Epilepsy is a multifaceted neurological disorder characterized by recurrent seizures and associated with molecular and immune alterations in key brain regions. The GASH/Sal (Genetic Audiogenic Seizure Hamster, Salamanca), a genetic model for audiogenic epilepsy, provides a powerful tool to study seizure mechanisms and resistance in predisposed individuals. This study investigates the proteomic and immune responses triggered by audiogenic kindling in the inferior colliculus, comparing non-responder animals exhibiting reduced seizure severity following repeated stimulation versus GASH/Sal naïve hamsters. To assess auditory pathway functionality, Auditory Brainstem Responses (ABRs) were recorded, revealing reduced neuronal activity in the auditory nerve of non-responders, while central auditory processing remained unaffected. Cytokine profiling demonstrated increased levels of proinflammatory markers, including IL-1 alpha (Interleukin-1 alpha), IL-10 (Interleukin-10), and TGF-beta (Transforming Growth Factor beta), alongside decreased IGF-1 (Insulin-like Growth Factor 1) levels, highlighting systemic inflammation and its interplay with neuroprotection. Building on these findings, a proteomic analysis identified 159 differentially expressed proteins (DEPs). Additionally, bioinformatic approaches, including Gene Set Enrichment Analysis (GSEA) and Weighted Gene Co-expression Network Analysis (WGCNA), revealed disrupted pathways related to metabolic and inflammatory epileptic processes and a module potentially linked to a rise in the threshold of seizures, respectively. Differentially expressed genes, identified through bioinformatic and statistical analyses, were validated by RT-qPCR. This confirmed the upregulation of six genes (Gpc1—Glypican-1; Sdc3—Syndecan-3; Vgf—Nerve Growth Factor Inducible; Cpne5—Copine 5; Agap2—Arf-GAP with GTPase domain, ANK repeat, and PH domain-containing protein 2; and Dpp8—Dipeptidyl Peptidase 8) and the downregulation of two (Ralb—RAS-like proto-oncogene B—and S100b—S100 calcium-binding protein B), aligning with reduced seizure severity. This study may uncover key proteomic and immune mechanisms underlying seizure susceptibility, providing possible novel therapeutic targets for refractory epilepsy.

Experimental evidence indicates that a high seizure burden can induce cerebral overexpression of P-glycoprotein (P-gp) at the blood–brain barrier, a phenomenon associated with drug-resistant epilepsy under the “transporter hypothesis”, but also at the neuronal level, linked to a reduced seizure threshold, increased seizure severity (SS), status epilepticus (SE), and a high spontaneous death (SD) rate. In contrast, we recently described a progressive reduction in SS and the absence of SE and SD in GASH/Sal hamsters subjected to 45 audiogenic seizures. Here, we examined SS, SE, and the SD, and the expression of P-gp, erythropoietin receptor (EPO-R), hypoxia-inducible factor 1 alpha subunit (HIF-1α) and cyclooxygenase 2 (COX-2), in the brains of GASH/Sal hamsters following 20 audiogenic kindling stimulations (AUK-20). SS was evaluated using the midbrain and limbic severity scales; gene expression was assessed by RT-qPCR and P-gp protein levels were measured by immunohistochemistry and Western blot (IHC/WB) analysis. A modest decrease in midbrain SS was observed, without an increase in the already low limbic SS scores, and no SE or SD events occurred. P-gp levels remained low in both IHC and WB analyses. At the mRNA level, we detected increased EPO-R expression, decreased HIF-1α, and increased COX-2 without an accompanying increased in Abcb1b. Unlike findings from other experimental epilepsy models, AUK-20 in GASH/Sal hamsters does not enhance limbic SS, trigger SE or SD, or induce P-gp overexpression in the brain. Independently of the implications for drug resistance, the lack of cerebral P-gp overexpression without increased SS in the AUK-20-GASH/Sal model supports a potential role of P-gp in modulating seizure severity and epilepsy-associated mortality risk.