Repeated Seizure Exposure in the SS Kcnj16‐/‐ Rat Causes Progressive Respiratory Suppression and Associated Brainstem Pathology

Abstract

Kir5.1 is an inwardly rectifying potassium (Kir) channel subunit highly expressed in the kidney and brain and encoded by the Kcnj16 gene. Knockout of Kcnj16 in a Dahl salt-sensitive rat (SSKcnj16-/-) results in susceptibility to audiogenic, generalized tonic-clonic seizures (GTCSs), which when repeated lead to spontaneous mortality. Patients with uncontrolled epilepsy are at high risk for Sudden Unexplained Death in Epilepsy (SUDEP), hypothesized to be a result of severe cardiorespiratory suppression and/or failure. We hypothesized that repeated audiogenic seizures in SSKcnj16-/- rats lead to progressively worsening cardiorespiratory suppression and associated brainstem pathology. SSKcnj16-/- and wild-type (WT) rats (10-12 weeks of age) were exposed to the seizure-inducing acoustic stimulus 1/day (d) for 10d and breathing measurement were made before, during and after exposure. Solitary GTSCs acutely led to apnea and decreases in breathing frequency for ~5 min postictally, with no change or an increase in total ventilation due to an increase in tidal volume. We also found a significant reduction in breath-to-breath variability throughout the postictal period. After exposure to repeated seizures, postictal frequency suppression became more pronounced and compensatory increases in tidal volume were reduced, such that total ventilation was also significantly reduced. Repeated seizures also further attenuated the ventilatory response to hypercapnia (p=0.0073). To gain insights into the effects of repeated seizures on cardiorespiratory circuits in the brainstem, we used immunofluorescence staining/confocal imaging to label and quantify total serotonin (5-HT) immunoreactivity (ir), 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=5/group). Repeated seizures resulted in reduced 5-HT-ir in multiple cardiorespiratory brainstem nuclei including the pre-Bötzinger Complex (pre-BötC), nucleus ambiguous (NA), and nucleus tractus solitarious (NTS) in addition to a decrease in 5-HT neurons. We also found increased Iba1-ir selectively within the pre-BötC and NA, whereas GFAP-ir was increased after 10 seizures in these same regions. This data suggests that inflammation and/or serotonergic dysregulation in cardiorespiratory brainstem nuclei may mediate progressive respiratory suppression following repeated seizures in this rat model.