Results

Score of convulsive behaviors

Seizure behavior scores are presented in Table 1 and Fig. 1.

Injections | Groups |  |  |  |  |  | 
 | Seizure | TPM (25 mg) | TPM (50 mg) | TPM (70 mg) | EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
1 | 0.4 ± 0.16 | 0.3 ± 0.15 | 0.3 ± 0.15 | 0.2 ± 0.13 | 0.66 ± 0.33 | 0.33 ± 0.21 | 0.16 ± 0.16
2 | 0.6 ± 0.16 | 0.3 ± 0.15 | 0.4 ± 0.16 | 0.6 ± 0.16 | 1.0 ± 0.25 | 0.16 ± 0.16 | 0.33 ± 0.21
3 | 1.1 ± 0.27 | 1.3 ± 0.15 | 0.4 ± 0.16 | 0.9 ± 0.23 | 1.16 ± 0.16 | 0.5 ± 0.22 | 0.66 ± 0.21
4 | 1.2 ± 0.13 | 1.3 ± 0.21 | 1.3 ± 0.21 | 1.0 ± 0.14 | 1.33 ± 0.21 | 1.16 ± 0.16 | 0.5 ± 0.22
5 | 1.9 ± 0.23 | 1.3 ± 0.15 | 1.7 ± 0.15 | 1.2 ± 0.13 | 1.83 ± 0.3 | 1.33 ± 0.21 | 0.83 ± 0.16
6 | 2.4 ± 0.26 | 2.1 ± 0.23 | 1.9 ± 0.17 | 1.6 ± 0.16 | 2.66 ± 0.21 | 1.5 ± 0.22 | 0.83 ± 0.3
7 | 2.8 ± 0.24 | 2.3 ± 0.15 | 2.1 ± 0.17 | 2.0 ± 0.14 | 2.83 ± 0.16 | 1.83 ± 0.16 | 1.0 ± 0.25
8 | 2.9 ± 0.27 | 2.8 ± 0.13 | 2.6 ± 0.26 | 1.8 ± 0.2 | 2.66 ± 0.33 | 2.33 ± 0.21 | 1.33 ± 0.21
9 | 3.5 ± 0.22 | 3.3 ± 0.15 | 2.8 ± 0.13 | 2.3 ± 0.15 | 2.83 ± 0.16 | 2.83 ± 0.16 | 1.16 ± 0.16
10 | 3.5 ± 0.16 | 3.6 ± 0.16 | 3.4 ± 0.13 | 2.6 ± 0.22 | 3.0 ± 0.4 | 3.5 ± 0 | 1.16 ± 0.16
11 | 4.3 ± 0.21 | 3.6 ± 0.16 | 3.4 ± 0.16 | 2.6 ± 0.16 | 3.0 ± 0.2 | 3.5 ± 0.22 | 1.16 ± 0.16
12 | 4.6 ± 0.16 | 4.1 ± 0.23 | 3.9 ± 0.1 | 3.0 ± 0.29 | 3.16 ± 0.1 | 3.66 ± 0.21 | 1.0 ± 0.25

Fig. 1: Seizure behavior scores: Temporal profile of seizure severity scores was assessed using the Racine scale. Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnov test, and individual data points are shown to illustrate variability. The combination of exercise and topiramate (EX+TPM) resulted in significantly lower seizure scores than those observed in both the Seizure group and the groups receiving topiramate alone (at doses of 25, 50, and 70 mg). The symbols *, × , + , †, æ, ^ and £ indicate a significant difference in comparison with the Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), and EX + TPM (50 mg) and EX + TPM (70 mg) groups, respectively. One, two, or three repetitions of symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Fig. 1 illustrates the temporal profile of seizure severity scores across the 12 injection days (every other day over a 4-week period). Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test, and individual data points are shown to illustrate variability.

Treatment with topiramate alone reduced seizure scores in a dose- and time-dependent manner. Compared to the Seizure group, the TPM (25 mg) and TPM (50 mg) groups did not show statistically significant differences until the final days of the experiment. In contrast, the TPM (70 mg) group showed a significant reduction from day 9 onward, with the effect becoming more pronounced over time (day 9: 3.5 ± 0.22 vs. 2.3 ± 0.15, p < 0.01; day 12: 4.6 ± 0.16 vs. 3.0 ± 0.29, p < 0.001; n = 10 per group). The detailed daily data for all groups are presented in Table 1 and Fig. 1.

Exercise combined with topiramate also led to reductions in seizure scores. The EX + TPM (25 mg) group showed a significant reduction on days 11 (3.0 ± 0.2 vs. 4.3 ± 0.21, p < 0.05, n = 10) and 12 (3.16 ± 0.1 vs. 4.6 ± 0.16, p < 0.05, n = 10) relative to the Seizure group. The EX + TPM (70 mg) group exhibited a significant reduction relative to the Seizure group from day 6 onward, with the effect becoming more pronounced over time (day 6: 0.83 ± 0.3 vs. 2.4 ± 0.26, p < 0.05; day 12: 1.0 ± 0.25 vs. 4.6 ± 0.16, p < 0.001; n = 10 per group). The detailed daily data for all groups are presented in Table 1 and Fig. 1.

A direct comparison between the combination and monotherapy groups revealed specific differences. The EX + TPM (70 mg) group had significantly lower seizure scores than the TPM (70 mg) group from day 9 onward, with the effect becoming more pronounced over time (day 9: 1.16 ± 0.16 vs. 2.3 ± 0.15, p < 0.05; day 12: 1.0 ± 0.25 vs. 3.0 ± 0.29, p < 0.001; n = 10 per group). Furthermore, seizure scores in the EX + TPM (70 mg) group were significantly lower than those in the TPM (25 mg) and TPM (50 mg) groups from day 9 onward (p < 0.001, n = 10 per group; see Table 1 and Fig. 1 for daily data).

A dose-dependent effect was observed within both treatment strategies. In the monotherapy groups, seizure scores in the TPM (70 mg) group were significantly lower than those in the TPM (25 mg) group from day 9 through day 12 (p < 0.05, n = 10 per group; see Table 1 for daily data). Similarly, among the combination therapy groups, the EX + TPM (70 mg) group showed significantly lower seizure scores than the EX + TPM (25 mg) group from day 6 onward, and significantly lower scores than the EX + TPM (50 mg) group from day 9 onward (p < 0.001, n = 10 per group; see Table 1 for daily data).

Latency of the first seizure appearance

Latency to seizure onset (mean ± S.E.M., in seconds) is presented in Table 2 and Fig. 2.

Injections | Groups |  |  |  |  |  | 
 | Seizure | TPM (25 mg) | TPM (50 mg) | TPM (70 mg) | EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
1 | 270.14 ± 2.34 | 292.2 ± 2.28 | 0 | 0 | 0 | 0 | 0
2 | 269.85 ± 2.85 | 280.8 ± 3.05 | 292.0 ± 2.3 | 0 | 0 | 0 | 0
3 | 256.37 ± 1.6 | 287.3 ± 2.31 | 282.5 ± 2.39 | 285.55 ± 1.63 | 0 | 0 | 0
4 | 262.8 ± 2.71 | 267 ± 2.75 | 279.5 ± 3.27 | 281.66 ± 2.35 | 290.5 ± 3.13 | 297.0 ± 3.18 | 0
5 | 246.3 ± 2.67 | 255.4 ± 2.96 | 274.1 ± 2.53 | 274.2 ± 0.9 | 278.0 ± 3.72 | 285.83 ± 3.97 | 0
6 | 244.9 ± 1.95 | 247.7 ± 2.43 | 266.0 ± 3.66 | 269.6 ± 1.46 | 272.16 ± 1.93 | 278.33 ± 1.6 | 280.66 ± 1.22
7 | 237.5 ± 2.29 | 239.5 ± 1.68 | 257.9 ± 2.91 | 260.5 ± 2.46 | 264.66 ± 2.23 | 272.66 ± 1.62 | 274.16 ± 1.24
8 | 227.7 ± 2.81 | 229.2 ± 1.39 | 248.3 ± 2.43 | 253.4 ± 2.81 | 258.16 ± 2.13 | 268.5 ± 1.56 | 269.5 ± 1.33
9 | 184.7 ± 1.94 | 200.9 ± 2.24 | 235.7 ± 2.05 | 245.4 ± 2.03 | 249.0 ± 2.19 | 254.33 ± 1.05 | 262.5 ± 0.88
10 | 158.1 ± 2.06 | 180.4 ± 1.95 | 216.7 ± 1.37 | 233.4 ± 3.03 | 240.16 ± 2.35 | 248.33 ± 1.42 | 254.66 ± 1.08
11 | 118.3 ± 1.28 | 144.6 ± 2.68 | 151.9 ± 1.55 | 154.0 ± 0.81 | 228.0 ± 2.64 | 236.66 ± 1.49 | 243.5 ± 0.92
12 | 94.1 ± 2.24 | 114.5 ± 2.83 | 130.8 ± 2.31 | 136.7 ± 2.91 | 152.66 ± 2.12 | 185.33 ± 1.78 | 227.33 ± 1.96

Fig. 2: Effects of monotherapy and combination therapy on seizure latency The line graph shows the time latency to the first PTZ-induced seizure. Combination therapy with moderate-intensity treadmill and topiramate (EX+TPM groups) significantly prolonged the seizure latency compared with the Seizure group and their respective topiramate monotherapy groups (TPM) at all dose levels (25, 50, and 70 mg). The most potent effect was observed in the EX + TPM (70 mg) group. Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnov test. The symbols *, × , + , †, æ, ^, and £ indicate a significant difference in comparison with the Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups, respectively.

Fig. 2 illustrates the time course of seizure latency across the 12 injection days. Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test.

No significant differences were observed between any groups during the first five injections.

From day 6 onward, all treatment groups showed significant increases in seizure latency compared to the Seizure group (p < 0.001). On day 6, the latencies for the TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups were 266.0 ± 3.66, 269.6 ± 1.46, 272.16 ± 1.93, 278.33 ± 1.6, and 280.66 ± 1.22 s, respectively, compared to 244.9 ± 1.95 s in the Seizure group. The effect became more pronounced over time, with the EX + TPM (70 mg) group reaching a latency of 227.33 ± 1.96 s on day 12, compared to 94.1 ± 2.24 s in the Seizure group (p < 0.001, n = 10 per group).

A dose-dependent increase in latency was observed among both monotherapy and combination therapy groups, with higher doses producing greater effects (p < 0.001, n = 10 per group). The combination therapy groups consistently showed longer latencies than their respective monotherapy groups. The detailed daily data for all groups are presented in Table 2 and Fig. 2.

Depression-like behaviors assessment by the Tail Suspension Test (TST)

Immobility time during the Tail Suspension Test, a behavioral index of depressive-like state, is presented as mean ± S.E.M. in Table 3 and Fig. 3.

Sham | EX | Seizure
15.5 ± 0.92 | 11.3 ± 0.76 | 65.8 ± 0.81
TPM (25 mg) | TPM (50 mg) | TPM (70 mg)
77.7 ± 0.36 | 89.4 ± 1.29 | 103.1 ± 1.33
EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
66.33 ± 0.71 | 78.83 ± 1.01 | 96.16 ± 0.47

Fig. 3: Immobility time in the Tail Suspension Test. Data on immobility time are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnovtest, and individual data points are shown to illustrate variability. The combination therapy of exercise and TPM significantly reduced immobility time compared to the monotherapy groups. The symbols #, Ҩ, *, × , + , †, æ, ^, and £ indicate a significant difference from Sham, EX, Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups respectively. One, two, and three repetitions of a symbol indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Fig. 3 illustrates the immobility time during the Tail Suspension Test across different experimental groups. Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test, and individual data points are shown to illustrate variability.

Animals in the Seizure group showed a significant increase in immobility time compared to the Sham group (65.8 ± 0.81 vs. 15.5 ± 0.92, p < 0.001, n = 10). All TPM monotherapy groups (25, 50, and 70 mg) also showed significantly longer immobility relative to the Sham group (77.7 ± 0.36, 89.4 ± 1.29, 103.1 ± 1.33 vs. 15.5 ± 0.92, p < 0.001, n = 10 for all groups).

Among the combination therapies, the EX + TPM (25 mg) and EX + TPM (50 mg) groups showed significantly lower immobility times compared to the Seizure group (66.33 ± 0.71 and 78.83 ± 1.01 vs. 65.8 ± 0.81, respectively), but both remained significantly higher than the Sham group (15.5 ± 0.92, p < 0.001, n = 10 for all comparisons). In contrast, the EX + TPM (70 mg) group exhibited a significant increase in immobility compared to the Sham group (96.16 ± 0.47 vs. 15.5 ± 0.92, p < 0.001, n = 10), while still showing a significant decrease relative to the Seizure group (96.16 ± 0.47 vs. 65.8 ± 0.81, p < 0.001).

The EX + TPM (25 mg) group showed a significant reduction in immobility compared with its corresponding monotherapy dose, TPM (25 mg), as well as compared with the higher-dose TPM (50 mg) and TPM (70 mg) groups (p < 0.001, n = 10 for all). Similarly, the EX + TPM (50 mg) and EX + TPM (70 mg) groups exhibited significantly shorter immobility than their respective monotherapy groups (p < 0.001, n = 10 for all). A comparison between the EX + TPM groups showed a dose-dependent effect; the EX + TPM (70 mg) group showed significantly longer immobility than both the EX + TPM (25 mg) and EX + TPM (50 mg) groups (p < 0.001, n = 10). The detailed data for all groups are presented in Table 3 and Fig. 3.

Effects of exercise and topiramate on anxiety-like behaviors

The number of entries into and the time spent in the closed arms were measured. Data are presented as mean ± S.E.M. in Table 4, Table 5, and Fig. 4, respectively.

Sham | EX | Seizure
3.7 ± 0.15 | 2.8 ± 0.13 | 4.6 ± 0.16
TPM (25 mg) | TPM (50 mg) | TPM (70 mg)
5.3 ± 0.15 | 5.7 ± 0.15 | 6.4 ± 0.16
EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
4.33 ± 0.21 | 5.16 ± 0.16 | 6.16 ± 0.16

Sham | EX | Seizure
285.3 ± 0.53 | 272.6 ± 0.42 | 309.8 ± 0.46
TPM (25 mg) | TPM (50 mg) | TPM (70 mg)
296.7 ± 0.26 | 317.0 ± 0.55 | 329.3 ± 1.83
EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
284.66 ± 0.98 | 308.83 ± 1.4 | 333.83 ± 0.47

Fig. 4: Assessment of anxiety-like behavior in the Elevated Plus Maze. (A) The mean number of entries into the closed arms is presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnovtest, and individual data points are shown to illustrate variability. The observed reduction in closed-arm entries in the combination therapy groups suggests an attenuation of the depression-like behavioral phenotype associated with Seizure and topiramate monotherapy. (B) The mean of time spent in the closed arms is presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnov test, and individual data points are shown to illustrate variability. Administration of topiramate (TPM) dose-dependently increased both parameters, an effect that was significantly counteracted by concurrent moderate-intensity treadmill exercise (EX+TPM), especially at higher doses. The symbols #, Ҩ, *, × , + , †, æ, ^, and £ indicate a significant difference in comparison with the Sham, EX, Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups respectively. One, two, or three repetitions of symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Fig. 4 illustrates the number of entries into and the time spent in the closed arms of the elevated plus maze across different experimental groups. Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test, and individual data points are shown to illustrate variability.

Animals in the Seizure group showed a significant increase in both the number of entries into (4.6 ± 0.16 vs. 3.7 ± 0.15, p < 0.001, n = 10) and time spent in (309.8 ± 0.46 vs. 285.3 ± 0.53, p < 0.001, n = 10) the closed arms compared with the Sham group. All TPM monotherapy groups also spent significantly more time in the closed arms compared with the Sham group (296.7 ± 0.26, 317 ± 0.55, 329.3 ± 1.83 for TPM 25, 50, and 70 mg, respectively; p < 0.001, n = 10).

Compared with the Seizure group, the TPM (50 mg) and TPM (70 mg) groups showed a significant increase in closed-arm time, while the TPM (25 mg) group showed a significant decrease (p < 0.01, n = 10). Similarly, the EX + TPM (25 mg) group showed a significant decrease in closed-arm time compared to the Seizure group, whereas the EX + TPM (70 mg) group showed a significant increase (p < 0.01, n = 10). No significant difference was observed between the Seizure and EX groups for either parameter.

Administration of TPM alone at all doses significantly increased the number of closed-arm entries compared with both the Sham and EX groups (p < 0.001, n = 10 for all). A dose-dependent effect was evident among monotherapy groups; the TPM (70 mg) group showed a significantly higher number of closed-arm entries than the TPM (25 mg) group (p < 0.01, n = 10).

The EX + TPM (25 mg) group showed a significantly lower number of closed-arm entries than the TPM (50 mg) and TPM (70 mg) groups (p < 0.05 and p < 0.001, respectively, n = 10), while the EX + TPM (70 mg) group exhibited a significantly higher number of closed-arm entries than the TPM (50 mg) group (p < 0.01, n = 10). A dose-response relationship was maintained within the combination therapy; the EX + TPM (70 mg) group made a significantly higher number of entries than the EX + TPM (25 mg) group (p < 0.001, n = 10). The detailed data for all groups are presented in Table 4, Table 5, and Fig. 4.

As shown in Table 4, Table 5**,** and Fig. 4**,** the EPM revealed significant changes in anxiety-like behavior across groups. The TST, which specifically measures depressive-like behavior, showed that exercise combined with topiramate significantly reduced immobility time. Taken together, these results suggest that the combination therapy improved both anxiety-like and depressive-like behaviors in epileptic rats.

5-HT1A receptor immunoreactive staining intensity in the CA1 subfields of the hippocampus

Immunofluorescence assessment using the anti−5-HT1A receptor antibody was performed to quantify the immunoreactive staining intensity in the CA1 subfields of the hippocampus. The results are presented in Table 6 and Fig. 5 (A, B).

Sham | EX | Seizure
1.58 ± 0.15 | 1.84 ± 0.02 | 0.75 ± 0.02
TPM (25 mg) | TPM (50 mg) | TPM (70 mg)
0.79 ± 0.02 | 0.97 ± 0.01 | 1.04 ± 0.02
EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
1.06 ± 0.06 | 1.5 ± 0.05 | 1.71 ± 0.03

Fig. 5: 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU) in the CA1 subfields of the hippocampus are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnov test, and individual data points are shown to illustrate variability. (A) Immunofluorescent photomicrographs of 5-HT1A receptor expression (Arbitrary Units, AU) in the CA1 subfields of the hippocampus. (B) Bar charts summarize the mean 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU). The 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU) was significantly upregulated in the EX + TPM (50 mg) and EX + TPM (70 mg) groups in the CA1 subfields of the hippocampus. The symbols #, Ҩ, *, × , + , †, æ, ^, and £ indicate a significant difference in comparison with the Sham, EX, Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups respectively. One, two, or three repetitions of symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test, and individual data points are shown to illustrate variability.

The immunoreactive staining intensity of 5-HT1A receptors in the EX group was significantly higher than the Sham group (1.84 ± 0.02 vs. 1.58 ± 0.15, p < 0.001, n = 10). In contrast, it was significantly lower in the Seizure group compared with both the Sham (0.75 ± 0.02 vs. 1.58 ± 0.15, p < 0.001, n = 10) and EX (0.75 ± 0.02 vs. 1.84 ± 0.02, p < 0.001, n = 10) groups.

Administration of TPM monotherapy significantly affected 5-HT1A receptor expression. All TPM groups (25, 50, and 70 mg) showed significantly lower staining intensity compared with the Sham group (0.79 ± 0.02, 0.97 ± 0.01, 1.04 ± 0.02 vs. 1.58 ± 0.15; p < 0.05 to p < 0.001, n = 10). Similarly, these groups were significantly lower than the EX group (p < 0.001 for TPM 25 mg and 50 mg; p < 0.01 for TPM 70 mg).

Combination therapy yielded a distinct profile. The EX + TPM (25 mg) group showed no significant difference from the Sham group but significantly higher expression levels than the Seizure group (1.06 ± 0.06 vs. 0.75 ± 0.02, p < 0.01, n = 10). Both the EX + TPM (50 mg) and EX + TPM (70 mg) groups demonstrated no significant difference compared with the Sham and EX groups. Crucially, these two combination groups (EX+TPM 50 mg and 70 mg) showed significantly higher 5-HT1A receptor expression compared with the Seizure group (1.5 ± 0.05, 1.71 ± 0.03 vs. 0.75 ± 0.02; p < 0.001 for both, n = 10).

Significantly higher 5-HT1A receptor expression was observed in all EX + TPM groups compared with their corresponding monotherapy doses (p < 0.001 for EX+TPM (25 mg) vs. TPM (25 mg); p < 0.01 for EX + TPM (50 mg) vs. TPM 50 mg and EX + TPM (70 mg) vs. TPM (70 mg); n = 10). Moreover, the EX + TPM (70 mg) group displayed significantly higher 5-HT1A receptor expression than both the EX + TPM (25 mg) (1.71 ± 0.03 vs. 1.06 ± 0.06, p < 0.05, n = 10) and the EX + TPM (50 mg) (1.71 ± 0.03 vs. 1.5 ± 0.05, p < 0.05, n = 10) groups. The detailed data for all groups are presented in Table 6 and Fig. 5.

5-HT1A receptor immunoreactive staining intensity in the CA3 subfields of the hippocampus

Effects of topiramate and exercise on 5-HT1A receptor expression in the CA3 subfields of the hippocampus, with full results, are depicted in Table 7 and Fig. 6 (A, B).

Sham | EX | Seizure
1.82 ± 0.005 | 2.49 ± 0.01 | 0.93 ± 0.005
TPM (25 mg) | TPM (50 mg) | TPM (70 mg)
1.1 ± 0.05 | 1.4 ± 0.05 | 1.7 ± 0.05
EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
1.35 ± 0.005 | 1.79 ± 0.01 | 1.98 ± 0.004

Fig. 6: 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU) in the CA3 subfields of the hippocampus is presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnov test, and individual data points are shown to illustrate variability. (A) Immunofluorescent photomicrographs of 5-HT1A receptor expression (green) in the CA3. subfields of the hippocampus (Arbitrary Units, AU). (B) Bar charts summarize the mean 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU). The 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU) was significantly higher in the EX, TPM (50 mg), TPM (70 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups in the CA3 subfields of the hippocampus than in the other experimental The symbols #, Ҩ, *, × , + , †, æ, ^, and £ indicate a significant difference in comparison with the Sham, EX, Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups respectively. One, two, or three repetitions of symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test, and individual data points are shown to illustrate variability.

The EX group showed a significantly higher 5-HT1A immunoreactive staining intensity compared with the Sham group (2.49 ± 0.01 vs. 1.82 ± 0.005, p < 0.001, n = 10). Conversely, the Seizure group displayed a significantly lower staining intensity relative to the Sham group (0.93 ± 0.005 vs. 1.82 ± 0.005, p < 0.001, n = 10). Moreover, the Seizure group showed significantly lower staining intensity than both the Sham (0.93 ± 0.005 vs. 1.82 ± 0.005, p < 0.001, n = 10) and the EX groups (0.93 ± 0.005 vs. 2.49 ± 0.01, p < 0.001, n = 10).

All TPM monotherapy groups had significantly lower 5-HT1A immunoreactive staining intensity compared with the Sham group (1.1 ± 0.05, 1.4 ± 0.05, 1.7 ± 0.05 vs. 1.82 ± 0.005; p < 0.001 for all, n = 10). Similarly, they had significantly lower staining intensity than the EX group (p < 0.001 for all, n = 10). When compared with the Seizure group, the TPM (25 mg) group showed no significant difference; however, both the TPM (50 mg) and TPM (70 mg) groups had significantly higher staining intensity relative to the Seizure group (1.4 ± 0.05, 1.7 ± 0.05 vs. 0.93 ± 0.005_; p < 0.001_ for both, n = 10).

A dose-dependent effect was observed among monotherapy groups. The staining intensity in the TPM (70 mg) group was significantly higher than in the TPM (50 mg) group (1.7 ± 0.05 vs. 1.4 ± 0.05, p < 0.01, n = 10), which in turn was significantly higher compared with the TPM (25 mg) group (1.4 ± 0.05 vs. 1.1 ± 0.05, p < 0.001, n = 10).

The EX + TPM (25 mg) group showed a significantly lower staining intensity compared with the Sham group (1.35 ± 0.005 vs. 1.82 ± 0.005, p < 0.001, n = 10) and the EX group (1.35 ± 0.005 vs. 2.49 ± 0.01, p < 0.001, n = 10), but a significantly higher one compared with the Seizure group (1.35 ± 0.005 vs. 0.93 ± 0.005, p < 0.001, n = 10). In contrast, both the EX + TPM (50 mg) and EX + TPM (70 mg) groups exhibited higher staining intensity, which was not significantly different from that in the Sham group. While still lower than the EX group (1.79 ± 0.01, 1.98 ± 0.004 vs. 2.49 ± 0.01, p < 0.001, n = 10), these two combination groups showed significantly higher staining intensity relative to the Seizure group (1.79 ± 0.01, 1.98 ± 0.004 vs. 0.93 ± 0.005; p < 0.001 for both, n = 10).

Combination therapy showed a superior effect compared to corresponding monotherapy doses. Both the EX + TPM (50 mg) and EX + TPM (70 mg) groups displayed significantly higher staining intensity than the TPM (50 mg) group (1.79 ± 0.01, 1.98 ± 0.004 vs. 1.4 ± 0.05; p < 0.001 for both, n = 10). Crucially, the EX + TPM (70 mg) group showed a significantly higher staining intensity compared to the TPM (70 mg) group (1.98 ± 0.004 vs. 1.7 ± 0.05_, p < 0.01_, n = 10). Furthermore, the EX + TPM (70 mg) group displayed a significantly higher staining intensity than both the EX + TPM (25 mg) (1.98 ± 0.004 vs. 1.35 ± 0.005, p < 0.001, n = 10) and EX + TPM (50 mg) groups (1.98 ± 0.004 vs. 1.79 ± 0.01, p < 0.01, n = 10). The detailed data for all groups are presented in Table 7 and Fig. 6.

5-HT1A immunoreactive staining intensity in the cerebral cortex

Analysis of 5-HT1A receptor expression in the cerebral cortex with immunofluorescence revealed the significant modulatory effects of both EX and TPM treatments. Results are presented in Table 8 and Fig. 7 (A, B).

Sham | EX | Seizure
0.86 ± 0.02 | 1.35 ± 0.06 | 0.57 ± 0.02
TPM (25 mg) | TPM (50 mg) | TPM (70 mg)
0.62 ± 0.01 | 0.88 ± 0.04 | 0.99 ± 0.02
EX + TPM (25 mg) | EX + TPM (50 mg) | EX + TPM (70 mg)
0.78 ± 0.01 | 1.36 ± 0.08 | 1.39 ± 0.15

Fig. 7: 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU) in the cerebral cortex is presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov-Smirnov test, and individual data points are shown to illustrate variability. (A) Immunofluorescent photomicrographs of 5-HT1A receptor expression (Arbitrary Units, AU) (green) in the cerebral cortex. (B) Bar charts summarize the mean 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU). The 5-HT1A receptor immunoreactive staining intensity (Arbitrary Units, AU) was significantly higher in the EX, EX + TPM (50 mg), and EX + TPM (70 mg) groups in the cerebral cortex than in the other experimental groups. The symbols #, Ҩ, *, × , + , †, æ, ^, and £ indicate a significant difference in comparison with the Sham, EX, Seizure, TPM (25 mg), TPM (50 mg), TPM (70 mg), EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups respectively. One, two, or three repetitions of symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Data are presented as mean ± S.E.M. (error bars). Normality of distribution was confirmed using the Kolmogorov–Smirnov test, and individual data points are shown to illustrate variability.

The EX group had a significantly higher 5-HT1A receptor expression level compared to the Sham group (1.35 ± 0.06 vs. 0.86 ± 0.02, p < 0.05, n = 10). In contrast, the Seizure group had a significantly lower expression level compared to the Sham group (0.57 ± 0.02 vs. 0.86 ± 0.02_, p < 0.01_, n = 10). The Seizure group had a significantly lower expression level than both the Sham (0.57 ± 0.02 vs. 0.86 ± 0.02, p < 0.01, n = 10) and the EX groups (0.57 ± 0.02 vs. 1.35 ± 0.06, p < 0.001, n = 10).

The TPM (25 mg) and TPM (50 mg) monotherapy groups had a significantly lower expression level compared to the Sham group (0.62 ± 0.01, 0.88 ± 0.04 vs. 0.86 ± 0.02; p < 0.05 for both, n = 10). The TPM (70 mg) group showed no significant difference from the Sham group. All TPM monotherapy groups had a significantly lower expression level than the EX group (p < 0.001 for TPM (25 mg); p < 0.05 for TPM (50 mg); p < 0.01 for TPM (70 mg), n = 10). Compared with the Seizure group, the TPM (25 mg) group showed no significant difference. In comparison, the TPM (50 mg) group and the TPM (70 mg) group had higher expression levels (0.57 ± 0.02 vs. 0.88 ± 0.04, p < 0.05, and 0.57 ± 0.02 vs. 0.99 ± 0.02, p < 0.01, respectively).

A dose-dependent trend was observed. The TPM (70 mg) group (0.99 ± 0.02) had a significantly higher expression level than both the TPM (25 mg) (0.62 ± 0.01) and TPM (50 mg) (0.88 ± 0.04) groups (p < 0.05 for both comparisons, n = 10).

The EX + TPM (25 mg) group showed no significant difference from the Sham group but had a significantly lower expression level than the EX group (0.78 ± 0.01 vs. 1.35 ± 0.06, p < 0.01, n = 10). Both the EX + TPM (50 mg) and EX + TPM (70 mg) groups had a significantly higher expression level compared to the Sham group (1.36 ± 0.08, 1.39 ± 0.15 vs. 0.86 ± 0.02; p < 0.01 for both, n = 10) and the Seizure group (0.57 ± 0.02; p < 0.001 for both, n = 10); however, they had a significantly lower expression level than the EX group (1.35 ± 0.06; p < 0.05 for both, n = 10).

Combination therapy demonstrated superior efficacy. The EX + TPM (50 mg) and EX + TPM (70 mg) groups had a significantly higher expression level than the TPM (25 mg) group (1.36 ± 0.08, 1.39 ± 0.15 vs. 0.62 ± 0.01; p < 0.001 for both, n = 10), as did the EX + TPM (50 mg) group compared with the TPM (50 mg) group (1.36 ± 0.08 vs. 0.88 ± 0.04, p < 0.05, n = 10). Moreover, the EX + TPM (70 mg) group had a significantly higher expression level compared with both the TPM (50 mg) (1.39 ± 0.15 vs. 0.88 ± 0.04, p < 0.01, n = 10) and TPM (70 mg) groups (1.39 ± 0.15 vs. 0.99 ± 0.02, p < 0.05, n = 10).

A clear dose-response effect was evident with combination therapy. Both the EX + TPM (50 mg) and EX + TPM (70 mg) groups had significantly higher expression levels than the EX + TPM (25 mg) group (1.36 ± 0.08, 1.39 ± 0.15 vs. 0.78 ± 0.01; p < 0.001 for all, n = 10). The EX + TPM (70 mg) group also had a significantly higher expression level compared with the EX + TPM (50 mg) group (1.39 ± 0.15 vs. 1.36 ± 0.08, p < 0.01, n = 10). The detailed data for all groups are presented in Table 8 and Fig. 7.