Abstracts

Rationale: Patients with new-onset refractory status epilepticus without explanation after initial evaluation (i.e., NORSE) often have poor outcomes. The understanding of the pathophysiological mechanisms underlying NORSE and its long-term consequences is crucial to improvement NORSE management and to prevent secondary neuronal injury. Several arguments suggest NORSE is a disorder of immunity or inflammation. Therefore, we hypothesize that NORSE patients have abnormal immune or inflammatory cells in brain parenchyma.

Methods: We used single-nucleus RNA sequencing to profile brain parenchyma cells. We analyzed post-mortem frontal and temporal cortex samples from a 27-year-old woman with cryptogenic NORSE. All serum and CSF tests were negative. The patient died 9 days after SE onset due to multiorgan failure. Data were compared to those obtained from the same brain regions of a control patient without history of neurological disorders. _x000D_ _x000D_ Post-mortem fresh frozen samples were obtained from the Department of Neuropathology Department of the Pitié-Salpêtrière Hospital (Paris, France). 50 mg of frozen tissue were used to isolate total brain nuclei. A total of 8,000 estimated nuclei for each sample were targeted on the 10X Genomics Chromium system and libraries were prepared using Single Cell 3’ V3.1 reagent kit. The generated single nucleus RNA-seq libraries were sequenced using Illumina NovaSeq 6000 and aligned to human reference genome GRCh38 using 10x Cell Ranger 6.0.1.

Results: The nuclei were of good quality with an average of 2400 genes and 4662 unique molecular identifiers per cell. We identified respectively 12 and 13 cell clusters in the temporal and the frontal cortex. The patient with NORSE had increased proportion of microglia and astrocytes compared with the control in both locations, while there was a lower proportion of oligodendrocytes in the temporal cortex. We found differentially expressed genes within these cell clusters, and a higher expression of C1QB and CSF1R (microglia) and SLCA12 and AQP4 (astrocytes) in the patient with NORSE. She also had an increased proportion of excitatory and GABAergic neurons in the temporal cortex and decreased proportion of GABAergic neurons in the frontal cortex. The excitatory/GABAergic neuron ratio was higher for the patient with NORSE than the control patient in both locations (temporal: 2.57 vs. 1.84; frontal: 12.4 vs. 2.53). We also found a cluster of brain-resident T cells mostly CD8+, in the temporal and the frontal cortex of the patient with NORSE._x000D_
Conclusions: The patient with NORSE presented higher activation of immune cells (microglia, T cells) and astrocytes; these could be involved in seizure persistence or adverse consequences of refractory SE. We also found an increase in the excitatory/GABAergic neuron ratio that might explain SE onset or persistence, or the common development of epilepsy after NORSE. New studies should be performed on analyzing the cells of blood and CSF drawn at the onset of NORSE and other forms of SE to distinguish mechanisms involved in SE development, refractoriness and consequences.

Funding: Institut Servier, NORSE Institute, Paratonnerre Association, Philippe Foundation