Evidence of Progressive Brainstem Pathology after Repeated Seizure Exposure in a Novel Rat Model of SUDEP

Abstract

Mutations in ion channel genes can alter neuronal excitability and have been linked to seizure disorders in humans. We previously established that a knockout rat model of Kcnj16, which encodes potassium channel subunit Kir5.1, on a Dahl SS background (SSKcnj16−/−) exhibits renal and respiratory impairments, in addition to audiogenic reflex epilepsy. While seizures can be considered benign acute events, patients with uncontrolled epilepsy are at high risk for Sudden Unexplained Death in Epilepsy (SUDEP), predicted to be due to cardiorespiratory failure following repeated seizures. The SSKcnj16−/− rat allows intractable epilepsy to be modeled with modifiable SUDEP risk by regulating seizure number and providing a controlled experimental environment with consistent occurrence of seizure‐induced death. During daily seizure exposure for up to 10 days, we observed progressive exacerbation of post‐ictal respiratory rate and heart rate suppression with spontaneous mortality in 33% of SSKcnj16−/− rats within hours of a seizure. We tested the hypothesis that repeated seizures induces neuroinflammation and dysregulation of adenosine (ADO) and serotonin (5HT) within brainstem cardiorespiratory nuclei, which may contribute to progressive cardiorespiratory suppression and subsequent mortalities in SSkcnj16−/− rats. Quantification of brainstem neurochemicals by HPLC revealed increased ADO and decreased 5HT in seizure‐exposed compared to naïve SSkcnj16−/− rats. These findings appeared to be brainstem‐specific as ADO levels in the cortex of seizure exposed rats were decreased. Western blot analyses of whole brainstem lysates showed trends toward decreased expression of ADO kinase (ADK) and A1 receptor (A1R) in seizure‐exposed rats. We used immunofluorescent staining and confocal imaging to label and quantify total 5HT, serotonergic neurons (tryptophan hydroxylase, TPH), microglia (Iba1), astrocytes (GFAP), total neurons (NeuN), and total nuclei (DAPI) in respiratory brainstem nuclei of SSkcnj16−/− rats exposed to 0, 3, or 10 seizures (n=2/group). Consistent with our HPLC data, 5HT staining was decreased in the pre‐Bötzinger Complex (preBötC), Nucleus Ambiguus (NA) and medullary Raphe (MR) of seizure‐exposed rats, which also showed fewer TPH‐positive neurons in MR. Staining also revealed that seizures may induce brainstem inflammation that resulted in transient increases reactive microglia and progressive astrogliosis in these rats. SSkcnj16−/− rats showed increased Iba1 after 3 seizures but no change after 10 seizures in the preBötC, NA, MR and nucleus of the solitary tract (NTS). GFAP progressively increased after 3 and 10 seizures in preBötC, NTS, and MR. Further studies will focus on validating these finding by increasing the number of animals in experimental groups and evaluating additional inflammatory markers. This preliminary data suggests that dysregulation of ADO and 5HT and/or inflammation in respiratory brainstem nuclei may mediate respiratory suppression and subsequent mortality associated with repeated seizure exposure in SSKcnj16−/− rats, which may have central relevance to the pathophysiology of SUDEP which remains poorly understood.Support or Funding InformationR01 HL122358