Probiotics by Modulating Gut-Brain Axis Together With Brivaracetam Mitigate Seizure Progression, Behavioral Incongruities, and Prevented Neurodegeneration in Pentylenetetrazole-Kindled Mice

Abstract

Background: The microbiota-gut-brain axis (MGBA) is a central nexus that integrates higher cognitive and emotional centers of the central nervous system (CNS) within the intricate functioning of the intestine. Accumulating evidence suggests that dysbiosis in the taxonomic diversity of gut flora plays a salient role in the progression of epilepsy and comorbid secondary complications.

Methods: In the current study, we investigated the impact of long-term oral bacteriotherapy (probiotics; 10 mL/kg; 10⁹ colony-forming unit/ml) as an adjunctive treatment intervention with brivaracetam (BRV; 10 mg/kg) over 21 days on pentylenetetrazole (PTZ) induced augmented epileptic response and associated electrographical and behavioral perturbations in mice. Moreover, we also unveiled antioxidant capacity and histopathologic changes in treated versus non-treated animals.

Results: Results revealed combination increases seizure threshold and prevented high ictal spiking. Additionally, it alleviated PTZ-induced neuropsychiatric disturbances such as anxiety and depressive-like phenotype along with cognitive deficits. Furthermore, dual therapy prompted physiological oxidant/antioxidant balance as evidenced by increased activity of antioxidant enzymes (SOD and catalase) and reduced levels of oxidative stressor (MDA). This therapeutic intervention with commensal species suppressed network-driven neuroinflammation and preserved normal cytoarchitecture with intact morphology in the pyramidal layers of cornu ammonis (CA1 and CA3).

Conclusion: Our study provides supporting evidence for the use of probiotics as adjunctive therapy with anti-seizure medications to modulate epileptogenic processes and related multimorbidities, particularly in individuals with drug-resistant seizures.

Keywords: EEG; Nissl's staining; PTZ kindling; epilepsy; gut–brain axis; oxidative stress; probiotics.

FIGURE 1

Experimental timeline for PTZ kindling and associated secondary comorbidities to assess the impact of BRV 10 + PRO 1 dual therapy on epileptogenic processes and plausible improvement in neuronal survival.

FIGURE 2

Graphical presentation of the impact of chronic probiotic supplementation alone and in combination with BRV on seizure score assessed as per Racine scale during the PTZ‐kindling process. The kindling was carried out by injecting PTZ (40 mg/kg) on alternate days for a total of 11 injections to all animals except healthy control mice. The results were evaluated by applying repeated‐measure two‐way ANOVA followed by Dunnet's test revealing the significant intergroup difference when compared with the PTZ group. The values are shown as the mean ± SEM (n = 6), while *p < 0.05, ***p < 0.001, and ****p < 0.0001 represents statistical significance.

FIGURE 3

Magnified electrograms presenting the impact of probiotics alone and in combination with BRV on the progression of epileptogenic foci. The animals were given their respective pretreatment with PRO 10 mL/kg and BRV 10 mg/kg alone and in combination and after post 30 min after therapy, they were challenged with PTZ 40 mg/kg. The EEG was recorded for 30 min after the administration of PTZ. The tracings represent ictal discharges in PTZ‐kindled mice during Stages 4–5 seizures; however, the high voltage spiking activity was reduced in combination therapy with BRV 10 + PRO 10.

FIGURE 4

Evaluation of the preventive impact of PRO 10 mL/kg and BRV 10 mg/kg treatment alone and in combination on the anxiety‐like behavior and locomotive activity of mice assessed by OFT on the 22nd day of the post‐kindling process. (A) Time spent in the central zone, (B) number of entries in the central zone, (C) distance traveled, (D) fast movements, and (E) fast rearing. (F) Representative track plots depicting reduced risk‐taking behavior as animals continued to roam around peripheries as compared to treated mice. The results were analyzed by applying one‐way ANOVA followed by Dunnett's test in comparison with the PTZ group. The data are represented as a mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 representing statistical significance.

FIGURE 5

Anxiolytic profiling of combination therapy with BRV 10 + PRO 10 in L/D test. Mice were allowed to explore the light and dark zones for 5 min on the 23rd day. (A) Time spent in the light zone, (B) number of entries in the light zone. (C) Representative track plots showing the impact of BRV 10 + PRO 1 alone and in combination on anxiety‐like behavior and exploration of a novel environment. The outcomes were evaluated by applying one‐way ANOVA by comparing the results of all groups with the PTZ group. Data are presented as mean ± SEM (n = 6) while *p < 0.05, **p < 0.01, and ****p < 0.0001 showing statistically significant significance.

FIGURE 6

The impact of BRV 10 and PRO 10 alone and in combination on the learning and memory aspects of animals. On the 24th day, animals were tested for their short‐term memory in the Y‐maze test noted by observing their percentage spontaneous alteration. Animals were further tested for episodic memory in the NOR test on the 25th day by monitoring their interaction with novel and previously familiarized objects. (A) % spontaneous alteration, (B) discrimination index. (C) Representative track plots showing improved alternation behavior in mice treated with BRV 10 + PRO 10 combination regime. (D) Representative track plots illustrating the impact of combination therapy on PTZ‐induced short‐term memory deficits. Moreover by the 26th–27th day epileptic and treated animals were tested for long‐term recognition memory by noticing their escape latencies shock zone in the PA test. (E) Step through latency during training (s). (F) Step through latency post 24 hr (s).  One way‐ANOVA following Dunnett's test was performed to analyze the data representing as mean ± SD (n = 6) *p < 0.05, **p < 0.01, and ***p < 0.001, as statistically significant in comparison with the PTZ group.

FIGURE 7

The impact of BRV 10 + PRO 10 alone and in combination on the long‐term spatial memory was assessed via MWM test from the 28th–33rd day. (A) Escape latencies on the testing days after training for initial 2 days with visible platform, (B) latency to enter the SW zone on probe day, (C) number of entries in the SW zone, (D) duration spent in the SWzone. (E) Track plots representing animal's learning aptitude and performance during experimental trials. Trajectory patterns unveiled positive impact of combinatorial regime of BRV 10 + PRO 10 to navigate hidden rescue platform with shorter escape latencies as compared to PTZ 40. Repeated two‐way ANOVA was applied for the escape latencies on the test days, while the other parameters of MWM were analyzed by applying one‐way ANOVA followed by Dunnett's test by comparing with the PTZ group. Data are presented as mean ± SEM (n = 6) while *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically significant.

FIGURE 8

Assessment of self‐care behavior via splash test. On the 34th day animal's behavioral despair was evaluated by splashing 10% sucrose solution on the back of epileptic and treated mice. (A) Grooming latency (s). (B) Grooming frequency. One way‐ANOVA following Dunnett's test was performed to analyze the data representing mean ± SEM (n = 6), *p < 0.05, **p < 0.01, and ****p < 0.0001 as statistically significant in comparison with the PTZ group.

FIGURE 9

Digital photomicrographs of Nissl's stained cornu‐ammonis (CA1 and CA3) represent the neuroprotective effect of BRV 10 + PRO 10 on PTZ‐induced neurodegeneration. Scale bar = 500 μm at 4×, 200 μm at 10×, and 50 μm at 40× magnification (n = 3).