency (28–40 Hz) (green line) (G).(After ANOVA followed by Tukey, * P < 0.05 ** P < 0.01 ***P < 0.001, n = 9)

Camphor had a Greater Preponderance in the Power of Beta and Gamma Oscillations and was Refractory to Treatment with Phenobarbital and Phenytoin

To assess seizure control observed after camphorated oil treatment, the animals were treated with anticonvulsants, and then ECoGs were recorded, and their power in beta (12–28 Hz) and gamma (28–40 Hz) oscillations was assessed. Seizure control after phenobarbital use was observed 10 min after recording. During the first 10 min, periods of ictal and interictal states could still be identified (Fig. 6C).

Phenytoin and sodium valproate were ineffective in controlling seizures, with the ictal and interictal states maintained throughout the recording, demonstrating that the chemoconvulsant model induced by camphorated oil was refractory to the treatments (Fig. 6D and E). Seizure control was effectively achieved with diazepam and propofol, which indicated better protection for the animals (Fig. 6A and B), with propofol being more effective. Recording patterns obtained with the use of anticonvulsants are shown for beta and gamma oscillations (Figs. 6F and G).

The mean beta oscillation in the control group was 0.013 ± 0.006 mV²/Hz x 10− 3, similar to the group treated with Diazepam (p = 0.959) and propofol (p = 0.999), but was lower than the other groups. The group treated with phenytoin (1.41 ± 0.29 mV²/Hz x 10− 3) was superior to the other groups treated with anticonvulsants. The group treated with phenobarbital (0.659 ± 0.178 mV²/Hz x 10− 3) and lower than the camphorated oil group, treated with phenytoin and sodium valproate. The diazepam-treated group (0.0942 ± 0.0193 mV²/Hz x 10− 3 was similar to the propofol-treated group (p = 0.999) (F(6, 56) = 90.02; p < 0.0001) (Fig. 6F). For gamma oscillations, the control group (0.005 ± 0.0011 mV²/Hz x 10− 3) was similar to the diazepam group (p = 0.998) and the propofol-treated group (p = 999). The phenobarbital group (0.523 ± 0.128 mV²/Hz x 10− 3) was similar to the phenytoin group (p = 0.923) and the sodium valproate-treated group (p = 0.169), but was inferior to the camphor group. The phenytoin-treated group (0.471 ± 0.094 mV²/Hz x 10− 3) was similar to the group treated with sodium valproate (p = 0.787). The group treated with Diazepam (0.0196 ± 0.004 mV²/Hz x 10− 3) was similar to the group treated with propofol (p = 0.999) (F(6, 56) = 77.56; p < 0.0001) (Fig. 6G).

Fig. 6

ECoG recordings demonstrating the action of anticonvulsants: Propofol (10 mg/kg, intravenous IV) (A), Diazepam (10 mg/kg IV) (B), Phenobarbital (10 mg/kg (IV) (C); Sodium valproate (10 mg/kg IV) (D), Phenytoin (10 mg/kg IV) (E), five minutes before the administration of camphor (470 mg/kg i.p.) and subsequently recorded for 15 min. Graph with mean power at beta (12–28 Hz) (F) and gamma (28–40 Hz) (G) frequencies. (After ANOVA followed by Tukey, * P < 0.05 ** P < 0.01 ***P < 0.001, n = 9)