GSE211407
Single Cell RNA-Sequence Analyses Reveal Uniquely   Expressed Genes and Heterogeneous Immune Cell   Involvement in the Rat Model of Intervertebral   Disc Degeneration
Purpose:  We used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture Methods: Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution Results: Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus Conclusions: . Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations Overall design: Rats (n=8, male Sprague-Dawley, 8 weeks) underwent surgery for retroperitoneal exposure of the L4-L6 -lumbar spine. The L4-5 and L5-6 IVDs each received a single stab injury via a 27G needle and allowed to recover postoperatively for either 2 (n=4) or 8 weeks (n=4). Animals were then  euthanized and the IVD tissues were isolated and pooled from L4-5 and L5-6 (LDP= lumbar disc puncture), or from L2-3 and L3-4 (CON= control) via dissection, with care to remove unwanted peripheral tissues by microdissection. The separated tissue was digested via pronase and collagenase (< 4h total) to obtain IVD cells (51,655 and 60,626 total cell yield from CON and LDP respectively).