Successful neuronal nuclei isolation from flash-frozen brain tissue of patients with mesial temporal lobe epilepsy with hippocampal sclerosis

RESUMO

INTRODUÇÃO: Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE+HS) is one of the most frequent and severe types of epilepsy. HS is characterized by neuronal death and gliosis at the hippocampus and dentate gyrus segments. Currently, there is great appreciation for the diversity of cell composition even within the same tissue since DNA, RNA, and regulatory elements have different dynamic properties in each cell of the body. Thus, in a complex disorder scenario, each cell type may be the key to understanding how a disruption in the epigenome landscape can contribute to disease development. Single-cell omics techniques allow for the analysis at an individual cell level and mapping of different cell populations. However, the most technically challenging step for single-cell multi-omics analyses today is cell/nuclei isolation. Nuclei isolation is especially difficult in surgical tissue presenting cell loss. Also, lipid-rich tissue like the brain may impose additional problems.

OBJETIVOS: In this context, we developed an original experimental protocol for nuclei isolation from frozen tissue of patients with pharmacoresistant MTLE+HS treated by epilepsy surgery.

MÉTODOS: We used three samples of the flash-frozen sclerotic hippocampus: two samples weighing 50mg; and one with 75mg. The protocol followed the steps: (a) mechanical lysis with twenty pumps of each pestle at a 2mL ounce; (b) chemical lysis with incubating the macerated tissue in time lyses tested; (c) mechanical lysis passing eight times the homogenate into a syringe and 23G needle; (d) filter the sample in 40µm and 30µm filter; (e) centrifugation for 5min, at 500g and 4 °C; (f) remove cell clumps and debris with optiprep in 50% and 29% solutions; (g) centrifugation for 30min, at 10.100g and 4 °C; (h) resuspension at a final volume of 25uL and counting the viable and inviable nuclei with trypan blue at Neubauer chamber. Then, we proceeded with one hippocampus at a time and performed three combinations: (i) 50mg of tissue, 500uL of lysis buffer, and five minutes of incubation; (ii) 50mg of tissue, 500uL of lysis buffer, and ten minutes of incubation and (iii) 75mg of tissue, 750uL of lysis buffer and ten minutes of incubation.

RESULTADOS: We determined that the best quality and nuclei counting was obtained with the combination of 50mg of tissue, 500uL of lysis buffer, and ten minutes of incubation: 2.9x10e6 nuclei/mL of viable nuclei and 0.35x10e6 nuclei/mL of inviable (89% viability) (Table 1). We also observed the presence of clumps and cell debris; however, this did not impair single-nuclei multi-omic sequencing experiments. In addition, we determined that the isolated neuronal nuclei are suitable for multi-omic single-nucleus Assay for Transposase-Accessible Chromatin using Sequencing (snATAC-Seq) and single-nucleus Sequencing of RNA (snRNA-seq) analysis.

CONCLUSÃO: Although the protocol has a core skeleton established by Maitra et al. 2021 [1], we added modifications that substantially improved the final output. Thus, we obtained a final concentration of 2.900 nuclei/µL that is satisfactory to initiate single-nuclei multi-omic sequencing, in which the minimum required concentration is 160 nuclei/uL. We also obtained material with excellent quality in terms of membrane nucleus stability and the number of clumps and debris. Our work improved two essential steps for nuclei dissociation. Furthermore, it established an efficient protocol to be used in surgically removed brain tissue presenting a limited number of viable cells, such as that found in HS. To our knowledge, this is the first nuclei isolation protocol specially designed and optimized for the surgical tissue of patients with MTLE+HS. We expect that by using this protocol, we and others may generate new information about the underlying mechanisms leading to cell death in MTLE+HS.

BIBLIOGRAFIA: [1] Maitra et al., DOI:10.1038/s41596-021-00514-4

PALAVRA-CHAVE: Epilepsy; Single-cell; Sequencing