Abstracts

Rationale: Sudden unexpected death in epilepsy (SUDEP) is a leading cause of premature death in patients with epilepsy. Postictal central respiratory depression is one patho-mechanism that could lead to SUDEP (Ryvlin P et al, 2013). Animal models of SUDEP have also recently shown spreading depolarization in the dorsal medulla (Aiba I & Noebels JL, 2015). Our previous work has identified inflammatory changes in the ventrolateral medulla (VLM) in some SUDEP post mortem (PM) cases suggesting pathological changes (Michalak Z et al, 2014). The pre-Botzinger complex (pBC) of the VLM is regarded as a critical nucleus for inspiratory rhythm generation. The precise localisation as well as the neuronal composition of the pBC is poorly defined in the human brain. Our aim was to study the pBC in a series of SUDEP PM cases compared to controls. ​Methods: 14 SUDEP PM cases (including 5 patients with Dravet syndrome) and 10 control individuals (sudden death patients without any history of epilepsy) were selected. In a proportion of SUDEP and controls, 9.4Tesla MRI of the whole or hemi-brainstem was carried out following formalin fixation. Serial sections through the rostral medulla (approximate obex level +3 to +12mm) were cut at 20 microns and stained with cresyl violet (CV), stepped at 200 micron intervals. 3D reconstructions of CV sections were built to delineate the boundaries of the pBC in a rostral-caudal direction and to facilitate co-registration with 9.4T MRI images of the brainstem. A panel of neuronal and interneuronal immunomarkers was trialed to delineate the neuronal composition of the pBC (including neurokinin 1 receptor, somatostatin, reelin, dbx1, ARVCF, galanin, KV1.1, SCN1a) as well as astroglial populations; these were carried out on intervening sections. Ethical guidelines: The project has been ethically approved and all brain samples are consented for use in research.Results: The pBC was delineated in the VLM, lying dorsal to the inferior olive, ventral to the nucleus amibiguus within the lateral reticular nucleus on CV sections and 3D reconstructions. In both SUDEP cases and controls, selective labelling of small and large lipofuscin rich neuronal cells in the pBC was noted with some markers, as somatostatin, in addition to labelling of neuronal processes and networks in the reticular formation and medulla. However, some putative pBC markers (as ARVCF, reelin) failed to identify neuronal populations in this region.Conclusions: Our method, utilizing serial sections, a specific immunohistochemistry panel with 3D reconstruction enables study of the extent and composition of this key inspiratory nucleus in PM brain tissues. This will enable comparison with structural MRI as well as control groups using stereological quantitative evaluation, to identify pathological brainstem alterations that could predispose to SUDEP. Source of funding: This study is supported by the Center for SUDEP Research through the National Institute of Neurological Disorders And Stroke of the National Institutes of Health under Award Numbers U01 NS090415